JP7263763B2 - Oligonucleotide used for detection of Legionella spp. and detection method thereof - Google Patents

Description

TECHNICAL FIELD The present invention relates to an oligonucleotide for rapid, highly sensitive and specific detection of Legionella contained in a sample, and a method for detecting Legionella using the oligonucleotide.

Legionella is a Gram-negative rod-shaped bacterium that exists in water or moist soil using protozoa such as amoeba as hosts. Legionella is classified into about 50 species, and it is believed that all species can cause legionellosis. In recent years, legionella bacteria proliferate in artificial environments such as cooling towers, circulating hot spring water, and 24-hour baths, and infection with legionellosis by inhalation of generated aerosols has become a problem. It is characteristic that the peak is seen mainly in July every year. The disease type includes transient Pontiac fever and fulminant pneumonia. The former is transient and cured, but the latter, Legionella pneumonia, progresses rapidly and requires early treatment, but it has no specific symptoms, so it is difficult to distinguish it from other pneumonia based on symptoms alone. be. In addition, since this bacterium mainly proliferates in cells such as macrophages, penicillin, cephem, and aminoglycodide antibiotics used for the treatment of other bacterial infections are effective because they have poor intracellular migration. Therefore, cell-translocating antibiotics such as erythromycin, rifampicin, and new quinolones are required. Without effective antibiotics, death often occurs within 7 days. Therefore, early diagnosis of Legionnaires' disease is necessary.

Clinically used methods for detecting Legionella spp. include a method of detection using an antibody against the urinary antigen of Legionella pneumophila serotype 1, and a gene that detects by amplifying a base sequence specific to Legionella spp. inspection methods, etc. However, the method of detecting using an antibody against the urinary antigen of Legionella pneumophila serotype 1 has a low sensitivity of about 60%, and can only detect Legionella pneumophila serotype 1, other bacteria Species sensitivity is poor. In genetic testing methods, detection methods based on nucleic acid amplification such as the LAMP method (Patent Document 1, Non-Patent Document 1) are used, and although both sensitivity and specificity are extremely high, the test takes time and lacks promptness. operation is complicated. Also, the LAMP method is insufficiently sensitive to some Legionella spp. and may miss them. Therefore, a highly sensitive, rapid and simple test is desired.

In the TRC method (Patent Document 2, Patent Document 3) used in the present invention, a device integrated from purification to detection is commercially available, and the result can be obtained in about 1 hour, so it is sufficiently rapid. I have it. In addition, both sensitivity and specificity are comparable to other detection methods based on nucleic acid amplification.

When the nucleic acid to be amplified is Legionella spp. 16S rRNA (ribosomal RNA) or its gene, there are many cross-reactive bacteria with high base sequence homology with 16S rRNA (or its genes) of other bacterial species. Therefore, it has been extremely difficult to design primer sets and oligonucleotide probes that detect Legionella spp. 16S rRNA or its genes with high sensitivity and specificity. Distinguishing from other pneumonia-causing bacteria was difficult. Particularly when using an amplification method capable of amplifying RNA under relatively low temperature constant temperature conditions (e.g., 40° C. to 50° C.), nucleic acids to be amplified tend to form higher-order structures. Oligonucleotide probe design was even more difficult.

JP 2009-131174 A Japanese Patent Application Laid-Open No. 2000-14400 JP-A-2001-37500

Japan Journal of Clinical Microbiology Vol. 23 No. 2 2013.

An object of the present invention is to provide a specific oligonucleotide that amplifies nucleic acids derived from Legionella spp. present in a sample with high sensitivity and speed and that is less prone to false positives, and a Legionella spp. using the oligonucleotide. is to provide a detection method for

The present inventor has completed the present invention as a result of earnest research in order to solve the above problems.

That is, the present invention is as follows.
(1) An oligonucleotide for detecting a specific base sequence of 23S rRNA or 23S rDNA of Legionella spp. Oligonucleotides for detecting Legionella characterized by containing bases.
(2) The oligonucleotide according to (1), which is labeled with a fluorescent dye.
(3) The oligonucleotide according to (2), wherein the oligonucleotide is configured to change its fluorescence properties when the oligonucleotide labeled with the fluorescent dye forms a complementary double strand. .
(4) The oligonucleotide according to (3), which is labeled with an intercalating fluorescent dye.
(5) A method for detecting a specific base sequence of 23S rRNA or 23S rDNA of a bacterium belonging to the genus Legionella or a complementary sequence thereof, characterized by using the oligonucleotide according to any one of (1) to (4) A method for detecting Legionella spp.
(6) The method for detecting Legionella spp. according to (5), which comprises the step of amplifying a specific base sequence or its complementary sequence using a set of primers.
(7) the set of primers is a first primer that hybridizes under stringent conditions to the sequence set forth in SEQ ID NO: 11 and/or hybridizes under stringent conditions to the sequence set forth in SEQ ID NO: 31; The method for detecting Legionella spp. according to (6), characterized by comprising a second primer that
(8) the first primer consists of 16 to 31 contiguous bases in the sequence set forth in SEQ ID NO: 11 or the complementary sequence, and the second primer is contiguous in the sequence set forth in SEQ ID NO: 31 or the complementary sequence; The method for detecting Legionella according to (7), which consists of 16 to 31 bases.

The present invention will be described in detail below.

Samples in the present invention include pharyngeal swab, sputum, bronchoalveolar lavage fluid, intratracheal aspirate, pleural effusion, blood, cerebrospinal fluid, culture fluid, environmental test water, and the like.

In the present invention, the specific base sequence of 23S rRNA or 23S rDNA of Legionella spp. is a sequence of 400 consecutive bases or less including the base sequence set forth in SEQ ID NO: 1 among the sequences of 23S rRNA or 23S rDNA of Legionella spp. Refers to a base sequence.
That is, in the present invention, a nucleic acid containing the specific base sequence or a sequence complementary to the specific base sequence is detected.
Conventionally known nucleic acid detection methods can be used to detect a nucleic acid containing a specific base sequence of Legionella 23S rRNA or a complementary sequence thereof using the oligonucleotide of the present invention. in particular,
(A) a method using electrophoresis or liquid chromatography;
(B) a hybridization method with an oligonucleotide probe labeled with a detectable label;
(C) Designed to change fluorescence properties by hybridizing a nucleic acid containing the specific nucleotide sequence or its complementary sequence with a part of the nucleotide sequence of an amplification product amplified using a set of primers a method using a fluorescent dye-labeled oligonucleotide;
etc. Examples of the (C) fluorescent dye-labeled oligonucleotide include fluorescent-labeled oligonucleotides utilizing FRET (fluorescence resonance energy transfer) and oligonucleotides labeled with an intercalating fluorescent dye.

As an example of an oligonucleotide labeled with an intercalating fluorescent dye, it specifically hybridizes under stringent conditions with a part of a nucleic acid containing a specific base sequence of Legionella 23S rRNA or a sequence complementary to the specific base sequence. There is an oligonucleotide labeled with an intercalating fluorescent dye through an appropriate linker at the 3′-end side, 5′-end side, phosphodiester portion or base portion of a lysable oligonucleotide. When the oligonucleotide forms a complementary double strand with a specific base sequence of Legionella spp. 23S rRNA (or a sequence complementary to the specific base sequence), the intercalating fluorescent dye portion intercalates into the complementary double strand portion. It is a probe whose fluorescence properties change when exposed to light. There is no particular limitation on the intercalating fluorescent dye to be labeled, and from widely used fluorescent dyes such as oxazole yellow, thiazole orange, ethidium bromide, hemicyanine, and derivatives thereof, considering the fluorescence intensity and fluorescence characteristics, It can be selected as appropriate. Except for the case where the 3' end side is labeled with a fluorescent dye, the 3' end side of the oligonucleotide may be appropriately modified with glycolic acid or the like in order to prevent the nucleic acid elongation reaction from the end side. .

As an oligonucleotide constituting the oligonucleotide labeled with the intercalating fluorescent dye, it is capable of specifically hybridizing under stringent conditions with a nucleic acid containing a specific base sequence or a sequence complementary to the specific base sequence, and has the sequence Among the base sequences described in No. 1, oligonucleotides containing a continuous base sequence consisting of at least 14 bases or a complementary sequence thereof can be mentioned. More preferably, an oligonucleotide that is hybridizable under stringent conditions with the nucleotide sequence set forth in SEQ ID NO: 3 (nucleotide sequence from 364493 to 364511 in GenBank No. AE017354.1) or its complementary sequence, SEQ ID NO: 5 (GenBank No. 364493rd to 364509th nucleotide sequence of AE017354.1) or an oligonucleotide hybridizable under stringent conditions with its complementary sequence, the nucleotide sequence described in SEQ ID NO: 7 (GenBank No. 364501st to 364515th nucleotide sequences of No. AE017354.1) or an oligonucleotide hybridizable with its complementary sequence under stringent conditions, the nucleotide sequence set forth in SEQ ID NO: 9 (GenBank No. 364501 of AE017354.1 364514) or its complementary sequence under stringent conditions.

"Stringent conditions" in the present invention refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. To give an example, polynucleotides with high homology (e.g., identity or similarity), e.g., 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95%, particularly preferably is a condition under which polynucleotides having a homology of 98% or more hybridize to each other and polynucleotides having a homology lower than 98% do not hybridize to each other. Specifically, such conditions include hybridization in 6×SSC (sodium chloride/sodium citrate) at about 45° C., followed by 0.5°C. 2×SSC, 0 . One or more washes at 50-65°C in 1% SDS are included. Another example of "stringent conditions" is 50% (v/v) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM phosphorous at 42°C. conditions in which sodium acid buffer (pH 6.5), 150 mM sodium chloride, 75 mM sodium citrate are present, and the nucleic acid amplification conditions described in the Examples herein. In addition, under the stringent conditions described above, if the specific nucleotide sequence can be hybridized sufficiently specifically and with high efficiency, the nucleotide sequences of the first and second primers can be compared with the specific nucleotide sequence. Substitutions, deletions, additions and modifications may be made. Although the length of the first and second primers can be set arbitrarily, it is preferably in the range of 10 to 50 bases. In the most preferred embodiment, the oligonucleotide that can specifically hybridize under stringent conditions is an oligonucleotide having a sequence complementary to the target base sequence.

According to the present invention, the first primer having a sequence complementary to the 3' end of the specific nucleotide sequence of Legionella 23S rRNA contained in the sample is the nucleotide sequence set forth in SEQ ID NO: 11 (GenBank No. AE017354. The nucleotide sequence from 364535th to 364611th of 1) and an oligonucleotide that can specifically hybridize under stringent conditions, and the 5' end of the specific nucleotide sequence of Legionella spp. 23S rRNA contained in the sample As a second primer having a homologous sequence, specifically hybridizable under stringent conditions with the nucleotide sequence set forth in SEQ ID NO: 31 (nucleotide sequence from 364329th to 364483rd of GenBank No. AE017354.1) are characterized by using different oligonucleotides, respectively.

Among them, it is preferable that the first primer and the second primer consist of 16 to 31 bases.

An example of the first primer is an oligonucleotide consisting of 16 to 31 consecutive bases in the nucleotide sequence set forth in SEQ ID NO: 11 or its complementary sequence. A more specific example is SEQ ID NO: 15 (GenBank No. Complementary sequence of base sequence from 364535th to 364553rd of AE017354.1), SEQ ID NO: 17 (GenBank No. Complementary sequence of base sequence from 364538th to 364553rd of AE017354.1), SEQ ID NO: 19 (GenBank No. Complementary sequence of base sequence from 364538th to 364557th of AE017354.1), SEQ ID NO: 21 (GenBank No. Complementary sequence of base sequence from 364554th to 364573rd of AE017354.1), SEQ ID NO: 23 (GenBank No. Complementary sequence of base sequence from 364570th to 364600th of AE017354.1), SEQ ID NO: 25 (GenBank No. Complementary sequence of base sequence from 364577th to 346600th of AE017354.1), SEQ ID NO: 27 (GenBank No. Complementary sequence of base sequence from 364581st to 364600th of AE017354.1) and SEQ ID NO: 29 (complementary sequence of base sequence from 364591st to 364611th of GenBank No. AE017354.1) Nucleotides are mentioned.

An example of the second primer is an oligonucleotide consisting of 16 to 31 consecutive bases in the nucleotide sequence set forth in SEQ ID NO: 31 or its complementary sequence, and a more specific example is SEQ ID NO: 33 (GenBank No. AE017354.1 364329th to 364348th nucleotide sequence) and SEQ ID NO: 35 (GenBank No. AE017354.1 364347th to 364368th nucleotide sequence), SEQ ID NO: 37 (GenBank No. AE017354.1 364388th to 364406th base sequence), SEQ ID NO: 39 (GenBank No. AE017354.1 364420th to 364449th base sequence) and SEQ ID NO: 41 (GenBank No. AE017354.1 364429th to 364449th bases sequence), SEQ ID NO: 43 (GenBank No. 364431st to 364451st nucleotide sequence of AE017354.1), SEQ ID NO: 45 (GenBank No. AE017354.1 364439th to 364459th nucleotide sequence), SEQ ID NO: 47 (Nucleotide sequence from 364455th to 364475th of GenBank No. AE017354.1), SEQ ID NO: 49 (nucleotide sequence from 364464th to 364483rd of GenBank No. AE017354.1), SEQ ID NO: 51 (GenBank No. AE017354. 364467th to 364482nd nucleotide sequences of 1).

Among them, the first primer is SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 in that it can rapidly and specifically detect Legionella 23S rRNA , 27, 28, 29, 30, and the second primer is SEQ ID NO: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 , 43, 44, 45, 46, 47, 48, 49, 50, 51, and 52, are preferred. Among them, the combination of the first primer and the second primer is SEQ ID NO: 19 and 38, 20 and 37, 19 and 42, 20 and 41, 19 and 44, 20 and 43, 25 and 34, 26 and 33, 25 and 38, 26 and 37, 25 and 38, 26 and 39, 25 and 42, 26 and 41, 25 and 44, 26 and 43, 25 and 46, 26 and 45, 25 and 48, 26 and 47, 25 and 50 , 26 and 49, 27 and 40, 28 and 39, 27 and 44, 28 and 43, 27 and 50, 28 and 49, 27 and 52, 28 and 51. A primer set is more preferred.

The primer set of the present invention is for amplifying a nucleic acid containing a specific nucleotide sequence of Legionella spp. 23S rRNA or a sequence complementary to the specific nucleotide sequence, using a nucleic acid amplification method commonly used by those skilled in the art, such as the RT-PCR method. Useful as a primer set. If an RNA polymerase promoter is further added to the 5′ end of either the first or second primer, the specific base sequence to which the RNA polymerase promoter is added is obtained using an RNA polymerase corresponding to the promoter. Alternatively, since a nucleic acid containing a sequence complementary to the specific base sequence is synthesized, a nucleic acid sequence based amplification (NASBA) method, a Transcription-Mediated Amplification (TMA) method, a Transcription-Reverse Transcription (TRC) method, etc. It is preferable in that RNA can be amplified using a method for amplifying RNA at a constant temperature. If the promoter added to the 5′ end of the primer is a promoter corresponding to the RNA polymerase used for RNA amplification (for example, T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase widely used in the field of molecular biology), good. In addition, the promoter may further include a transcription initiation region known to affect transcription efficiency. A specific example of the promoter (T7 promoter) added to the 5' end of the primer when T7 RNA polymerase is used as the RNA polymerase for RNA amplification is an oligonucleotide consisting of the sequence set forth in SEQ ID NO:55.

Detection of Legionella 23S rRNA using the oligonucleotide of the present invention can be carried out, for example, by the following steps (1) to (6).
(1) A first primer, which is an oligonucleotide specifically hybridizable with the nucleotide sequence of SEQ ID NO: 11 or its complementary sequence under stringent conditions, hybridizes to Legionella spp. 23S rRNA, and RNA-dependent DNA a step of synthesizing a cDNA complementary to a specific nucleotide sequence with an enzyme having polymerase activity to generate an RNA-DNA double strand with the RNA;
(2) degrading the RNA of the RNA-DNA double strand by an enzyme having ribonuclease H (RNase H) activity (generation of single-stranded DNA);
(3) The single-stranded DNA is hybridized with a second primer, which is an oligonucleotide specifically hybridizable with the base sequence of SEQ ID NO: 31 or its complementary sequence under stringent conditions (here, Either the first or second primer has an RNA polymerase promoter added to its 5′ end), and an enzyme having DNA-dependent DNA polymerase activity is used to generate a specific nucleotide sequence or complementary to a specific nucleotide sequence. generating a double-stranded DNA containing a promoter capable of transcribing the RNA of the sequence;
(4) producing an RNA transcript using the double-stranded DNA as a template by an enzyme having RNA polymerase activity;
(5) step of generating RNA transcripts in a chain reaction by using the RNA transcripts as templates for cDNA synthesis in the reaction of (1);
(6) an oligonucleotide containing SEQ ID NO: 1 or its complementary sequence, which is hybridizable under stringent conditions to a specific base sequence or a sequence complementary to the specific base sequence, and an intercalating fluorescent dye for the oligonucleotide; using an oligonucleotide labeled with to measure the amount of the RNA transcript over time.

An enzyme having RNA-dependent DNA polymerase activity used in step (1), an enzyme having RNase H activity used in step (2), and an enzyme having DNA-dependent DNA polymerase activity used in step (3) The enzymes can be added individually or in various combinations, and a retrovirus-derived reverse transcriptase having the above activities can also be used. The reverse transcriptase is not particularly limited, but includes AMV (Avian Myeloblastosis Virus) reverse transcriptase, MMLV (Molony Murine Leukemia Virus) reverse transcriptase, and RAV (Rous Associated Virus) reverse transcriptase, which are commonly used in the field of molecular biology. , HIV (Human Immunodeficiency Virus) reverse transcriptase and the like can be used.

The reaction temperature in the Legionella spp. 23S rRNA detection method according to the above-described embodiment depends on the heat resistance and activity of each enzyme used, the Tm of the primer/probe, etc., but the enzymes used are AMV reverse transcriptase and T7 RNA polymerase. When the length of the primer/probe is in the range of 17 to 31 bases, the reaction temperature may be set in the range of 35 to 65°C, more preferably in the range of 40 to 50°C.

In the method for detecting Legionella spp. 23S rRNA according to the above-described aspect, since the fluorescence intensity is measured over time, the measurement can be terminated at any time when a significant increase in fluorescence is observed, and nucleic acid amplification and measurement can be performed. In addition, it is possible to finish within 20 minutes in general.

The Legionella spp. 23S rRNA detection method according to the above aspect includes adding a sample to a Legionella spp. 23S rRNA reagent comprising the above-described first primer, second primer, and an oligonucleotide labeled with an intercalating fluorescent dye. By placing it on a temperature control block that can detect fluorescence over time, it is possible to automatically amplify and detect Legionella 23S rRNA. Oligonucleotides for detecting Legionella 23S rRNA of the present invention preferably consist of the base sequences of SEQ ID NOS:3-10.

The oligonucleotide of the present invention hybridizes to a sequence specific to Legionella spp. 23S rRNA (specific base sequence) and a part of its complementary sequence, thereby generating a specific base sequence of Legionella spp. 23S rRNA or its complementary sequence. It can be specifically detected.

INDUSTRIAL APPLICABILITY The oligonucleotide of the present invention and the method for detecting Legionella spp. using the oligonucleotide can rapidly and highly sensitively detect Legionella spp. contained in a sample, and have high specificity with extremely low occurrence of false positives. Therefore, it is possible to quickly present the test results to a doctor, which is thought to contribute to preventing the spread of infection and preventing the emergence of resistant bacteria by administering appropriate drugs.

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of Standard RNA or Dilution of Bacterial Culture A standard RNA of Legionella pneumophila, a species of the genus Legionella, used in the examples described later was prepared by the following method. Quantification of the prepared standard RNA was performed based on the absorbance at 260 nm.
(1) Legionella pneumophila standard RNA
By artificially preparing a 23S rRNA gene based on the Legionella pneumophila 23S rRNA sequence (nucleotide sequence from 361720th to 364621st of GenBank No. AE017354.1), in vitro transcription, and then purifying the transcript, Legionella pneumophila standard RNA was prepared.

Example 2 Preparation of Oligonucleotide Labeled with Intercalating Fluorescent Dye An oligonucleotide labeled with an intercalating fluorescent dye (hereinafter referred to as INAF probe) shown in (A) below was prepared in JP-A-2000-316587. It was produced based on the method disclosed in the publication.
(A) the nucleotide sequence set forth in SEQ ID NO: 3 (GenBank No. 364493rd to 364511th nucleotide sequence of AE017354.1) or its complementary sequence, the nucleotide sequence set forth in SEQ ID NO: 5 (GenBank No. AE017354.1 364493rd to 364509th nucleotide sequence) or its complementary sequence, the nucleotide sequence set forth in SEQ ID NO: 7 (GenBank No. AE017354.1 364501st to 364515th nucleotide sequence) or its complementary sequence, SEQ ID NO: 9 An oligonucleotide consisting of the described base sequence (GenBank No. 364501st to 364514th base sequence of AE017354.1) or its complementary sequence, wherein in SEQ ID NOS: 3 and 5, the 13th guanine from the 5' end and the 14th adenine, and in SEQ ID NOS: 7 and 9, thiazole orange is labeled via a linker between the 11th cytosine and the 12th guanine from the 5' end.

Example 3 Examination of Oligonucleotides for Legionella pneumophila 23S rRNA Detection It was evaluated by the method shown in . The INAF probes listed in Table 1 are the probes prepared in Example 2.
(1) Of the standard RNAs prepared in Example 1, the Legionella pneumophila standard RNA (Example 1 (1)) was diluted with an RNA diluent (10 mM Tris-HCl buffer (pH 8.0), 1 mM EDTA, 0 02% sodium cholate) to 1000 copies/15 μL, and these were used as RNA samples.
(2) A reaction solution having the following composition was dispensed into an evaporative drying tube and evaporated to dryness.

Composition of reaction solution: concentration is RNA sample, starting solution, final concentration after addition (in 30 μL) 60 mM Tris-HCl buffer (pH 8.35)
300 mM trehalose 0.39 mM each dATP, dCTP, dGTP, dTTP
2.1 mM each ATP, CTP, UTP
1.5mM GTP
3.2mM ITP
0.2 μM First primer (T7 promoter (SEQ ID NO: 55) added to the 5′ end of the oligonucleotide consisting of the base sequence of each SEQ ID NO)
0.2 μM Second primer 25 nM INAF probe (prepared in Example 2)
0.025 mg/mL bovine serum albumin 200 T7 RNA polymerase 6.4 U AMV reverse transcriptase (3) After the above evaporation drying, add 15 μL of the RNA sample, incubate at 46° C. for 5 minutes, then start with the following composition: 15 μL of liquid was added and stirred.

Composition of enzyme solution: final concentration at reaction (in 30 μL) 9.00% dimethyl sulfoxide 20 mM magnesium chloride 130 mM potassium chloride (4) While reacting at 46° C., the fluorescence intensity of the reaction solution was measured over time for 20 minutes.

The time when the starting solution was added and the stirring was completed was defined as 0 minutes, and the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value for a predetermined time by the background fluorescence intensity ratio) exceeded 1.45. and the time at that time was defined as the detection time. In the experiments whose results are shown in Tables 1, 2 and 3, each combination was measured twice and the average value was used. "N.D." means that the fluorescence intensity ratio 20 minutes after the start of the reaction was 1.45 or less (negative determination).

Regarding the detection performance of Legionella pneumophila 23S rRNA (Table 1), among the oligonucleotide combinations (A01 to A43) examined in this example, A11, A18, A26, A27, A36, A37, and A39 Both detected 1000 copies/test of Legionella pneumophila 23S rRNA within 10 minutes, and it can be said that the combination of oligonucleotides can rapidly detect Legionella pneumophila 23S rRNA.

Among the combinations of oligonucleotides examined in this example, using A28 from among the combinations whose detection time was within 10 minutes,
Legionella detection (Table 2) and cross-reactivity (Table 3) were performed.
Regarding the detection of Legionella spp., ⁹ species of Legionella spp. of 1 CFU/test were detected. The combination of oligonucleotides examined in this example can be said to be a combination of oligonucleotides capable of specifically detecting 23S rRNA of Legionella spp.

Claims (4)

A fluorescence-labeled oligonucleotide probe having a sequence homologous to a specific base sequence of Legionella spp. 23S rRNA or 23S rDNA or a part of its complementary sequence, and a sequence homologous to a part of said specific base sequence or its complementary sequence and a second primer having a sequence homologous to the specific base sequence or a part of the complementary sequence thereof, a nucleic acid comprising the specific base sequence or a complementary sequence of the specific base sequence A primer/probe set for amplification under constant temperature conditions,
Any of the following (1) to (4),
The primer/probe set:
(1) the fluorescent-labeled oligonucleotide probe is the nucleotide sequence set forth in SEQ ID NO: 5 or 7 or its complementary sequence, the first primer is the nucleotide sequence set forth in SEQ ID NO: 25 or its complementary sequence, and the second a primer set in which the primers are the nucleotide sequences set forth in SEQ ID NOS: 33, 39, 41, 43, 45, 47, 49 or 51;
(2) the fluorescent-labeled oligonucleotide probe is the nucleotide sequence set forth in SEQ ID NO: 5 or 7 or its complementary sequence, the first primer is the nucleotide sequence set forth in SEQ ID NO: 27 or its complementary sequence, and the second a primer set in which the primers are the nucleotide sequences set forth in SEQ ID NOS: 39, 41, 43, 49 or 51 or their complementary sequences;
(3) the fluorescent-labeled oligonucleotide probe is the base sequence set forth in SEQ ID NO: 5 or 7 or a complementary sequence thereof, the first primer is the base sequence set forth in SEQ ID NO: 17 or a complementary sequence thereof, and the second a primer set in which the primers are the nucleotide sequence set forth in SEQ ID NO: 43 or a complementary sequence thereof;
(4) the fluorescent-labeled oligonucleotide probe is the base sequence set forth in SEQ ID NO: 5 or 7 or a complementary sequence thereof, the first primer is the base sequence set forth in SEQ ID NO: 19 or a complementary sequence thereof, and the second A primer set in which the primers are the nucleotide sequences set forth in SEQ ID NOS: 39, 43 or 45 or their complementary sequences. 2. The primer-probe set according to claim 1 , wherein the fluorescently-labeled oligonucleotide probe is an oligonucleotide probe configured to change its fluorescent properties when it forms a complementary double strand. 3. The primer-probe set according to claim 2 , wherein said oligonucleotide probe is labeled with an intercalating fluorescent dye. A method for detecting a specific base sequence of 23S rRNA or 23S rDNA of Legionella spp . A method for detecting Legionella , comprising the step of amplifying a specific base sequence of 23S rRNA or 23S rDNA or a complementary sequence thereof under constant temperature conditions .