JP2001231564A - Oligonucleotide for detecting bacteria and method of detection using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a gene for detecting a bacterium belonging to the genus Pediococcus which grows in beer and muddies beer and a method for detecting the bacterium by the use of this gene. SOLUTION: This method is a method for detecting Pediococcus damnosus rapidly with high sensitivity by the use of a gene sequence in the spacer domain constituted between 16S rRNA gene and 23S rRNA gene specific to Pediococcus damnosus participating in beer muddiness.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a Pediococcus damnosu of the genus Pediococcus which grows in beer and renders beer turbid.
The present invention relates to a gene for detecting s ) and a method for detecting the bacterium using the gene.

[0002]

2. Description of the Related Art As microorganisms (bacteria) that make beer turbid, Pediococcus damnos
us ) is known. For this detection, the bacteria must be isolated through growth culture and separation culture, and it takes at least 7 days. Thereafter, the isolated bacteria are grown and identified by performing many property tests such as morphological observation, gram staining, catalase test, and sugar utilization. These diverse tests are cumbersome, time consuming and expensive. In addition to these commonly used identification tests, DNA is extracted from isolated bacteria, immobilized on a membrane or other support, and a hybridization test is performed using DNA of a standard bacterium as a probe. Thus, there is a method for identifying a bacterial species. However, this method also requires several days, and it is difficult to obtain sufficient detection sensitivity and selectivity.

Therefore, recently, as a method for detecting Pediococcus damnosus, a method using an antibody that specifically reacts with Pediococcus damnosus (Brauwelt: v.
ol. 131, NO.41 1797-1798, 1800-1802, 1991).
In this method, Pediococcus damnosus is stained by the fluorescent antibody method, and then is caused to flow through a laser irradiation optical path to be colored and detected and counted. However, there is a problem in specificity.

[0004] Another detection method will be described below. A method for identifying Pediococcus damnosus by obtaining a mass spectrum of the fatty acid methyl ester derivatized in the cells using pyrolysis mass spectrometry (J. ASBC: vol.55, NO.2, 79 -82 (1997))
There is. However, this method requires isolation of the bacterium and has problems in terms of speed and specificity.

A method for identifying Pediococcus damnosus using PCR and temperature gradient gel electrophoresis targeting the 5S rRNA gene (J. ASBC: vol. 52, NO. 3, 95-99)
(1994)). In this method, a region of the 5S rRNA gene highly conserved in bacteria is amplified by a PCR reaction, and a second PCR reaction is performed using a primer having a high GC content. The obtained PCR product is subjected to temperature gradient gel electrophoresis, and Pediococcus damnosus is identified based on the electrophoresis pattern. However, this method requires isolation of the bacterium and has problems in terms of speed and specificity.

[0006] Also, the 16S of Pediococcus damnosus
A method for specifically detecting Pediococcus damnosus by hybridization using the rRNA gene and the base sequence of the 23S rRNA gene as a target (Tokuhei 8-503620)
Is being developed. However, the 16S rRNA gene and 23S rRNA gene used in these technologies may be similar across microorganisms, and may erroneously detect microorganisms other than the specific microorganism to be detected. However, there was a problem in terms of specificity.

On the other hand, it is known that the gene of the spacer region formed between the 16S rRNA gene and the 23S rRNA gene has a base sequence specific to the microorganism species. / JP99 / 04341 and Japanese Patent Application No. 10-286697, but the nucleotide sequence of the gene for the spacer region of Pediococcus damnosus has not been clarified.

[0008]

Accordingly, the present invention provides a gene sequence of a spacer region comprised between the 16S rRNA gene and the 23S rRNA gene specific to Pediococcus damnosus, which is involved in beer turbidity. It is an object of the present invention to provide a method capable of rapidly and sensitively detecting a sequence.

[0009]

Means for Solving the Problems (1) The first invention relates to a 16-membered Pediococcus damnosus containing a part or all of the nucleotide sequence shown in SEQ ID NO: 1.
This is the gene sequence of the spacer region between the gene encoding S rRNA and the gene encoding 23S rRNA. (2) the second invention, Pejiokokkasu-Damunosasu (Pedi include some or all of the nucleotide sequence shown in SEQ ID NO: 2
ococcus damnosus ) and the gene encoding 16S rRNA
This is the gene sequence of the spacer region between the genes encoding S rRNA. (3) The third aspect of the present invention, Pejiokokkasu-Damunosasu (Pedi
ococcus damnosus ) and the gene encoding 16S rRNA
An oligonucleotide, wherein the gene sequence of the spacer region between the genes encoding S rRNA has at least one of the sequences of SEQ ID NO: 3 or has a corresponding complementary sequence. (4) The fourth invention provides a method for making an oligonucleotide sequence prepared from the gene sequence described in (1) or (2) as a primer for nucleic acid synthesis, and subjecting the oligonucleotide sequence to gene amplification treatment to obtain a Pediococcus dammnosus.
mnosus ) is a way to detect. (5) The fifth invention provides an oligonucleotide prepared from the gene sequence described in (1) or (2) or (3)
The nucleotide sequence encoding the 16S rRNA gene of Pediococcus damnosus and the oligonucleotide described in the above is used as a primer for nucleic acid synthesis and subjected to gene amplification treatment to detect Pediococcus damnosus. is there. (6) The sixth invention is Pedioccus damnosus.
(5) The method according to (5), wherein the nucleotide sequence encoding the 16S RNA gene of S. occus damnosus has the sequence of SEQ ID NO: 4.

[0010] Techniques for gene amplification are already known, and are based on the polymerase chain reaction (hereinafter abbreviated as PCR) developed by R. Saiki et al., Science 230, 1350 (1985). You can do it.

[0011] This method is a reaction for amplifying a specific gene sequence and has a rapid, high sensitivity, high specificity, and simplicity. It is also being applied to rapid determination and rapid detection of harmful bacteria in the food field. By performing the PCR method, the target nucleotide sequence between the two primers is amplified hundreds of times, even if only a small amount is present in the sample, and a large number of copies are produced before detection is possible. You. In addition, in order to perform the PCR method, it is necessary to release nucleic acid components from bacteria present in the sample.
In this method, the amplification reaction proceeds if the target sequence is present in a number of molecules or more. Therefore, a simple pretreatment using a lytic enzyme or a surfactant can be sufficient for the test. Therefore, the utility value is extremely high as compared with the conventional bacteria detection method.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION In order to take advantage of these facts, the present invention provides a gene sequence of a spacer region between a gene encoding 16S rRNA and a gene encoding 23S rRNA of Pediococcus damnosus, and selects from these. Sequence of the spacer region between the isolated oligonucleotide or the gene encoding 16S rRNA and the gene encoding 23S rRNA and the 16S rRNA of Pediococcus damnosus
PCR targeting the nucleotide sequence encoding the gene
We developed a method to quickly and highly sensitively determine whether or not Pediococcus damnosus is present in a sample by functioning as a primer for nucleic acid synthesis in the method and subjecting it to gene amplification.

The specimen may be beer or a semi-finished product during beer production, or a sample collected from the environment such as sewage. Oligonucleotides used as primers may be chemically synthesized or natural, and any of them may be used.

Hereinafter, a method for detecting Pediococcus damnosus using the PCR method will be described. The nucleotide sequences used in the PCR method are shown in SEQ ID NOs: 3 and 4 as an example, and are not limited thereto. The primer length used in the PCR method was 23 nucleotides in the primers described in SEQ ID NOS: 3 and 4, but is not limited thereto. Preferably, one having a length of 10 to 50 bases is used.

The gene encoding the 16S rRNA of Pediococcus damnosus defined by the two primers and the 23S rR
The nucleotide length of the target sequence on the nucleotide sequence of the spacer region between the gene encoding NA and the nucleotide sequence of the 16S rRNA gene is 1537 base pairs and 1728 base pairs when SEQ ID NOS: 3 and 4 are combined. When two bands are detected by gel electrophoresis, it can be determined that Pediococcus damnosus was present. Since this combination of primers is specific to Pediococcus damnosus, it can be used for detection of this strain. PCR
By changing the base sequence of the primer used in the method,
The length of the nucleotide sequence to be amplified will vary.

The temperature condition in the PCR reaction is as follows.
90-98 ° C by heat denaturation reaction to make the main strand
37-65 by annealing reaction to hybridize to A
℃, 50-75 ℃ by chain length reaction with DNA polymerase
The target sequence can be amplified by performing several tens of cycles in which this is performed as one cycle. After the PCR reaction, the reaction product is separated by electrophoresis, and nucleic acid staining is performed with ethidium bromide or cyber green.If the base length of the amplified nucleotide sequence is equal to the base length of the above-described target sequence, the target substance is detected in the sample. It can be determined that bacteria exist. Chromatography is also effective for detecting the amplified nucleotide sequence.

[0017]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of Specimen The strain was Pediococcus damnosa.
osus ) (JCM5886) was used. In order to confirm the specificity of the Pediococcus damnosus primers shown in SEQ ID NOs: 3 and 4 of the present invention, other bacteria shown in Table 1 were used. After culturing them in an appropriate growth medium, the cells were collected by centrifugation. After that, DNA extraction from the cells was performed in the Shinsei Kagaku Experiment Course 2
~ 21 (edited by The Biochemical Society of Japan, Tokyo Doujinshi)
A DNA solution was obtained.

[0019]

【table 1】

Example 2 Cloning of the spacer region between the gene encoding 16S rRNA of Pediococcus damnosus and the gene encoding 23S rRNA and determination of the nucleotide sequence (1) Amplification of the 16S / 23S rRNA spacer region by PCR Selection and Synthesis of Oligonucleotide Primers for Most of the 16S rRNA gene of Pediococcus damnosus has been identified (DDBJ accession No.
D87678], and primers were selected based on the nucleotide sequences at positions 1402 to 1421.

Although the nucleotide sequence of the 23S rRNA gene of Pediococcus damnosus has been determined, it has been reported that the nucleotide sequence at positions 21 to 38 of the 23S rRNA gene is highly conserved among bacteria (Microbiology: vol. .142, 3-16 (199
6)), primers were selected based on the sequence to obtain a corresponding complementary sequence.

(2) Amplification of 16S / 23S rRNA spacer region by PCR method 0.1 μg of the DNA solution of Pediococcus damnosus prepared in Example 1 was placed in a 0.2 ml tube (PerkinElmer), and TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) ), Registered trademark)
Ex Taq (Registered trademark) buffer r, 5 μl, 2.5 mM dNTP
4 μl of the mixture (dATP, dGTP, dCTP, DTTP), 5 U / μl of T
0.25 μl of aKaRa Ex Taq (registered trademark), Example 2- (1)
0.5 μl of each of the primers having a concentration of 100 mM prepared in the above, and sterilized distilled water were added thereto to obtain a 50 μl solution. The tube was set in an automatic gene amplifier Thermal Cycler (Perkin Elmer) to perform an amplification reaction. The reaction conditions were as follows: denaturation at 94 ° C for 2.5 minutes, denaturation at 94 ° C for 30 seconds → 55
30 ° C., 30 seconds of primer annealing → 72 ° C., 30 seconds of synthesis reaction cycle. 5 μl after reaction
Was subjected to agarose gel electrophoresis, and the DNA was stained with Cyber Green to confirm the amplified DNA. As a result, about 600 bps (hereinafter referred to as “long”)
450bps (hereinafter referred to as "short") DNA was amplified.

(3) Cloning and Sequencing of Long Spacer Region The reaction solution after completion of PCR is used for High Pure PCR Product Purific
Unreacted dNTPs were removed using an ation Kit (Boehringer Mannheim, trade name). Amplification prepared in this way
To 30 ng of DNA, 2 μl of the pCR 2.1 vector contained in the TA Cloning Kit (Invitrogen, trade name) and 1 part of T4 DNA ligase
1 μl of 10 × ligation buffer, 1 μl of sterile water to a total volume of 10 μl, and after reacting at 14 ° C. overnight, add 2 μl of the mixture and 2 μl of 0.5 M β-mercaptoethanol to E. coli INVαF ′ competent cells. In the ice,
After standing for 30 minutes, the mixture was heat-treated at 42 ° C for 30 seconds to transform Escherichia coli with the plasmid. For transformed E. coli
SOC medium (2.0% tryptone, 0.5% yeast extract, 10.0m
M NaCl, 2.5 mM KCl, 10.0 mM MgCl ₂ -6H ₂ O, 20.0 mM glucose) 250 μl, and shake at 37 ° C. for 60 minutes.
L containing μg / ml ampicillin and 40 μg / ml X-Gal
The cells were inoculated on a B plate medium and cultured overnight at 37 ° C. The white colonies that appeared were inoculated into 3 ml of LB liquid medium containing 50 μg / ml ampicillin, and cultured at 37 ° C. overnight. After the culture, plasmids were extracted from E. coli using an automatic plasmid extraction device. Take a part of the obtained plasmid and use the restriction enzyme Ec
After reaction at 37 ° C for 60 minutes with oRI (Takara Shuzo Co., Ltd.), agarose gel electrophoresis, DN by Cyber Green
A staining confirmed that the long was inserted.

Using the thus obtained plasmid as a template, a sequencing reaction was performed. Sequencing primers are IRD41 Infrared Dye Labeled M13 Forward primer and IRD800 Infrared Dye Labeled M13 Revers
e Primer (manufactured by Nisshinbo Industries, sold by Aloka Co., Ltd.) and the reaction solution are Sequi Therm (registered trademark) Long-Read (registered trademark)
Cycle Sequencing Kit-LC (EPICENTER TECHNOLOGIES) was used. For nucleotide sequencing, use the 4000L Long ReadIR
(Registered trademark) DNA sequencing system (manufactured by LI-COR) was used.

The Pediococcus damnosus obtained (JC
SEQ ID NO: 1 shows the gene sequence of the spacer region (long) between the gene encoding 16S rRNA and the gene encoding 23S rRNA of M5886).

(4) Cloning and Sequencing of Short Spacer Region The reaction solution obtained after completion of the PCR in Example 2- (2) was subjected to High Pure PCR.
Unreacted dNTPs were removed using a Product Purification Kit (Boehringer Mannheim, trade name). 30 ng of the amplified DNA thus prepared is added to a TA Cloning Kit (Invitrog
2 μl of pCR 2.1 vector contained in
1 μl of DNA ligase, 1 × 10 × ligation buffer
μl, sterilized water was added to a total volume of 10 μl, and reacted at 14 ° C. overnight.Then, 2 μl and 2 μl of 0.5 M β-mercaptoethanol were both added to E. coli INVαF ′ competent cells and left on ice for 30 minutes. After that, heat treated at 42 ° C for 30 seconds,
Transformation of the plasmid into E. coli was performed. SOC medium (2.0% tryptone, 0.5% yeast extract, 10.0 mM NaCl, 2.5 mM KCl, 10.0 mM MgCl ₂ -6H ₂ O, 2%
After adding 250 μl of 0.0 mM glucose) and shaking at 37 ° C. for 60 minutes, 50 μg / ml ampicillin and 40 μg / ml X-
The cells were inoculated on an LB plate medium containing Gal and cultured at 37 ° C. overnight.
The white colonies that appeared were inoculated into 3 ml of LB liquid medium containing 50 μg / ml ampicillin, and cultured at 37 ° C. overnight. After the culture, plasmids were extracted from E. coli using an automatic plasmid extraction device. A portion of the obtained plasmid was removed, reacted with the restriction enzyme EcoR I (Takara Shuzo) at 37 ° C. for 60 minutes, and then agarose electrophoresis and DNA staining with Cyber Green confirmed that the short was inserted. .

Using the thus obtained plasmid as a template, a sequencing reaction was performed. Sequencing primers are IRD41 Infrared Dye Labeled M13 Forward primer and IRD41 Infrared Dye Labeled M13 Reverse
Primer (manufactured by Nisshinbo, sold by Aloka Co., Ltd.) and the reaction solution were SequiTherm (registered trademark) Long-Read (registered trademark)
Cycle Sequencing Kit-LC (EPICENTRE TECHNOLOGIES) was used. For nucleotide sequencing, use the 4000L Long ReadIR
(Registered trademark) DNA sequencing system (manufactured by LI-COR) was used.

The obtained Pediococcus damnosus 16
SEQ ID NO: 2 shows the gene sequence of the spacer region (short) between the gene encoding S rRNA and the gene encoding 23S rRNA.

Example 3 Detection of Pediococcus damnosus by PCR (1) Selection and synthesis of primers for detection of Pediococcus damnosus DNASIS (Hitachi Software Engineering Co., Ltd., trade name) based on SEQ ID NOS: 1 and 2 Was used to analyze sequences specific to Pediococcus damnosus. As a result, the sequence from the 378th to the 400th position on the gene sequence of the spacer region between the gene encoding 16S rRNA and the gene encoding 23S rRNA of Pediococcus damnosus of SEQ ID NO: 1, and Pediococcus damnosus of SEQ ID NO: 2 Of 1
Sequences from position 183 to position 205 on the gene sequence of the spacer region between the gene encoding 6S rRNA and the gene encoding 23S rRNA were selected. (SEQ ID NO: 3)

Further, the 16S of Pediococcus damnosus
From the 166th to 188th positions of the gene sequence encoding rRNA,
The specific primer shown in SEQ ID NO: 4 was selected. These oligonucleotides were chemically synthesized in the same manner as in Example 2- (1).

(2) Detection and identification of Pediococcus damnosus using primers having the sequences of SEQ ID NO: 3 and SEQ ID NO: 4 The DNA solution of each bacterium prepared in Example 1 was synthesized with the primer (SEQ ID NO: 3) synthesized in Example 3. No. 3 and SEQ ID No. 4) using PC
R performed. PCR was performed under the following temperature conditions: heat denaturation; annealing at 94 ° C. for 30 seconds; chain reaction at 48 ° C. for 1 minute; After completion of PCR, the reaction solution was subjected to electrophoresis on an agarose gel at a constant voltage of 100 V for 30 minutes. In addition to the reaction solution, a pHY marker was also electrophoresed as a molecular weight marker. After electrophoresis, after staining in Cyber Green solution for 30 minutes,
When the gel is observed under UV irradiation, bands of about 1700 bps and about 1500 bps are detected only in Pediococcus damnosus using SEQ ID NOs: 3 and 4. 1 and 2 show the results of electrophoresis.

From these results, when the oligonucleotides of SEQ ID NO: 3 and SEQ ID NO: 4 of the present invention were used as PCR primers, a band of the desired length was detected only in Pediococcus damnosus. From this, each oligonucleotide of the present invention, the 16S of Pediococcus damnosus
It was shown that the gene sequence of the spacer region between the gene encoding rRNA and the gene encoding 23S rRNA and the target base sequence on the gene encoding 16S rRNA were correctly recognized. In addition, since no band of the target length was detected in any other staphylococci or gram-positive bacteria that may cause turbidity of beer, including other Pediococcus genus, the present invention detects Pediococcus damnosus in a species-specific manner. It was shown that Pediococcus damnosus could be detected and identified at the same time.

[0033]

Industrial Applicability The present invention provides a 16S rRNA gene specific to Pediococcus damnosus associated with beer turbidity and a 23S rRNA.
It is intended to provide a gene sequence of a spacer region constituted between RNA genes, and to provide a method capable of rapidly and sensitively detecting the sequence using the sequence.

[0034]

[Sequence List] SEQUENCE LISTING <110> Asahi Breweries, Ltd <120> Genes for detecting bacteria and a detection method using them <130> 99T3-45 <160> 4 <210> 1 <211> 479 <212> DNA <213 > Pediococcus damnosus <400> 1 ggggtgaagt cgtaacaagg tagccgtagg agaacctgcg gctggatcac 50 ctcctttcta aggatattta gaaacggaaa cctacacata tgtcgagact 100 ttgtttagtt ttgagaggtc tactctcaaa tgtataagcg cgaacgggcc 150 tatagctcag ctggtttaga gcgcacgcct gataagcgtg aggtcgatgg 200 ttcaagtcca tttaggccca taggtacatt tttggggaat tagctcagct 250 gggagagcgc ctgccttgca cgcaggaggt cagcggttcg atcccgctat 300 tctccattga cggcttagcc gtcgaatttg ttctttgaaa actaaataat 350 atcgaaaaat tttctaattt taattatcag ataattaaac cgagaacatt 400 gcgttttata gagttttaaa acaagattag ttcaaaaata atcgctaaac 450 tcgaaaacca ctttatcttt gataaagtt 479 <210> 2 <211> 284 <212> DNA <213> Pediococcus damnosus <400> 2 ggggtgaagt cgtaacaagg tagccgtagg agaacctgcg gctggatcac 50 ctcctttcta aggatattta gaaacggaaa cctacacata tgtcgaaact 100 ttgtttagtt ttgagaggtc tactctcaaa atttgttctt tgaaaactaa 150 ataatatcga aaaattttct aattttaatt atcagataat taaaccgaga 200 acattgcgtt ttatagagtt ttaaaacaag attagttcga aaaataatct atta <210atta <210atta <210a gtatta <210a gt> acct aca t aca>211> 23 <212> DNA <400> 4 tgctaatacc gcataataaa atg 23

[Brief description of the drawings]

FIG. 1 is an electrophoretogram of various strains shown in Example 1.
Lane 1: pHY marker, Lane 2: Pediococcus damnosus
(JCM5886), Lane 3: Pediococcus acidilactici (JC
M5885), Lane 4: Pediococcus dextrinicus (JCM588)
7), Lane 5: Pediococcus halophilus (JCM5888),
Lane 6: Pediococcus parvulus (JCM5889), Lane 7:
Pediococcus pentosaceus (JCM5890), Lane 8: Leuco
nostoc oenos (JCM6125), lane 9: Leuconostoc mese
nteroides subsp. mesenteroides (JCM6124), lane 1
0: Leuconostoc lactis (JCM6123), Lane 11: Leucon
ostoc paramesenteroides (NCDO803), lane 12: Leuc
onostoc gelidum (NCDO2775), lane 13: Leuconostoc
carnosum (NCDO2776), lane 14: Leuconostoc ameli
biosum (NCDO2787), lane 15: Megasphaera cerevisai
ae (DSM20462), lane 16: Lactococcus lactis (JCM5
805), Lane 17: Staphylococcus aureus subsp.aureus
(IAM1011) The pHY marker bands are 4870 bps in order from the top,
2016bps, 1360bps, 1107bps, 926bps, 658bps, 489bp
s, 267 bps.

FIG. 2 is an electrophoretogram of various strains shown in Example 1.
Lane 1: pHY marker, Lane 2: Pediococcus damnosus
(JCM5886), Lane 3: Lactobacillus casei (JCM113
6), Lane 4: Lactobacillus brevis (JCM1059), Lane 5: Lactobacillus plantarum (JCM1149), Lane 6: La
ctobacillus acidophilus (IFO13951), lane 7: Lact
obacillus lindneri (DSM20690), lane 8: Lactobaci
llus coryniformis (JCM1164) pHY marker bands are 4870bps, 2016bps, 1360bps, 1107bps, 926bp in order from the top
s, 658 bps, 489 bps, and 267 bps.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B024 CA01 4B063 QA01 QA18 QQ06 QQ16 QQ42 QR08 QR32 QR39 QR42 QR62 QS25 QX02

Claims (6)

[Claims] 1. A Pejiokokkasu-Damunosasu include some or all of the nucleotide sequence shown in SEQ ID NO: 1 (Pediococ
cus damnosus ) gene encoding 16S rRNA and 23S rR
Gene sequence of the spacer region between the genes encoding NA. Wherein Pejiokokkasu-Damunosasu include some or all of the nucleotide sequence shown in SEQ ID NO: 2 (Pediococ
cus damnosus ) gene encoding 16S rRNA and 23S rR
Gene sequence of the spacer region between the genes encoding NA. (3) Pediococcu damnosus
s damnosus ) gene encoding 16S rRNA and 23S rRNA
An oligonucleotide characterized in that the gene sequence of the spacer region between the genes coding for has the sequence of SEQ ID NO: 3 or the corresponding complementary sequence. 4. A method for detecting Pediococcus damnosus by causing an oligonucleotide prepared from the gene sequence according to claim 1 or 2 to function as a primer for nucleic acid synthesis and subjecting it to gene amplification treatment. 5. An oligonucleotide prepared from the gene sequence according to claim 1 or 2 or the oligonucleotide according to claim 3, and a nucleotide sequence encoding a 16S rRNA gene of Pediococcus damnosus. A method of detecting Pediococcus damnosus by functioning as a primer for nucleic acid synthesis and performing gene amplification treatment. 6. The Pejiokokkasu-Damunosasu (Pediococcu
6. The method according to claim 5, wherein the nucleotide sequence encoding the 16S rRNA gene of S. damnosus ) is an oligonucleotide having the sequence of SEQ ID NO: 4.