AU2003220730B2 - Genes for detecting bacteria and detection method by using the same - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): ASAHI BREWERIES, LTD.

Invention Title: GENES FOR DETECTING BACTERIA AND DETECTION METHOD BY USING THE SAME The following statement is a full description of this invention, including the best method of performing it known to me/us: 1 Genes for detecting bacteria and a method for detecting bacteria by using the genes Field of the Invention The present invention relates to genes for detecting Pectinatus frisingensis or Pectinatus cerevisiiphilus of the genus Pectinatus, which is known as beer-spoilage bacteria, and a method for detecting the bacteria by using the genes.

Description of the Prior Art Bacteria of the genus Pectinatus have been known as beer-spoilage bacteria. In the genus, two kinds of Pectinatusfrisingensis and Pectinatus cerevisiiphilus have been known. For detecting the bacteria of the genus Pectinatus, the bacteria must be isolated after multiplication culture and separation culture. It takes at least seven days. Then, isolated bacteria are multiplied and tested by many qualitative tests such as morphological observation, gram stainability, a catalase test, utilization of various carbon sources and the like to identify the bacteria.

These tests are very troublesome, and it takes much time and it costs much. In addition to these common identification tests, there is a method that DNAis extracted from isolated bacteria, fixed on a membrane, and conducted a hybridization test by using standard bacteria DNA as a probe to identify the class. However, it takes some days, and it is difficult to obtain necessary detective sensitivity and selectivity.

Lately, a method for detection of bacteria of the genus Pectinatusis disclosed by using a monoclonal antibody that specifically reacts with Pectinatus cerevisiiphilus (ASBC Journal: 51(4)158-163, 1993). However, the method is insufficient to the detective sensitivity. The method has a problem that Pectinatus frisingensis can not be detected.

The other detection method has been reported. It can detect Pectinatus fisingensis and Pectinatus cerevisiiphilus by a Ribotyping method that 2 polymorphism of a ribosomal RNA gene is detected Am. Soc. Chem.: 56 19-23, 1998). However, since the method needs operation for isolating the bacteria, it has problems of detective sensitivity and speed.

Considering these problems, further quick detection methods have been studied. W097/20071 discloses a method for detecting Pectinatus comprising extracting DNA of the test microorganism, and using a PCR method that a complementary oligonucleotide of the DNA functionates as a primer. However, the base sequences of 16S rRNA gene used in the technique are sometimes similar to those of microorganisms of the other genera, so that there are problems that the other microorganisms are detected in addition to particular microorganisms to be detected.

The gene in a spacer between a 16S rRNA gene and a 23S rRNA gene has a specific gene sequence. Though methods for detecting microorganisms using the gene sequence are disclosed in Japanese Jozo Ronbunshu 50, 22-31 (1995), APPL.

ENVIRON. MICROBIOL. VOL.62, NO.5, 1683-1688(1996), FEMS MICROBIOL LETT. VOL. 84, NO.3, 307-312(1991), Japanese Patent Kokai Publication No. 6-98800 and the like, gene sequences of the spacers of the genus Pectinatus have not been found.

Summary of the Invention The present invention seeks to provide gene sequences of a spacer region that is constituted between a 16S rRNA gene and a 23S rRNA gene specific for the genus Pectinatus relating to beer-spoilage, and to provide a method for sensitively and quickly detecting the genus by using the sequences.

In a first aspect, the invention provides an isolated nucleic acid molecule comprising a base sequence of a spacer region between a gene coding 16S rRNA and a gene coding 23S rRNA of Pectinatusfrisingensis containing a part of the base sequence or the whole base sequence represented by SEQ ID NO: 1.

In a second aspect, the invention provides an isolated nucleic acid molecule comprising a base sequence of a spacer region between a gene coding 16S rRNA and a gene coding 23S rRNA of Pectinatusfrisingensis containing a part of the base sequence or the whole base sequence represented by SEQ ID NO: 2.

In a third aspect, the invention provides an isolated nucleic acid molecule comprising a base sequence of a spacer region between a gene coding 16S rRNA and a 3 gene coding 23S rRNA ofPectinatus cerevisiiphilus containing a part of the base sequence or the whole base sequence represented by SEQ ID NO: 3.

In a fourth aspect, the invention provides an isolated nucleic acid molecule comprising a base sequence of a spacer region between a gene coding 16S rRNA and a gene coding 23S rRNA of Pectinatus cerevisiiphilus containing a part of the base sequence or the whole base sequence represented by SEQ ID NO: 4.

In a fifth aspect, the invention provides an oligonucleotide characterized in that the gene sequence of a spacer region between a gene coding 16S rRNA and a gene coding 23S rRNA of Pectinatusfrisingensis has at least one of the following sequence group or the corresponding complementary sequence: 5'-CCATCCTCTTGAAAATCTC-3' 5'-TCTCRTCTCACAAGTTTGGC-3' In a sixth aspect, the invention provides an oligonucleotide that comprises a base sequence of a spacer region between a gene coding 16S rRNA and a gene coding 23S rRNA of Pectinatus cerevisiiphilus having at least one of the following sequence group or the corresponding complementary sequence: 5'-CACTCTTACAAGTATCTAC-3' 5'-CCACAATATTTCCGACCAGC-3' 5'-AGTCTTCTCTACTGCCATGC-3' In a seventh aspect, the invention provides a method for detecting Pectinatus frisingensis, wherein the oligonucleotide made from the base sequence of the nucleic acid molecule according to the first or second aspects uses as a primer for synthesis of nucleic acids, and the nucleic acid is treated by gene amplification to detect the bacteria.

In an eighth aspect, the invention provides a method for detecting Pectinatus cerevisiiphilus, wherein the oligonucleotide made from the base sequence of the nucleic acid molecule according to the third or fourth aspects uses as a primer for synthesis of nucleic acids, and the nucleic acid is treated by gene amplification to detect the bacteria.

In a ninth aspect, the invention provides a method for detecting Pectinatus frisingensis, wherein the oligonucleotide made from the gene sequence according to the first or second aspects, or the oligonucleotide made from the gene sequence according to the fifth aspect, and a nucleotide sequence coding 16S rRNA gene of Pectinatus 4 frisingensis use as primers for synthesis of nucleic acids, and the nucleic acid is treated by gene amplification to detect the bacteria.

In a tenth aspect, the invention provides a method for detecting Pectinatus cerevisiiphilus, wherein the oligonucleotide made from the base sequence of the nucleic acid molecule according to the third or fourth aspects or the oligonucleotide made from the base sequence of the nucleic acid molecule according to the sixth aspect, and a nucleotide sequence coding 16S rRNA gene of Pectinatus cerevisiiphilus use as primers for synthesis of nucleic acids, and the nucleic acid is treated by gene amplification to detect the bacteria.

(11) In an eleventh aspect, the invention provides a method according to the ninth aspect, wherein the nucleotide sequence coding the 16S rRNA gene of Pectinatus frisingensis has the following sequence: 5'-CGTATCCAGAGATGGATATT-3' (12) In a twelfth aspect, the invention provides a method according to the tenth aspect, wherein the nucleotide sequence coding the 16S rRNA gene of Pectinatus cerevisiiphilus has the following sequence: 5'-CGTATGCAGAGATGCATATT-3' Brief Description of Drawings Figure 1. It shows Electrophoretogram in Example 3.

Figure 2. It shows Electrophoretogram in Example Detailed Description of the Invention Since the technique of gene amplification is well known, it is conducted under the polymerase chain reaction method which has been developed by Saiki et al. (abbreviated as PCR method hereinafter; Science 230, 1350, 1985).

This method is conducted by amplification reaction of particular gene sequences. Since the method shows quick reaction, high sensitivity and specificity and convenience, applications has been tried to quickly judge viruses in medical fields or quickly detect noxious bacteria in food fields. By the PCR method, even if only a few nucleotide sequences are present in test samples, the target nucleotide sequence between two primers is amplified several hundred times, and the copies are produced in large quantities to be detectable. For conducting the PCR method, the nucleic acid ingredient should be liberated from the bacteria in the test samples. However, in the PCR method, when several or more molecules exist in the target sequence, the amplification reaction proceeds.

Accordingly, samples of the PCR method can be provided by a simple pretreatment of the bacteria with a lytic enzyme or a surfactant. For this reason, the method for detecting bacteria has merits higher than conventional methods.

The present invention provides gene sequences of a spacer region between a gene coding 16SrRNA and a gene coding 23SrRNA in each Pectinatus Irisingensis or Pectinatus cerevisiiphilus. By using a nucleotide sequence coding a 16SrRNA gene or oligonucleotide which is selected from the sequence as a primer for nucleic acid synthesis in the PCR method, and by gene amplification treatment, the present inventors have developed a quick and high sensitive method for judging the existence of Pectinatus frisingensis or Pectinatus cerevisiiphilus in samples.

The test samples may be beer or semi-products of beer, or a sample extracted from sewage and the like. The oligonucleotide for a primer may be a chemical synthetic or natural product.

Description of the Preferred Embodiments As shown hereinafter, in the method of the present invention, 6 Pectinatusfrisingensis or Pectinatus cerevisiiphilus is detected by the PCR method.

The base sequences used in the PCR method are, not by way of limitation, for example, above-mentioned fifth, sixth, eleventh and twelfth aspects. The primer length used in the PCR method is, not by way of limitation, 19-20 base length in above-mentioned fifth, sixth, eleventh and twelfth aspects, preferably, 10-50 base length.

When Pectinatusfrisingensis is detected by the PCR method, the existence of the bacteria is judged by that the DNA fragments amplified in case of the combination of and as the primer are about 700 base pairs and about 900 base pairs, and the DNA fragments amplified in case of the combination of and as the primer are about 700 base pairs and about 900 base pairs. When these bands are detected by electrophoresis, it is judged that Pectinatusfrisingensis is present. Since the combination of the primers, in any cases, is specific for Pectinatusfrisingensis bacteria, the genus can be detected. By parallel using two of the combination, further precise determination becomes possible. By changing the base sequences of the primers used in the PCR method, the length of the nucleotide sequences amplified can be changed.

On the other hand, when Pectinatus cerevisiiphilus is detected by the PCR method, the existence of the bacteria is judged by that the DNA fragments amplified in case of the combination of and are about 600 base pairs, the DNA fragments amplified in case of the combination of@ and are about 650 base pairs, and the DNA fragments amplified in case of the combination of and are about 700 base pairs.

When these bands are detected by electrophoresis, it is judged that Pectinatus cerevisiiphilus is present. Since the combination of the primers, in any cases, is specific for Pectinatus cerevisiiphilus bacteria, the genus can be detected. By parallel using two or more of the combination, further precise determination becomes possible. By changing the base sequences of the primers used in the PCR method, the length of the nucleotide sequences amplified can be changed.

The temperature conditions of one cycle in the PCR method are 90-98 0

C

in a thermal denaturation reaction in which double-stranded DNA is changed to single-stranded DNA, 37-65oC in an annealing reaction in which DNA is hybridized into primer template DNA, and 50-75°C in a chain elongation reaction in which DNA polymerase is reacted. The target sequences can be amplified by several ten cycles. After PCR reaction, the reactant is separated by electrophoresis, and the nucleic acid is stained with ethidium bromide or the like. When the base length of the amplified nucleotide sequence is equal to the base length of the above target sequence, it can be judged that the bacteria to be detected are in the test sample. To detect the amplified nucleotide sequence, chromatography is usable.

The sequences of the present invention are described in the following: SEQ ID NO: 1 The sequence length is 624, the sequence type is nucleic acid, the strandness is double, the topology is linear, the molecule type is genomic DNA, and the original source is Pectinatus risingensis DSM6306.

SEQ ID NO: 2 The sequence length is 442, the sequence type is nucleic acid, the strandness is double, the topology is linear, the molecule type is genomic DNA, and the original source is Pectinatus frisingensis DSM6306.

SEQ ID NO: 3 The sequence length is 724, the sequence type is nucleic acid, the strandness is double, the topology is linear, the molecule type is genomic DNA, and the original source is Pectinatus cerevisiiphilus DSM20467.

SEQ ID NO: 4 The sequence length is 399, the sequence type is nucleic acid, the strandness is double, the topology is linear, the molecule type is genomic DNA, and the original source is Pectinatus cerevisiiphilus DSM20467.

SEQ ID NO: 5 The sequence length is 19, the sequence type is nucleic acid, the strandness is single, the topology is linear, the molecule type is genomic DNA, and the original source is Pectinatus lfisingensis DSM6306.

SEQ ID NO: 6 The sequence length is 20, the sequence type is nucleic acid, the strandness is single, the topology is linear, the molecule type is genomic DNA, and the original source is Pectinatus risingensis DSM6306.

8 SEQ ID NO: 7 The sequence length is 19, the sequence type is nucleic acid, the strandness is single, the topology is linear, the molecule type is genomic DNA, and the original source is Pectinatus cerevisiiphilus DSM20467.

SEQ ID NO: 8 The sequence length is 20, the sequence type is nucleic acid, the strandness is single, the topologyis linear, the molecule type is genomic DNA, and the original source is Pectinatus cerevisiiphilus DSM20467.

SEQ ID NO: 9 The sequence length is 20, the sequence type is nucleic acid, the strandness is single, the topology is linear, the molecule type is genomic DNA, and the original source is Pectinatus cerevisiiphilus DSM20467.

SEQ ID NO: 10 The sequence length is 20, the sequence type is nucleic acid, the strandness is single, the topology is linear, the molecule type is genomic DNA, and the original source is Pectinatus frisingensis DSM6306.

SEQ ID NO: 11 The sequence length is 20, the sequence type is nucleic acid, the strandness is single, the topology is linear, the molecule type is genomic DNA, and the original source is Pectinatus cerevisiiphilus DSM20467.

The present invention is described by working examples in the following. The present invention is not limited by these examples.

Example 1 Preparation of test samples Pectinatus frisingensis DSM6306 and Pectinatus cerevisiiphilus DSM20467 were used as bacterial strains belonging to Pectinatus. To confirm the specificity of Pectinatus fisingensis and Pectinatus cerevisiiphilus primers shown in SEQ ID NO:5, 6, 7, 8, 9, 10 and 11 in the present invention, the other bacteria shown in Table 1 were used. These bacteria were cultivated on suitable culture mediums, and the strains were collected by centrifugation. The DNA from the strains were extracted in accordance with the description of SHIN-SEIKAGAKU-JIKKEN-KOZA 2, Nucleic acid I, Separation and Purification, p.p. 20-21 (edited by Japan Biochemical Learned Society, Tokyo- Kagaku-Dojin), and a DNA solution was obtained.

[Table 1] Bacteria7 No.

2 3 4 6 7 8 9 1 0 1 2 1 3 Bacteria type Strain name Pectinatus friSingenlsis DSM6306 Pectinatus cer'evisiiphilus DSM20467 Selenomonas lacticifex DSM20757 Zymophilus raffinosivora2s DSM20765 Zynlophilus paucivorans DSM20756 Escherichia ccli IF03301 lvegasphaera cerevisiae DSM20462 Lactobacillus acidophilus IF013951 Lactobacillus plan tarwn JCM1149 Lactobacillus brevis JCM1059 Lactococcus lactis JCM5805 Leuconostoc mesenteroides JCM6 124 Pediococcus damnosus JCM5886 Remarks type strain type strain type strain type strain type strain K- 12 type strain type strain type strain type strain type strain type strain type strain Example 2 Cloning of spacer regions between the gene coding 16S rRNA and the gene coding 23S rRNAof Pectinatus frisingensis, and determination of the base sequences Selection and synthesis of oligonucleotide primers for amplification of 16S/23S rRNA spacer region by the PCR method Since the base sequences of the 16S ribosomal RNA gene of Pectinatus fn'singensis were apparent (International Journal of Systematic Bacteriology, Vol. 40, p.p. 19-27 (1990)), the primers were selected on the basis of the 557-576" base sequences.

Since the base sequences of the 23S ribosomal RNA gene of Pectinatus frisingensis were apparent (Systematic Applied Microbiology, Vol. 15, p.p. 487- 501 (1990), EMBL Accession Number X48423), the primers were selected on the basis of the 1 2 0 th base sequences to obtain corresponding comprehensive sequences. The synthesis was entrusted to Sawady Technology Co., Ltd.

Amplification of 16S/23S rRNA spacer regions by the PCR method The Pectinatus frisingensis DNA solution 0.1 u g, which was prepared in Example 1, was placed in a 0.2 ml tube (manufactured by Perkin-Elmer), 5 g 1 of 10X buffer in a rTaq DNA Polymerase Kit (Toyobo Co., Ltd.), 3/1 of MgCl 2 5gl of a 2mM dNTP mixture solution (dATP, dGTP, dCTP and dTTP), gl of 5 units of rTaq polymerase, and each 0.5 gl of 100 mM primers prepared in Example were added to the solution, and then sterilized distilled water was added to obtain final volume of 50 Ul. The tube was set on a thermal cycler of an automatic gene amplification device (Perkin Elmer) and the amplification method was conducted. The reaction was repeated by cycles, and one cycle had the following conditions: Denaturation at 94°C for 2.5 minutes; Denaturation at 94°C for seconds; Annealing of primers at 55°C for 30 seconds; and synthetic reaction at 72°C for 30seconds. After the reaction, using 5 gl of the solution, 11 electrophoresis was conducted by agarose gel. DNA was dyed with ethidium bromide, and amplified DNA was observed. The result shows that about 1600 bp (abbreviated as "long" hereinafter) DNA and about 1400 bp (abbreviated as "short" hereinafter) DNA were amplified.

Cloning and sequencing of the spacer region "long" Using a high pure PCR product purification kit (Baringer Manhaim), unreactive dNTPs was removed from the solution after the PCR reaction. To the resulting amplified DNIA 100 ng, 2 /ul ofplasmid pCR 2.1 contained in a TA cloning kit (INVITROGEN), 1 Iul ofligase and 1 gil of buffer were added, and then sterilized water was added to obtain the total volume of 10 gl. After the solution was reacted at 14°C for 4 hours, 2 ul of the solution and 2 Ll of 0.5 M 8 -mercaptoethanol were added to Eschenrchia coliINVa 'F competent cells, and placed in ice for 30 minutes. Then, the solution was heated at 42°C for seconds, and plasmid transformation to the bacteria was conducted. To the transformed bacteria, 250 gul of a SOC culture Tryptone, 0.5% yeast extract, 10.0 mM NaC1, 2.5 mM KC1, 10.0 mM MgCl 2 -6H 2 0, and 20.0 mM glucose) was added, and the mixture was shaken at 37°C for 60 minutes, then transferred to a LB plate culture medium containing 50 g g ml of ampicillin and 40 t g ml X-Gal, and cultured at 37°C overnight. The expressed white colony was transferred to 3 ml of a LB liquid culture medium containing 50 g g ml of ampicillin, and cultured at 37°C overnight.

After the cultivation, plasmids were extracted from the bacteria with a plasmid mini kit (QIAGEN). Apart of the resulting plasmids was taken out and reacted with a restriction enzyme EcoRI (manufactured by Takara Shuzo) at 37 °C for 60 minutes, and separated by agarose electrophoresis. The DNA was dyed with ethidium bromide, and insertion of"long" was confirmed. 500 ng of the residual plasmid was reacted with restriction enzyme SmaI (manufactured by TOYOBO Co., Ltd.) at 30°C for 60 minutes. To the reactant, 2 ul of 3 M sodium acetate and 500AlI of 100% ethanol were added, and the mixture was 12 placed in ice for 15 minutes and centrifuged at 15000 rpm for 15 minutes, and the supernatant was removed. To the precipitate, 500 gl of 70% ethanol was added, the mixture was centrifuged at 15000 rpm for 15 minutes, and the supernatant was removed, and dried for 10 minutes under reduced pressure.

Sterilized water was added to dissolve the precipitate, and the mixture was reacted with restriction enzyme XbaI (Baringer Manhaim) at 37°C for minutes. To the reactant, equivalent phenol chloroform (equivalent mixture liquid) was added and gently mixed, the mixture was centrifuged at 15000 rpm for 15 minutes, and the water layer (upper layer) was recovered.

To the recovery liquid, equivalent water-saturated ether was added and gently mixed, and the mixture was centrifuged at 15000 rpm for 15 minutes to remove the ether layer (upper layer). To the remaining water layer, 2 Al of 3M sodium acetate and 500 1 of 100% ethanol were added, and the mixture was placed in ice for 15 minutes and centrifuged at 15000rpm for 15 minutes to remove the supernatant. To the precipitate, 500/tl of 70% ethanol was added, and the mixture was centrifuged at 15000 rpm for 15 minutes to remove the supernatant, and the residue was dried under reduced pressure for 10 minutes, and 20 /1l of sterilized distillation water was added. To 5 ul of the solution, 1 ,l of 10X buffer contained in a blunting kit (Takara Shuzo Co., Ltd.) and 3 gl of sterilized distillation water were added, and the mixture was maintained at °C for 5 minutes, 1 gl ofT4 DNApolymerase was added, and the mixture was maintained at 37°C for 5 minutes to obtain blunt ends. After T4 DNA polymerase was inactivated by stirring, 40 ul of ligation solution Aand 10 ptl of ligation solution B were added, and the mixture was maintained at 16°C for 30 minutes to conduct internal ligation.

The reactant 2 A.1 and 2 ul of 0.5M /3-mercaptoethanol were added to a Escherichia co'INVa'F competent cell, and the mixture was placed in ice for minutes and heated at 42°C for 30 seconds, and the plasmid was transformed to the Escherichia coli. To the transformed Escherichia col, a SOC culture 13 medium Tryptone, 0.5% Yeast extract, 10.0 mM NaC1, 2.5 mM KC1, 10.0 mM MgC12-GH 2 0, 20.0 mM glucose) 250/zl was added, and the mixture was shaken at 37°C for 60 minutes and spread on a LB plate culture medium containing 50 ig ml ampicillin to culture at 37°C overnight. Appeared white colonies were inoculated into 3 ml of a LB liquid culture medium containing i g/ ml of ampicillin and cultured at 37°C overnight. After the culture, the plasmid was extracted from the Escherichia coliwith a plasmid mini kit (QIAGEN Company).

Using such obtained plasmid as a template, a sequence reaction was conducted. As the sequencing primers, an IRD41 Infrared Dye Labeled M13 Forward primer and an IRD41 Infrared Dye Labeled M13 Reverse primer (manufactured by Nisshinbo, sold by Aroka Co., Ltd.) were used. As the reaction liquid, SequiTherm (trademark) Long-Read (trademark) Cycle Sequencing Kit-LC (manufactured by EPICENTRE TECHNOLOGIES) was used. 4000L Long ReadIR (trademark) DNA Sequencing System (manufactured by LI-COR) was used for the determination of the base sequences.

The gene sequence of spacer region "long" between the gene coding 16S rRNA and the gene coding 23S rRNA of Pectinatus frisingensis DSM6306 bacteria is shown in SEQ ID NO: 1.

Cloning and sequencing of spacer region "short" Using a high pure PCR product purification kit (Baringer Manhaim), unreactive dNTPs was removed from the solution after the PCR reaction in Example To the resulting amplified DNA 100 ng, 2 gl ofplasmid pCR 2.1 contained in a TA cloning kit (INVITROGEN), 1 ul of ligase and 1 /l of buffer were added, and then sterilized water was added to obtain the total volume of 10 pl. After the solution was reacted at 140C for 4 hours, 2 /l of the solution and 2 gl of 0.5 M 8 -mercaptoethanol were added to Escherichia coi INVa'F competent cells, and placed in ice for 30 minutes. Then, the 14 solution was heated at 42°C for 30 seconds, and plasmid transformation to the bacteria was conducted. To the transformed bacteria, 250 gl of a SOC culture Tryptone, 0.5% yeast extract, 10.0 mM NaCI, 2.5 mM KC1, 10.0 mM MgC1 2 -6H 2 0, and 20.0 mM glucose) was added, and the mixture was shaked at 37°C for 60 minutes, then transferred to a LB plate culture medium containing /g ml of ampicillin and 40 g ml X-Gal, and cultured at 37°C overnight.

The appeared white colony was transferred to 3 ml of a LB liquid culture medium containing 50 ug ml of ampicillin, and cultured at 37°C overnight.

After the cultivation, plasmid was extracted from the bacteria with a plasmid mini kit (QIAGEN).

Apart of the resulting plasmid was taken out and reacted with a restriction enzyme EcoRI (manufactured by Takara Shuzo) at 37°C for minutes, and separated by agarose electrophoresis. The DNA was dyed with ethidium bromide, and insertion of "short" was confirmed. 500 ng of the residual plasmid was reacted with restriction enzyme SmaI (manufactured by TOYOBO Co., Ltd.) at 30°C for 60 minutes. To the reactant, 2 /l of 3 M sodium acetate and 500 gl of 100% ethanol were added, and the mixture was placed in ice for 15 minutes and centrifuged at 15000 rpm for 15 minutes, and the supernatant was removed. To the precipitate, 500 al of 70% ethanol was added, the mixture was centrifuged at 15000 rpm for 15 minutes, and the supernatant was removed, and dried for 10 minutes under reduced pressure.

Sterilized water was added to dissolve the precipitate, and the mixture was reacted with restriction enzyme XbaI (Baringer Manhaim) at 37°C for minutes. To the reactant, equivalent phenol chloroform (equivalent mixture liquid) was added and gently mixed, the mixture was centrifuged at 15000 rpm for 15 minutes, and the water layer (upper layer) was recovered. To the recovery liquid, equivalent water-saturated ether was added and gently mixed, and the mixture was centrifuged at 15000 rpm for 15 minutes to remove the ether layer (upper layer).

To the remaining water layer, 2 j l of 3M sodium acetate and 500 pl of 100% ethanol were added, and the mixture was placed in ice for 15 minutes and centrifuged at 15000rpm for 15 minutes to remove the supernatant. To the precipitate, 500 ul of 70% ethanol was added, and the mixture was centrifuged at 15000 rpm for 15 minutes to remove the supernatant, and the residue was dried under reduced pressure for 10 minutes, and 20 pul of sterilized distilled water was added. To 5 ul of the solution, 1 jIl of 10X buffer contained in a blunting kit (Takara Shuzo Co., Ltd.) and 3 upl of sterilized distilled water were added, and the mixture was maintained at 70°C for 5 minutes, 1 /l ofT4 DNA polymerase was added, and the mixture was maintained at 37°C for 5 minutes to obtain blunt ends. After T4 DNA polymerase was inactivated by stirring, 40 g 1 of ligation solution A and 10 jl of ligation solution B were added, and the mixture was maintained at 16°C for 30 minutes to conduct internal ligation. 2 pul of the reactant and 2 jl1 of 0.5M j3 -mercaptoethanol were added to a Escherichia co'INVc 'F competent cell, and the mixture was placed in ice for minutes and heated at 42°C for 30 seconds, and the plasmid was transformed to the Escherichia coi.

To the transformed Escherichia col, 250 pul of SOC culture medium Tryptone, 0.5% Yeast extract, 10.0 mM NaC1, 2.5 mM KC1, 10.0 mM MgC12-6H 2 0, 20.0 mM glucose) was added, and the mixture was shaken at 37°C for 60 minutes and spread on a LB plate culture medium containing 50 j g ml ampicillin to culture at 37°C overnight. Appeared white colonies were inoculated into 3 ml of a LB liquid culture medium containing 50 jug ml of ampicillin and cultured at 37°C overnight. After the culture, the Plasmid was extracted from the Escherichia coliwith a plasmid mini kit (QIAGEN Company).

Using such obtained plasmid as a template, a sequence reaction was conducted. As the sequencing primers, an IRD41 Infrared Dye Labeled M13 Forward primer and an IRD41 Infrared Dye Labeled M13 Reverse primer (manufactured by Nisshinbo, sold by Arok co., Ltd.) were used. As the reaction 16 liquid, SequiTherma (trademark) Long-Read (trademark) Cycle Sequencing Kit- LC (manufactured by EPICENTRE TECHNOLOGIES) was used. 4000L Long ReadIR (trademark) DNA Sequencing System (manufactured by LI-COR) was used for the determination of the base sequences.

The gene sequence of spacer region "short" between the gene coding 16S rRNA and the gene coding 23S rRNA of Pectinatus frnsingensis is shown in SEQ ID NO: 2.

Example 3 Detection of Pectinatus frisingensis by the PCR method Selection and synthesis of a primer for Pectinatus frisingensis The sequences specific for Pectinatus frisingensisby using DNASIS (tradename of Hitachi Soft Engineering Ltd., Co.) on the basis of SEQ ID NO: 1 and SEQ ID NO: 2 were analyzed. The result selected a sequence of 377 t to 395" on the gene sequence of the spacer region between the gene coding 16S rRNA and the gene coding 23S rRNA of Pectinatus fisingensis of SEQ ID NO: 1, and a sequence of 19 5 th to 213 h on the gene sequence of the spacer region between the gene coding 16S rRNA and the gene coding 23S rRNA of Pectinatus risingensis of SEQ ID NO: 2. (SEQ ID NO: In addition, the similar analysis selected a sequence of 361 s to 380 h on the gene sequence of the spacer region between the gene coding 16S rRNA and the gene coding 23S rRNAof Pectinatus frisingensis of SEQ ID NO: 1, and a sequence of 17 9 th to 19 8 h on the gene sequence of the spacer region between the gene coding 16S rRNA and the gene coding 23S rRNA of Pectinatus fisingensis of SEQ ID NO: 2. (SEQ ID NO: 6.) Further, specific primer showing in SEQ ID NO: 10 was selected by a gene sequence coding 16S rRNA of Pectinatus frisingensis. The oligonucleotides were chemically synthesized by the same method as in Example Detection and identification of Pectinatus frisingensis by the primers 17 having the sequences of SEQ ID NO: 6 and SEQ ID NO: The DNA solutions of bacteria prepared in Example 1 were treated with the primers synthesized in Example 3 (SEQ ID NO:6 and SEQ ID NO:10) by PCR. The temperature conditions of the PCR were as follows: Thermal denaturation; 94°C, 30 seconds Annealing; 55°C, 30 seconds Chain elongation reaction; 72°C, 30 seconds One cycle of the conditions was repeated 35 times. After the PCR, the reactant was electrophoresed with agalose gel at constant 100 V for 30 minutes.

ApHY marker was also electrophoresed at the same time as a molecular weight marker. After the electrophoresis, the agarose gel was stained in about g/ml of an ethidium bromide solution for 20 minutes, and ultraviolet was applied to observe the gel and take a photograph of the gel. By the observation or the photography of the gel, the base length of the amplified products was determined from the relative migration distance of the molecular marker.

As shown in Fig. 1, bands of about 700 bps and about 900 bps were detected only in case of Pectinatus frisingensis.

From the results, when the oligonucleotides of SEQ ID NO: 6 and SEQ ID NO: 10 were used as PCR primers, the bands having objective length were detected only in case of Pectinatus fisingensis. Accordingly, it was shown that each oligonucleotide of the present invention correctly recognized the gene sequences of the spacer region between the gene coding 16S rRNA and the gene coding 23S rRNA of Pectinatus risingensis, and the base sequence targeted on the gene coding 16S rRNA. Moreover, the bands having the aimed length were not observed in the same genus Pectinatus cerevisiiphilus, and relative strictly anaerobic bacteria and Gram-positive bacteria. Accordingly, Pectinatus hisingensis can be specifically detected, and at the same time also determined by the present invention.

Example 4 18 Cloning and determination of the base sequence of the spacer regions between the gene coding 16S rRNA and the gene coding 23S rRNA of Pectinatus cerevisiiphilus Selection and synthesis of oligonucleotide primers for amplifying 16S/23S rRNA spacer regions by PCR As the base sequence of 16S ribosome RNA gene of Pectinatus cerevisiiphilus is disclosed in International Journal of Systematic Bacteriology, Vol. 40, pages 19-27 (1990), the primers were selected on the basis of the base sequence of 55 7 th -5 76 The base sequence of 23 ribosome RNA gene of Pectinatus cerevisiiphilus had not been disclosed, but the base sequence of 23 ribosome RNA gene of Pectinatus fisingensis had been disclosed in Systematic Applied Microbiology, Vol. 15, pages 487-501 (1990), EMBL Accession Number X48423.

The primer was selected to obtain the complementary sequence corresponding to the base sequence of 1"-20' of 23 ribosome RNA gene of Pectinatus fisingensis.

Sawaday Technology was entrusted with the synthesis.

Amplification of 16S/23S rRNAby PCR The DNA solution 0.1 g g of Pectinatus cerevisiiphilus prepared in Example 1 was charged in a 0.2 ml tube (Perkin-Elmer 5 pl of 10x buffer in rTaq DNA Polymerase Kit (TOYOBO Co., Ltd.), 3 il of 25mM MgCl 2 5 gl of 2mM dNTP mixture solution (dATP, dGTP, dCTP and dTTP), 0.5 /ul of 5 unit/Il 1 rTaq-polymerase, each 0.5pl of the 100mM primers prepared in Example 2-(1) were added, and sterilized water was added to obtain final volume of50/l. The tube was set in a thermal cycler of an automatic gene amplification device (Perkin-Elmer Co.) and amplification reaction was conducted. 30 cycles were carried under the reaction conditions of one cycle of denaturation at 94°C for minutes, denaturation at 94°C for 30 seconds, primer annealing at 55°C for seconds and synthetic reaction at 72°C for 30 seconds. After the reaction, 5 /l of the reactant was used in agarose gel electrophoresis, the DNA was stained 19 with ethidium bromide, and the amplified DNA was observed. As a result, DNA of about 1700 bp (abbreviated as "long") and DNA of about 1400 bp (abbreviated as "short") were amplified.

Cloning and sequencing of the spacer region "long" Using a high pure PCR product purification kit (Baringer Manhaim), unreactive dNTPs was removed from the solution after the PCR reaction. To ng of the resulting amplified DNA, 2 pl of plasmid pCR 2.1 contained in a TA cloning kit (INVITROGEN), 1 ul ofligase and 1 gl of buffer were added, and then sterilized water was added to obtain the total volume of 10 After the solution was reacted at 14 0 C for 4 hours, 2 gul of the solution and 2 gl of 0.5 M /3-mercaptoethanol were added to Escherichia coliINVa'F competent cells, and placed in ice for 30 minutes. Then, the solution was heated at 42°C for seconds, and plasmid transformation to the bacteria was conducted. To the transformed bacteria, 250 #l of a SOC culture Tryptone, 0.5% yeast extract, 10.0 mM NaC1, 2.5 mM KC1, 10.0 mM MgCl 2 -6H 2 0, and 20.0 mM glucose) was added, and the mixture was shaked at 37°C for 60 minutes, then transferred to a LB plate culture medium containing 50 g g ml of ampicillin and 40 gg ml X-Gal, and cultured at 37°C overnight. The expressed white colony was transferred to 3 ml of a LB liquid culture medium containing 50 i g ml of ampicillin, and cultured at 37°C overnight.

After the cultivation, plasmids were extracted from the bacteria with a plasmid mini kit (QIAGEN). Apart of the resulting plasmids was taken out and reacted with a restriction enzyme EcoRI (manufactured by Takara Shuzo) at 37 °C for 60 minutes, and.separated by agarose electrophoresis. The DNA was dyed with ethidium bromide, and insertion of"long" was confirmed. 500 ng of the residual plasmid was reacted with restriction enzyme SmaI (manufactured by TOYOBO Co., Ltd.) at 30°C for 60 minutes. To the reactant, 2 ll of 3 M sodium acetate and 500 ul of 100% ethanol were added, and the mixture was placed in ice for 15 minutes and centrifuged at 15000 rpm for 15 minutes, and the supernatant was removed. To the precipitate, 500 Ul of 70% ethanol was added, the mixture was centrifuged at 15000 rpm for 15 minutes, and the supernatant was removed, and the residual was dried for 10 minutes under reduced pressure. Sterilized water was added to dissolve the precipitate, and the mixture was reacted with restriction enzyme Xbal (Baringer Manhaim) at 37 °C for 60 minutes. To the reactant, equivalent phenol chloroform (equivalent mixture liquid) was added and gently mixed, the mixture was centrifuged at 15000 rpm for 15 minutes, and the water layer (upper layer) was recovered. To the recovery liquid, equivalent water-saturated ether was added and gently mixed, and the mixture was centrifuged at 15000 rpm for 15 minutes to remove the ether layer (upper layer). To the remaining water layer, 2 /l of 3M sodium acetate and 500 ul of 100% ethanol were added, and the mixture was placed in ice for 15 minutes and centrifuged at 15000rpm for 15 minutes to remove the supernatant.

To the precipitate, 500 gl of 70% ethanol was added, and the mixture was centrifuged at 15000 rpm for 15 minutes to remove the supernatant, and the residue was dried under reduced pressure for 10 minutes, and 20 gl of sterilized distillation water was added. To 5 gl of the solution, 1 u.l of 10X buffer contained in a blunting kit (Takara Shuzo Co., Ltd.) and 3 g l of sterilized distillation water were added, and the mixture was maintained at 70°C for minutes, 1 /l of T4 DNA polymerase was added, and the mixture was maintained at 37°C for 5 minutes to obtain blunt ends. After T4 DNA polymerase was inactivated by stirring, 40 /ul of ligation solution A and 10 Atl of ligation solution B were added, and the mixture was maintained at 16°C for 30 minutes to conduct internal ligation. 2 a.l of the reactant and 2 /tl of 3 -mercaptoethanol were added to Escherichia coliINVa'F competent cells, and the mixture was placed in ice for 30 minutes and heated at 42°C for 30 seconds, and the plasmid was transformed to the Escherichia col.

To the transformed Escherichia col, 250 gl of a SOC culture medium 21 Tryptone, 0.5% Yeast extract, 10.0 mM NaCL, 2.5 mM KC1, 10.0 mM 20.0 mM glucose) was added, and the mixture was shaken at 37°C for 60 minutes and spread on a LB plate culture medium containing 50 g ml of ampicillin to culture at 37°C overnight. Appeared white colonies were inoculated into 3 ml of a LB liquid culture medium containing 50 g g ml of ampicillin and cultured at 37°C overnight. After the culture, the plasmid was extracted from the Escheichia coElwith a plasmid mini kit (QIAGEN Company).

Using such obtained plasmid as a template, a sequence reaction was conducted. As the sequencing primers, an IDRD41 Infrared Dye Labeled M13 Forward primer and an IRD41 Infrared Dye Labeled M13 Reverse primer (manufactured by Nisshinbo, sold by Aroka Co., Ltd.) were used. As the reaction liquid, SequiTherm (trademark) Long-Read (trademark) Cycle Sequencing Kit-LC (manufactured by EPICENTRE TECHNOLOGIES) was used. 4000L Long ReadIR (trademark) DNA Sequencing System (manufactured by LI-COR) was used for the determination of the base sequences.

The gene sequence of spacer region "long" between the gene coding 16S rRNA and the gene coding 23S rRNA of Pectinatus cerevisiiphilus is shown in SEQ ID NO: 3.

Cloning and sequencing of spacer region "short" Using a high pure PCR product purification kit (Baringer Manhaim), unreactive dNTPs was removed from the solution after the PCR reaction in Example To 100 ng of the resulting amplified DNA, 2 igl of plasmid pCR 2.1 contained in a TA cloning kit (INVITROGEN), 1 Ail of ligase and 1 Iul of buffer were added, and then sterilized water was added to obtain the total volume of 10 ul. After the solution was reacted at 14°C for 4 hours, 2 Jl of the solution and 2 ul of 0.5 M -mercaptoethanol were added to Escherichia coliINVa 'F competent cells, and placed in ice for 30 minutes. Then, the solution was heated at 42°C for 30 seconds, and plasmid transformation to the 22 bacteria was conducted. To the transformed bacteria, 250 gl of a SOC culture Tryptone, 0.5% yeast extract, 10.0 mM NaC1, 2.5 mM KC1, 10.0 mM MgCl -6H 2 O, and 20.0 mM glucose) was added, and the mixture was shaked at 37°C for 60 minutes, then transferred to a LB plate culture medium containing 50 ig ml of ampicillin and 40 u g ml X-Gal, and cultured at 37°C overnight.

The appeared white colony was transferred to 3 ml of a LB liquid culture medium containing 50 u g ml of ampicillin, and cultured at 37°C overnight.

After the cultivation, plasmid was extracted from the bacteria with a plasmid mini kit (QIAGEN).

A part of the resulting plasmid was taken out and reacted with a restriction enzyme EcoRI (manufactured by Takara Shuzo) at 37°C for minutes, and the reactant was separated by agarose electrophoresis. The DNA was dyed with ethidium bromide, and insertion of"short" was confirmed. 500 ng of the residual plasmid was reacted with restriction enzyme SmaI (manufactured by TOYOBO Co., Ltd.) at 30°C for 60 minutes. To the reactant, 2 gl of 3 M sodium acetate and 500 ul of 100% ethanol were added, and the mixture was placed in ice for 15 minutes and centrifuged at 15000 rpm for minutes, and the supernatant was removed. To the precipitate, 500 gl of ethanol was added, the mixture was centrifuged at 15000 rpm for 15 minutes, and the supernatant was removed, and the residue was dried for 10 minutes under reduced pressure. Sterilized water was added to dissolve the precipitate, and the mixture was reacted with restriction enzyme BamHI (Takara Shuzo Co.) at 37°C for 60 minutes. To the reactant, equivalent phenol chloroform (equivalent mixture liquid) was added and gently mixed, the mixture was centrifuged at 15000 rpm for 15 minutes, and the water layer (upper layer) was recovered. To the recovery liquid, equivalent water-saturated ether was added and gently mixed, and the mixture was centrifuged at 15000 rpm for 15 minutes to remove the ether layer (upper layer). To the remaining water layer, 2 upl of 3M sodium acetate and 500 gl of 100% ethanol were added, and the mixture 23 was placed in ice for 15 minutes and centrifuged at 15000rpm for 15 minutes to remove the supernatant.

To the precipitate, 500 P l of 70% ethanol was added, and the mixture was centrifuged at 15000 rpm for 15 minutes to remove the supernatant, and the residue was dried under reduced pressure for 10 minutes, and 20 gl of sterilized distilled water was added. To 5gl of the solution, 1 l of 10X buffer contained in a blunting kit (Takara Shuzo Co., Ltd.) and 3 ul of sterilized distilled water were added, and the mixture was maintained at 70°C for 5 minutes, 1 ul ofT4 DNApolymerase was added, and the mixture was maintained at 37°C for minutes to obtain blunt ends. After T4 DNA polymerase was inactivated by stirring; 40 ul of ligation solution A and 10 /l1 of ligation solution B were added, and the mixture was maintained at 16°C for 30 minutes to conduct internal ligation. 2 ul of the reactant and 2 gl of 0.5M 6-mercaptoethanol were added to a Escherichia coliNVr'F competent cell, and the mixture was placed in ice for 30 minutes and heated at 42°C for 30 seconds, and the plasmid was transformed to the Escherichia coli To the transformed Escherichia cob, 250 gl of a SOC culture medium Tryptone, 0.5% Yeast extract, 10.0 mM NaC1, 2.5 mM KC1, 10.0 mM MgCl2-6H 2 O, 20.0 mM glucose) was added, and the mixture was shaken at 37°C for 60 minutes and spread on a LB plate culture medium containing 50 A g ml ampicillin to culture at 37°C overnight.

Appeared white colonies were inoculated into 3 ml of a LB liquid culture medium containing 50 u g ml of ampicillin and cultured at 37°C overnight. After the culture, the plasmid was extracted from the Eschericbua coliwith a plasmid kit (QIAGEN Company).

Using such obtained plasmid as a template, a sequence reaction was conducted. As the sequencing primers, an IRD41 Infrared Dye Labeled M13 Forward primer and an IRD41 Infrared Dye Labeled M13 Reverse primer (manufactured by Nisshinbo, sold by Aroka Co., Ltd.) were used. As the reaction liquid, SequiTherm (trademark) Long-Read (trademark) Cycle 24 Sequencing Kit-LC (manufactured by EPICENTRE TECHNOLOGIES) was used. 4000L Long ReadIR (trademark) DNA Sequencing System (manufactured by LI-COR) was used for the determination of the base sequences.

The gene sequence of spacer region "short" between the gene coding 16S rRNA and the gene coding 23S rRNA of Pectinatus cerevisiiphilus is shown in SEQ ID NO: 4.

Example Detection of Pectinatus cerevisi'philus by the PCR method Selection and synthesis of a primer for Pectinatus cerevisiiphilus The sequences specific for Pectinatus cerevisiiphilus using DNASIS (tradename of Hitachi Soft Engineering Ltd., Co.) on the basis of SEQ ID NO: 3 were analyzed. The result selected a sequence of 135 h to 15 3 rd on the gene sequence of the spacer region between the gene coding 16S rRNA and the gene coding 23S rRNA of Pectinatus cerevisiiphilus of SEQ ID NO: 3. (SEQ ID NO: 7.) In addition, the similar analysis selected a sequence of 172 n to 191 on the gene sequence of the spacer region between the gene coding 16S rRNA and the gene coding 23S rRNA of Pectinatus cerevisiiphilus of SEQ ID NO: 3. (SEQ ID NO: 8.) The similar analysis also selected a sequence of 2 0 3 rd to 2 2 2 nd on the gene sequence of the spacer region between the gene coding 16S rRNA and the gene coding 23S rRNA of Pectinatus cerevisiiphilus of SEQ ID NO: 3. (SEQ ID NO: 9.) Further, specific primer showing in SEQ ID NO: 11 was selected by a gene sequence coding 16S rRNA of Pectinatus cerevisiiphilus. The oligonucleotides were chemically synthesized by the same method as in Example Detection and identification of Pectinatus cerevisiipbilus by the primers having the sequences of SEQ ID NO: 7 and SEQ ID NO: 11.

The DNA solutions of bacteria prepared in Example 1 were treated with the primers synthesized in Example (SEQ ID NO: 7 and SEQ ID NO: 11) by PCR. The temperature conditions of the PCR were as follows: Thermal denaturation; 94°C, 30 seconds Annealing; 55*C, 30 seconds Chain elongation reaction; 72°C, 30 seconds One cycle of the conditions was repeated 35 times. After the PCR, the reactant was electrophoresed with agalose gel at constant 100 V for 30 minutes.

A pHY marker was also electrophoresed at the same time as a molecular weight marker. After the electrophoresis, the gel was stained with 5g/g/ml of an ethidium bromide solution for 20 minutes, and ultraviolet was applied to observe the gel and take a photograph of the gel. By the observation or the photography of the gel, the base length of the amplified products was determined from the relative migration distance with a molecular weight marker.

As shown in Fig. 2, a band of about 600 bps was detected only in case of Pectinatus cerevisiiphilus.

From the results, when the oligonucleotides of SEQ ID NO: 7 and SEQ ID NO: 11 were used as PCR primers, the band having objective length was detected only in case of Pectinatus cerevisihphilus. Accordingly, it was shown that each oligonucleotide of the present invention correctly recognized the gene sequences of the spacer region between the gene coding 16S rRNA and the gene coding 23S rRNA of Pectinatus cerevisiiphilus, and the base sequence targeted on the gene coding 16S rRNA. Moreover, the bands having the aimed length were not observed in the same genus Pectinatus fn'singensis, and relative strictly anaerobic bacteria and Gram-positive bacteria. Accordingly, Pectinatus cerevisiiphilus can be specifically detected, and at the same time also determined by the present invention.

By the present invention, the genes of the spacer region constituted between the 16S rRNA genes and the 23S rRNA genes of Pectinatus fisingensis -26- 0 0 and Pectinatus cerevisiiphilus have been proved, and a method for quickly and reliably detecting Pectinatusfrisingensis and Pectinatus cerevisiiphilus can be provided by Susing a part or all of the gene sequences.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to C preclude the presence or addition of further features in various embodiments of the 10 invention.

It is to be understood that a reference herein to a prior art document does not constitute an admission that the document forms part of the common general knowledge in the art in Australia or in any other country.

Claims (14)

1. An isolated nucleic acid molecule comprising a base sequence of a spacer region between a gene coding 16S rRNA and a gene coding 23S rRNA of Pectinatusfrisingensis containing a part of the base sequence or the whole base sequence represented by SEQ ID NO: 1.

2. An isolated nucleic acid molecule comprising a base sequence of a spacer region between a gene coding 16S rRNA and a gene coding 23S rRNA of Pectinatusfrisingensis containing a part of the base sequence or the whole base sequence represented by SEQ ID NO: 2.

3. An isolated nucleic acid molecule comprising a base sequence of a spacer region between a gene coding 16S rRNA and a gene coding 23S rRNA of Pectinatus cerevisiiphilus containing a part of the base sequence or the whole base sequence represented by SEQ ID NO: 3.

4. An isolated nucleic acid molecule comprising a base sequence of a spacer region between a gene coding 16S rRNA and a gene coding 23S rRNA of Pectinatus cerevisiiphilus containing a part of the base sequence or the whole base sequence represented by SEQ ID NO: 4. An oligonucleotide characterized in that the base sequence of a spacer region between a gene coding 16S rRNA and a gene coding 23S rRNA of Pectinatus frisingensis has at least one of the following sequence group or the corresponding complementary sequence: 5'-CCATCCTCTTGAAAATCTC-3' 5'-TCTCRTCTCACAAGTTTGGC-3'.

6. An oligonucleotide that comprises a base sequence of a spacer region between a gene coding 16S rRNA and a gene coding 23S rRNA of Pectinatus cerevisiiphilus having at least one of the following sequence group or the corresponding complementary sequence: 5'-CACTCTTACAAGTATCTAC-3' 5'-CCACAATATTTCCGACCAGC-3' 5'-AGTCTTCTCTACTGCCATGC-3'.

7. A method for detecting Pectinatusfrisingensis, comprising employing an oligonucleotide made from the base sequence of the nucleic acid molecule described Cin claim 1 or 2 as a primer for synthesis of nucleic acids, and treating the nucleic acid <c by gene amplification to detect the bacteria.

8. A method for detecting Pectinatus cerevisiiphilus, comprising C employing the oligonucleotide made from the base sequence of the nucleic acid molecule described in.claim 3 or 4 as a primer for synthesis of nucleic acids, and treating the nucleic acid by gene amplification to detect the bacteria.

9. A method for detecting Pectinatusfrisingensis, comprising employing N the oligonucleotide made from the base sequence of the nucleic acid molecule described in claim 1 or 2, or the oligonucleotide made from the base sequence described in claim C 10 5, and a nucleotide sequence coding 16S rRNA gene of Pectinatusfrisingensis as primers for synthesis of nucleic acids, and treating the nucleic acid by gene amplification to detect the bacteria. A method for detecting Pectinatus cerevisiiphilus, comprising employing the oligonucleotide made from the base sequence of the nucleic acid molecule described in claim 3 or 4 or the oligonucleotide made from the base sequence of the nucleic acid molecule described in claim 6, and a nucleotide sequence coding 16S rRNA gene of Pectinatus cerevisiiphilus as primers for synthesis of nucleic acids, and treating the nucleic acid by gene amplification to detect the bacteria.

11. A method as claimed in claim 9, wherein the nucleotide sequence coding the 16S rRNA gene of Pectinatusfrisingensis has the following sequence: 5'-CGTATCCAGAGATGGATATT-3'.

12. A method as claimed in claim 10, wherein the nucleotide sequence coding the 16S rRNA gene of Pectinatus cerevisiiphilus has the following sequence: 5'-CGTATGCAGAGATGCATATT-3'.

13. An isolated nucleic acid molecule comprising a base sequence of a spacer region between a gene coding 16s rRNA and a gene coding 23s rRNA of Pectinatusfrisingensis substantially as hereinbefore described with reference to Examples 2 or 3.

14. An isolated nucleic acid molecule comprising a base sequence of a 3 0 spacer region between a gene coding 16s rRNA and a gene coding 23s rRNA of Pectinatus cerevisiiphilus substantially as hereinbefore described with reference to Examples 4 or An oligonucleotide substantially as herein before described with reference to any one of Examples 2 to

16. A method for detecting Pectinatusfrisingensis substantially as herein before described with reference to Example 3.

17. A method for detecting Pectinatus cerevisiiphilus substantially as herein before described with reference to Example Dated this 12th day of December 2002 ASAHI BREWERIES, LTD By their Patent Attorneys GRIFFITH HACK