JP2001145492A - Nucleic acid probe for detecting bacterium belonging to the genus pectinatus and method of detecting beer muddiness causal bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a nucleic acid sequence which can be used on an assay for detecting the presence of the genus Pectinatus bacteria and its evolutionary relative bacteria and combines preferentially with the rRNA or rDNA of microorganisms causing beer muddiness. SOLUTION: This nucleic acid sequence is a sequence targeting the 16 S rDNA and 16 S rRNA of the genus Pectinatus bacteria in order to selectively detect the bacteria belonging to the genus Pectinatus bacteria and its oligonucleotide is a single strand oligonucleotide characterized by containing a specific sequence or a correspondent complementary chain.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to the detection of a bacterium of the genus Pectinatus, which is a beer harmful bacterium, or a bacterium closely related to its evolution, or the identification or quantification of the bacterium.

[0002]

2. Description of the Related Art In recent years, the trend toward beer production has brought a new sense of beer freshness. Against this background, there is an increasing need for beer manufacturers to quickly and accurately determine the contamination of beer harmful bacteria in order to dramatically shorten the time from product manufacture to shipping.

[0003] An obligately anaerobic bacterium, Pectinatus (Pe
ctinatus) and its evolutionary relatives, Megasphaera, Selenomonas
The genus Zymophilus increases in the frequency of detection as the anaerobicity of product beer increases with the development of beer brewing technology. In particular, pectinatas is the causative agent of several product accidents that occurred in the 1990s. Has been identified as

[0004] It is therefore important to develop a sufficiently specific and sensitive bacterial diagnostic test that can be used in the food industry, especially in the beer industry, to enable rapid and selective detection of Pectinatus species. The identification of classic beer harmful bacteria is based on morphological observation, gram stainability, catalase test, and many other properties as well as biochemical tests, finally re-ingesting the isolated bacteria in fresh beer, its growth It was determined by observing Noh. Such a series of operations requires a lot of time and effort, and it is often difficult to make an accurate determination. Recently, several methods for more rapid detection and identification of Pectinatus species have been studied. For example, a method of detecting Pectinatus species using an antigen-antibody reaction (J. Am. Soc. Brew. Chem .: 51).
(4) 158-163, 1993) and DNA extracted from Pectinatus spp.
After amplifying the target sequence by performing R (Polymerase Chain Reaction), there is a method of judging the presence or absence of a nucleic acid peculiar to the genus Pectinatus (Japanese Patent Publication No. 09-820071).

[0005]

However, in these methods, if an antigen or a target nucleic acid is present, a positive determination is made. Bacteria that have no viability or have a very low activity and have a low beer growth potential. Indistinguishable from bacteria that still retain Also, good reproducibility by PCR method,
Sufficient sensitivity requires appropriate cultivation to obtain the required minimum amount of bacterial nucleic acid and preliminary steps of nucleic acid extraction and gene amplification. This is because the amount of bacterial nucleic acids is extremely small, and very low concentrations of nucleic acids cannot be efficiently extracted, or the target nucleic acid is present in only a few copies per bacterial genome.

[0006]

SUMMARY OF THE INVENTION One object of the present invention is to provide:
Organism ribosomal RNA (rRNA) and ribosomal DNA that cause beer turbidity but are not present in unturbed beer
(RDNA) to provide an oligonucleotide complementary to the unique nucleic acid sequence.

Another object of the present invention is to provide a nucleic acid oligonucleotide capable of hybridizing to a target region which can be made accessible under nucleic acid hybridization having both specificity, sensitivity and speed. To provide.

Another object of the present invention is to provide a method for performing hybridization by selectively using these oligonucleotides labeled with an appropriate tracer as a probe to selectively detect and identify and quantify the bacterium. It is.

[0009]

DETAILED DESCRIPTION OF THE INVENTION Bacterial ribosomes contain at least three RNs, designated 5S, 16S and 23S rRNA.
A molecule. Historically, these names have been related to the size of the RNA molecule as determined by the rate of sedimentation. In the present invention, bacterial ribosomal RNA (rRNA) can be used as a target. One of the advantages of this use is that rRNA
Is abundant in all living cells.

Before disclosing the present invention in more detail, various terms used herein are defined as follows. "Oligonucleotide" or "oligonucleotide fragment"
Is characterized by the information sequence of the natural (or optionally modified) nucleic acid and is capable of hybridizing, under predetermined conditions, with a complementary or substantially complementary nucleotide fragment, similar to a naturally occurring nucleic acid. Are two synonyms that indicate a chain of nucleotide units The strand may comprise nucleotide units of a different structure than the natural nucleic acid. For example, a nucleic acid which is a constituent unit of an oligonucleotide is not a phosphodiester found in a naturally occurring nucleic acid, but is bonded by another ester bond or an amide bond (generally called PNA), and hybridizes to a target region. Any material that can soy may be used. Oligonucleotides (or nucleotide fragments) can contain, for example, up to 100 nucleotide units. Generally, at least 10
Containing a single nucleotide unit and can be obtained from a naturally occurring nucleic acid molecule or by genetic recombination or by chemical synthesis.

"Hybridization" is understood to mean that under appropriate conditions, nucleotide fragments having sufficiently complementary sequences are bonded to each other by stable and specific hydrogen bonding to form a double strand. . Hybridization conditions are determined by "stringency", that is, the severity of the reaction conditions. The higher the stringency when performing hybridization, the higher the specificity. That is, appropriate hybridization conditions include, in particular, the base composition of the probe / target duplex, and the
It is determined from the degree of mismatch between individual nucleic acids and is expressed as a function of hybridization reaction parameters such as the concentration and type of ionic species present in the hybridization solution, the nature and concentration of denaturing agents, or the hybridization temperature. The stringency of the conditions under which the hybridization reaction is carried out depends in particular on the probe used. All these data are well known and the appropriate conditions can be determined in each case by routine experimentation, within the skill of the ordinary technician.

[0012] Further, "homologous" means to indicate the degree of similarity between two or more nucleic acid sequences, and does not mean the taxonomic degree of similarity between organisms. The degree of similarity is expressed as a percentage, for example, 2
The term "90% homologous" means that 90% of the bases in the first sequence match identically to the bases in the second sequence.

[0013] A "probe" has hybridization specificity under defined conditions.
Ribosomal RNA or ribosomal DNA (rDN
Form a hybridization complex with the target nucleic acid having the nucleotide sequence contained in A), for example, from 10 to
A nucleotide fragment comprising 100 nucleotide units, especially 12 to 35 nucleotide units. Probes can be used for detection, identification, and quantification purposes. For example,
Radioisotopes, enzymes, especially enzymes capable of acting on chromogenic, fluorescent or luminescent substrates (especially peroxidase or alkaline phosphatase), chromogenic chemical compounds,
It can be labeled with a marker selected from chromogenic, fluorescent or luminescent compounds, analogues of nucleotide bases and ligands such as biotin.

A "primer" comprises, for example, 10 to 100 nucleotide units and is a defined condition for the initiation of enzymatic polymerization, for example in amplification methods such as PCR (polymerase chain reaction), in sequencing processes, in reverse transcription methods and the like. Oligonucleotides with hybridization specificity below.

The oligonucleotide according to the present invention can be used for testing the presence or absence of a target nucleic acid in a sample in all known hybridization techniques, in particular, a technique of spot attachment on a filter called "dot-blot" (MANIAT).
IS et al., Molecular Cloning, Cold Spring Harbor, 198
2), a DNA transfer technique called "Southern blot" (SOUTHERN EM, Mol. Biol., 98, 503 (1975)),
It can be used according to the technique of RNA transfer called "Northern blot".

According to the present invention, (1) Pectinatus flisingensis (P.
nsis), and (2) Pectinatus cerevisiphyllus (P. cere) in comparison with other species.
visiiphilus); (3)
Compared to other genera, Pectinatus species (P. frisingens
is, P. cerevisiiphilus); (4) Pectinatus, Megasphaera, Zymophilus, or Zymophilus, which causes beer turbidity, as compared to other genera. About 10-100 nucleotide fragments are provided which hybridize preferentially to Selenomonas spp. The nucleotide fragments of the present invention are useful for detecting the presence of organisms that cause beer turbidity. Probes complementary to or at least 90% homologous to at least 10 contiguous nucleic acids of the above nucleotides are also included in the invention.

[0017] One of the present inventions is a beer turbid harmful bacterium.
For oligonucleotides and probes that are complementary to or hybridize to a particular region of S rRNA or rDNA. Specifically, the nucleic acid is used for at least 90% of a sequence containing any 10 consecutive oligonucleotides in a sequence selected from the group of sequences defined by the probe groups shown in SEQ ID NOs: 1 to 8 in the sequence listing. Or homologous thereto. Specifically, the nucleic acid of (1) has SEQ ID NOS: 1 to 3
The nucleic acid of (2) is an oligonucleotide of SEQ ID NO: 4 and / or 5, the nucleic acid of (3) is an oligonucleotide of SEQ ID NO: 6 and / or 7, and the nucleic acid of (4) is an oligonucleotide of SEQ ID NO: 8 . Probe design was performed using the 16S rRNA database (RDP, Larsen et al., 1993) and Gene Ban.
This was performed based on the gene sequence information published from the k data library.

Another aspect of the present invention is a method for detecting the presence of beer turbid bacteria. The method includes contacting a sample suspected of containing a target bacterium with at least one nucleic acid. This nucleic acid can be obtained from (1) Pectinatus flisingensis; (2) Pectinatus cerevisiphyllus; (3) Pectinus species (P. frisingensis, P. cerevisiiphilus); (4) Pectinase causing beer turbidity. A genus, Megasfera, Zymophilus, or Selenomonas; having about 10-100 nucleotides that hybridize preferentially to rRNA or rDNA.

In this method, the nucleic acid probe is used for the target rRNA.
Or applying hybridization conditions to the sample such that it binds preferentially to rDNA to form a nucleic acid complex;
And detecting the complex as an indicator of the presence of the target organism (one or more). Preferably, the nucleic acid probe is any one of the sequences selected from the group of sequences defined by the probe group shown in SEQ ID NOs: 1 to 8.
Homologous or homologous to at least 90% of the sequence comprising 0 consecutive oligonucleotides.

The probe of the present invention can be used to develop a nucleic acid hybridization assay for the specific detection of beer turbid organisms in beer, semi-finished products during beer production, or samples collected from environments such as breweries and sewage. Provides the basis for The probes of the present invention also provide a basis for confirming the presence of beer turbid harmful bacteria. The first step in the development of the probes of the present invention is to target a specific nucleic acid probe with one of the targeted beer harmful bacteria.
It was to identify unique regions within the 6S rRNA. This means that any target region in the 16S rRNA
(1) of Pectinatus flisingensis causing beer turbidity; (2) of Pectinatus cerevisiphilus; (3) Pectinatus genus (P. frisingensis,
P. cerevisiiphilus); (4) To find out which is specific to pectinatas, Megasfera, Zymophilus or Selenomonas, which causes beer turbidity.

Therefore, in order to realize these, the sequence of 16S rDNA of Pectinatus flisinginsis and Pectinatus cerevisiae was determined by a known experimental protocol, and Megasfera sp., Zymophilus sp., Which are evolutionary neighbors of Pectinatus sp. Bacteria as well as Lactobacillus spp., Known as selenomonas spp. Or beer harmful fungi, or other bacteria including E. coli 16S rDNA.
Was compared with the sequence. Based on this result, (1) Pectinatus flisingensis causing beer turbidity;
(2) Pectinatus cerevisiiphilus; (3) Pectinatus genus (P. frisingensis, P. cerevisiiphilu)
s); (4) Probes specific to any of pectinus, Megasfera, Zymophilus, or Selenomonas causing beer turbidity were designed as shown in Sequence Listings 1 to 8. . In particular,
The nucleic acid of (1) is any one of SEQ ID NOs: 1 to 3, (2)
Is the oligonucleotide of SEQ ID NO: 4 or 5, the nucleic acid of (3) is the oligonucleotide of SEQ ID NO: 6 or 7, and the nucleic acid of (4) is the oligonucleotide of SEQ ID NO: 8.

The probe of the present invention is used for fluorescence in situ
It can be used for hybridization (hereinafter abbreviated as FISH). Beer and semi-finished products during beer production, or samples collected from environments such as breweries or sewage, are subjected to centrifugation or membrane filtration to capture beer harmful bacteria, and targets that may be present in the sample. The beer turbid harmful bacteria and the detection probe are brought into contact under appropriate hybridization conditions. The detection probe penetrates into the cytoplasm of the target bacterium, and the 16S rRNA present there.
The target site within is accessed and hybridized under appropriate hybridization conditions. At this time, specific hybridization phenomena can be monitored by an appropriate technique by labeling the detection probe with a tracer such as a radioisotope, a fluorescent substance, or a chemiluminescent substance. For example, when the detection probe is labeled with a radioisotope, an assay is performed by a method such as autoradiography, and when the detection probe is labeled with a fluorescent substance, an assay using a fluorescence microscope or a CCD camera is used. Digital analysis is performed, and when labeled with a chemiluminescent substance, analysis using a photosensitive film or digital analysis using a CCD camera can be performed to determine the presence or absence of a target organism.

The oligonucleotide as defined above may also be used in a known manner in the presence of a thermostable polymerase in the presence of at least one bacterium of the genus Pectinatus.
Species or 16 evolutionarily related bacteria of the genus Pectinatus
It can be used as a specific primer for amplifying and synthesizing S rDNA or 16S rRNA (PCR, RT-PCR, etc.). By the combination of the primers used and the setting of an appropriate reaction program, the presence or absence of the target bacterium can also be determined from the result of whether or not the target nucleic acid is amplified.

[0024]

The present invention will be described with reference to the following examples, but the present invention is not limited to these examples. Example 1 Detection and identification of beer harmful bacteria by FISH method The following two methods are used to collect bacteria from beer and semi-finished products during beer production, or samples collected from environments such as breweries and sewage. It was performed using.

(1) Collection by centrifugation method 50 ml of the test sample is placed in a centrifuge tube, centrifuged (10,000 Xg, 10 minutes, 4 ° C), the supernatant is discarded, and after collecting bacterial pellets, another 30 ml is collected. , And centrifuged under the same conditions to wash. After washing, remove 1 bacterial pellet
ml of fixative (P with 4% w / v paraformaldehyde)
BS) and fixed at 4 ° C for 5 hours. After the bacteria are fixed, the bacterial cells are washed twice by centrifugation,
The suspension was resuspended in PBS containing an appropriate amount of 50% v / v ethanol to obtain a bacterial solution to be subjected to FISH. One drop of each of these bacterial liquids was placed on a slide glass coated with gelatin, air-dried, and then allowed to stand in a 70% ethanol liquid and then a 99% ethanol liquid for 2 minutes each, dehydrated, and used as a test specimen to be subjected to FISH. . The probe used for FISH has a 5 '
An ITC (fluorescein isothiocyanate) -labeled fluorescent probe synthesized by a conventional method was used. FISH is 50
20 μl of a hybridization solution (0.9 M NaCl, 0.05% SDS, 20 mM Tris, 5 mM EDTA, 10% formamide, pH 7.6) containing pmol of the fluorescent probe was brought into contact with the above-mentioned test sample, and the solution was placed in a chamber maintained at a high humidity. C. for 2 hours. Washing was performed as follows. The test specimen was allowed to stand in the above hybridization solution at 42 ° C. for 20 minutes, and then rinsed with sterile distilled water at room temperature to remove excess fluorescent probe. After the hybridization and washing, the test specimen was air-dried, and all cells were stained with an anti-fading agent (SlowFade ^™ ; Molecular Probe) containing 10 μg / ml of DAPI, and observed with a fluorescence microscope.

(2) Collection by Membrane Filter 150 ml of the test sample was suction-filtered through a polycarbonate membrane filter (47 mm in diameter, 0.4 μm in pore diameter), and washed twice with 50 ml of PBS. Subsequently, 10 ml of hybridization buffer (0.9 MN
The cells were washed with aCl, 0.05% SDS, 20 mM Tris, 5 mM EDTA, 10% formamide, pH 7.6) and subjected to FISH. FISH is 5
200 μl of a hybridization solution (0.9 M NaCl, 0.05% SDS, 20 mM Tris, 5 mM EDTA, 10% formamide, pH 7.6) containing 0 pmol of the fluorescent probe was dropped on a plastic petri dish, and the surface on which the bacterial cells were captured was dropped. The filter was brought into contact with the filter so that it was on top, and the temperature was kept at 40 ° C. for 2 hours in a chamber maintained at high humidity. Washing was performed as follows. The filter after the hybridization was set in a suction filtration apparatus, and the filter was suction-washed with 100 ml of a hybridization buffer at 42 ° C., and subsequently with 50 ml of sterilized water filtered at room temperature. After the hybridization and washing, the membrane filter is aseptically air-dried, and placed on a non-fluorescent slide glass with the bacteria-capturing surface facing up. Further, 10 μg / ml DAPI is directly applied to the surface.
Anti-fading agent (SlowFade®; Molecular)
Probe), all cells were stained, the filter was covered with a cover glass, and observed with a fluorescence microscope. Fig. 1 Observed image
Shown in

FISH using FITC-labeled specific probe
When analysis is performed and observed under a fluorescence microscope, it can be seen that the entire target bacterium emits fluorescence unique to FITC. For example, when in situ hybridization is performed using a FITC-labeled complementary-strand probe of SEQ ID NO: 1, as shown in the figure, Pectinatus furysingensis is detected by fluorescent labeling, but Pectinatus cerevisophilus is not detected. . The two methods described above completely corresponded, and as a result, the specificity of the probe in the FISH method was as shown in Table 1. In the table, ○ indicates reactivity, and × indicates no reactivity.

[0028]

[Table 1]

There is also a method of using a plurality of probes at the same time. For example, probe 1 and probe 2
When used for analysis, probe 1 and probe 2
Since the target region in the 16S rRNA was different, the signal could be enhanced additively. As described above, it was useful to use the probes obtained according to the present invention in combination according to the target target to be detected.

Example 2 Quantification of Pectinatus sp. By FISH method The quantification of Pectinatus sp. In a sample was performed as follows. Immediately before collecting bacteria in the sample as in Example 1,
Escherichia coli, which was directly counted and controlled using a bactometer, was added to the sample, and FISH analysis results were analyzed.
For example, beer contaminated with Pectinatus furysingensis is mixed with E. coli (C) controlled by a direct counting method, and the beer contaminated with Pectinatus furysingensis has SEQ ID NO: 1 in the sequence list specific to 16S rRNA of Pectinatus furysingensis. FISH analysis was carried out as in Example 1 using the fluorescently labeled probe shown in FIG. From the obtained fluorescence images, the number of bacteria stained with DAPI (A) and the number of FITC-labeled bacteria (B) were determined as shown in FIG.
One can estimate the number of Pectinatus flisingensis present in the beer.

By observation with a fluorescence microscope, Pectinatus furysingensis can be quantified from the ratio of cells that do not emit fluorescence (Escherichia coli) to cells that emit (Pectinus furysingensis). For example, Pectinatus-flip Shin Gen to cis alone contaminated samples 10 ⁷ E. coli specimens recovered by adding, SEQ ID NO: 1 fluorescently labeled
When FISH analysis was performed using the complementary strand probe of the above, if 10 fluorescently labeled cells (Pectinus furysingensis) were observed in 1000 cells of Escherichia coli not fluorescently labeled, This means that 10 ⁵ were present in the original sample. That is, the number of pectinus flisingensis (N) present in the beer subjected to the test can be represented by the following relational expression.

N = B × C / AB Further, for a sample contaminated with bacteria of a genus Pectinatus and a plurality of bacteria, a bacterium not present in the sample is used as a counter strain instead of the above-mentioned E. coli. This makes it possible to estimate the number of bacteria in a sample of a specific pectinus genus. Whether or not the counter strain to be used was present in the sample could be easily known from the determination by the PCR method. For example, if you want to know the number of Pectinatus furysingensis in beer that is contaminated by Pectinatus furysingensis and other microorganisms, but is not contaminated by Pectinatus cerevisiaus, This will be described below. A plurality of microbes contaminated with microbes including Pectinatus furysingensis are mixed with C. pectinatus cerevisifiras controlled by a direct counting method to obtain 16S rRNA specific to 16S rRNA of Pectinatus furysingensis. Example 1 in the presence of the fluorescently labeled probe shown in Example 1 and the probe labeled with rhodamine shown in SEQ ID NO: 4 specific to 16S rRNA of Pectinus cerevisiaus.
FISH analysis was performed as described above. The number of rhodamine-labeled bacteria (A) and the number of FITC-labeled bacteria (B) were determined from the obtained fluorescent images, and it was possible to estimate the number of Pectinatus furysingensis present in the contaminated beer. . That is, the number of pectinus flisingensis (N) present in the beer subjected to the test can be represented by the following relational expression.

N = B × C / A Here, the labeling substance of the probe that specifically hybridizes with the 16S rRNA of the counter strain may be any substance that can be distinguished from the target substance of the probe specific to the target bacterium.

Example 3 Identification of Pectinatus by Dot Blot Nitrocellulose, nylon,
The nucleic acid extracted from the bacterial sample by a known method was immobilized on a filter such as PVDF. Depending on the purpose, the probe was labeled using a DIG oligonucleotide tailing kit manufactured by Boehringer Mannheim and subjected to hybridization. Detection was performed by Boehringer Mannheim.
Using a DIG luminescence detection kit, follow the procedure manual,
Finally, the filter was exposed to an X-ray film for an appropriate time, and the species of the genus Pectinatus was identified based on the presence or absence of a hybridization signal. Table 2 shows the results of the dot blot. In the table, ●: reactive. ：: No reactivity.

[0035]

[Table 2]

[0036]

[Sequence List] <110> Asahi Breweries Ltd. <120> A nucleic acid probe for detecting Pectinatus bacteria and amethod for detecting beer turbidity bacteria <130> 99T3-33 <160> 8 <210> 1 <211> 20 < 212> DNA <213> Pectinatus frisingensis <400> 1 cggaacatta agcggatctt <210> 2 <211> 20 <212> DNA <213> Pectinatus frisingensis <400> 2 gggtccgaac tgaggttctt <210> 3 <211> 20 <212> DNA < 213> Pectinatus frisingensis <400> 3 gagaccgcga ggtggagcga <BR> <210> 4 <211> 20 <212> DNA <213> Pectinatus cerevisiiphilus <400> 4 cggatgacta agcggatctt <210> 5 <211> 20 <212> DNA <213 > Pectinatus cerevisiiphilus <400> 5 ggattcgaac tggtcatctt <210> 6 <211> 20 <212> DNA <213> Pectinatus frisingensis <400> 6 cggctaaggg ccgcaaggca <210> 7 <211> 20 <212> DNA <213> Pectinatus frisingensis < 400> 7 ggcttttagc tatcgcttgg <210> 8 <211> 20 212> DNA <213> Pectinatus frisingensis <400> 8 cgaaagtcat ccacacccga

[Brief description of the drawings]

FIG. 1 is a fluorescence microscopic image of a result of detection of a genus Pectinatus by the FISH method.

FIG. 2 is a diagram showing a method for quantifying Pectinatus spp.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) (C12Q 1/68 C12N 15/00 ZNAA C12R 1:01) C12R 1:01)

Claims (17)

[Claims] The present invention relates to a bacterium of the genus Pectinatus belonging to the genus Pectinatus.
is) In order to selectively detect bacteria,
A sequence targeting 16S rDNA and 16S rRNA,
A single-stranded oligonucleotide, wherein the oligonucleotide has at least one of SEQ ID NOs: 1 to 3 in the sequence listing or has a corresponding complementary strand. 2. A bacterium belonging to the genus Pectinatus, wherein the bacterium is P. cerevisiiphi.
lus) A sequence targeting 16S rDNA and 16S rRNA of a bacterium belonging to the genus Pectinatus for selective detection of the bacterium, and the oligonucleotides thereof are represented by SEQ ID NOs: 4 and 5 in the sequence listing.
A single-stranded oligonucleotide having at least one of the following, or having a corresponding complementary strand. 3. A sequence targeting the 16S rDNA and 16S rRNA of the genus Pectinatus for selective detection of the genus Pectinatus, wherein the oligonucleotide is represented by SEQ ID NO: 6 or 7 in the sequence listing. A single-stranded oligonucleotide having at least one or a corresponding complementary strand. 4. A method for selectively detecting a genus Pectinatus and its evolutionary relatives such as Megasphaera, Zymophilus and Selenomonas.
A single-stranded oligonucleotide which targets 16S rDNA and 16S rRNA of a bacterium belonging to the genus Pectinatus, wherein the oligonucleotide has SEQ ID NO: 8 in the sequence listing or has a corresponding complementary strand. 5. An oligonucleotide comprising at least 10 contiguous nucleotide units in a sequence selected from the sequence of oligonucleotides according to claims 1 to 4, or being at least 90% homologous. A single-stranded oligonucleotide, characterized in that: 6. The oligonucleotide according to claim 1, which is labeled with a tracer. 7. The oligonucleotide according to claim 1, wherein the oligonucleotide is immobilized on a solid support. 8. A method for collecting bacteria from a sample, the method comprising:
One or more nucleic acid probes that are oligonucleotides selected from those described in any one of claims 7 to 7,
A step of contacting under appropriate hybridization conditions, and a step of measuring the presence or absence of the formation of a hybridization complex between the probe and the nucleic acid of the sample, the genus Pectinatus, or an evolutionary relative thereof. How to detect bacteria. 9. A method for collecting bacteria from a sample, the method comprising:
One or more nucleic acid probes that are oligonucleotides selected from those described in any one of claims 7 to 7,
A step of contacting under appropriate hybridization conditions, and a step of measuring the presence or absence of the formation of a hybridization complex between the probe and the nucleic acid of the sample. Identification method. 10. The method for detecting a bacterium belonging to the genus Pectinatus or an evolutionarily related bacterium according to claim 8, wherein the method for collecting bacteria is either a centrifugation method or a membrane filter collection method. 11. The method for identifying a bacterium belonging to the genus Pectinatus or an evolutionarily related bacterium according to claim 9, wherein the method for collecting the bacteria is one of a centrifugation method and a membrane filter collection method. 12. A method for measuring the presence or absence of the formation of a hybridization complex is a FISH method (fluorescence in
The method for detecting a bacterium belonging to the genus Pectinatus or an evolutionarily related bacterium according to claim 8, which is any one of a situ hybridization, a dot-blot method, a colony hybridization method, a southern blot method, and a northern blot method. 13. A method for measuring the presence or absence of the formation of a hybridization complex is a FISH method (fluorescence in
The method for identifying a bacterium belonging to the genus Pectinatus or an evolutionarily related bacterium according to claim 9, which is any one of a situ hybridization, a dot-blot method, a colony hybridization method, a southern blot method, and a northern blot method. 14. A step of intentionally mixing a control bacterium numerically controlled into a sample, and an oligonucleotide 1 selected from those described in any one of claims 1 to 7.
Contacting one or more nucleic acid probes under suitable hybridization conditions; measuring the presence or absence of hybridization complexes between the probes and the bacterial nucleic acid of the sample; and A method for quantifying a pectinus or a closely related evolutionary bacterium, comprising estimating the number of target bacteria from the numbers of existing bacteria and control bacteria. 15. A method for measuring the presence or absence of the formation of a hybridization complex is a FISH method (fluorescence in si
15. The method for quantifying a pectinus genus or an evolutionarily related bacterium according to claim 14, which is a tu hybridization. 16. A method for using the oligonucleotide according to any one of claims 1 to 5 as a nucleotide primer for amplifying a nucleic acid in the presence of a polymerase. 17. A method for detecting a nucleic acid amplified by the method according to claim 10 by an electrophoresis method and a nucleic acid staining method.