AU737017B2 - Nucleic acid probes and amplification oligonucleotides for neisseria species - Google Patents

Description

S F Ref: 400640D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant: a a a Actual Inventor(s): Address for Service: Invention Title: Gen-Probe Incorporated 10210 Genetic Center Drive San Diego California 92121 UNITED STATES OF AMERICA Yeasing Yang, Gary Bee and Sherrol McDonough Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Nucleic Acid Probes and Amplification Olgonucleotides for Neisseria Species The following statement is a full description of this invention, Including the best method of performing it known to me/us:- 5845

DESCRIPTION

NUCLEIC ACID PROBES AND AMPLIFICATION

OLIGONUCLEOTIDES

FOR NEISSERIA SPECIES FIELD OF THE INVENTION The inventions described and claimed herein relate to the design and use of amplification oligonucleotides and nucleic acid probes to Neisseria qonorrhoeae and Neisseria meninaitidis which allow detection of these organisms in test samples.

Backaround of the Invention **e 10 The genus Neisseria includes two gram-negative species of pyogenic cocci that are pathogenic for man, and that have no other known reservoir: the meningococcus _(Neisseria meninqitidis) and the gonococcus (Neisseria gonorrhoeae). A number of non-pathogenic species also inhabit 15 the upper respiratory tract of humans and may be easily confused with meningococci. Meningococcal meningitis was recognized as a contagious disease early in the 19th century and is especially prevalent among military personnel. The causative agent of meningococcal meningitis is Neisseria meningitidis Neisseria gonorrhoeae is one of the main causes of epidemic sexually transmitted disease and is prevalent in the United States. Infection with Neisseria qonorrhoeae causes many common symptoms including urethritis, cervicitis, and proctitis. In addition, chronic infection with Neisseria gonorrhoeae can cause pelvic inflammatory disease.

The Neisseria are gram-negative cocci which grow in meningococci have polysaccharide-containing capsules.

Gonococcis may also possess capsules, but the exact chemical composition of such a capsule is unknown. In addition, both gonococci and meningococci may have pili which play a role in virulence.

Meningococci and gonococci are difficult to cultivate and require special techniques to grow the organisms from body fluids. In addition, selective culture medium, (for example, Thayer-Martin medium) and growth in 3-10% carbon dioxide at approximately 35 0 C is required to maximize the culture of organisms.

In addition to the difficult cultivation, the gonococcus and meningococcus detection by immunoassay suffers a lack of sensitivity and specificity. This appears to be due to the cross reaction between various other pathogens and non-pathogenic microorganisms often found in the same clinical specimens.

Oligonucleotides for the amplification of nucleic acid for detection of Neisseria have been described. Biken- 20 meyer and Armstrong, J. Clin. Microbiol. '30:3089-3094 (1992), describe probe sets for use in the ligase chain reaction directed to the Opa and pilin genes of Neisseria gonorrhoeae. Kristiansen et al. Lancet 340:1432-1434 (1992) describe primers directed to an insertion element 25 referred to as IS1106 for amplification and detection of Neisseria meninqitidis. McLaughlin et al., Mol. and Cell Probes 7:7-17 (1993) describe primers for use in the polymerase chain reaction directed to the 16S-23S rRNA internal transcribed spacer and a set of primers directed to a subregion of the 16S rRNA of Neisseria menincitidis.

Probes for. the detection of rRNA or. rDNA sequences of Neisseria connorhoeae and/or Neisseria meninqitidis have been described by Granato and Franz J. Clin. Microbiol.

28:944-948, (1990), Wolff, U.S. Patent 5,173,401 (Dec. 22, 1992), Rossau and Van Heuverswijn, European Patent Application Publication No. 0 337 896, Hogan et al. PCT/US87/- 03-009, and Barns et al., U.S. Patent 5,217,862 (Jun. 8, 1993).

SUMMARY OF INVENTION The featured invention discloses and claims novel and useful amplification oligonucleotides, helper oligonucleotides, and oligonucleotide hybridization assay probes which are designed to be complementary to specific regions of the rRNA (ribosomal RNA) or rDNA (ribosomal

DNA)

nucleotide sequences of Neisseria, or oligonucleotides having a nucleic acid sequence substantially corresponding to a specific portion of Neisseria rRNA or rDNA nucleotide sequence or its complement. Because these amplification oligonucleotides, helper oligonucleotides and hybridization assay probes are derived from the rRNA of pathogenic Neisseria, a superior detection assay is obtained due to the higher level of RNA expressed from these rRNA genes and the lack of lateral transfer of the rRNA sequences between organisms.

The amplification oligonucleotides and oligonucleotide hybridization assay probes function by hybridizing to target Neisseria 16S and 23S rRNA. and/or rDNA gene sequences under stringent hybridization assay conditions.

In preferred embodiments, the probes and amplification oligonucleotides described herein, when used together, can 25 distinguish Neisseria meningitidis from other microorganisms found in clinical samples such as blood or tissues and from Neisseria aonorrhoeae species. Accordingly, the 'amplification oligonucleotides and hybridization assay probes may be used in an assay to specifically detect 30 and/or amplify Neisseria meninitidis-derived nucleic acids. In preferred embodiments, the hybridization assay probes described herein are able to selectively hybridize to nucleic acids from Neisseria meninqitidis over those from Neisseria gonorrhoeae under stringent hybridization conditions. In some embodiments of the present invention, the hybridization assay probe comprises an oligonucleotide that contains a reporter group such as an acridinium ester or a radioisotope to help identify hybridization of the probe to its target sequence. In some embodiments, the amplification oligonucleotide optionally has a nucleic acid sequence recognized by an RNA polymerase or which enhances transcription initiation by an RNA polymerase.

There is herein disclosed hybridization nucleic acids from Neisseria. Preferably, the following nucleotide sequences: assay probes useful for detecting the presence of hybridization assay probes are selected from the SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO:

GGCTGTTGCT

GGCTGTTGCT

CGCTGATATT

CGCTGGTATT

GGCUGUUGCU

GGCUGUUGCU

CGCUGAUAUU

CGCUGGUAUU

GAACGTACCG

GCCAATATCG

CCGCTACCCG

CATCGGCCGC

GAACGUACCG

GCCAAUAUCG

CCGCUACCCG

CAUCGGCCGC

AATATCAGCG

AATACCAGCG

AGCAACAGCC

AGCAACAGCC

AAUAUCAGCG

AAUACCAGCG

AGCAACAGCC

AGCAACAGCC

GGTAGCGG

GCGGCCGATG

GTACGTTC

CGATATTGGC

GGUAGCGG

GCGGCCGAUG

GUACGUUC

CGAUAUUGGC

There is further disclosed hybridization assay probes useful for detecting nucleic acids from Neisseria meningitidis. These hybridization assay probes are preferably selected from the following nucleotide sequences: SEQIDNO: 1V SEQ ID NO: 1, SEQIDNO: 1, SEQIDNO: 1( SEQ ID NO: 2, SEQ ID NO: 2( SEQ ID NO: 27 SEQ ID NO: 2E

GGCTGTTGCT

GGCTGTTGCT

CGCTGATATT

CGCTGGTATT

GGCUGUUGCU

GGCUGUUGCU

CGCUGAUAUU

CGCUGGUAUU

AATATCAGCG

AATACCAGCG

AGCAAOAGCC

AGCAACAGCC

AAUAUCAGCG

AAUACCAGCG,

AGCAACAGCC, and

AGCAACAGCC.

11ADav1Jh\T 1RT7P1()r 1 A In addition, there is disclosed hybridization assay probes useful for detecting Neisseria gonorrhoeae nucleic acids. Preferably, these hybridization assay probes have a nucleotide sequence selected from one of the following nucleotide sequences: SEQ ID NO: 1 SEQ ID NO: 3 SEQIDNO: 29 SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 34 In addition, there is GAACGTACCG

GGTAGCGG

GCCAATATCG

GCGGCCGATG

CCGCTACCCG

GTACGTTC

CATCGGCCGC

CGATATTGGC

GAACGUACCG

GGUAGCGG

GCCAAUAUCG

GCGGCCGAUG

CCGCUACCCG

GUACGUUC

CAUCGGCCGC

CGAUAUUGGC

disclosed a probe mix comprising a hybridization assay probe together o o*o.

*o o *oo oooooo* *oe oooo* oo* *ooo oo *oo* o with a helper oligonucleotide (probe). Preferably, helper oligonucleotides are used to facilitate the specific hybridization of the assay probe to its target nucleic acid; helper oligonucleotides are 15 described by Hogan and Milliman U.S. Patent No. 5,030,557 which is hereby incorporated by reference and enjoys common ownership with the present invention. Oligonucleotides used as helper probes described herein include the following sequences: SEQ ID NO: 2 SEQ ID NO: 4 20 SEQ ID NO: 39 SEQ ID NO: 40 SEQ ID NO: 13 SEQ ID NO: 14 SEQ ID NO: 35 SEQ ID NO: 36 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 37 SEQ ID NO: 38

GGGATAACTG

ACGGTACCTG

GGGAUAACUG

ACGGUACCUG

GCCTTCGGGT

GCTGATGACG

GCCUUCGGGU

GCUGAUGACG

TTTTCTTCCC

GCCGGTGCTT

UUUUCUUCCC

GCCGGUGCUU

ATCGAAAGAT

AAGAATAAGC

AUCGAAAGAU

AAGAAUAAGC

TGTAAAGGAC

GTACCTGAAG

UGUAAAGGAC

GUACCUGAAG

TGACAAAAGT

ATTCTTCAGG

UGACAAAAGU

AUUCUUCAGG

CAGCTAATAC

ACCGGCTAAC

CAGCUAAUAC

ACCGGCUAAC

TTTTGTCAGG

AATAAGCACC

UUUUGUCAGG

AAUAAGCACC

CCTTTACAAC

TACCGTCATC

CCUUUACAAC

UACCGUCAUC

CGCATACG

TACGTG

CGCAUACG

UACGUG

GAAGAAAA

GGC

GAAGAAAA

GGC

CCGAAGGC

AGC

CCGAAGGC, and

AGC

There is further disclosed compositions for detecting Neisseria meningitidis and Neisseria gonorrhoeae that are nucleic acid hybrids formed between an oligonucleotide as herein disclosed and a specific region of a nucleotide polymer from a Neisseria meningitidis or Neisseria gonorrhoeae. Generally, the nucleotide polymer contains a nucleic acid sequence that substantially corresponds to an oligonucleotide sequence as herein disclosed or its complement and is derived ftom the rRNA or the rDNA encoding the ribosomal RNA of the Neisseria meningitidis or Neisseria J:\D,-jvf jh\1 IRFRAM 70-, gonorrhoeae. The oligonucleotide present in these compositions may be an amplification oligonucleotide, a helper oligonucleotide, a hybridization assay probe, or a combination thereof.

Thus, compositions described herein may contain one or more amplification oligonucleotides, one or more helper oligonucleotides, and one or more hybridization assay probes.

The compositions herein disclosed containing a probe hybridized to its target sequence are useful for detecting the presence of a nucleic acid sequence. Compositions herein disclosed containing a helper oligonucleotide hybridized to its target nucleic acid sequence are useful for making a particular portion of the target nucleic acid available for hybridization. Compositions herein disclosed containing an oligonucleotide primer hybridized to its target sequence are useful for creating an initiation site for a polymerase at the 3' end of the primer, and/or providing a template for extension of the 3' end of the target sequence.

There is further disclosed methods for detecting the presence of Neisseria in which a test sample is contacted with a nucleic acid hybridization assay probe under stringent hybridization assay conditions wherein the nucleic acid hybridization assay probe is capable of hybridizing to 15 Neisseria meningitidis target nucleic acid sequences and not to the nucleic acid sequences from °Neisseria gonorrhoeae. There is further disclosed oligonucleotides and the equivalents thereof used in these methods that optionally contain a reporter molecule that aids in the identification of the hybridization of the probe to its target sequence. This invention is useful for detecting the presence of Neisseria nucleic acids in test samples from humans such as blood, blood derived e*i. 20 samples, tissues, tissue derived samples, other body fluids and body samples.

There is further disclosed methods for detecting the presence of Neisseria meningitidis in which the nucleic acid is amplified using at least one amplification oligonucleotide as herein disclosed. In preferred embodiments, that amplification is then followed by a detection step in which the amplified nucleic acid is detected using an oligonucleotide hybridization assay probe as herein disclosed. The methods disclosed herein also contemplate the use of amplification oligonucleotides which include the nucleotide sequence for an RNA promoter.

In additiorihere is featured amplification oligonucleotides useful for detection of organisms of the genus Neisseria in an amplification assay. Such oligomers preferably substantially correspond to one of the following nucleotide sequences: SEQ ID NO: 5 GTCCCCTGCT TTCCCTCTCA

AGAC

SEQ ID NO: 6 GGCGAGTGGC GAACGGGTGA

GTAACATA

SEQ ID NO: 7 GCTGCTGCAC GTAGTTAGCC GGTGCTTATT

CTTCAG

SEQ ID NO: 8 GTTAGCCGGT GCTTATTCTT CAGGTACCGT

CATCG

SSEQ ID NO: 9 CGGGTTGTAA AGGACTTTTG TCAGGGAAGA AAAGGCCGTT SEQ ID NO: 10 GAAGGCCTTC GGGTTGTAAA GGAC O^ -4 F1ADav1Jh\T SEQ ID NO:41 GUCCCCUGCU UUCCCUCUCA AGAC SEQ ID NO: 42 GGCGAGUGGC GAACGGGUGA GUAACAUA SEQ ID NO: 44 GUUAGCCGGU GCUUAUUCUU CAGGUACCGU CAUCG SEQ ID NO: 45 CGGGUUGUAA AGGACUUUUG UCAGGGAAGA AAGGCCGUU, and SEQ ID NO: 46 GAAGGCCUUC GGGUUGUAAA GGAC where the oligomer may be unmodified or contain a modification such as addition of a specific nucleic acid sequence to 5' terminus that is recognized by an RNA polymerase, (including but not limited to the promoter sequence for T7, T3, or SP6 RNA polymerase), and/or sequences which enhance initiation of RNA transcription by an RNA polymerase. One example of a promoter sequence includes the sequence SEQ ID NO: 53 5'-AATTTAATACGACTCACTATAGGGAGA-3'.

Other examples of useful promoter sequences are contained in various commercially available vectors including, for example, pBluescript@ vectors from Strata-gene Cloning Systems (San Diego, CA) or the pGEM T M vectors from Promega Corp. (Madison, WI).

In another aspect, there is disclosed the amplification oligonucleotides bind to or cause elongation through sequences substantially corresponding to the following sequences:

S..

0 *5 SEQ ID NO: 23 SEQ ID NO: 24 SEQ ID NO: 19 20 SEQ ID NO: 21 SEQ ID NO: 20 SEQ ID NO: 22 SEQ ID NO: 47 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 50 SEQ ID NO: 51 SEQ ID NO: 52

GTCTTGAGAG

TATGTTACTC

CTGAAGAATA

CGATGACGGT

AACGGCCTTT

GTCCTTTACA

GUCUUGAGAG

UAUGUUACUC

CUGAAGAAUA

CGAUGACGGU

AACGGCCUUU

GUCCUUUACA

GGAAAGCAGG

ACCCGTTCGC

AGCACCGGCT

ACCTGAAGAA

TCTTCCCTGA

ACCCGAAGGC

GGAAAGCAGG

ACCCGUUCGC

AGCACCGGCU

ACCUGAAGAA

UCUUCCCUGA

ACCCGAAGGC

that contain one or

GGAC

CACTCGCC

AACTACGTGC AGCAGC TAAGCACCGG CTAAC CAAAAGTCCT TTACAACCCG

CTTC

GGAC

CACUCGCC

AACUACGUGC AGCAGC UAAGCACCGG CUAAC CAAAAGUCCU UUACAACCCG

CUUC

more of the oligonucleotides as herein There is further disclosed kits disclosed including amplification oligonucleotides, helper oligonucleotides and hybridisation assay probes. In preferred embodiments, a kit disclosed herein includes at least one amplification oligonucleotide and one hybridisation assay probe capable of distinguishing Neisseria, Neisseria Smeningitidis or Neisseria gonorrhoeae from other microorganisms.

S Background descriptions of the use of nucleic acid hybridisation to detect particular nucleic 3s zacid sequences are given in Kohne, U.S. Patent No. 4,851,330 issued July 25, 1989, and by OF:\ v FF61 r \av .ih\LIIRFF106179snecdnr-,-- 8 Hogan et al., International Patent Application No. PCT/US87/03009, entitled "Nucleic Acid Probes for Detection and/or Quantitation of Non-Viral Organisms", both references hereby incorporated by reference herein. Hogan et al., supra, describe methods for determining the presence of a non-viral organism or a group of non-viral organisms in a sample sputum, urine, blood and tissue sections, food, soil and water).

In the most preferred embodiments, the compositions, probe mixes, probes, amplification primers, helper oligonucleotides and the like have a nucleotide sequence that consists of the specified nucleic acid sequence rather than substantially corresponding to the nucleic acid sequence.

These most preferred embodiments use the sequence listed in the sequence listing which forms part of the present disclosure.

According to a first embodiment of the invention, there is provided a probe mix for detecting Neisseria gonorrhoeae comprising a nucleic acid hybridisation assay probe for detecting Neisseria gonorrhoeae under stringent hybridisation assay conditions which substantially corresponds to an S. oligonucleotide having a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1 GAACGTACCG

GGTAGCGG,

SEQ ID NO: 31 GAACGUACCG

GGUAGCGG,

SEQ ID NO: 3 GCCAATATCG GCGGCCGATG, and SEQ ID NO: 32 GCCAAUAUCG

GCGGCCGAUG,

and a helper oligonucleotide 10-100 nucleotides in length which hybridises to a Neisseria gonorrhoeae nucleic acid sequence under stringent hybridisation conditions, said helper oligonucleotide having a nucleic acid sequence substantially corresponding to: sequence SEQ ID .NO: 2 or SEQ ID NO: 39 when said nucleic acid hybridisation assay probe substantially 20 corresponds to SEQ ID NO: 1 or SEQ ID NO: 31; and to SEQ ID NO: 4 or SEQ ID NO: 40 when said nucleic acid hybridisation assay probe substantially corresponds to SEQ ID NO: 3 or SEQ ID NO: 32.

According to a second embodiment of the invention, there is provided a composition for detecting Neisseria gonorrhoeae comprising a nucleic acid hybrid formed under stringent hybridisation conditions between a Neisseria gonorrhoeae nucleic acid and an oligonucleotide having a nucleic acid sequence substantially corresponding to a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1 GAACGTACCG

GGTAGCGG,

SEQ ID NO: 31 GAACGUACCG

GGUAGCGG,

>SEQ ID NO: 29 CCGCTACCCG

GTACGTTC,

Q ID NO: 33 CCGCUACCCG

GUACGUUC,

f1ADav1Jh\f Int7piorr7o.- SEQ ID NO: 3 GCCAATATCG

GCGGCCGATG,

SEQ ID NO: 32 GCCAAUAUCG

GCGGCCGAUG,

SEQ ID NO: 30 CATCGGCCGC CGATATTGGC, and SEQ ID NO: 34 CAUCGGCCGC

CGAUAUUGGC.

According to a third embodiment of the invention, there is provided a method for amplifying Neisseria gonorrhoeae nucleic acid in a test sample comprising amplifying said nucleic acid with one or more amplification oligonucleotides that will bind to or cause elongation through a nucleic acid sequence substantially corresponding to one or more of the following nucleic acid sequences: SEQ ID NO: 23 GTCTTGAGAG GGAAAGCAGG

GGAC,

SEQ ID NO: 47 GUCUUGAGAG GGAAAGCAGG

GGAC,

SEQ ID NO: 24 TATGTTACTC ACCCGTTCGC CACTCGCC, and SEQ ID NO: 48 UAUGUUACUC ACCCGUUCGC

CACUCGCC,

"5 wherein at least one of said amplification oligonucleotides optionally has a nucleic acid sequence recognised by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase.

According to a fourth embodiment of the invention, there is provided a method for amplifying Neisseria gonorrhoeae nucleic acid in a test sample comprising amplifying said nucleic acid with one or more amplification oligonucleotides that will bind to or cause elongation through a nucleic acid sequence substantially corresponding to one or more of the following nucleic acid sequences: SEQ ID NO: 19 CTGAAGAATA AGCACCGGCT AACTACGTGC

AGCAGC,

SEQ ID NO: 49 CUGAAGAAUA AGCACCGGCU AACUACGUGC

AGCAGC,

SEQ ID NO: 20 AACGGCCTTT TCTTCCCTGA CAAAAGTCCT

TTACAACCCG,

SEQ ID NO: 51 AACGGCCUUU UCUUCCCUGA CAAAAGUCCU

UUACAACCCG,

SEQ ID NO: 21 CGATGACGGT ACCTGAAGAA TAAGCACCGG

CTAAC,

SEQ ID NO: 50 CGAUGACGGU ACCUGAAGAA UAAGCACCGG

CUAAC,

SEQ ID NO: 22 GTCCTTTACA ACCCGAAGGC CTTC, and SEQ ID NO: 52 GUCCUUUACA ACCCGAAGGC

CUUC,

wherein at least one of said amplification oligonucleotides optionally has a nucleic acid sequence recognised by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase.

According to a fifth embodiment of the invention, there is provided a kit containing four oligonucleotides, each said oligonucleotide having a nucleic acid sequence substantially corresponding to one of the following nucleic acid sequences: EQ ID NO: 5 GTCCCCTGCT TTCCCTCTCA AGAC, or SEQ ID NO: 41 GUCCCCUGCU UUCCCUCUCA AGAC, and [i:\Dav[.,ih\I IRFF1() I170- SEQ ID NO: 6 GGCGAGTGGC GAACGGGTGA GTAACATA, or SEQ ID NO: 42 GGCGAGUGGC GAACGGGUGA GUAACAUA, and SEQ ID NO: 2 GGGATAACTG ATCGAAAGAT CAGCTAATAC CGCATACG, or SEQ ID NO: 39 GGGAUAACUG AUCGAAAGAU CAGCUAAUAC CGCAUACG, and SEQ ID NO: 1 GAACGTACCG GGTAGCGG, or SEQ ID NO: 31 GAACGUACCG

GGUAGCGG,

wherein at least one of said oligonucleotides optionally has a nucleic acid sequence recognised by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase.

According to a sixth embodiment of the invention, there is provided a kit containing three oligonucleotides, each said oligonucleotide having a nucleic acid sequence substantially corresponding to one of the following nucleic acid sequences: SEQ ID NO: 5 GTCCCCTGCT TTCCCTCTCA AGAC, or SEQ ID NO: 41 GUCCCCUGCU UUCCCUCUCA AGAC, and SEQ ID NO: 2 GGGATAACTG ATCGAAAGAT CAGCTAATAC CGCATACG, or SEQ ID NO: 39 GGGAUAACUG AUCGAAAGAU CAGCUAAUAC CGCAUACG, and SEQ ID NO: 1 GAACGTACCG GGTAGCGG, or SEQ ID NO: 31 GAACGUACCG

GGUAGCGG,

wherein at least one of said oligonucleotides optionally has a nucleic acid sequence recognised by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase.

rr:\r-Mv ib\1 TAFF1flfl7Qqnercdoncc Detailed Description of the Invention A. Definitions The following terms have the indicated meanings in the specification unless expressly indicated to have a different meaning.

By "target nucleic acid" is meant a nucleic acid having a target nucleotide sequence.

By "oligonucleotide" is meant a single-stranded nucleotide polymer made of more than 2 nucleotide subunits covalently joined together. Preferably between 10 and 100 nucleotide units are present, most preferably between 12 and 50 nucleotides units are joined together. The sugar groups of the nucleotide subunits may be ribose, deoxyribose or modified derivatives thereof such as 2'-O-methyl ribose. The nucleotide subunits of an oligonucleotide may be joined by phosphodiester linkages, phosphorothioate linkages, methyl phosphonate linkages or by other rare or non-naturally-occurring linkages that do not prevent hybridization of the oligonucleotide. Furthermore, an 20 oligonucleotide may have uncommon nucleotides or nonnucleotide moieties. An oligonucleotide as defined herein is a nucleic acid, preferably DNA, but may be RNA or have a combination of ribo- and deoxyribonucleotides covalently linked. Oligonucleotide probes and amplification oligo- 25 nucleotides of a defined sequence may be produced by techniques known to those of ordinary skill in the art, such as by chemical or biochemical synthesis, and by in vitro or in vivo expression from recombinant nucleic acid molecules, bacterial or retroviral vectors. As 30 intended by this disclosure, an oligonucleotide does not consist of wild-type chromosomal DNA or the in vivo transcription products thereof. One use of a probe is as a hybridization assay probe; probes may also be used as in vivo or in vitro therapeutic amplification oligomers or antisense agents to block or inhibit gene transcription, or translation in diseased, infected, or pathogenic cells.

By "target nucleic acid sequence", "target nucleotide sequence" or "target sequence" is meant a specific deoxyribonucleotide or ribonucleotide sequence comprising all or a part of the nucleotide sequence of a single-stranded nucleic acid molecule, and the deoxyribonucleotide or ribonucleotide sequence complementary thereto.

Nucleic acid hybridization is the process by which two nucleic acid strands having completely or partially complementary nucleotide sequences come together under predetermined reaction conditions to form a stable, double-stranded hybrid with specific hydrogen bonds.

Either nucleic acid strand may be a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), or an analog of one of these nucleic acids; thus hybridization can involve RNA:RNA hybrids, DNA:DNA hybrids, or RNA:DNA hybrids.

The term "hybridization" as used in this application, refers to the ability of two completely or partly complementary single nucleic acid strands to come together in an antiparallel orientation to form a stable structure having a double-stranded region. The two constituent strands of this double-stranded structure, sometimes called a hybrid, are held together with hydrogen bonds. Although these hydrogen bonds most commonly form between nucleotides containing the bases adenine and thymine or uracil (A and :25 T or U) or cytosine and guanine (C and G) on single nucleic acid strands, base pairing can form between bases who are not members of these "canonical" pairs. Noncanonical base pairing is well-known in the art. See e The Biochemistry of the Nucleic Acids (Adams et al., eds., 1992).

"Stringent" hybridization assay conditions refer to conditions wherein a specific hybridization assay probe is able to hybridize with target nucleic acids (preferably rRNA or rDNA of a Neisseria, Neisseria menincitidis or Neisseria conorrhoeae) over other nucleic acids present in the test sample derived either from other microorganisms or from humans. It will be appreciated that these conditions may vary depending upon factors including the GC content and length of the probe, the hybridization temperature, the composition of the hybridization reagent or solution, and the degree of hybridization specificity sought. Specific stringent hybridization conditions are provided in the disclosure below.

As an example of specific stringent hybridization conditions useful in detecting Neisseria, Neisseria meninqitidis or Neisseria gonorrhoeae, for the hybridization assay probes of this invention, a set of preferred stringent hybridization assay conditions was used. One preferred set comprised hybridizing the target nucleic acid and hybridization probe together in 100 Al of 0.05 M lithium succinate (pH 6 M LiCI, 1% lithium lauryl sulfate, 10 mM ethylene diamine tetraacetic acid (EDTA), 10 mM ethylene glycol bis (beta-amino ethyl ether) tetraacetic acid (EGTA) at 600C for minutes, then adding 300l of 0.15 M sodium tetraborate (pH 1% TRITON® X-100 at 600C for 5-7 minutes.

20 Additional sets of stringent hybridization conditions can be determined after reading the present disclosure by those or ordinary skill in the art.

By "probe" is meant a single-stranded oligonucleotide having a sequence partly or completely complementary to a nucleic acid sequence sought to be detected, so as to hybridize thereto under stringent hybridization conditions. The term "probe" is meant to exclude nucleic acids normally existing in nature. Purified oligonucleotide probes may be produced by techniques known in the art such as chemical synthesis and by in vitro or in vivo expression from recombinant nucleic acid molecules, e.g., retroviral vectors. Preferably probes are 10 to 100 nucleotides in length. Probes may or may not have regions which are not complementary to a target sequence, so long as such sequences do not substantially affect hybridization under stringent hybridization conditions. If such regions exist they may contain a 5' promoter sequence 13 and/or a binding site for RNA transcription, a restriction endonuclease recognition site, or may contain sequences which will confer a desired secondary or tertiary structure, such as a catalytic active site or a hairpin structure on the probe, on the target nucleic acid, or both.

A probe may be labeled with a reporter group moiety such as a radioisotope, a fluorescent or chemiluminescent moiety, with an enzyme or ligand, which can be used for detection or confirmation that the probe has hybridized to the target sequence.

As used in this disclosure, the phrase "a probe (or oligonucleotide) having a nucleic acid sequence consisting essentially of a sequence selected from" a group of specific sequences means that the probe, as a basic and novel characteristic, is capable of stably hybridizing to a nucleic acid having the exact complement of one of the listed nucleic acid sequences of the group under stringent hybridization conditions. An exact complement includes the corresponding DNA or RNA sequence.

The phrase "substantially corresponding to a nucleic acid sequence" means that the referred-to nucleic acid is sufficiently similar to the nucleic acid sequence such that the referred-to nucleic acid has similar hybridization properties to a nucleic acid sequence in that it would hybridize with the same target nucleic acid sequences under stringent hybridization conditions.

One skilled in the art will understand that substantially corresponding probes and primers of the invention can vary from the referred-to sequence and still hybridize to the same target nucleic acid sequence. This variation from the nucleic acid may be stated in terms of a percentage of identical bases within the sequence or the percentage of perfectly complementary bases between the probe or primer and its target sequence. One skilled in the art will also understand that this variation could be expressed as the number of bases in a probe or primer or the number of mismatched bases of a probe that do not hybridize to a corresponding base of a target nucleic acid sequence. Probes or primers of the present invention substantially correspond to a nucleic acid sequence if these percentages are from 100% to 80% or from 0 base mismatches in a 10 nucleotide target sequence to 2 bases mismatched in a 10 nucleotide target sequence.

In preferred embodiments, the percentage is from 100% to 85%. In more preferred embodiments, this percentage is from 90% to 100%; in other preferred embodiments, this percentage is from 95% to 100%. One skilled in the art will understand the various modifications to the hybridization conditions that might be required at various percentages of complementarity to allow hybridization to a specific target sequence without causing an unacceptable level of non-specific hybridization.

By "nucleic acid hybrid" or "hybrid" is meant a nucleic acid structure containing a double-stranded, hydrogen-bonded region, preferably of between 10 and 100 nucleotides in length, most preferably of between about 12 20 and 50 nucleotides in length, wherein each strand is complementary to the other and wherein the region is sufficiently stable under stringent hybridization conditions to be detected by means including but not limited to chemiluminescent or fluorescent light detection, autoradiography, or gel electrophoresis. Such hybrids may comprise RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules or duplex molecules containing analogs of these nucleic acids.

By "complementary" is meant that the nucleotide sequences of similar regions of two single-stranded nucleic acids, or to different regions of the same singlestranded nucleic acid have a nucleotide base composition S" that allow the single strands to hybridize together in a stable double-stranded hydrogen-bonded region under stringent hybridization conditions. When a contiguous sequence of nucleotides of one single-stranded region is able to form a series of "canonical" hydrogen-bonded base pairs with an analogous sequence of nucleotides of the other single-stranded region, such that A is paired with U or T and C is paired with G, the nucleotides sequences are "perfectly" complementary.

By "conservatively modified variants" is meant nucleic acids or oligonucleotides having a nucleotide sequence that is complementary to a nucleic acid region of another nucleic acid, such region in turn being perfectly complementary to a reference nucleic acid. Such conservatively modified variants are able to stably hybridize to a target nucleic acid region having a Neisseria, Neisseria meninoitidis or Neisseria qonorrhoeae nucleotide sequence under stringent hybridization conditions.

By "amplification oligonucleotide" is meant an oligonucleotide capable of hybridizing to a target nucleic acid sequence and acting as a primer for nucleic acid synthesis or a promoter template for synthesis of a complementary strand, thereby forming a functional promoter sequence) or both, for the initiation of nucleic acid 20 synthesis. If the amplification oligonucleotide is designed to initiate RNA synthesis, the oligonucleotide may contain nucleotide sequences which are non-complementary to the target sequence, but are recognized by an RNA polymerase (such as T7, T3 and SP6 RNA polymerase) An :25 amplification oligonucleotide may or may not have a 3' terminus which is modified to prevent or lessen the amount of primer extension. An amplification oligonucleotide as defined herein will preferably be between 10 and 100 nucleotides in length; most preferably between about 12 and 50 nucleotides in length. While the amplification oligonucleotides of the present invention may be chemically synthesized or derived from a vector, such oligonucleotides are not naturally-occurring nucleic acids.

By "nucleic acid amplification" or "target amplification" is meant increasing the number of nucleic acid molecules having at least one target nucleic acid sequence.

By "antisense" or "negative sense" is meant having a nucleic sequence complementary to that of a reference nucleic acid sequence.

By "sense", "same-sense" or "positive sense" is meant having a nucleic acid sequence analogous to that of a reference nucleic acid sequence.

By "helper oligonucleotide" is meant a nucleic acid probe designed to hybridize with the target nucleic acid at a different locus than that of a labeled probe, thereby either increasing the rate of hybridization of the labeled probe, increasing the melting temperature (Tm) of the target:labeled probe hybrid, or both.

"Phylogenetically closely related" means that the organisms are closely related to each other in an evolutionary sense and therefore would have a higher total nucleic acid sequence homology than organisms that are more distantly related. Organisms occupying adjacent and next to adjacent positions on the phylogenetic tree are closely related. Organisms occupying positions further 20 away than adjacent or next to adjacent positions on the phylogenetic tree will still be closely related if they have significant total nucleic acid sequence homology.

B. Hybridization Conditions and Probe/Primer Design Hybridization reaction conditions, most importantly the temperature of hybridization and the concentration of salt in the hybridization solution, can be selected to allow the amplification oligonucleotides or hybridization probes of the present invention to preferentially hybridize to nucleic acids having a target Neisseria nucleotide 30 sequence, and not to other non-target nucleic acids suspected of being present in the test sample. At decreased salt concentrations and/or increased temperatures (called increased stringency) the extent of nucleic acid hybridization decreases as hydrogen bonding between paired nucleotide bases in the double-stranded hybrid molecule is disrupted; this process is called "melting".

Generally speaking, the most stable hybrids are those having the largest number of contiguous perfectly matched hydrogen-bonded) nucleotide base pairs. Thus, such hybrids would usually be expected to be the last to melt as the stringency of the hybridization conditions increases. However, a double-stranded nucleic acid region containing one or more mismatched, "non-canonical", or imperfect base pairs (resulting in weaker or non-existent base pairing at that position in the nucleotide sequence of a nucleic acid) may still be sufficiently stable under conditions of relatively high stringency to allow the nucleic acid hybrid to be detected in a hybridization assay without cross reacting with other, non-selected nucleic acids present in the test sample.

Hence, depending on the degree of similarity between the nucleotide sequences of the target nucleic acid and those of non-target nucleic acids belonging to phylogenet- 20 ically distinct, but closely-related organisms on one **hand, and the degree of complementarity between the nucleotide sequences of a particular amplification oligonucleotide or hybridization probe and those of the target and non-target nucleic acids on the other, one or more mismatches will not necessarily defeat the ability of the oligonucleotide to hybridize to that nucleic acid and not to non-target nucleic acids.

The hybridization assay probes of the present invention were chosen, selected, and/or designed to maximize 30 the difference between the melting temperatures of the probe:target hybrid (Tm, defined as the temperature at which half of the potentially double-stranded molecules in a given reaction mixture are in a single-stranded, denatured state) and the Tm of a mismatched hybrid formed between the probe and the rRNA or rDNA of the phylogenetically most closely-related organisms expected to be present in the test sample, but not sought to be detected.

While the unlabeled amplification oligonucleotides and helper oligonucleotides need not have such an extremely high degree of specificity as the labeled hybridization assay probe to be useful in the present invention, they are designed in a similar manner to preferentially hybridize to one or more target nucleic acids over other nucleic acids.

Probes specific for Neisseria meninqitidis were designed using sequences determined in prospective target areas using primers complementary to the 16S rRNAs of strains of Neisseria including Neisseria gonorrhoeae

(ATCC

No. 19424), Neisseria meninqitidis serogroup A (ATCC No.

13077), serogroup C (ATCC No. 23248) and serogroup L (ATCC No. 43828), clinical isolates of Neisseria meiniqitidis, Neisseria lactamica (ATCC NO. 29193), Neisseria cinerea (ATCC NO. 14685), Neisseria mucosa (ATCC NO. 19696), Neisseria sicca (ATCC NO. 29193) and Kinqella kingae (ATCC No. 23330). The nucleic acid sequence from phylogenetically near neighbors, including the published sequence of 20 Neisseria conorrhoeae NCTC 8375, Rossau et al. Nuc. Acids Res. 16:6227 were also used as comparisons with the nucleic sequences from Neisseria meninqitidis to determine variable regions.

To facilitate the identification of nucleic acid 25 sequences to be used as probes and amplification oligonucleotides, the nucleotide sequences from different species of organisms were first aligned to maximize homology.

Within the rRNA molecule there is a close relationship "between the overall structure and function. This imposes restrictions on evolutionary changes in the primary sequence so that the secondary structure is maintained.

For example, if a base is changed on one side of a helix, a compensating change may be evolutionarily made to the other side to preserve the complementarity (this is referred to as co-variance) This allows two very different sequences to be aligned using the conserved primary sequence and also the conserved secondary structure 19 elements as points of reference. Potential target sequences for the hybridization probes were identified by noting variations in the homology of the aligned sequences in certain discrete regions (variable regions) of the rRNA and rDNA sequences.

The sequence evolution at each of the variable regions is mostly divergent. Because of the divergence, more distant phylogenetic relatives of Neisseria meninaitidis or Neisseria aonorrhoeae tend to show greater variability in a given variable region than phylogenetically closer relatives. The observed sufficient variation between Neisseria meninaitidis and Neisseria aonorrhoeae species was used to identify preferred target sites and design useful probes.

We have identified sequences which vary between Neisseria meninaitidis and Neisseria aonorrhoeae, between these and other Neisseria species, and between members of the genus Neisseria and other organisms by comparative analysis of rRNA sequences published in the literature or determined in the laboratory. Computers and computer programs which may be used or adapted for the purposes herein disclosed are commercially available. We have seen .sufficient variation between the target organisms and the closest phylogenetic relative likely to be found in the same sample to design the present probes. The Neisseria meninqitidis strains have been classified into three sequence groups in the probe region represented by serogroups A, C and L.

e. Merely identifying putatively unique potential target nucleotide sequences does not guarantee that a functionally species-specific hybridization assay probe may be made to hybridize to Neisseria' rRNA or rDNA comprising that sequence. Various other factors will determine the suitability of a nucleic acid locus as a target site for species-specific probes. Because the extent and specificity of hybridization reactions such as those described herein are affected by a number of factors, manipulation of one or more of those factors will determine the exact sensitivity and specificity of a particular oligonucleotide, whether perfectly complementary to its target or not. The importance and effect of various assay conditions are known to those skilled in the art as described in Hogan et al., PCT/US87/03009, and Hogan and Hammond, U.S. Patent No. 5,216,143, and Kohne, U.S. Patent No.

4,851,330 which share the same assignee as the present application and are hereby incorporated by reference herein.

The desired temperature of hybridization and the hybridization solution composition (such as salt concentration, detergents and other solutes) can also greatly affect the stability of double-stranded hybrids. Conditions such as ionic strength and the temperature at which a probe will be allowed to hybridize to target must be taken into account in constructing a group- or speciesspecific probe. The thermal stability of hybrid nucleic _acids generally increases with the ionic strength of the 20 reaction mixture. On the other hand, chemical reagents which disrupt hydrogen bonds, such as formamide, urea, dimethyl sulfoxide and alcohols, can greatly reduce the thermal stability of the hybrids.

To maximize the specificity of a probe for its target, the subject probes of the present invention were designed to hybridize with their targets under conditions of high stringency. Under such conditions only single nucleic acid strands having a high degree of complementarity will hybridize to each other; single nucleic acid strands without such a high degree of complementarity will not form hybrids. Accordingly, the stringency of the assay :'".conditions determines the amount of complementarity which should exist between two nucleic acid strands in order to form a hybrid. Stringency is chosen to maximize the difference in stability between the hybrid formed between the probe and the target nucleic acid and potential hybrids between the probe and any non-target nucleic acids present.

Proper specificity may be achieved by minimizing the length of the probe having perfect complementarity to sequences of non-target organisms, by avoiding G and C rich regions of homology to non-target sequences, and by constructing the probe to contain as many destabilizing mismatches to nontarget sequences as possible. Whether a probe sequence is useful to detect only a specific type of organism depends largely on the thermal stability difference between probe:target hybrids versus potential probe:nontarget hybrids. In designing probes, the differences in the T, values between these hybrids should be made as large as possible (preferably about 5SC or more).

Manipulation of the T, can be accomplished by changes to probe length and probe composition (GC content vs. AT content).

In general, the optimal hybridization temperature for oligonucleotide probes of about 10-50 nucleotides in 20 length is approximately 5 0 C below the melting temperature for a given duplex. Incubation at temperatures below the optimum temperature may allow mismatched base sequences to hybridize and can therefore decrease specificity. The longer the probe, the more hydrogen bonding between base 25 pairs and, in general, the higher the Increasing the percentage of G and C also increases the Tm because G-C base pairs exhibit additional hydrogen bonding and therefore greater thermal stability than A-T base pairs.

A preferred method to determine T, measures hybridization using a Hybridization Protection Assay (HPA) according to Arnold et al., U.S. Patent No. 5,283,174 which S: enjoys exclusive ownership with the present application *0 and is incorporated by reference herein. Tm can be measured using HPA in the following manner. A probe:target hybrid is formed in lithium succinate buffered solution (0.1 M lithium succinate buffer, pH 5.0, 2 mM ethylenediamine tetraacetic acid (EDTA), 2 mM ethylene glycol-bis (3-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), 10% (w/v) lithium lauryl sulfate) using an excess amount of target.

Aliquots of the hybrid are then diluted in the lithium succinate buffered solution and incubated for five minutes at various temperatures starting below that of the anticipated Tm for example, 55 0 C and increasing in 2-5 0 C increments. This solution is then diluted with a mild alkaline borate buffer (0.15 M sodium tetraborate, pH 7.6, 5% (v/v) TRITON® X-100) and incubated at a lower temperature (for example 50 0 C) for ten minutes. Under these conditions the acridinium ester attached to a single-stranded probe is hydrolyzed while the acridinium ester attached to hybridized probe is relatively protected from hydrolysis. Thus, the amount of acridinium ester remaining is proportional to the amount of hybrid and can be measured by the chemiluminescence produced from the acridinium ester upon the addition of hydrogen peroxide followed by alkali. Chemiluminescence can be measured in a luminometer Gen- Probe LEADER® I or LEADER® 50). The resulting data are 20 plotted as percent of maximum signal (usually from the lowest temperature) versus temperature. The Tm is defined as the temperature at which 50% of the maximum signal remains. In addition to the method above, Tm may be determined by isotopic methods well known to those skilled in the art Hogan et al., supra).

It should be noted that the Tm for a given hybrid varies depending on the hybridization solution used.

Factors such as the salt concentration, detergents, and other solutes can effect hybrid stability during thermal 30 denaturation Sambrook, E.F. Fritsch and T. Maniatis, 2 Molecular Cloning, ch. 11 (2d ed. 1989)). Conditions a a such as ionic strength and incubation temperature under which a probe will be used to hybridize to target should be taken into account in constructing a probe. On the other hand, chemical reagents which disrupt hydrogen bonds such as formamide, urea, dimethylsulfoxide and alcohols, can greatly reduce the thermal stability of the hybrids.

To ensure the probe is specific for its target, it is desirable to have probes which hybridize only under conditions of high stringency. Under conditions of high stringency only highly complementary nucleic acid hybrids will form; hybrids without a sufficient degree of complementarity will not form. Accordingly, the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands to form a hybrid. Stringency is chosen to maximize the difference in stability between the hybrid formed with the target and other nucleic acid sequences.

The length of the target nucleic acid sequence and, accordingly, the length of the probe sequence can also be important. In some cases, there may be several sequences from a particular region, for example, a variable region varying in location and length, which yield probes with the desired hybridization characteristics. In other cases, one probe may be significantly better than another probe with a nucleotide sequence differing by a single 20 base. While it is possible for nucleic acids that are not perfectly complementary to hybridize, the longest stretch of perfectly homologous base sequence will generally determine hybrid stability, with the composition of the base pairs also playing a role.

25 Regions of rRNA which form strong internal structures S. inhibitory to hybridization are less preferred target regions at least in assays in which helper probes are not used. Likewise, probe designs which result in extensive self complementarity should be avoided. If one of the two strands is wholly or partially involved in an intramolecular or intermolecular hybrid it will be less able to participate in the formation of a new intermolecular S" probe:target hybrid. Ribosomal RNA molecules are known to form very stable intramolecular helices and secondary structures by hydrogen bonding. By designing a hybridization assay so that a substantial portion of the targeted sequence remains in a single-stranded state until hybridization with the probe, the rate and extent of hybridization between probe and target may be greatly increased.

One way this may be accomplished is by choosing as a target nucleotide sequence a sequence that is relatively uninvolved in intramolecular hydrogen-bonding Alternatively or additionally, the hybridization assay probe may be used in a probe mix with helper oligonucleotides which can make the target site more accessible for hybridization with the hybridization assay probe.

A DNA target occurs naturally in a double-stranded form as does the product of the polymerase chain reaction (PCR). These double-stranded targets are naturally inhibitory to hybridization with a probe and require denaturation prior to hybridization. Appropriate denaturation and hybridization conditions are known in the art E. M. Southern, J. Mol. Bio. 98:503 (1975)).

A number of formulae are available which will provide an estimate of the melting temperature for perfectly matched oligonucleotides to their target nucleic acids.

One such formula, Tm 81.5 16.6(loglo[Na])+ 0.41(fraction G C)-(600/N) (where N the length of the oligonucleotide in number of nucleotides) provides a good estimate for the Tm for oligonucleotides between 14 and 60 or 70 nucleotides in 3' length. From such calculations, subsequent empirical verification or "fine tuning" of the Tm may be made using screening techniques well known in the art. For further *-.information on hybridization and oligonucleotide probes see, Sambrook et al., 2 Molecular Cloning: A Laboratory Manual (Cold Springs Harbor Laboratory Press 1989) hereby incorporated by reference herein (at Chapter 11) This reference, among others well known in the art, also provides estimates of the effect of mismatches on the Tm of a hybrid. Thus, from the known nucleotide sequence of a given region of the ribosomal RNA (or rDNA) of two or more organisms, oligonucleotides may be designed which will distinguish these organisms from one another.

C. Nucleic Acid Amplification Preferably, the amplification oligonucleotides of the present invention are oligodeoxynucleotides and are sufficiently long to be used as a substrate for the synthesis of extension products by a nucleic acid polymerase. Optimal primer length should take into account several factors, including the temperature of reaction, the structure and base composition of the primer, and how the primer is to be used. For example, for optimal specificity the oligonucleotide primer generally should contain at least about 12 nucleotides depending on the complexity of the target nucleic acid sequence. If such specificity is not essential, shorter primers may be used; in such a case, it may be desirable to carry out reaction at cooler temperatures in order to form stable hybrid complexes with the template nucleic acid.

Useful guidelines for designing amplification oligonu- 20 cleotides and probes with desired characteristics are described herein. Our best mode target regions contain at least two and preferably three conserved regions of Neisseria meninqitidis or Neisseria qonorrhoeae nucleic acid. These regions are about 15-350 in length; prefera- 25 bly 15-150 nucleotides in length.

The degree of amplification observed with a set of primers or promoter primers depends on several factors, including the ability of the oligonucleotides to hybridize to their complementary sequences and their ability to be 30 extended or copied enzymatically. While oligonucleotides of different lengths and base composition may be used, oligonucleotides preferred in this invention have target binding regions of 18-40 bases with a predicted Tm to target of about 65 0

C.

Parameters which affect hybridization of a probe such as Tm, complementarity and secondary structure of the target sequence also affect primer hybridization and therefore performance. The degree of non-specific extension (primer-dimer or non-target copying) can also affect amplification efficiency, therefore primers are selected to have low self- or cross-complementarity, particularly at the 3' ends of the sequence. Long homopolymer tracts and high GC content are avoided to reduce spurious primer extension. Computer programs are available to aid in this aspect of the design.

A nucleic acid polymerase used in conjunction with the amplification oligonucleotides of the present invention refers to a chemical, physical or biological agent which incorporates either ribo- or deoxyribonucleotides, or both, into a nucleic acid polymer, or strand, in a template-dependent manner. Examples of nucleic acid polymerases include DNA-directed DNA polymerases, RNA-directed DNA polymerases, and RNA-directed RNA polymerases.

DNA

polymerases bring about nucleic acid synthesis in a template-dependent manner and in a 5' to 3' direction.

20 Because of the antiparallel orientation of the two strands in a double-stranded nucleic acid, this direction is from a 3' region on the template to a 5' region on the template. Examples of 'DNA-directed DNA polymerases include E. coli DNA polymerase I, the thermostable DNA polymerase 25 from Thermus aquaticus (Taq), and the large fragment of DNA polymerase I from Bacillus stearothermophilus (Bst).

Examples of RNA directed DNA polymerases include various retroviral reverse transcriptases, such as Moloney murine leukemia virus (MMLV) reverse transcriptase or avian myeloblastosis virus (AMV) reverse transcriptase.

During most nucleic acid amplification reactions, a nucleic acid polymerase adds nucleotide residues to the 3' end of the primer using the target nucleic acid as a template, thus synthesizing a second nucleic acid strand having a nucleotide sequence partially or completely complementary to a region of the target nucleic acid. In many nucleic acid amplification reactions, the two strands comprising the resulting double-stranded structure must be separated by chemical or physical means in order to allow the amplification reaction to proceed. Alternatively, the newly-synthesized template strand may be made available for hybridization with a second primer or promoter-primer by other means e.g. through strand displacement or the use of a nucleolytic enzyme which digests part or all of the original target strand. In this way the process may be repeated through a number of cycles, resulting in a large increase in the number of nucleic acid molecules having the target nucleotide sequence.

Either the first or second amplification oligonucleotide, or both, may be a promoter-primer. Such a promoterprimer usually contains nucleotide sequences that are not complementary to those of the target nucleic acid molecule, or primer extension product(s). For example, Kacian and Fultz, U.S. Patent No. 5,399,491 which is hereby incorporated by reference, describes various such oligonucleotides. These non-complementary sequences may be located 5' to the complementary sequences on the amplification oligonucleotide, and may provide a locus for initiation of RNA synthesis when made double-stranded through the action of a nucleic acid polymerase. The promoter thus provided may allow for the in vitro transcription of multiple RNA copies of the target nucleic acid sequence. It will be appreciated that when reference is made to a primer in this specification, such reference is intended to include the primer aspect of a promoterprimer as well unless the context of the reference clearly 30 indicates otherwise.

In some amplification systems, for example the amplification method of Dattagupta et al., supra, the amplification oligonucleotides may contain 5' non-complementary nucleotides which assist in strand displacement. Furthermore, when used in conjunction with a nucleic acid polymerase having 5' exonuclease activity, the amplification oligonucleotides may have modifications at their 5' end to prevent enzymatic digestion. Alternatively, the nucleic acid polymerase may be modified to remove the 5' exonuclease activity, such as by treatment with a protease that generates an active polymerase fragment with no such nuclease activity. In such a case the oligonucleotides need not be modified at their 5' end.

1. Preparation of Oliconucleotides All of the amplification oligonucleotides of the present invention can be readily prepared by methods known in the art. Preferably, the primers are synthesized using solid phase methods. For example, Caruthers, et al., describe using standard phosphoramidite solid phase chemistry to join nucleotides by phosphodiester linkages.

Automated solid-phase chemical synthesis using cyanoethyl phosphoramidite precursors has been described by Barone, et al., Nucleic Acids Research, 12:405 (1984). (Methods in Enzymoloqv, Volume 143, pg. 287 (1987)). Likewise, Bhatt describes a procedure for synthesizing oligonucleotides containing phosphorothioate linkages. (WO92/04358, 20 entitled "Method and Reagent for Sulphurization of Organophosphorous Compounds", which enjoys common ownership with the present invention.) Also, Klem et al., entitled "Improved Process for the Synthesis of Oligomers", PCT WO 92/07864, describe the synthesis of oligonucleotides 25 having different linkages including methylphosphonate linkages. The latter three references are hereby incorporated by reference herein. In addition, methods for the organic synthesis of oligonucleotides are known to those of skill in the art, and are described in Sambrook, et 30 al., supra, previously incorporated by reference herein.

Following synthesis and purification of a particular oligonucleotide, several different procedures may be utilized to purify and control the quality of the oligonucleotide. Suitable procedures include polyacrylamide gel electrophoresis or high pressure liquid chromatography.

29 Both of these procedures are well known to those skilled in the art.

All of the oligonucleotides of the present invention, whether hybridization assay probes, amplification oligonucleotides, or helper oligonucleotides, may be modified with chemical groups to enhance their performance or to facilitate the characterization of amplification products.

For example, backbone-modified oligonucleotides such as those having phosphorothioate or methylphosphonate groups which render the oligonucleotides resistant to the nucleolytic activity of certain polymerases or to nuclease enzymes may allow the use of such enzymes in an amplification or other reaction. Another example of modification involves using non-nucleotide linkers Arnold, et al., "Non-Nucleotide Linking Reagents for Nucleotide Probes", EP 0 313 219 hereby incorporated by reference herein) incorporated between nucleotides in the nucleic acid chain which do not interfere with hybridization or the elongation of the primer. Amplification oligonucleot- 20 ides may also contain mixtures of the desired modified and natural nucleotides.

The 3' end of an amplification oligonucleotide may be blocked to prevent initiation of DNA synthesis as described by McDonough, et al., entitled "Nucleic Acid 25 Sequence Amplification", W094/03472 which enjoys common ownership with the present invention and is hereby incorporated by reference herein. A mixture of different 3' oblocked amplification oligonucleotides, or of 3' blocked and unblocked oligonucleotides may increase the efficiency of nucleic acid amplification, as described therein.

As disclosed above, the 5' end of the oligonucleotides may be modified to be resistant to the activity present in some nucleic acid polymerases. Such modifications can be carried out by adding a non-nucleotide group to the terminal 5' nucleotide of the primer using techniques such as those described by Arnold, et al., supra, entitled "Non-Nucleotide Linking Reagents for Nucleotide Probes", previously incorporated by reference herein.

Once synthesized, selected oligonucleotide probes may be labeled by any of several well known methods J. Sambrook, supra). Useful labels include radioisotopes as well as non-radioactive reporting groups. Isotopic labels include 3 H, 32 P, 1251, S"Co and 14C. Isotopic labels can be introduced into the oligonucleotide by techniques known in the art such as nick translation, end labeling, second strand synthesis, the use of reverse transcription, and by chemical methods. When using radiolabeled probes hybridization can be detected by autoradiography, scintillation counting, or gamma counting. The detection method selected will depend upon the particular radioisotope used for labeling.

Non-isotopic materials can also be used for labeling and may be introduced internally into the nucleic acid sequence or at the end of the nucleic acid sequence.

Modified nucleotides may be incorporated enzymatically or 20 chemically. Chemical modifications of the probe may be performed during or after synthesis of the probe, for example, through the use of non-nucleotide linker groups as described by Arnold, et al., supra "Non-Nucleotide Linking Reagents for Nucleotide Probes", previously 25 incorporated by reference herein. Non-isotopic labels include fluorescent molecules, chemiluminescent molecules, enzymes, cofactors, enzyme substrates, haptens or other ligands.

Preferably, the probes are labeled with an acridinium ester. Acridinium ester labeling may be performed as described by Arnold et al., U.S. Patent No. 5,185,439, entitled "Acridinium Ester Labeling and Purification of Nucleotide Probes" issued February 9, 1993 and hereby incorporated by reference herein.

2. Amplification of Neisseria rRNA and rDNA The amplification oligonucleot ides of the present invention are directed to particular Neisseri-a 16S rRNA nucleotide sequences, or their rDNA counterparts. These amplification ol igonuc leot ides may flank, overlap or be contained within at least one of the target nucleotide sequences used as a hybridization assay probe to detect the presence of Neisseria in a nucleic acid amplification assay. The amplification ol igonucleot ides described and claimed herein comprise two sets of amplification oligonucleotides. Members of the set of amplification oligonucleotides are able to hybridize with a nucleic acid having or substantially corresponding to one of the following nucleotide

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

20 SEQ

SEQ

SEQ

SEQ

SEQ

2 5 and ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: sequences: 23 GTCTTGAGAG GGAAAGCAGG GGAC 24 TATGTTACTC ACCCGTTCGC CACTCGCC 19 CTGAAGA.ATA AGCACCGGCT AACTACGTGC AGCAGC 21 CGATGACGGT ACCTGAAGAA TAAGCACCGG CTAAC 20 AACGGCCTTT TCTTCCCTGA CAAAAGTCCT

TTACAACCCG

22 GTCCTTTACA ACCCGAAGGC CTTC 47 GUCUTJGAGAG GGAAAGCAGG GGAC 48 UAtJGUUACUC ACCCGUUCGC

CACUCGCC

49 CUGAAGAAUA AGCACCGGCU AACtJACGUGC

AGCAGC

50 CGAUGACGGtJ ACCUGAAGAA UAAGCACCGG CUAAC ID NO: 51 AACGGCCUUU UCUUJCCCUGA CAAAAGUCCU tJUACAACCCG SEQ ID NO: 52 GUCCUtJUACA ACCCGAAGGC CUUC In preferred embodiments, these amplification oligonucleotides have or substantially correspond to the following sequences: SEQ ID NO: 5 GTCCCCTGCT TTCCCTCTCA AGAC SEQ ID NO: 6 GGCGAGTGGC GAACGGGTGA GTAACATA SEQ ID NO: 7 GCTG.CTGCAC GTAGTTAGCC GGTGCTTATT CTTCAG SEQ ID NO: 8 GTTAGCCGGT GCTTATTCTT CAGGTACCGT CATCG SEQ ID NO: 9 CGGGTTGTAA AGGACTTTTG TCAGGGAAGA AAAGGCCGTT SEQ ID NO: 10 GAAGGCCTTC GGGTTGTAAA GGAC SEQ ID NO: 41 GUCCCCUGCU UUCCCUCUCA AGAC SEQ ID NO: 42 GGCGAGUGGC GAACGGGUGA GUAACAUA SEQ ID NO: 43 GCUGCUGCAC GUAGUUAGCC GGUGCUUAUU

CUUCAG

SEQ ID NO: 44 GUUAGCCGGU GCUUAUUCUU CAGGUACCGU

CAUCG

SEQ ID NO: 45 CGGGUUGUAA AGGACUUUUG UCAGGGAAGA

AAAGGCCGUU

SEQ ID NO: 46 GAAGGCCUUC GGGUUGUAAA GGAC These oligonucleotides may also have additional, noncomplementary bases at their 5' end comprising a promoter sequence able to bind an RNA polymerase and direct RNA transcription using the target nucleic acid as a template.

For example the promoter, SEQ ID NO: 53 AATTTAATAC GACTCACTAT AGGGAGA may be used.

All of the amplification oligonucleotides of the present invention may have sequences which do not contain modifications or additions to these sequences. The amplification oligonucleotides may also or alternatively have modifications, such as blocked 3' and/or 5' termini or additions including but not limited to the addition of a specific nucleotide sequence that is recognized by an RNA polymerase, the promoter sequence for T7, T3, or SP6 RNA polymerase), addition of sequences which 20 enhance initiation or elongation of RNA transcription by S: an RNA polymerase, or sequences which may provide for intramolecular base pairing and encourage the formation of secondary or tertiary nucleic acid structures.

Amplification oligonucleotides are used in a nucleic 25 acid amplification procedure, such as the polymerase chain reaction or an amplification reaction using RNA polymerase, DNA polymerase and RNAse H or its equivalent, as described by Kacian and Fultz supra, Dattagupta et al., supra, and by Sninsky et al., US. Patent No. 5,079,351; all hereby incorporated by reference herein, the first two of which enjoy common ownership with the present invention.

A wide variety of methods are available to detect an amplified target sequence. For example, the nucleotide substrates or the primers can include a detectable label which is incorporated into newly synthesized DNA. The resulting labeled amplification product is then separated from the unused labeled nucleotides or primers and the label is detected in the separated product fraction.

Substances which can serve as useful detectable labels are well known in the art and include radioactive isotopes, fluorescent compounds, chemiluminescent compounds, chromophores, as well as ligands such as biotin and haptens which, while not directly detectable, can be readily detected by a reaction with labeled forms of their specific binding partners, avidin and antibodies, respectively.

Another approach is to detect the amplification product by hybridization with a detectably labeled nucleic acid probe and measuring the resulting hybrids in any conventional manner. In particular, the product can be assayed by hybridizing a chemiluminescent acridinium ester-labeled nucleic acid probe to the target sequence, selectively hydrolyzing the acridinium ester present on unhybridized probe, and measuring the chemiluminescence produced from the remaining acridinium ester in a luminom- ,20 eter. (see, Arnold, et al., supra, U.S. Patent No.

5,283,174, and Nelson, et al., "Non-Isotopic DNA Probe Technologies", Academic Press, San Diego (Kricka, ed.

:co 1992) both references hereby incorporated by reference herein.) 25 D. Oliqonucleotide Hybridization Assay Probes to Neisseria meningitidis or Neisseria conorrhoeae rRNA and rDNA The oligonucleotide hybridization assay probes disclosed and claimed herein are able to preferentially hybridize to target nucleic acids of Neisseria meningitidis rRNA or rDNA nucleotide sequences over nucleic acids of phylogenetically closely related bacterial species. These hybridization assay probes were designed, selected and/or chosen based upon a comparison of the nucleotide sequences of corresponding regions of the ribosomal RNA of Neisseria meninqitidis and said phylogenetically closely-related species. In preferred embodiments these probes selectively hybridize to the nucleic acids of Neisseria meninQitidis over the nucleic acids of Neisseria conorrhoeae.

The present invention contemplates oligonucleotide hybridization probes that selectively hybridize to the nucleic acids of Neisseria meningitidis and not to the nucleic acids of Neisseria gonnorhoeae and include Neisseria menigitidis nucleic acid sequences having or substantially corresponding to the following nucleic acid sequences: SEQ ID NO: 11 GGCTGTTGCT

AATATCAGCG

SEQ ID NO: 12 GGCTGTTGCT

AATACCAGCG

SEQ ID NO: 15 CGCTGATATT

AGCAACAGCC

SEQ ID NO: 16 CGCTGGTATT

AGCAACAGCC

SEQ ID NO: 25 GGCUGUUGCU

AAUAUCAGCG

SEQ ID NO: 26 GGCUGUUGCU

AAUACCAGCG

SEQ ID NO: 27 CGCUGAUAUU

AGCAACAGCC

SEQ ID NO: 28 CGCUGGUAUU

AGCAACAGCC

A number of oligonucleotide hybridization assay probes 20 of the present invention preferably hybridize to target nucleic acids containing Neisseria qonorrhoeae rRNA or rDNA nucleotide sequences over nucleic acids of other phylogenetically closely related bacterial species. In preferred embodiments, these hybridization assay probes 25 can distinguish Neisseria conorrhoeae nucleic acids from Neisseria meninqitidis.

The hybridization probes of the present invention that selectively hybridize to nucleic acids derived from Neisseria conorrhoeae and not to the nucleic acids of 30 Neisseria meningitidis have or substantially correspond to the following nucleotide sequences: SEQ ID NO 1: GAACGTACCG GGTAGCGG o' SEQ ID NO 3: GCCAATATCG GCGGCCGATG SEQ ID NO 29: CCGCTACCCG

GTACGTTC

SEQ ID NO 30: CATCGGCCGC CGATATTGGC SEQ ID NO 31: GAACGUACCG

GGUAGCGG

SEQ ID NO 32: GCCAAUAUCG

GCGGCCGAUG

SEQ ID NO 33: CCGCUACCCG

GUACGUUC

SEQ ID NO 34: CAUCGGCCGC

CGAUAUUGGC

The oligonucleotide hybridization assay probes of the present invention are preferably labeled with a reporter group moiety such as a radioisotope, a fluorescent or chemiluminescent moiety, with an enzyme or other ligand, which can be used for detection or confirmation that the probe has hybridized to the target sequence. The Applicant most prefers the use of chemiluminescent acridinium esters as labels. See e.a. Arnold et al., U.S. Patent No.

5,185,439, previously incorporated by reference herein.

The assay probe is mixed with a sample suspected of containing a nucleic acid having the target sequence under hybridization conditions suitable for allowing annealing of the two strands by hydrogen bonding in the region of complementarity.

The probe may also be combined with one or more unlabeled helper oligonucleotides to facilitate binding to the nucleic acid having the target Neisseria menincitidis 20 or Neisseria qonorrhoeae nucleotide sequence. The probe then hybridizes to the target nucleic acid present in the sample; the resulting hybrid duplex may be separated and detected by various techniques well known in the art, such as hydroxyapatite adsorption and radioactive monitoring.

25 Also included among these techniques are those that involve selectively degrading the label present on unhybridized probe and then measuring the amount of label associated with the remaining hybridized probe, as disclosed in Arnold et al., U.S. Patent No. 5,283,174, which 30 enjoys common ownership with the present application and is incorporated by reference herein. This latter technique is presently preferred by the Applicants.

E. Helper Oliconucleotides Used in the Detection of Neisseria Specific helper oligonucleotides were used to facilitate the hybridization of the hybridization assay probes to the target nucleic acid. Helper oligonucleotides are described in Hogan and Milliman, U.S. Patent No. 5,030,557 entitled Means and Method for Enhancing Nucleic Acid Hybridization, which enjoys common ownership with the present application and is hereby incorporated by reference herein.

Helper probes are selected to hybridize to nucleic acid sequences located near the region targeted by the hybridization assay probe. Hybridization of the helper probe alters the secondary and tertiary structure of the target nucleic acid, facilitating the hybridization of the probe to the target nucleic acid.

Specific helper oligonucleotides for facilitating the specific detection of Neisseria meningitidis nucleic acids have or substantially correspond to one of these nucleotide sequences: SEQ ID NO: 13 GCCTTCGGGT TGTAAAGGAC TTTTGTCAGG GAAGAAAA SEQ ID NO: 14 GCTGATGACG GTACCTGAAG AATAAGCACC GGC SEQ ID NO: 17 TTTTCTTCCC TGACAAAAGT CCTTTACAAC CCGAAGGC 20 SEQ ID NO: 18 GCCGGTGCTT ATTCTTCAGG TACCGTCATC AGC SEQ ID NO: 35 GCCUUCGGGU UGUAAAGGAC UUUUGUCAGG GAAGAAAA SEQ ID NO: 36 GCUGAUGACG GUACCUGAAG AAUAAGCACC GGC SEQ ID NO: 37 UUUUCUUCCC UGACAAAAGU CCUUUACAAC CCGAAGGC SEQ ID NO: 38 GCCGGUGCUU AUUCUUCAGG UACCGUCAUC AGC 25 In preferred embodiments, hybridization probes directed to Neisseria meningitidis nucleic acids substantially correspond to SEQ ID NOS: 11, 12, 25 or 26 used in a probe mixture together with a helper oligonucleotide having or substantially corresponding to the nucleotide sequence of: SEQ ID NOS: 13, 14, 35 and 36 In other embodiments, a hybridization assay probe directed to Neisseria meninaitidis nucleic acids substantially corresponding to SEQ ID NOS: 15, 16, 27 or 28 is used in a probe mixture together with a helper oligonucleotide having or substantially corresponding to a nucleotide sequence of: SEQ ID NOS: 17, 18, 37 and 38 In a preferred embodiment, a hybridization probe directed to Neisseria conorrhoeae ribosomal nucleic acid substantially corresponding to SEQ ID NOS: 1 or 31 is used in a mixture together with a helper oligonucleotide having or substantially corresponding to the nucleotide sequence of: SEQ ID NOS: 2 or 39 In other preferred embodiments, a hybridization probe directed to Neisseria qonorrhoeae nucleic acids substantially corresponding to SEQ ID NOS: 3 or 32 is used in a probe mixture together with a helper oligonucleotide having or substantially corresponding to a nucleotide sequence of: :20 SEQ ID NOS: 4 or Helper oligonucleotides generally may be used under stringent hybridization conditions, but are not necessarily species specific.

F. Nucleic Acid Compositions In another related aspect, the invention features compositions comprising a nucleic acid hybrid between a hybridization assay probe and a nucleic acid sequence substantially complementary thereto (probe:target). One ***use of the hybrid formed between probe and target is to 30 detect the presence of a target sequence. For example, acridinium ester present in hybrids is resistant to hydrolysis in alkali solution whereas AE present in single-stranded nucleic acid is hydrolyzed in alkali solution (Arnold et al., entitled "Homogenous Protection Assay," EPO application number 88308767.8, publication number 309230, and by U.S. Patent No. 5,238,174 hereby incorporated by reference). Thus, presence of target nucleic acids can be detected, after hydrolysis of the unbound AE-labeled probe, by measuring chemiluminescence of acridinium ester remaining associated with the nucleic acid hybrid.

The present invention also contemplates compositions comprising a nucleic acid hybrid between an amplification oligonucleotide and a nucleic acid sequence substantially complementary thereto (primer:target). One use the nucleic acid hybrid formed between primer and target is to provide an initiation site for a nucleic acid polymerase at the 3' end of the amplification oligonucleotide. For example, hybrids may form an initiation site for reverse transcriptase, DNA polymerases such as Taq polymerase or T4 DNA polymerase and RNA polymerases such as, T7 polymerase, SP6 polymerase, T3 polymerases and the like.

The present invention also features compositions comprising nucleic acid hybrids between a helper oligonucleotide and a nucleic acid sequence substantially comple- 20 mentary thereto (helper oligonucleotide:target). One use of the hybrid between the helper oligonucleotide and target is to make available a particular nucleic acid sequence for hybridization. For example, a hybrid between a helper oligonucleotide and its target may make a nucleic *25 acid sequence capable of hybridizing to the target sequence available for hybridization with a hybridization probe. A full description of the use of helper oligonucleotides is provided in Hogan and Milliman, U.S. Patent No. 5,030,557.

30 Compositions of the present invention include compositions for detecting Neisseria meninqitidis nucleic acid comprising a nucleic acid hybrid formed between a nucleic acid derived from Neisseria meninitidis and an oligonucleotide having a nucleic acid sequence substantially corresponding to at least one of the nucleic acid sequences that follows: SEQ ID NO: 11 GGCTGTTGCT

AATATCAGCG

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

20 SEQ

SEQ

SEQ

SEQ

SEQ

25 SEQ

SEQ

SEQ

ID NO: 12 ID NO: 15 ID NO: 16 ID NO: 25 ID NO: 26 ID NO: 27 ID NO: 28 ID NO: 13 ID NO: 14 ID NO: 17 ID NO: 18 ID NO: 35 ID NO: 36 ID NO: 37 ID NO: 38 ID NO: 5 ID NO: 6 ID NO: 7 ID NO: 8 ID NO: 9 ID NO: 10 ID NO: 41 ID NO: 42 ID NO: 43 ID NO: 44 ID NO: 45 ID NO: 46 Preferred GGCTGTTGCT

AATACCAGCG

CGCTGATATT

AGCAACAGCC

CGCTGGTATT

AGCAACAGCC

GGCUGUUGCU

AAUAUCAGCG

GGCUGUEJGCU

AAUACCAGCG

CGCUGAUAUU AGCAACAGCC CGCUGGUAUJ

AGCAACAGCC

GCCTTCGGGT TGTAAAGGAC TTTTGTCAGG

GAAGAAAA

GCTGATGACG GTACCTGAAG AATAAGCACC

GGC

TTTTCTTCCC TGACAAAAGT CCTTTACAAC

CCGAAGGC

GCCGGTGCTT ATTCTTCAGG TACCGTCATC AGC GCCUIJCGGGU UGUAAAGGAC UUUUGUCAGG

GAAGAAAA

GCUGAUGACG GUACCUGAAG AAUAAGCACC

GGC

UUtJUCUUCCC UGACAAAAGU CCUIJUACAAC

CCGAAGGC

GCCGGUGCUU AtJUCUUCAGG UACCGUCAUC

AGC

GTCCCCTGCT TTCCCTCTCA

AGAC

GGCGAGTGGC GAACGGGTGA GTAACATA GCTGCTGCAC GTAGTTAGCC GGTGCTTATT

CTTCAG

GTTAGCCGGT GCTTATTCTT CAGGTACCGT CATCG CGGGTTGTAA AGGACTTTTG TCAGGGAAGA AAAGGCCGTT GAAGGCCTTC GGGTTGTAAA

GGAC

GUCCCCUGCU UUCCCUCUCA

AGAC

GGCGAGUGGC GAACGGGJGA

GUAACAUA

GCUGCUGCAC GUAGUtJAGCC GGUGCJUAUJ CUUCAG GUIJAGCCGGU GCUUAUUCUU CAGGUACCGU CAUCG CGGGLTUGUAA AGGACUUtUUG UCAGGGAAGA

AAAGGCCGUU

GAAGGCCUUC GGGUUGUAAA

GGAC

compositions of the present invention include compositions for detecting Neisseria menincritidis 30 comprising a nucleic acid hybrid formed between a nucleic acid derived from Neisseria meninaitidis and an oligonucleotide having a nucleic acid sequence substantially corresponding to at least one of the nucleic acid sequ-ences that follows: SEQ ID NO: 11 GGCTGTTGCT AATATCAGCG SEQ ID NO: 12 GGCTGTTGCT AATACCAGCG SEQ ID NO: 15 CGCTGATATT AGCAACAGCC SEQ ID NO: 16 CGCTGGTATT AGCAACAGCC SEQ ID NO: 25 GGCUGUUGCU AAUAUCAGCG SEQ ID NO: 26 GGCUGUUGCU AAUACCAGCG SEQ ID NO: 27 CGCUGAUAUU AGCAACAGCC SEQ ID NO: 28 CGCUGGUAUU AGCAACAGCC The present invention also contemplates compositions for detecting Neisseria meninqitidis having a nucleic acid hybrid formed between a Neisseria meninqitidis-derived nucleic acid and a hybridization assay probe having a nucleic acid sequence substantially corresponding to: SEQ ID NO: 11 or SEQ ID NO: and which may also optionally contain a helper oligonucleotide hybridized to said nucleic acid which has a nucleic acid sequence which substantially corresponds to one of the following nucleic acid sequences: SEQ ID NOS: 13, 14, 35 or 36.

The present invention also contemplates compositions for detecting Neisseria meninqitidis having a nucleic acid hybrid formed between a Neisseria meningitidis-derived 20 nucleic acid and a hybridization assay probe having a nucleic acid sequence substantially corresponding to: SEQ ID NO: 12 or SEQ ID NO: 26; and which may also optionally contain a helper oligonucleotide hybridized to said nucleic acid which has a nucleic 25 acid sequence which substantially corresponds to one of the following nucleic acid sequences: SEQ ID NOS: 13, 14, 35 or 36.

The present invention also contemplates compositions for detecting Neisseria menincitidis having a nucleic acid 30 hybrid formed between a Neisseria meninqitidis-derived nucleic acid and a hybridization assay probe having a nucleic acid sequence substantially corresponding to: SEQ ID NO: 15 or SEQ ID NO: 27; and which may also optionally contain a helper oligonucleotide hybridized to said nucleic acid which has a nucleic acid sequence which substantially corresponds to one of the following nucleic acid sequences: SEQ ID NOS: 17, 18, 37 or 38.

The present invention also contemplates compositions for detecting Neisseria meningitidis having a nucleic acid hybrid formed between a Neisseria menincitidis-derived nucleic acid and a hybridization assay probe having a nucleic acid sequence substantially corresponding to: SEQ ID NO: 16 or SEQ ID NO: 28; and which may also optionally contain a helper oligonucleotide hybridized to said nucleic acid which has a nucleic acid sequence which substantially corresponds to one of the following nucleic acid sequences: SEQ ID NOS: 17, 18, 37 or 38.

The present invention also contemplates compositions for detecting Neisseria aonorrhoeae having a nucleic acid hybrid formed between a Neisseria aonorrhoeae-derived nucleic acid and a hybridization assay probe having a nucleic acid sequence substantially corresponding to: SEQ ID NO: 1 or SEQ ID NO: 31; and which may also optionally contain a helper oligonucle- 20 otide hybridized to said nucleic acid which-has a nucleic acid sequence which substantially corresponds to one of the following nucleic acid sequences: SEQ ID NOS: 2 or 39.

The present invention also contemplates compositions 25 for detecting Neisseria gonorrhoeae having a nucleic acid hybrid formed between a Neisseria gonorrhoeae-derived nucleic acid and a hybridization assay probe having a nucleic acid sequence substantially corresponding to: SEQ ID NO: 3 or SEQ ID NO: 32; and which may also optionally contain a helper oligonucleotide hybridized to said nucleic acid which has a nucleic acid sequence which substantially corresponds to one of the following nucleic acid sequences: SEQ ID NOS: 4 or The present invention also contemplates compositions for detecting Neisseria meninqitidis having a nucleic acid derived from Neisseria meninqitidis and an oligonucleotide having a nucleic acid sequence substantially corresponding to SEQ ID NOS: 7 or 43 and/or an oligonucleotide having nucleic acid sequence substantially corresponding to at least one nucleic acid sequence that follows: SEQ ID NOS: 9 or and optionally has a hybridization assay probe capable of hybridizing to a Neisseria meninaitidis nucleic acid and which has a nucleic acid sequence substantially corresponding to one of the following nucleic acid sequences: SEQ ID NOS: 11, 15, 25 or 27 and which may also optionally contain a helper oligonucleotide which has a nucleic acid sequence which substantially corresponds to one of the following nucleic acid sequences: SEQ ID NOS: 13, 14, 35 or 36.

The present invention also contemplates compositions for detecting Neisseria meninqitidis having a nucleic acid 20 derived from Neisseria meninqitidis and an oligonucleotide with a nucleic acid sequence substantially corresponding to SEQ ID NOS: 7 or 43 and/or which also has an oligonucleotide having nucleic 25 acid sequence substantially corresponding to at least one nucleic acid sequence that follows: SEQ ID NOS: 9 or and optionally has a hybridization assay probe capable of hybridizing to Neisseria meninqitidis nucleic acids and which has a nucleic acid sequence substantially corresponding to one of the following nucleic acid sequences: "SEQ ID NOS: 12, 16, 26 or 28 and which may also optionally contain a helper oligonucleotide which has a nucleic acid sequence which substantially corresponds to one of the following nucleic acid sequences: SEQ ID NOS: 13, 14, 35 or 36.

The present invention also contemplates compositions for detecting Neisseria meningitidis having a nucleic acid derived from Neisseria menincitidis and an oligonucleotide with a nucleic acid sequence substantially corresponding to SEQ ID NOS: 7 or 43 and/or which also has an oligonucleotide having nucleic acid sequence substantially corresponding to at least one nucleic acid sequence that follows: SEQ ID NOS: 9 or and optionally has a hybridization assay probe capable of hybridizing to a Neisseria meninaitidis nucleic acid which has a nucleic acid sequence substantially corresponding to one of the following nucleic acid sequences: SEQ ID NOS: 15, 11, 27 or and which may also optionally contain a helper oligonucleotide which has a nucleic acid sequence which substantially corresponds to one of the following nucleic acid sequences: SEQ ID NOS: 17, 18, 37 or 38.

The present invention also contemplates compositions for detecting Neisseria meninqitidis having a nucleic acid derived from Neisseria meninqitidis and an oligonucleotide with a nucleic acid sequence substantially corresponding to SEQ ID NOS: 7 or 43 and/or which also has an oligonucleotide having nucleic acid sequence substantially corresponding to at least one nucleic acid sequence that follows: 30 SEQ ID NOS: 9 or and optionally has a hybridization assay probe capable of hybridizing to a Neisseria meninaitidis nucleic acid which has a nucleic acid sequence substantially corresponding to one of the following nucleic acid sequences: SEQ ID NOS: 16, 12, 28 or and which may also optionally contain a helper oligonucleotide which has a nucleic acid sequence which substantial- 44 ly corresponds to one of the following nucleic acid sequences: SEQ ID NOS: 17, 18, 37 or 38.

The present invention also contemplates compositions for detecting Neisseria meninitidis having a nucleic acid derived from Neisseria meninitidis and an oligonucleotide with a nucleic acid sequence substantially corresponding to SEQ ID NOS: 8 or 44 and/or which also has an oligonucleotide having nucleic acid sequence substantially corresponding to at least one nucleic acid sequence that follows: SEQ ID NOS: 10 or 46 and optionally has a hybridization assay probe capable of hybridizing to a Neisseria menin itidis nucleic acid which has a nucleic acid sequence substantially corresponding to one of the following nucleic acid sequences: SEQ ID NOS: 15, 11, 27 or and which may also optionally contain a helper oligonucleotide which has a nucleic acid sequence which substantially corresponds to one of the following nucleic acid sequences: SEQ ID NOS: 17, 18, 37 or 38.

The present invention also contemplates compositions for detecting eissria eninitidi having a nucleic acid derived from Neisseria mentin itidi and an oligonucleotide with a nucleic acid sequence substantially corresponding to SEQ ID NOS: 8 or 44 30 and/or which also has an oligonucleotide having nucleic acid sequence substantially corresponding to at least one nucleic acid sequence that follows: SEQ ID NOS: 10 or 46 and optionally has a hybridization assay probe capable of hybridizing to a Neisseria meninqitidi nucleic acid which has a nucleic acid sequence substantially corresponding to one of the following nucleic acid sequences: S. SEQ ID NOS: 16, 12, 28 or 26 and which may also optionally contain a helper oligonucleotide which has a nucleic acid sequence which substantially corresponds to one .of the following nucleic acid sequences: SEQ ID NOS: 17, 18, 37 or 38.

Preferred compositions of the present invention include compositions for detecting Neisseria gronorrhoeae comprising a nucleic acid hybrid formed between a nucleic acid derived from Neisseria gonorrhoeae and an oligonucleotide having a nucleic acid sequence substantially corresponding to at least one of the nucleic acid sequences that follow: SEQ ID NO 1: GAACGTACCG

GGTAGCGG

SEQ ID NO 3: GCCAATATCG

GCGGCCGATG

SEQ ID NO 29: CCGCTACCCG

GTACGTTC

SEQ ID NO 30: CATCGGCCGC

CGATATTGGC

SEQ ID NO 31: GAACGUACCG

GGUAGCGG

SEQ ID NO 32: GCCAAUAUCG

GCGGCCGAUG

SEQ ID NO 33: CCGCUACCCG

GUACGUUC

SEQ ID NO 34 CAUCGGCCGC

CGAUALTUGGC

V SEQ ID NO: 5 GTCCCC TGCT TTCCCTCTCA AGAC SEQ ID NO: 6 GGCGAGTGGC GAACGGGTGA

GTAACATA

S..SEQ ID NO: 7 GCTGCTGCAC GTAGTTAGCC GGTGCTTATT

CTTCAG

SEQ ID NO: 8 GTTAGCCGGT GCTTATTCTT CAGGTACCGT

CATCG

SEQ ID NO: 9 CGGGTTGTAA AGGACTTTTG TCAGGGAAGA

AAAGGCCGTT

SEQ I D NO: 10 GAAGGCCTTC GGGTTGTAAA

GGAC

SEQ ID NO: 41 GUCCCCUGCU UCJCCCUCIJCA

AGAC

OV. SEQ ID NO: 42 GGCGAGUGGC GAACGGGUGA

GUAACAUA

SEQ ID NO: 43 GCUGCUGCAC GUAGUUAGCC GGUGCUTIU

CLUCAG

SEQ ID NO: 44 GUTUAGCCGGU GCUTUAUUtCUU CAGGUACCGU

CAUCG

*SEQ ID NO: 45 CGGGtJUGUAA AGGACUUUJG UCAGGGAAGA

AAAGGCCGUU

SEQ ID NO: 46 GAAGGCCUUC GGGUTJGUAAA

GGAC

SEQ ID NO: 2 GGGATAACTG ATCGAAAGAT CAGCTAATAC

CGCATACG

SEQ ID NO: 4 ACGGTACCTG AAGAATAAGC ACCGGCTAAC

TACGTG

SEQ ID NO: 39 GGGAUAACTJG AUCGAAAGAU CAGCUAAUAC

CGCAUACG

SEQ ID NO: 40 ACGGUACCUG AAGAAUAAGC ACCGGCUAAC

UACGUG

More preferred compositions of the present invention include compositions for detecting Neisseria conorrhoeae comprising a nucleic acid hybrid formed between a nucleic acid derived from Neisseria qonorrhoeae and an oligonucleotide having a nucleic acid sequence substantially corresponding to at least one of the nucleic acid sequences that follow: SEQ ID NO: 1 GAACGTACCG

GGTAGCGG

SEQ ID NO: 3 GCCAATATCG

GCGGCCGATG

SEQ ID NO: 31 GAACGUACCG

GGUAGCGG

SEQ ID NO: 32 GCCAAUAUCG

GCGGCCGAUG

SEQ ID NO: 2 GGGATAACTG ATCGAAAGAT CAGCTAATAC

CGCATACG

SEQ ID NO: 4 ACGGTACCTG AAGAATAAGC ACCGGCTAAC

TACGTG

SEQ ID NO: 39 GGGAUAACUG AUCGAAAGAU CAGCUAAUAC

CGCAUACG

SEQ ID NO: 40 ACGGUACCUG AAGAAUAAGC ACCGGCUAAC

UACGUG

The present invention also contemplates compositions for detecting Neisseria conorrhoeae having a nucleic acid derived from Neisseria gonorrhoeae and an oligonucleotide with a nucleic acid sequence substantially corresponding to SEQ ID NOS: 5 or 41 "and/or which also optionally has an oligonucleotide having nucleic acid sequence substantially corresponding to at least one nucleic acid sequence that follows: SEQ ID NOS: 6 or 42 and optionally has a hybridization assay probe capable of hybridizing to a Neisseria gonnorhoeae nucleic acid and which has a nucleic acid sequence substantially corresponding to one of the following nucleic acid sequences: 30 SEQ ID NOS: 1, 29, 31 or 33 and which may also optionally contain a helper oligonucleotide which as a nucleic acid sequence which substantially corresponds to one of the following nucleic acid sequences: SEQ ID NOS: 2 or 39 The present invention also contemplates compositions for detecting Neisseria connorhoeae having a nucleic acid derived from Neisseria gonnorhoeae and an oligonucleotide with a nucleic acid sequence substantially corresponding to SEQ ID NOS: 7 or 42 and/or which optionally has an oligonucleotide nucleic acid sequence substantially corresponding to at least one nucleic acid sequence that follows: SEQ ID NOS: 9 or and which optionally has a hybridization assay probe capable of hybridizing to a Neisseria qonnorhoeae nucleic acid and which has a nucleic acid sequence substantially corresponding to one of the following nucleic acid sequences: SEQ ID NOS: 3, 30, 32 or 34 and which may also optionally contain a helper oligonucleotide which has a nucleic acid sequence which substantially corresponds to one of the following nucleic acid sequences: SEQ ID NOS: 4 or 20 and which also optionally has an oligonucleotide having a nucleic acid sequence substantially corresponding to at least one nucleic acid sequence that follows: SEQ ID NOS: 8 or 44 The present invention also contemplates compositions for detecting Neisseria connorhoeae having a nucleic acid derived from Neisseria connorhoeae and an oligonucleotide with a nucleic acid sequence substantially corresponding to SEQ ID NOS: 10 or 46 30 and optionally has a hybridization assay probe capable of hybridizing to Neisseria connorhoeae nucleic acids and which has a nucleic acid sequence substantially corresponding to one of the following nucleic acid sequences: SEQ ID NOS: 3, 30, 32 or 34 and/or which may also optionally contain a helper oligonucleotide which has a nucleic acid sequence which substantially corresponds to one of the following nucleic acid sequences: SEQ ID NOS: 4 or The present invention also contemplates nucleic acid hybrids comprising probes of the present invention and also at least one helper oligonucleotide that has a nucleic acid sequence substantially corresponding to at.

least one of the nucleic acid sequences that follows:

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

20 SEQ ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: GCCTTCGGGT TGTAAAGGAC TTTTGTCAGG GAAGAAAA GCTGATGACG GTACCTGAAG AATAAGCACC

GGC

TTTTCTTCCC TGACAAAAGT CCTTTACAAC

CCGAAGGC

GCCGGTGCTT ATTCTTCAGG TACCGTCATC

AGC

GCCIJUCGGGU UGUAAAGGAC UtJUUGUCAGG

GAAGAAAA

GCUGAUGACG GUACCUGAAG AAUAAGCACC GGC UUUEJCUUCCC UGACAAAAGU CCUUEJACAAC

CCGAAGGC

GCCGGUGCUTJ AtUUCUTJCAGG UACCGUCAUC

AGC-

GGGATAACTG ATCGAAAGAT CAGCTAATAC

CGCATACG

ACGGTACCTG AAGAATAAGC ACCGGCTAAC

TACGTG

GGGAUAACTJG AUCGAAAGAU CAGCtIAAUAC

CGCAUACG

ACGGUACCUG AAGAAUAAGC ACCGGCUAAC UACGUG The present invention also contemplates compositions for amplifying Neisseria nucleic acids. comprising a nucleic acid hybrid formed between a Neisseria nucleic acid and an oligonucleotide having a nucleic acid sequence substantially corresponding to a nucleic acid sequence selected from the group consisting of: I. SEQ ID NO: SEQ ID NO: SEQ ID NO: 30 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 5 GTCCCCTGCT TTCCCTCTCA AGAC 6 GGCGAGTGGC GAACGGGTGA GTAACATA 41 GUCCCCUGCU UtJCCCIJCUCA AGAC 7 GCTGCTGCAC GTAGTTAGCC GGTGCTTATT CTTCAG 8 GTTAGCCGGT GCTTATTCTT CAGGTACCGT CATCG 43 GCUGCUGCAC GUAGUUAGCC GGUGCUUAUU CUUCAG 44 GUUAGCCGGU GCUUAUtJCUU CAGGUACCGU CAUCG 9 CGGGTTGTAA AGGACTTTTG TCAGGGAAGA AAAGGCCGTT 10 GAAGGCCTTC GGGTTGTAAA

GGAC

45 CGGGUUGUAA AGGACUUtUUG UCAGGGAAGA AAAGGCCGUEJ 46 GAAGGCCTJUC GGGULJGUAAA GGAC G. ASSAY METHODS The present invention contemplates various methods for assaying for the presence of Neisseria meninqitidis or Neisseria gonorrhoeae nucleic acid within a sample. One skilled in the art will understand that the exact assay conditions, probes or primers used will vary depending on the particular assay format used and the source of the sample.

Generally, the present invention contemplates methods for detecting the presence of Neisseria meninqitidis by contacting a test sample under stringent hybridization conditions with a nucleic acid hybridization assay probe capable of preferentially hybridizing under stringent hybridization assay conditions to a Neisseria meninqitidis target nucleic acid over nucleic acids from Neisseria gonorrhoeae, said target nucleic acid having a nucleic acid sequence substantially corresponding to a sequence selected from the group consisting of: SEQ ID NO: 11 GGCTGTTGCT AATATCAGCG 20 SEQ ID NO: 12 GGCTGTTGCT AATACCAGCG *SEQ ID NO: 15 CGCTGATATT AGCAACAGCC SEQ ID NO: 16 CGCTGGTATT AGCAACAGCC SEQ ID NO: 25 GGCUGUUGCU AAUAUCAGCG SEQ ID NO: 26 GGCUGUUGCU AAUACCAGCG SEQ ID NO: 27 CGCUGAUAUU AGCAACAGCC SEQ ID NO: 28 CGCUGGUAUU AGCAACAGCC Preferred methods for detecting the presence of Neisseria meninqitidis include the step of contacting a test sample under stringent hybridization conditions with a nucleic acid hybridization assay probe capable of preferentially hybridizing under stringent hybridization assay conditions to a Neisseria meninqitidis target nucleic acid sequence over nucleic acid sequences of Neisseria qonorrhoeae, said target nucleic acid sequence substantially corresponding to a sequence selected from the group consisting of: SEQ ID NO: 11 GGCTGTTGCT AATATCAGCG SEQ ID NO: 12 GGCTGTTGCT

AATACCAGCG

SEQ ID NO: 15 CGCTGATATT

AGCAACAGCC

SEQ ID NO: 16 CGCTGGTATT

AGCAACAGCC

Preferred methods for detecting the presence of Neisseria aonorrhoeae include the step of contacting a test sample under stringent hybridization conditions with a nucleic acid hybridization assay probe capable of preferentially hybridizing under stringent hybridization assay conditions to a Neisseria gonorrhoeae target nucleic acid sequence over a nucleic acid sequence of Neisseria menincitidis, said target nucleic acid sequence substantially corresponding to a sequence selected from the group consisting of: SEQ ID NO 1: GAACGTACCG

GGTAGCGG

SEQ ID NO 3: GCCAATATCG

GCGGCCGATG

SEQ ID NO 29: CCGCTACCCG

GTACGTTC

SEQ ID NO 30: CATCGGCCGC

CGATATTGGC

SEQ ID NO 31: GAACGUACCG

GGUAGCGG

SEQ ID NO 32: GCCAAUAUCG

GCGGCCGAUG

SEQ ID NO 33: CCGCUACCCG

GUACGUUC

SEQ ID NO 34: CAUCGGCCGC

CGAUAUUGGC

In other embodiments, the present invention also contemplates methods for detecting the presence of Neisseria qonorrhoeae microorganisms by contacting a test sample under stringent hybridization conditions with a nucleic acid hybridization assay probe capable of preferentially hybridizing under stringent hybridization assay conditions to a Neisseria conorrhoeae nucleic acid sequence over nucleic acid sequences from Neisseria menincitidis, said 30 target nucleic acid sequences substantially corresponding to a sequence selected form the group consisting of: SEQ ID NO 1: GAACGTACCG GGTAGCGG SEQ ID NO 3: GCCAATATCG GCGGCCGATG SEQ ID NO 31: GAACGUACCG

GGUAGCGG

SEQ ID NO 32: GCCAAUAUCG

GCGGCCGAUG

The present invention also contemplates methods of detecting Neisseria by first amplifying a portion of the Neisseria nucleic acid and then optionally using a hybridization assay probe of the present invention to assay for a specific Neisseria-derived nucleic acid amplified by the primers of the present invention. The amplified nucleic acid can be detected by a number of methods including gel electrophoresis.

In preferred embodiments, the present invention contemplates methods of detecting Neisseria-derived nucleic acid by first amplifying said nucleic acid with at least one amplification oligonucleotide that will bind to or cause elongation through one or more of the following sequences: SEQ ID NO: 47 GUCUUGAGAG GGAAAGCAGG

GGAC

SEQ ID NO: 48 UAUGUUACUC ACCCGUUCGC

CACUCGCC

SEQ ID NO: 49 CUGAAGAAUA AGCACCGGCU AACUACGUGC

AGCAGC

SEQ ID NO: 50 CGAUGACGGU ACCUGAAGAA UAAGCACCGG

CUAAC

SEQ ID NO: 51 AACGGCCUUJ UCUUCCCUGA CAAAAGUCCU

UUACAACCCG

SEQ ID NO: 52 GUCCUUUACA ACCCGAAGGC

CUUC

wherein said amplification oligonucleotide optionally has a nucleic acid sequence recognized by an RNA polymerase or which enhances initiation of elongation by an RNA polymerase.

This first method step is then optionally followed by detecting the amplified nucleic acid produced in the amplification step with an oligonucleotide hybridization assay probe able to specifically hybridize to nucleic acids derived from Neisseria species, Neisseria cinerea, Neisseria meninitidis or Neisseria ,onorrhoeae under stringent hybridization conditions.

The amplification oligonucleotide used in the methods of the present invention may optionally have a nucleic acid sequence for example, a promoter sequence, recognized by an RNA polymerase or which enhances initiation by an RNA polymerase.

In other preferred embodiments, the present invention contemplates a method for amplifying Neisseria nucleic acids in a test sample by amplifying the nucleic acid with 52 one or more amplification oligonucleotide that will bind to or cause elongation through a nucleic acid sequence substantially corresponding to the following nucleotide sequences: SEQ ID NOs: 19 or 49, SEQ ID NOs: 21 or 50, or with a second amplification oligonucleotide that will bind to or cause elongation through a nucleic acid sequence substantially corresponding to one of the following sequences: SEQ ID NOs: 20 or 51, SEQ ID NOs: 22 or 52 or both, said amplification oligonucleotides, wherein at least one of said amplification oligonucleotides optionally has a nucleic acid sequence recognized by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase.

In other more preferred embodiments, the present invention contemplates a method for amplifying Neisseriaderived nucleic acids in a test sample comprising amplifying said nucleic acid with one or more .amplification oligonucleotide that will bind to or cause elongation through a nucleic acid sequence substantially correspond- **0 ing to one of the following nucleotide sequences: SEQ ID NOS: 19 or 49, or with a second amplification oligonucleotide that will bind to or cause elongation through a nucleic acid sequence substantially corresponding to the following sequences: SEQ ID NOS: 20 or 51, or both said amplification oligonucleotides, wherein at least one of said amplification 30 oligonucleotides optionally has a nucleic acid sequence recognized by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase.

In other preferred embodiments, the present invention contemplates a method for increasing the number of Neisseria-derived nucleic acid sequences in a test sample comprising amplifying said nucleic acid with one or more amplification oligonucleotides that will bind to or cause elongation through a nucleic acid sequence substantially corresponding to the following nucleotide sequences: SEQ ID NOS: 21 or or with a second amplification oligonucleotide that will bind to or cause elongation through a nucleic acid sequence substantially corresponding to the following sequences: SEQ ID NOS: 22 or 52, or both said amplification oligonucleotides, wherein at least one of said amplification oligonucleotides optionally has a nucleic acid sequence recognized by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase.

The above methods may also include the further step of detecting the amplified nucleic acid with an oligonucleotide hybridization assay probe able to specifically hybridize to Neisseria menincitidis nucleic acids under stringent hybridization conditions.

Specifically, the methods may detect Neisseria menincitidis using oligonucleotide hybridization assay probes 20 which will hybridize under stringent hybridization conditions to a nucleic acid sequence substantially corresponding to a sequence selected from the group consisting of: SEQ ID NO: 11 GGCTGTTGCT AATATCAGCG SEQ ID NO: 27 CGCUGAUAUU AGCAACAGCC SEQ ID NO: 12 GGCTGTTGCT

AATACCAGCG

SEQ ID NO: 28 CGCUGGUAUU AGCAACAGCC SEQ ID NO: 15 CGCTGATATT AGCAACAGCC SEQ ID NO: 25 GGCUGUUGCU

AAUAUCAGCG

SEQ ID NO: 16 CGCTGGTATT AGCAACAGCC SEQ ID NO: 26 GGCUGUUGCU

AAUACCAGCG

The present invention also contemplates methods for increasing the number of Neisseria conorrhoeae-derived nucleic acids in a test sample by amplifying said nucleic acid with one or more amplification oligonucleotides that will bind to or cause elongation through a nucleic acid sequence substantially corresponding to one or more of the following nucleotide sequences: SEQ ID NOs 23 or 47, SEQ ID NOs 24 or 48, and where the amplification oligonucleotide optionally has a nucleic acid sequence recognized by an RNA polymerase or which enhances initiation or elongation by an RNA polymer- Additional methods are contemplated for amplifying Neisseria onorrhoea-derived nucleic acids in a test sample with a first amplification oligonucleotide that will bind to or cause elongation through a nucleic acid sequence substantially corresponding to one of the following nucleotide sequences: SEQ ID NOs: 23 or 47, or with a second amplification oligonucleotide that will bind to or cause elongation through a nucleic acid sequence substantially corresponding to one of the following sequences: SEQ ID NOs: 24 or 48, or with both said first and second amplification oligonucleotides wherein one of the amplification oligonucleotides optionally has a nucleic acid sequence recognized by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase.

These methods of amplifying a Neisseria connorhoeaederived nucleic acid may be followed by the step of detecting the amplified nucleic acid with an oligonucleode hybridizatio assay probe able to specifically hybridize to Neisseria onorrhoea nucleic acids under stringent hybridization conditions.

30 Preferably the oligonucleotide hybridization assay probe has a nucleic acid sequence substantially corresponding to a sequence selected from the group consisting of: SEQ ID NOS: 1, 29, 31, and 33.

The detecting of Neisseri-a oorrhoeae nucleic acid may include the use of a helper oligonucleotide having a nucleic acid sequence substantially corresponding to a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 2, and SEQ ID NO: 39.

Other methods of detecting Neisseria gonorrhoeae nucleic acid are contemplated by increasing the number of Neisseria qonorrhoeae-derived nucleic acids in a test sample by amplifying said nucleic acid with one or more amplification oligonucleotide that will -bind to or cause elongation through a nucleic acid sequence substantially corresponding to one or more of the following nucleotide sequences: SEQ ID NOS: 19 or 49, SEQ ID NOS: 20 or 51, SEQ ID NOS: 21 or 50, SEQ ID NOS: 22 or 52, and where the amplification oligonucleotide optionally has a nucleic acid sequence recognized by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase.

Preferred methods for amplifying Neisseria nucleic acids in a test sample include amplifying the nucleic acid 20 with one or more amplification oligonucleotide that will bind to or cause elongation through a nucleic acid sequence substantially corresponding to one of the following nucleotide sequences: SEQ ID NOS: 19 or 49, or 25 with a second amplification oligonucleotide that will bind to or cause elongation through a nucleic acid sequence substantially corresponding to one of the following sequences: SEQ ID NOS: 20 or 51, or with both said first and second amplification oligonucleotides.

Alternatively the present invention contemplates amplifying Neisseria nucleic acids in a test sample comprising amplifying the nucleic acid with one or more amplification oligonucleotide that will bind to or cause elongation through a nucleic acid sequence substantially corresponding to one of the following nucleotide sequences: SEQ ID NOS: 21 or 50, or with a second amplification oligonucleotides that will bind to or cause elongation through a nucleic acid sequence substantially corresponding to one of the following sequences: SEQ ID NOS: 22 or 52, or with both said first and second amplification oligonucleotides.

The amplification of the Neisseria nucleic acid is preferably followed by detecting the amplified nucleic acid with an oligonucleotide hybridization assay probe able to specifically hybridize to Neisseria aonorrhoeae nucleic acids under stringent hybridization conditions.

The oligonucleotide hybridization assay probe used preferably has a nucleic acid sequence substantially corresponding to a sequence selected from the group consisting of: SEQ ID NOS: 3, 32, 30, 34 20 H. DIAGNOSTIC SYSTEMS The present invention also contemplates diagnostic systems in kit form.' A diagnostic system of the present invention may include a kit which contains, in an amount sufficient for at least one assay, amplification primers 25 and/or hybridization assay probes of the present invention in a packaging material. Typically, the kits would also include instructions for use of the packaged primers and/or probes.

The various components of the diagnostic system may be provided in various forms. For example, the required enzymes, the nucleotide triphosphates, the primers and probes may be provided as a lyophilized reagent. These lyophilized reagents may be premixed before lyophilization so that when reconstituted form a complete mixture with the proper ratio of each of the components ready for use in the assay. In addition, the diagnostic systems of the present invention may contain a reconstitution reagent for reconstituting the lyophilized reagents of the kit. In preferred kits, the enzymes, nucleotides, triphosphates and required cofactors for the enzymes are provided as a single lyophilized reagent that when reconstituted forms a proper reagent for use in the present methods. In these preferred kits, a lyophilized primer agent may also be provided. In other preferred kits, lyophilized probe reagents are provided.

Typical packaging materials would include solid matrices such as glass, plastic, paper, foil, micro particles and the like, capable of holding within fixed limits hybridization assay probe or amplification primer of the present invention. Thus, for example, a package made from packaging materials can be a glass vial used to contain sub-milligram picogram, nanogram etc.) quantities of a contemplated primer or hybridization assay probe or it could be a microtiter plate well to which the probes and/or primers of the present invention have been operatively affixed, linked so as to be capable of participating in a detection method of the present invention.

oInstructions for use typically include a tangible expression describing the various reagents and/or concentrations of reagents and at least one assay method parameter which, for example, would be the relative amounts of reagents to use per amount of sample. In addition, such specifics as maintenance, time periods, temperature and buffer conditions may also be included.

30 The present invention contemplates diagnostic systems or kits containing the oligonucleotides of a composition of the present invention. The present invention also contemplates diagnostic systems or kits containing the oligonucleotides required to perform a method of the present invention.

This method preferably also uses a helper oligonucleotide having a nucleic acid sequence substantially corresponding to a nucleic acid sequence selected from the group consisting of: SEQ ID NO. 4: and SEQ ID NO. The present invention contemplates diagnostic systems or a kit containing at least one oligonucleotide having a nucleic acid sequence substantially corresponding to a nucleic acid sequence selected from the group consisting of: SEQ ID NOS: 1, 3, 11, 12, 15, 16, 29, 30, 33, 34, 27, 28, 26.

The present invention contemplates diagnostic systems or a kit having an oligonucleotide hybridization assay probe having at least one helper probe having a nucleic acid sequence substantially corresponding to the sequence selected from the group consisting of: SEQ ID NOS: 2 or 39, when said oligonucleotide substantially corresponds to SEQ ID NOS: 1 or 31; or SEQ ID NOS: 4 or 0 when said oligonucleotide substantially corresponds to SEQ ID NOS: 3 or 32; or SEQ ID NOS: 13 or 35, or SEQ ID NOS: 14 or 36, when said oligonucleotide substantially corresponds to SEQ ID NOS: 11 or 25, or SEQ ID NOS: 12 or 26; 30 or SEQ ID NOS: 17 or 37, SEQ ID NOS: 18 or 38, when said oligonucleotide substantially corresponds to SEQ ID NOS: 15 or 27, or SEQ ID NOS: 16 or 28.

The present invention contemplates diagnostic systems or a kit containing two oligonucleotides having a nucleic acid sequence substantially corresponding to a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 7 GCTGCTGCAC GTAGTTAGCC GGTGCTTATT

CTTCAG

SEQ ID NO: 8 GTTAGCCGGT GCTTATTCTT CAGGTACCGT CATCG SEQ ID NO: 9 CGGGTTGTAA AGGACTTTTG TCAGGGAAGA AAAGGCCGTT SEQ ID NO: 10 GAAGGCCTTC GGGTTGTAAA GGAC SEQ ID NO: 43 GCUGCUGCAC GUAGUUAGCC GGUGCUUAUU CUUCAG SEQ ID NO: 44 GUUAGCCGGU GCUUAUUCUU CAGGUACCGU CAUCG SEQ ID NO: 45 CGGGUUGUAA AGGACUUUUG UCAGGGAAGA AAAGGCCGUU SEQ ID NO: 46 GAAGGCCUUC GGGUUGUAAA GGAC optionally having a 5' sequence recognized by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase.

The present invention contemplates diagnostic systems or a kit containing oligonucleotides having a nucleic acid sequence substantially corresponding to the following sequences: SEQ ID NOS: 7 or 43, SEQ ID NOS: 9 or SEQ ID NOS: 11 or SEQ ID NOS: 13 or SEQ ID NOS: 14 or 36.

The present invention contemplates diagnostic systems or a kit containing oligonucleotides having a nucleic acid sequence substantially corresponding to the following sequences: •SEQ ID NOS: 15 or 27, SEQ ID NOS: 16 or 26, SEQ ID NOS: 17 or 37, 30 SEQ ID NOS: 18 or 38.

The present invention contemplates diagnostic systems or a kit containing oligonucleotides having a nucleic acid sequence substantially corresponding to the following sequences: SEQ ID NOS: 7 or 43, SEQ ID NOS: 9 or SEQ ID NOS: 15 or 27, SEQ ID NOS: 16 or 28, SEQ ID NOS: 17 or 37, SEQ ID NOS: 18 or 38.

The present invention contemplates diagnostic systems or a kit containing oligonucleotides having a nucleic acid sequence substantially corresponding to the following sequences: SEQ ID NOS: 5 or 41, SEQ ID NOS: 6 or 42, SEQ ID NOS: 2 or 39, SEQ ID NOS: 1 or 31.

The present invention contemplates diagnostic systems or a kit containing oligonucleotides having a nucleic acid sequence substantially corresponding to the following sequences: SEQ ID NOS: 5 or 41, SEQ ID NOS: 2 or 39, SEQ ID NOS: 1 or 31.

EXAMPLES

20 Examples are provided below to illustrate different aspects and embodiments of the present invention. These examples are not intended in any way to limit the disclosed invention, which is limited only by the claims.

Probes specific for Neisseria meningitidis were 25 designed using sequences determined in prospective target areas using primers complementary to the 16S rRNAs of Neisseria qonorrhoeae (ATCC NO. 19424), Neisseria meninq- S itidis sero group A (ATCC NOs 13077), serogroup C (ATCC No. 23248) and serogroup L (ATCC No. 43828), and clinical isolates, Neisseria lactamica (ATCC NO. 23970), Neisseria 0* cinerea (ATCC NO. 14685), Neisseria mucosa (ATCC NO.

19696), Neisseria sicca (ATCC NO. 29193) and Kincaella kinaae (ATCC NO. 23330). The nucleic acid sequence from phylogenetically near neighbors, including the published sequence of Neisseria gonorrhoeae NCTC 8375 Rossau et al., 61 Nuc. Acids Res. 16:6227 were also used as comparisons with the nucleic sequence from Neisseria menincgitidis to determine variable regions.

An example of such an alignment follows: A specific sequence in which, Neisseria meninqitidis varied from E.

coli and Neisseria qonorrohoeae was chosen for probe design. Two different probes were designed to Neisseria meninqitidis (SEQ ID NO: 11) and (SEQ ID NO: 12). The rRNA sequences are shown below: E. coli GAGUAAAG(UUAAUAC)CUUUG SEQ ID NO: 54 GGCTGTTG(CTAATAC)CAGCG SEQ ID NO: 12 0 GGCTGTTG(CTAATAT)CAGCG SEQ ID NO: 11 N. meninqitidis.c GGCUGUUG(CUAAUAU)CAGCG SEQ ID NO: 5 N. gonorrhoeae. P GGCUGUUG (CCAAUAU)CGGGG SEQ ID NO: 56 1 The following hybridization assay probe featured in the examples described below: sequences are SEQ ID NO: SEQ ID NO: 20 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 1 GAACGTACCGGGTAGCGG, 3 GCCAATATCGGCGGCCGATG, 11 GGCTGTTGCTAATATCAGCG, 12 GGCTGTTGCTAATACCAGCG, 15 CGCTGATATTAGCAACAGCC, and 16 CGCTGGTATTAGCAACAGCC Example 1.

25 In this experiment, purified N. conorrhoeae rRNA (ATCC NO. 19424) was amplified with oligonucleotides containing sequences complementary to N. qonorrhoeae rRNA using the techniques described in Kacian et al. U.S.

Patent No. 5,399,491. Two promoter primers were synthe- 30 sized, each containing a T7 RNA polymerase promoter sequence 5'-AATTTAATACGACTCACTATAGGGAGA-3' SEQ ID NO. 53 at the 5' end, covalently attached to a target complementary sequence 5'-GTCCCCTGCTTTCCCTCTCAAGAC-3' (SEQ ID NO.

at the 3' end. One promoter primer was synthesized with a free 3' OH group, and was used at two pmol per reaction. The second promoter primer was synthesized with an alkane diol group at the 3' end and was used at 13 pmol 63 per reaction. The target nucleic acid and primers were heated to 95 0 C for 15 minutes and cooled to 42w. Moloney Murine Leukemia Virus reverse transcriptase (MMLV RT), 900 units, and 400 units of T7 RNA polymerase were added. The final amplification mixture contained 50 mM Tris C (pH 35 mM potassium chloride, 4 mM GTP, 4 mM ATP, 4 mM UTP, 4 mM CTP, 1 mM dATP, 1 mM dTTP, 1 mM dCTP, 1 mM dGTP, mM MgC 2 20 mM N-Acetyl-L-Cysteine, and (w/v) glycerol. After a two hour incubation at 42 0 C, the entire one hundred ul amplification reaction was assayed by hybridization with an acridinium ester labeled probe of sequence 5'-GAACGTACCGGGTAGCGG- 3 (SEQ. ID. NO. 1) and an unlabeled helper probe of sequence TCAGCTATACCGCATACG-3rfor (SEQ. ID. NO. 2) Hybridizati on was performed in 200 H of a solution containing 0.05

M

lithium succinate (pH 0.6 M LiCI, 1% lithium lauryl sulfate, 10 mM EDTA, 0 mM EGTA, at 60C for 10 t h i u m minutes, followed by addition of 300 pl of 0.15 M sodium tetraborate pH 8.5, 1% TRITON® X-100. This mixture was 20 incubated at 60C for 10 minutes, and cooled to room temperature There n u t e s and cooled to room t emperature. The remaining chemiluminescence in each tube was assayed in a Gen-Probe LEADER® I luminometer equipped with automatic l u mi n o e t e r qctpp with automatic injection of 1 mM nitric acid and 0.1% 0.0 hydrogen peroxide followed by injection of a solu- ion containing 1 N sodium hydroxide. Results were given in Relative Light Units (RLU), a measure of the photons detected by the luminometer.

abe Amplification of Neisseria norrhoea nucleic acid with amplification oligonucleotides comprising SEQ

ID

I30 NO. 5 followed by detection with.a probe comprising

SEQ

ID NO. 1.

0 0: 0.1 pg* 103,596 99,931 123,512 0.025 pg 25,636 39,454 29,594 0 pg 1,084 1,012 640 pg picogram Example 2.

This experiment demonstrates amplification of N.

gonorrhoeae rRNA with two primers of opposite sense. The promoter-primer described in Example 1 containing a T7 RNA polymerase promoter sequence and a 3' target hybridizing 1 0 region of sequence 5'-GTCCCCTGCTTTCCCTCTCAAGAC-3'

(SEQ.

ID. NO. 5) was used at 15 pmol per reaction and a primer containing a sequence of the same sense as N. conorrhoeae rRNA, 5'-GGCGAGTGGCGAACGGGTGAGTAACATA-3' (SEQ. ID. NO. 6) was used at 15 pmol per reaction. Reactions were performed in triplicate. The amplification conditions were as described in Example 1, and samples were heated to 95 0 C for 5 minutes, then cooled to 42 0 C. Enzymes were- added, and after a two hour incubation at 42 0 C, 20 il of the amplification reaction was assayed by hybridization with an 20 acridinium ester labeled probe synthesized with sequence SEQ. ID. NO. 1 and an unlabeled helper probe synthesized with sequence SEQ. ID. NO. 2. The primers amplified

N.

onorrhoeae RNA and allowed detection of less than 100 copies of target.

Table 2 Amplification of N. Qonorrhoeae rRNA with primers comprising SEQ. ID. NOs. 5 and 6 and detection with a probe comprising SEQ ID NO. 1.

Amount of rRNA target added

RLU

500 copies (0.0012 pg) 334,838 343,107 1,320,194 copies (0.0002 pg) 255,898 1,512,486 377,938 0 pg 2,354 2,454 2,440 Example 3.

In this experiment, two promoter primers of identical sequence were again used. Each promoter primer was synthesized with a 5' T7 RNA polymerase promoter sequence 5-AATTTAATACGACTCACTATAGGGAGA-3' SEQ ID NO: 53 at the end and a target hybridizing region CCGGTGCTTATTCTTCAG-3' (SEQ ID NO. 7) at the 3' end. The promoter primers were synthesized either with a 3'-hydroxyl group and used at 2 pmol per reaction, or with a 3'alkane diol and used at 13 pmol per reaction. Samples were heated to 95 0 C for 5 minutes and cooled to 42 0 C prior :to enzyme addition. The amplification conditions were as described in Example I. After a two hour incubation at 42 0 C, 100 kl of the amplification reaction was assayed by hybridization with an acridinium ester labeled probe synthesized with sequence 5'GCCAATATCGGCGGCCGATG-3'

(SEQ.

ID. NO. 3) and an unlabeled helper probe with the sequence 5'-ACGGTACCTGAAGAATAAGCACCGGCTAACTACGTG-3, (SEQ. ID. NO.

4) using the conditions described in Example 1.

Table 3. Amplification of N. qonorrhoeae rRNA using primers comprising SEQ ID NO. 7.

Amount of rRNA target added RLU with probe SEQ ID NO. 3 0.025 pg 95,905 49,717 59,774 0.0125 pg 10,520 12,576 12,322 0.005 pg 19,498 40,962 21,722 0 pg 2,888 2,792 2,777 10 Example 4.

In this experiment, N. conorrhoeae rRNA was amplified with a mixture of two oligonucleotides, one a promoter primer complementary to N. qonorrhoeae rRNA and one primer of the same sense as N. conorrhoeae rRNA. The promoter 15 primer contained a T7 RNA polymerase promoter sequence at the 5' end and a target hybridizing region TAGTTAGCCGGTGCTTATTCTTCAG-3' (SEQ ID NO. 7) at the 3' end and was used with a primer of sequence TTTTGTCAGGGAAGAAAAGGCCGTT-3' (SEQ. ID. NO. 9) at 30 pmol per reaction. Alternatively, a promoter primer containing a target hybridizing region of sequence 00 p.

S

S

S

S..

67 5'-GTTAGCCGGTGCTTATTCTTCAGGTACCGTCATCG-3' (SEQ. ID. NO. 8) was used at 15 pmol per reaction with the promoter primer with a sequence 5'-GAAGGCCTTCGGGTTGTAAAGGAC-3' (SEQ. ID.

NO. 10), at 15 pmol per reaction. Amplification conditions were as described for Example 1. Twenty Al'of the product was assayed by hybridization with an acridinium ester labeled probe synthesized with sequence 5'GCCAATATCGGCGGCCGATG-3' (SEQ. ID. NO. 3) and an unlabeled helper probe synthesized with the sequence ACGGTACCTGAAGAATAAGCACCGGCTAACTACGTG-3' (SEQ. ID. NO. 4) as described in Example 1.

Table 4. Amplification primers comprising SEQ ID of N. conorrhoeae rRNA using NOs. 7 and 9 or 8 and Amount of rRNA target RLU with probe SEQ ID added NO. 3 Primer sequences: SEQ ID SEQ ID NOS. NOS.

7 and 9 8 and pg 817,344 680,836 802,901 603,811 785,206 619,969 0.05 p9 188,661 132,359 192,656 157,509 204,878 87,161 0.005 pg 25,276 51,398 26,451 40,032 24,915 55,968 0 pg 3,600 2,189 3,366 2,205 888 68 Example This example demonstrates the reactivity of the amplification and detection assay. Fresh cultures of thirteen strains of N. qonorrhoeae were suspended in 0.9% sodium chloride at a density of approximately 1010 cells/ml and lysed in a solution containing 3 lithium lauryl sulfate, 30 mM sodium phosphate pH 6.8, 1 mM EDTA and 1 mM EGTA. Release of nucleic acid was confirmed by hybridization with a probe directed to a conserved region of ribosomal RNA of all bacteria. The cell lysates were further diluted in water and added to amplification reactions containing 30 pmol of a promoter-primer containing a 5' T7 RNA promoter sequence SEQ ID NO: 53 ATACGACTCACTATAGGGAGA-3' and a 3' target binding sequence comprising SEQ. ID. NO. 7, and 30 pmol of primer comprising sequence SEQ. ID. NO. 9. Duplicate reactions containing lysate from at least 10 s cells were performed using an amplification mixture containing 50 mM Tris HC1 (pH 35 mM potassium chloride, 4 mM GTP, 4 mM ATP, 4 mM UTP, 4 mM CTP, 1 mM dATP, 1 mM dTTP, 1 mM dCTP, 1 mM dGTP, 20 mM MgC1 2 20 mM N-acetyl-L-cysteine, 5% glycerol and the oligonucleotide primers described above. The mixture was heated to 95 0 C for 5 minutes, cooled to 42 0 C and 900 units of MMLV reverse transcriptase and 400 units of T7 RNA 25 polymerase were added. After a one hour incubation at 42 0

C,

ul of amplification reaction was assayed by hybridization with an acridinium ester labeled probe synthesized with sequence 5'GCCAATATCGGCGGCCGATG-3' (SEQ. ID. NO. 3) and an unlabeled helper probe containing sequence ACGGTACCTGAAGAATAAGCACCGGCTAACTACGTG-3' (SEQ. ID. NO. 4).

69 Table 5. Amplification of different strains of N. gonorrhoeae using primers comprising SEQ ID NOs. 7 and 9.

N. qonorrhoeae ATCC No.

9793 9826 9827 9828 9830 RLU with probe SEQ ID NO. 3 1,150,477 1,162,284 1,173,586 1,149,251 1,093,440 1,080,405 1,143,960 1,149,465 10150 1,165,108 1,143,063 1,105,754 1,131,598 10874 1,139,487 1,103,912 11688 1,024,195 1,112,160 11689 1,141,404 1,116,069 19424 1,104,256 1,116,832 27628 1,133,696 1,117,624 27630 1,132,496 1,146,161 27631 1,089,105 1,070,058 Example 6.

Sequence analysis of other Neisseria species indicated that the amplification oligonucleotides of this invention could amplify nucleic acids of other species. This example demonstrates the utility of the amplification oligonucleotides of this invention to amplify nucleic acid from another Neisseria species, N. meninqitidis. In the course of development of a specific probe for N. meninqitidis, it became clear that the members of the species N_ menincitidis were not homogeneous in the probe region of choice. The sequences of 16S rRNAs of representative

N.

meninqitidis species which showed low reactivity to the initial probe were determined and a second probe was 15 designed. These data demonstrate the differential reactivity of three N. meninqitidis species to the two probes.

In this example, purified RNA from Neisseria qonorrhoeae (ATCC No. 19424), or lysates from Neisseria meninqitidis serogroup A (ATCC No. 13077) serogroup C (ATCC No. 13102) and serogroup L, (ATCC No. 43828) representing approximately 1,000 cells were amplified with a promoter-primer and primer described in Example 5 under the conditions described in Example 5. Ten Al samples of the 100 gl amplification reactions were assayed by hybridization with 25 an acridinium ester labeled probe synthesized with sequence 5'GCCAATATCGGCGGCCGATG-3' (SEQ ID NO. 3 and an unlabeled helper probe synthesized with the sequence ACGGTACCTGAAGAATAAGCACCGGCTAACTACGTG-3, (SEQ ID NO. or an acridinium ester labeled probe synthesized with the sequence 5'-GGCTGTTGCTAATATCAGCG-3' (SEQ ID NO. 11) and two unlabeled helper probes, one synthesized with sequence 5'-GCCTTCGGGTTGTAAAGGACTTTTGTCAGGGAAGAAAA-3' (SEQ ID NO.

13) and one synthesized with the sequence 5'-GCTGATGACGGTACCTGAAGAATAAGCACCGGC-3' (SEQ ID NO. 14), or an acridinium ester labeled probe synthesized with sequence 5'-GGCTGTTGCTAATACCAGCG-3' (SEQ ID NO. 12) with unlabeled helper probes SEQ ID NO: 13 and 14 or with a combination of labeled probes SEQ ID NO: 11 and 12 used with unlabeled helper probes SEQ ID NO: 13 and 14.

Sequence analysis indicated that other strains of Neisseria will also amplify with these primers.

Table 6. Amplification of Neisseria aonorrhoeae and Neisseria meningitidis strains using primers comprising SEQ ID NOs. 7 and 9.

RLU

Probe SEQ ID 3 11 12 11+12 15 NOs: Helper probe 4 13+14 13+14 13+14 SEQ ID NOs: Organism N. gonorrhoeae 1,017,626 1,660 820 1,603 994,788 1,448 809 1,559 1,030,242 1,743 805 1,792 20 N. meningitidis 2,059 1,208,967 3,534 829,251 Serogroup A 1,861 1,115,956 3,700 760,360 2,183 1,138,675 3,546 775,675 N. meningitidis 1,931 1,164,254 2,819 749,502 Serogroup C 2,130 1,068,489 2,477 687,517 1,963 1,110,933 3,103 803,732 N. meningitidis 1,833 85,321 1,206,045 1,537,314 Serogroup L 1,972 79,555 1,199,815 1,474,016 1,814 77,797 1,211,022 1,645,742 The data show that strains of N. menincitidis and N.

aonorrhoeae can be amplified using primers comprising

SEQ

ID NOs. 7 and 9 and detected with probes of SEQ ID NOs. 3, 11, and 12.

Example 7.

The sensitivity of the amplification and detection assay for N. meninqitidis were demonstrated in this experiment. In this example, Neisseria meninqitidis serogroup C cells were cultured and suspended in 0.9% sodium chloride to a density of approximately 10' cells per ml. Cells were lysed following addition of an equal volume of a solution containing 3 lithium lauryl sulfate, 30 mM sodium phosphate (pH 1 mM EDTA,. 1 mM EGTA and diluted with water prior to addition to the amplification reactions. Amplifications were performed as described for Example 5 using the promoter primer and primer described in Example 5 (SEQ ID NOs. 7 and 9, respectively). Twenty Al of the reaction was analyzed by hybridization in the HPA format using an acridinium ester labeled probe synthesized with the sequence 5'-GGCTGTTGCTAATATCAGCG-3' (SEQ ID NO. 11) and two unlabeled helper probes, one synthesized with the sequence 5' -GCCTTCGGGTTG- 25 TAAAGGACTTTTGTCAGGGAAGAAAA-3' (SEQ ID NO. 13) and one synthesized with the sequence 5'-GCTGATGACGGTACCTGAAGAATAAGCACCGGC-3' (SEQ ID NO. 14).

Table 7. Amplification of N. menincitidis serogroup A with amplification oligomers comprising SEQ ID NOs. 7 and 9, followed by detection with probe SEQ ID NO. 11.

Amount of RLU with target added probe SEQ ID NO. 11 cells 723,645 648,069 686,492 4 cells 195,370 189,451 162,128 0.4 cells 28,585 23,253 824,742 64,945 0 cells 1,432 1,202 1,258

S

C

ExamDle 8.

To demonstrate the reactivity and specificity of the probes directed to N. meningitidis 16S rRNA, a mixture of probes containing acridinium ester labeled oligonucleotides synthesized with the sequence (SEQ ID NO. 15) or sequence (SEQ ID NO. 16), and unlabeled helper probes synthesized with the sequence 5' -TTTTCTTCCCTGACAAAAGTCCTTTACAAC- CCGAAGGC-3' (SEQ ID NO. 17 and CGTCATCAG-3' (SEQ ID NO. 18), were hybridized to nucleic acid in lysates prepared from fresh cultures of the Neisseria species listed below. Each lysate was tested with a probe directed to a conserved region of 23S rRNA to confirm the lysis of the organism and integrity of the rRNA.

74 Table 8. Reactivity and specificity of probes directed to N. meninaitidis 16S rRNA.

organism ATCC RLU with RLU with No. probe mix* conserved probe N. cinerea 14685 736,927 59,831 N. denitrificans 14686 581 50,391 N. elonaata 25295 1,511 52,017 N. elnrata subspe- 29315 618 53,312 cies glycolytica N. flavescens 13120 1,316 53,397 N. gonorrhoeae 9793 1,826 62,658 N. aonorrhoeae 9827 753 60,252 N. gonorrhoeae 9830 4,832 58,346 N. aonorrhoeae 10150 1,139 61,573 N. cronorrhoeae 10874 759 58,291 N. gonorrhoeae 11689 4,824 60,039 N. gonorrhoeae 19088 910 53,594 N. cronorrhoeae 19424 851 60,372 N. cronorrhoeae 21824 746 62,153 N. gonorrhoeae 27630 1,829 53,241 N. cronorrhoeae 33084 784 62,696 N. cronorrhoeae 35541 431 59,229 N. lactamica 23970 3,497 54,255 I r N. meningitidia serogroup A 13077 844, 739 54,292 9* a a N. meningitidis ser- 23255 722,108 61,439 ogroup B N. meriingitidis ser- 13090 704,890 57,321 ogroup B N. meningitidis ser- 23251 761,475 58,545 ogroup B N. meninqitidis, ser- 13103 770-,221 63,704 ogroup C N. meriinq~itidis ser- 13106 761,099 60,928 ogroup C N. meninitidia ser- 13102 752,743 62,351 ogroup C N. meningitidis ser- 13111 711,196 59,635 ogroup C N. meninqitidis ser- 13109 768,874 63,295 ogroup C N. meninqitidia ser- 13110 676, 060 58,150 ogroup C N. menincitidis ser- 13112 543,492 54,921 ogroup C N. menincritidis se- 23248 321,600 59,308 rogroup C N. meningitidis ser- 13113 770,893 56,429 ogroup D N. meninqitidis 35558 797,072 58,882 group E N. meinqtitidis ser- 43828 559,406 61,534 ogroup L N. meninaitidis ser- 43744 705,798 62,152 ogroup W-135 N. meningitidis ser- 35561 778,600 .54,938 ogroup Y N. meningitidis s- 35562 749,756 61,793 erogroup Z N. meninqitidis 13095 726,612 52,614 N. meninqitidis 13101 775,912 59,839 N. meninqitidis 13804 785,737 61,790 N. meninqitidis 43743 734,400 61,357 N. mucosa 19696 1,560 53,427 N. mucosa subspecies 25999 1,761 59,306 heidelbergensis N. sicca 29193 1,205 58,260 15 N. sicca 9913 2,203 57,764 N. subflava 14799 2,046 50,832 Negative sample 5,251 124 467 132 1,691 138 *probe mix contained acridinium ester labeled probes synthesized with sequences of SEQ ID NO. 15 and SEQ ID NO.

20 16 and unlabeled helper probes synthesized with sequences of SEQ ID NO. 17 and SEQ ID NO. 18.

The data show that the mixture of probes allowed detection of all of the N. meninqitidis strains tested. The probe mix did show a cross reaction with N. cinerea, an organism unlikely to be found in the same clinical samples as N_.

meninqitidis. Treatment of patients with N. cinerea infections would be the same as for patients infected with N. meninqitidis.

Example 9.

This example demonstrates the specificity of the amplification and detection assay. Thirty pmol of the promoter-primer comprising SEQ. ID. NO. 7 and 30 pmol of the primer comprising SEQ. ID. NO. 9 were used in the assay with eleven different Neisseria species. Cell lysates were prepared as described in Example 5 and amplified and analyzed by hybridization using the conditions described in Example 1. Twenty microliters of the amplification reactions were hybridized to an acridinium ester labeled probe synthesized with sequence CGGCGGCCGATG-3' (SEQ ID NO. 3) and an unlabeled helper probe synthesized with the sequence AGCACCGGCTAACTACGTG-31 (SEQ ID NO. or an acridinium ester labeled probe synthesized with the sequence GGCTGTTGCTAATATCAGCG-3' (SEQ ID NO. 11) in the presence of unlabeled helper probes synthesized with sequences comprising SEQ ID NOs. 13 and 14, or an acridinium ester labeled probe synthesized with the sequence TAATACCAGCG-3' (SEQ ID NO. 12) in the presence of unlabeled helper probes of SEQ ID NOs. 13 and 14.

Table 9. Specificity of an assay usinq amplification with oliqonucleotides comprising SEQ ID NOs. 7 and 9 followed by detection with probes comprising SEO ID NOs. 3, 11 or 12. oo-

L

RLU

SEQ ID Probe Probe to 11 12 3 NOs.. conserved regions of bacterial. rRNA Help- 13+14 13+14 4 ers organism ATCC No.

Neisseria 14685 2,468,721 540,699 1,804 1,633 cinerea 609,648 2,484 1,536 575,050. 1,943 1,494 Neisseria 14686 2,339,034 740 644 1,563 denitrifi- 659 578 1,539 cans Neisseria 25295 2,486,745 772 428 1,521 eloncrata 738 3,297 1,.528 Neisseria 29315 2,397,697 697 431 1,443 eloncrata 954 813 1,528 subspecies orlycolytica Neisseria 13120 2,622,452 780 493 1,547 flavescens 874 481 1,610 969 429 1,589 Neisseria 23970 2,299,619 736 410 1,621 lactamica 839 425 1,544 1,583 428 1,559 Neisseria 19696 2,565,699 1,021 981 1,596 mucosa 1,408 559 6,781 1851 5,260 A1,574 4. 5

S.

S S S. 4.

*S

S S .5 i

S

.5 S S

S

Neisseria mucosa heidelbergensis 25999 2,927,147 653 664 367 390 1,430 1,971 Neisseria 9913 2,427,561 699 777 1,609 sicca 847 477 1,552 834 437 1,642 Neisseria 29193 2,804,642 954 423 1,588 sicca 615 388 1,505 Neisseria 19424 N.T. 3,826 419 586,358 conor- 1,092 411 564,987 rhoeae* 2,390 388 554,134 Neisseria 13077 N.T. 557,656 1,287 1,492 meningiti- 621,180 1,009 1,509 dis* Sero- 539,592 954 1,617 group A

CC

be S *5

C

en.

*purified RNA used at N.T. Not tested.

500 pg per reaction.

C

C. e.

49 Ce C S C S. C

**CC

C

eeC.

*e C.

S C

S

0

S

The data shown in the examples described above confirm that the novel amplification oligonucleotides herein 20 described and claimed are capable of amplifying Neisseria nucleic acid and can be used in an assay to distinguish Neisseria meninqitidis or Neisseria qonorrhoeae from each other, the closest known phylogenetic neighbours. None of the examples described herein are intended to limit the 25 present invention to the embodiments of this disclosure, said invention being limited exclusively by the claims which follow.

SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

Gen-Probe Incorporated 9880 Campus Point Drive San Diego, California 92121 NUCLEIC ACID PROBES AND AMPLIFICATION

OLIGO-

NUCLEOTIDES FOR NEISSERIA SPECIES (ii) TITLE OF INVENTION: (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS:

ADDRESSEE:

STREET:

CITY:

STATE:

COUNTRY:

ZIP:

Lyon Lyon 633 West Fifth Street Suite 4700 Los Angeles California

U.S.A.

90071-2066 .00 *m 099 COMPUTER READABLE FORM: MEDIUM TYPE:

COMPUTER:

OPERATING SYSTEM:

SOFTWARE:

3.5" Diskette, 1.44 Mb storage IBM Compatible IBM P.C. DOS Word Perfect 5.1

**SS

S

69 *55*

SO

55 9.95 0*

C

S@ Se S S O 0

*SSOOO

0 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: To Be Assigned 30 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: FILING DATE: U.S. 08/475,460 June 7, 1995 (viii) ATTORNEY/AGENT INFORMATION:

NAME:

Guise, Jeffrey

W.

REGISTRATION NUMBER: REFERENCE/DOCKET

NUMBER:

(ix) TELECOMMUNICATION

INFORMATION:

34,613 2 07 /230-PCT

TELEPHONE:

TELEFAX:

TELEX:

(213) 489-1600 (213) 955-0440 67-3510 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAACGTACCG GGTAGCGG INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

38 base pairs nucleic acid single linear SEQ ID NO: 2: (xi) SEQUENCE DESCRIPTION: GGGATAACTG ATCGAAAGAT CAGCTAATAC CGCATACG INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GCCAATATCG GCGGCCGATG INFORMATION FOR SEQ ID NO: 4: SEQUENCE

CHARACTERISTICS:

LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: ACGGTACCTG AAGAATAAGC ACCGGCTAAC

TACGTG

INFORMATION FOR SEQ ID NO: SEQUENCE

CHARACTERISTICS:

LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GTCCCCTGCT TTCCCTCTCA

AGAC

INFORMATION FOR SEQ ID NO: 6: SEQUENCE

CHARACTERISTICS:

LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GGCGAGTGGC GAACGGGTGA

GTAACATA

25 INFORMATION FOR SEQ ID NO: 7: SEQUENCE

CHARACTERISTICS:

LENGTH: 36 base.pairs TYPE: nucleic acid STRANDEDNESS: single 30 TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GCTGCTGCAC GTAGTTAGCC GGTGCTTATT

CTTCAG

83 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GTTAGCCGGT GCTTATTCTT CAGGTACCGT CATCG INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CGGGTTGTAA AGGACTTTTG TCAGGGAAGA AAAGGCCGTT INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GAAGGCCTTC GGGTTGTAAA GGAC 24 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: .GGCTGTTGCT AATATCAGCG INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 20 bas TYPE: nuclei STRANDEDNESS: single TOPOLOGY: linear e pairs c acid ID NO: 12: (xi) SEQUENCE DESCRIPTION: GGCTGTTGCT AATACCAGCG

SEQ

INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GCCTTCGGGT TGTAAAGGAC TTTTGTCAGG GAAGAAAA INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GCTGATGACG GTACCTGAAG AATAAGCACC GGC INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CGCTGATATT AGCAACAGCC INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 20 bas TYPE: nuclei STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION:

SEQ

CGCTGGTATT

AGCAACAGCC

INFORMATION FOR SEQ ID NO: 17: 0 SEQUENCE CHARACTERISTICS: LENGTH: 38 bas TYPE: nuclei STRANDEDNESS: single TOPOLOGY: linear e pairs c acid ID NO: 16: e pairs c acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: TTTTCTTCCC TGACAAAAGT CCTTTACAAC

CCGAAGGC

INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GCCGGTGCTT ATTCTTCAGG TACCGTCATC AGC INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: CTGAAGAATA AGCACCGGCT AACTACGTGC AGCAGC INFORMATION FOR SEQ ID NO: SEQUENCE

CHARACTERISTICS:

LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AACGGCCTTT TCTTCCCTGA CAAAAGTCCT TTACAACCCG INFORMATION FOR SEQ ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: CGATGACGGT ACCTGAAGAA TAAGCACCGG CTAAC INFORMATION FOR SEQ ID NO: 22: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: GTCCTTTACA ACCCGAAGGC

CTTC

25 INFORMATION FOR SEQ ID NO: 23: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: GTCTTGAGAG GGAAAGCAGG GGAC 0.00 INFORMATION FOR SEQ ID NO: 24: SEQUENCE CHARACTERISTICS: LENGTH: 28 bas TYPE: nuclei STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION:

SEQ

TATGTTACTC ACCCGTTCGC CACTCGCC INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 bas TYPE: nuclei STRANDEDNESS: single TOPOLOGY: linear e pairs c acid ID NO: 24: e pairs c acid ID NO: (xi) SEQUENCE DESCRIPTION: GGCUGUUGCU AAUAUCAGCG

SEQ

a INFORMATION FOR SEQ ID NO: 26: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: GGCUGUUGCU AAUACCAGCG 25 INFORMATION FOR SEQ ID NO: 27: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: CGCUGAUAUU AGCAACAGCC INFORMATION FOR SEQ ID NO: 28: SEQUENCE

CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

20 base pairs nucleic acid single linear SEQ ID NO: 28: (xi) SEQUENCE

DESCRIPTION:

CGCUGGUAUU

AGCAACAGCC

INFORMATION FOR SEQ ID NO: 29: SEQUENCE

CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

18 base pairs nucleic acid single linear SEQ ID NO: 29: (xi) SEQUENCE

DESCRIPTION:

CCGCTACCCG

GTACGTTC

INFORMATION FOR SEQ ID NO: SEQUENCE

CHARACTERISTICS:

9* a..

S

C

S. *5

S

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

20 base pairs nucleic acid single linear SEQ ID NO: (xi) SEQUENCE

DESCRIPTION:

CATCGGCCGC

CGATATTGGC

INFORMATION FOR SEQ ID NO: 31: SEQUENCE

CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

18 base pairs nucleic acid single linear SEQ ID NO: 31: (xi) SEQUENCE

DESCRIPTION:

GAACGUACCG

GGUAGCGG

INFORMATION FOR SEQ ID NO: 32: SEQUENCE

CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: GCCAAUAUCG GCGGCCGAUG INFORMATION FOR SEQ ID NO: 33: SEQUENCE

CHARACTERISTICS:

LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: CCGCUACCCG GUACGUUC 18 INFORMATION FOR SEQ ID NO: 34: SEQUENCE

CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: CAUCGGCCGC CGAUAUUGGC 25 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single 30 TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GCCUUCGGGU UGUAAAGGAC UUUUGUCAGG GAAGAAAA 6 0 0 00 S. .0 S S 0 0 *0S00* 0 5 INFORMATION FOR SEQ ID NO: 36: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: GCUGAUGACG GUACCUGAAG AAUAAGCACC GGC 33 INFORMATION FOR SEQ ID NO: 37: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: UUUUCUUCCC UGACAAAAGU CCUUUACAAC CCGAAGGC 38 INFORMATION FOR SEQ ID NO: 38: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: GCCGGUGCUU AUUCUUCAGG UACCGUCAUC AGC 33 INFORMATION FOR SEQ ID NO: 39: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: GGGAUAACUG AUCGAAAGAU CAGCUAAUAC CGCAUACG 38 INFORMATION FOR SEQ ID NO: SEQUENCE

CHARACTERISTICS:

LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ACGGUACCUG AAGAAUAAGC ACCGGCUAAC UACGUG 36 INFORMATION FOR SEQ ID NO: 41: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: GUCCCCUGCU UUCCCUCUCA AGAC 24 INFORMATION FOR SEQ ID NO: 42: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: So GGCGAGUGGC GAACGGGUGA GUAACAUA 28 25 INFORMATION FOR SEQ ID NO: 43: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: GCUGCUGCAC GUAGUUAGCC GGUGCUUAUU CUUCAG 36 coo* INFORMATION FOR SEQ ID NO: 44: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: GUUAGCCGGU GCUUAUUCUU CAGGUACCGU

CAUCG

INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CGGGUUGUAA AGGACUUUUG UCAGGGAAGA

AAAGGCCGUU

INFORMATION FOR SEQ ID NO: 46: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs 20 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: GAAGGCCUUC GGGUUGUAAA

GGAC

25 INFORMATION FOR SEQ ID NO: 47: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: GUCUUGAGAG GGAAAGCAGG GGAC INFORMATION FOR SEQ ID NO: 48: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48: UAUGUUACUC ACCCGUUCGC

CACUCGCC

INFORMATION FOR SEQ ID NO: 49: SEQUENCE

CHARACTERISTICS:

LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: CUGAAGAAUA AGCACCGGCU AACUACGUGC

AGCAGC

-INFORMATION FOR SEQ ID NO: SEQUENCE

CHARACTERISTICS:

LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CGAUGACGGU ACCUGAAGAA UAAGCACCGG

CUAAC

INFORMATION FOR SEQ ID NO: 51: SEQUENCE

CHARACTERISTICS:

LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single 30 TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: AACGGCCUUU UCUUCCCUGA CAAAAGUCCU UUACAACCCG INFORMATION FOR SEQ ID NO: 52: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52: GUCCUUUACA ACCCGAAGGC CUUC INFORMATION FOR SEQ ID NO: 53: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53: AATTTAATAC GACTCACTAT AGGGAGA 20 *o *oo *oooo INFORMATION FOR SEQ ID NO: 54: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: GAGUAAAGUU AAUACCUUUG INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GGCUGUUGCU AAUAUCAGCG INFORMATION FOR SEQ ID NO: 56: SEQUENCE

CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56: GGCUGUUGCC

AAUAUCGGGG

Claims (23)

1. A probe mix for detecting Neisseria gonorrhoeae comprising a nucleic acid hybridisation assay probe for detecting Neisseria gonorrhoeae under stringent hybridisation assay conditions which substantially corresponds to an oligonucleotide having a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1 GAACGTACCG GGTAGCGG, SEQ ID NO: 31 GAACGUACCG GGUAGCGG, SEQ ID NO: 3 GCCAATATCG GCGGCCGATG, and SEQ ID NO: 32 GCCAAUAUCG GCGGCCGAUG, and a helper oligonucleotide

10-100 nucleotides in length which hybridises to a Neisseria gonorrhoeae nucleic acid sequence under stringent hybridisation conditions, said helper oligonucleotide having a nucleic acid sequence substantially corresponding to: sequence SEQ ID NO: 2 or SEQ ID NO: 39 when said nucleic acid hybridisation assay probe substantially 10 corresponds to SEQ ID NO: 1 or SEQ ID NO: 31; and to SEQ ID NO: 4 or SEQ ID NO: 40 when said nucleic acid hybridisation assay probe substantially corresponds to SEQ ID NO: 3 or SEQ ID :NO: 32. 2. The probe mix of claim 1, wherein said oligonucleotide has a nucleic acid sequence substantially corresponding to the following nucleic acid sequence: SEQ ID NO: 1 GAACGTACCG GGTAGCGG. s 3. The probe mix of claim 1, wherein said oligonucleotide has a nucleic acid sequence substantially corresponding to the following nucleic acid sequence: SEQ ID NO: 31 GAACGUACCG GGUAGCGG. 4. The probe mix of claim 1, wherein said oligonucleotide has a nucleic acid sequence substantially corresponding to the following nucleic acid sequence: SEQ ID NO: 3 GCCAATATCG GCGGCCGATG. 5. The probe mix of claim 1, wherein said oligonucleotide has a nucleic acid sequence substantially corresponding to the following nucleic acid sequence: SEQ ID NO: 32 GCCAAUAUCG GCGGCCGAUG. 6. The probe mix of any one of claims 2-5, wherein said mix comprises an oligonucleotide consisting of a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1 GAACGTACCG GGTAGCGG, SEQ ID NO: 31 GAACGUACCG GGUAGCGG, j o.^SEQ ID NO: 3 GCCAATATCG GCGGCCGATG, and i( LEQ ID NO: 32 GCCAAUAUCG GCGGCCGAUG, 11ADavIih\T and a helper oligonucleotide 10-100 nucleotides in length which hybridises to a Neisseria gonorrhoeae nucleic acid sequence under stringent hybridisation conditions, said helper oligonucleotide having a nucleic acid sequence substantially corresponding to: sequence SEQ ID NO: 2 or SEQ ID NO: 39 when said nucleic acid hybridisation assay probe substantially corresponds to SEQ ID NO: 1 or SEQ ID NO: 31; and to SEQ ID NO: 4 or SEQ ID NO: 40 when said nucleic acid hybridisation assay probe substantially corresponds to SEQ ID NO: 3 or SEQ ID NO: 32. 7. A composition for detecting Neisseria gonorrhoeae comprising a nucleic acid hybrid formed under stringent hybridisation conditions between a Neisseria gonorrhoeae nucleic acid and an oligonucleotide having a nucleic acid sequence substantially corresponding to a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1 GAACGTACCG GGTAGCGG, SEQ ID NO: 31 GAACGUACCG GGUAGCGG, SEQ ID NO: 29 CCGCTACCCG GTACGTTC, SEQ ID NO: 33 CCGCUACCCG GUACGUUC, SEQ ID NO: 3 GCCAATATCG GCGGCCGATG, SEQ ID NO: 32 GCCAAUAUCG GCGGCCGAUG, SEQ ID NO: 30 CATCGGCCGC CGATATTGGC, and SEQ ID NO: 34 CAUCGGCCGC CGAUAUUGGC. 8. The composition of claim 7 further comprising a helper oligonucleotide which has a nucleotide sequence that substantially corresponding to a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 4 ACGGTACCTG AAGAATAAGC ACCGGCTAAC TACGTG, SEQ ID NO: 40 ACGGUACCUG AAGAAUAAGC ACCGGCUAAC UACGUG, SEQ ID NO: 2 GGGATAACTG ATCGAAAGAT CAGCTAATAC CGCATACG, and SEQ ID NO-39 GGGAUAACUG AUCGAAAGAU CAGCUAAUAC CGCAUACG. 9. A method for amplifying Neisseria gonorrhoeae nucleic acid in a test sample comprising amplifying said nucleic acid with one or more amplification oligonucleotides that will bind to or cause elongation through a nucleic acid sequence substantially corresponding to one or more of the following nucleic acid sequences: SEQ ID NO: 23 GTCTTGAGAG GGAAAGCAGG GGAC, SEQ ID NO: 47 GUCUUGAGAG GGAAAGCAGG GGAC, SEQ ID NO: 24 TATGTTACTC ACCCGTTCGC CACTCGCC, and S' SEQ ID NO: 48 UAUGUUACUC ACCCGUUCGC CACUCGCC, i~ I in or, wherein at least one of said amplification oligonucleotides optionally has a nucleic acid sequence recognised by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase. The method of claim 9, wherein said amplification oligonucleotides include an amplification oligonucleotide having a nucleic acid sequence substantially corresponding to the following nucleic acid sequence: SEQ ID NO: 23 GTCTTGAGAG

11. The method of claim amplification oligonucleotide having following nucleic acid sequence: SEQ ID NO: 47 GUCUUGAGAG

12. The method of claim amplification oligonucleotide having following nucleic acid sequence: SEQ ID NO: 24 TATGTTACTC

13. The method of claim 0 0**0 GGAAAGCAGG GGAC. 9, wherein said amplification oligonucleotides include an a nucleic acid sequence substantially corresponding to the GGAAAGCAGG GGAC. 9, wherein said amplification oligonucleotides include an a nucleic acid sequence substantially corresponding to the ACCCGTTCGC CACTCGCC. 9, wherein said amplification oligonucleotides include an amplification oligonucleotide having a nucleic acid sequence substantially corresponding to the following nucleic acid sequence: SEQ ID NO: 48 UAUGUUACUC ACCCGUUCGC CACUCGCC.

14. The method of any one of claims 10-13, wherein said amplification oligonucleotides include an amplification oligonucleotide consisting of a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 23 GTCTTGAGAG GGAAAGCAGG GGAC, SEQ ID NO: 47 GUCUUGAGAG GGAAAGCAGG GGAC, SEQ ID NO: 24 TATGTTACTC ACCCGTTCGC CACTCGCC, and SEQ ID NO: 48 UAUGUUACUC ACCCGUUCGC CACUCGCC, wherein said amplification oligonucleotide optionally has a nucleic acid sequence recognised by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase. The method of any one of claims 9-14 further comprising the step of detecting the amplified nucleic acid with an oligonucleotide hybridization assay probe able to specifically hybridise to the amplified Neisseria gonorrhoeae nucleic acid under stringent hybridization conditions. FI-\ 1 i h\i InpP r1f6i70cr-- i

16. The method of claim 15 wherein said oligonucleotide hybridization assay probe has a nucleic acid sequence substantially corresponding to a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1 GAACGTACCG GGTAGCGG, SEQ ID NO: 29 CCGCTACCCG GTACGTTC, SEQ ID NO: 31 GAACGUACCG GGUAGCGG, and SEQ ID NO: 33 CCGCUACCCG GUACGUUC.

17. The method of claim 16 wherein said detecting step includes the use of a helper oligonucleotide having a nucleic acid sequence substantially corresponding to a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 2 GGGATAACTG ATCGAAAGAT CAGCTAATAC CGCATACG, and SEQ ID NO: 39 GGGAUAACUG AUCGAAAGAU CAGCUAAUAC CGCAUACG.

18. A method for amplifying Neisseria gonorrhoeae nucleic acid in a test sample comprising amplifying said nucleic acid with one or more amplification oligonucleotides that will bind to or cause elongation through a nucleic acid sequence substantially corresponding to one or more 10o of the following nucleic acid sequences: SEQ ID NO: 19 CTGAAGAATA AGCACCGGCT AACTACGTGC AGCAGC, SEQ ID NO: 49 CUGAAGAAUA AGCACCGGCU AACUACGUGC AGCAGC, SEQ ID NO: 20 AACGGCCTTT TCTTCCCTGA CAAAAGTCCT TTACAACCCG, SEQ ID NO: 51 AACGGCCUUU UCUUCCCUGA CAAAAGUCCU UUACAACCCG, SEQ ID NO: 21 CGATGACGGT ACCTGAAGAA TAAGCACCGG CTAAC, SEQ ID NO: 50 CGAUGACGGU ACCUGAAGAA UAAGCACCGG CUAAC, SEQ ID NO: 22 GTCCTTTACA ACCCGAAGGC CTTC, and SEQ ID NO: 52 GUCCUUUACA ACCCGAAGGC CUUC, wherein at least one of said amplification oligonucleotides optionally has a nucleic acid sequence recognised by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase.

19. The method of claim 18, wherein said amplification oligonucleotides include an amplification oligonucleotide having a nucleic acid sequence substantially corresponding to the following nucleic acid sequence: SEQ ID NO: 19 CTGAAGAATA AGCACCGGCT AACTACGTGC AGCAGC. The method of claim 18, wherein said amplification oligonucleotides include an piplification oligonucleotide having a nucleic acid sequence substantially corresponding to the following nucleic acid sequence: (TAn.,vi iI,\1 IPFF1061'70- 100 SEQ ID NO: 49 CUGAAGAAUA

21. The method of claim amplification oligonucleotide having following nucleic acid sequence: SEQ ID NO: 20 AACGGCCTTT

22. The method of claim amplification oligonucleotide having following nucleic acid sequence: SEQ ID NO: 51 AACGGCCUUU AGCACCGGCU AACUACGUGC AGCAGC. 18, wherein said amplification oligonucleotides include an a nucleic acid sequence substantially corresponding to the TCTTCCCTGA CAAAAGTCCT TTACAACCCG. 18, wherein said amplification oligonucleotides include an a nucleic acid sequence substantially corresponding to the UCUUCCCUGA CAAAAGUCCU UUACAACCCG.

23. The method of claim 18, wherein said amplification oligonucleotides include an amplification oligonucleotide having a nucleic acid sequence substantially corresponding to the 9 9 9 *9**99 9 *99* 9* 9 99*e *9*9 .9 following nucleic acid sequence: SEQ ID NO: 21 CGATGACGGT 10o 24. The method of claim amplification oligonucleotide having following nucleic acid sequence: SEQ ID NO: 50 CGAUGACGGU

25. The method of claim amplification oligonucleotide having 15 following nucleic acid sequence: SEQ ID NO: 22 GTCCTTTACA

26. The method of claim amplification oligonucleotide having ACCTGAAGAA TAAGCACCGG CTAAC. 18, wherein said amplification oligonucleotides include an a nucleic acid sequence substantially corresponding to the ACCUGAAGAA UAAGCACCGG CUAAC. 18, wherein said amplification oligonucleotides include an a nucleic acid sequence substantially corresponding to the ACCCGAAGGC CTTC. 18, wherein said amplification oligonucleotides include an a nucleic acid sequence substantially corresponding to the following nucleic acid sequence: SEQ ID NO: 52 GUCCUUUACA ACCCGAAGGC CUUC.

27. The method of any one of claims 18-26 further comprising the step of detecting the amplified .nucleic acid with an oligonucleotide hybridization assay probe able to specifically hybridise to the amplified Neisseria gonorrhoeae nucleic acid under stringent hybridization conditions.

28. The method of claim 27 wherein said oligonucleotide hybridization assay probe has a nucleic acid sequence substantially corresponding to a nucleic acid sequence selected from the group consisting of: TR SEQ ID NO: 3 GCCAATATCG GCGGCCGATG, Q ID NO: 32 GCCAAUAUCG GCGGCCGAUG, [I:\DayLib\LIBFF1061 70snrne.r -r 101 SEQ ID NO: 30 CATCGGCCGC CGATATTGGC, and SEQ ID NO: 34 CAUCGGCCGC CGAUAUUGGC.

29. The method of claim 28 wherein said detecting step includes the use of a helper oligonucleotide having a nucleic acid sequence substantially corresponding to a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 4 ACGGTACCTG AAGAATAAGC ACCGGCTAAC TACGTG, and SEQ ID NO: 40 ACGGUACCUG AAGAAUAAGC ACCGGCUAAC UACGUG. The method of claim 9 or 18 wherein said optional nucleic acid sequence substantially corresponds to SEQ ID NO: 53.

31. A kit containing four oligonucleotides, each said oligonucleotide having a nucleic acid sequence substantially corresponding to one of the following nucleic acid sequences: SEQ ID NO: 5 GTCCCCTGCT TTCCCTCTCA AGAC, or SEQ ID NO: 41 GUCCCCUGCU UUCCCUCUCA AGAC, and SEQ ID NO: 6 GGCGAGTGGC GAACGGGTGA GTAACATA, or SEQ ID NO: 42 GGCGAGUGGC GAACGGGUGA GUAACAUA, and SEQ ID NO: 2 GGGATAACTG ATCGAAAGAT CAGCTAATAC CGCATACG, or SEQ ID NO: 39 GGGAUAACUG AUCGAAAGAU CAGCUAAUAC CGCAUACG, and SEQ ID NO: 1 GAACGTACCG GGTAGCGG, or SEQ ID NO: 31 GAACGUACCG GGUAGCGG, 9° wherein at least one of said oligonucleotides optionally has a nucleic acid sequence recognised by an RNA polymerase or which enhances initiation or elongation by an 1 0 RNA polymerase.

32. A kit containing three oligonucleotides, each said oligonucleotide having a nucleic acid sequence substantially corresponding to one of the following nucleic acid sequences: SEQ ID NO: 5 GTCCCCTGCT TTCCCTCTCA AGAC, or SEQ ID NO: 41 GUCCCCUGCU UUCCCUCUCA AGAC, and SEQ ID NO: 2 GGGATAACTG ATCGAAAGAT CAGCTAATAC CGCATACG, or SEQ ID NO: 39 GGGAUAACUG AUCGAAAGAU CAGCUAAUAC CGCAUACG, and SEQ ID NO: 1 GAACGTACCG GGTAGCGG, or SEQ ID NO: 31 GAACGUACCG GGUAGCGG, wherein at least one of said oligonucleotides optionally has a nucleic acid sequence recognised by an RNA polymerase or which enhances initiation or elongation by an RK- 4 RNA polymerase. :\DavLi M I B FF106I 79srec. doc: 2ce a 102

33. A probe mix for detecting Neisseria gonorrhoeae, substantially as hereinbefore described with reference to any one of the examples.

34. A composition for detecting Neisseria gonorrhoeae, substantially as hereinbefore described with reference to any one of the examples. A method for amplifying Neisseria gonorrhoeae nucleic acids in a test sample, substantially as hereinbefore described with reference to any one of the examples.

36. A kit containing at least three oligonucleotides, substantially as hereinbefore described with reference to any one of the examples. Dated 19 June, 2001 Gen-Probe Incorporated Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON peg 0 0 1 a S 54*0 so&* Go** so a f1:\Davhb\TARFF106 1 7 Qkrec H