JP2002034578A - Base sequence for detecting bacteria belonging to lactobacillus and pediococcus, and method for detecting the same bacteria - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an oligonucleotide capable of preferentially hybridizing to bacteria belonging to Lactobacillus and Pediococcus. SOLUTION: An oligonucleotide complementary to 23S rRNA or rDNA region of bacteria causing turbidity of beer or hybridizing to the region is disclosed. More particularly, an oligonucleotide containing either oligonucleotide having a continuous eight bases in sequence groups represented by the sequence number 1-82 (refer to the specification) and at least 90% homology to the sequence is disclosed. A method for detecting containing a process making a sample suspected of containing a target bacterium contact to at least one kind of a nucleotide fragment is disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a bacterium belonging to the genus Lactobacillus and to Pediococcus.
ccus) The gene sequence of 23S rRNA of the genus Bacillus and detection of the bacterium using the sequence, identification of the bacterium, or quantification of the bacterium.

[0002]

2. Description of the Related Art In recent years, the trend toward beer drafting has been
It brought a new value of beer freshness. Against this background, there is an increasing need for beer manufacturers to dramatically reduce the time from product manufacture to shipping and to quickly and accurately determine contamination of beer turbid bacteria.
One of the representative bacteria causing beer turbidity is lactobacilli, which are facultative anaerobic bacteria.

[0003] 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 lactic acid bacteria. The identification of classic beer turbid bacteria is based on morphological observation, Gram stainability, catalase test and many other properties as well as biochemical tests, and finally re-ingests the isolated bacteria in fresh beer and its growth Noh was determined by observation. Such a series of operations requires a lot of time and effort, and it is often difficult to make an accurate determination. Recently, a nucleic acid has been extracted from a lactic acid bacterium and a specific oligonucleotide has been used as a primer or a probe in a PCR (Polymerase Chain Reaction) (Japanese Patent Laid-Open No.
-210980, JP-A-6-41899, JP-A-6-113888) or hybridization (Japanese Patent Application Laid-Open No. 8-503620). However, these methods require a step of extracting nucleic acids from the cells, and cannot detect individual cells.

[0004]

One object of the present invention is to provide ribosomal RNA (rRN) of an organism that causes beer turbidity.
A) and unique nucleic acid sequences in ribosomal DNA (rDNA),
And an oligonucleotide complementary thereto. It is another object of the present invention to provide oligonucleotides capable of hybridizing to target regions of rRNA and rDNA that can be made accessible under nucleic acid hybridization with the combined specificity, sensitivity and speed. It is another object of the present invention to provide a method for performing hybridization by using these oligonucleotides labeled with an appropriate tracer as a probe to selectively detect, identify and quantify the bacterial individual. .

[0005]

SUMMARY OF THE INVENTION In order to achieve these objects, the present invention provides a nucleic acid sequence of a gene encoding 23S rRNA of Lactobacillus and Pediococcus. The present invention also provides a method for selectively detecting Lactobacillus and Pediococcus spp.
Oligonucleotides or corresponding complementary strand oligonucleotides that target rRNA and DNA, wherein the oligonucleotides have at least 8 consecutive bases or are at least 90% homologous in any of the SEQ ID NOs in the Sequence Listing It is. The present invention also relates to the identification of Lactobacillus or Pediococcus sp.
A method for determining in a sample containing or capable of containing a nucleic acid of said species of bacteria, characterized in that any of said oligonucleotides is used as a probe.

Further, the present invention is a method for using the nucleotide as a primer for amplifying a nucleic acid in the presence of a polymerase. Bacterial ribosomes are 5S, 16S and 2
It contains at least three types of RNA molecules called 3S rRNA. Historically, these names have been related to the size of the RNA molecule as determined by sedimentation velocity. In the present invention, bacterial ribosomal rRNA can be used as a target. One advantage of using this is that rRNA is abundant in all living cells.

Before disclosing the present invention in more detail, the 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 that is a constituent unit of an oligonucleotide is not a phosphodiester bond found in a naturally occurring nucleic acid, but may be another ester bond or an amide bond (generally a PNA).
), As long as it can hybridize to the target region. Oligonucleotides (or nucleotide fragments) can contain, for example, up to 100 nucleotide units. Generally, at least
It contains eight nucleotide units and, from natural nucleic acid molecules,
Or it can be obtained by genetic recombination or by chemical synthesis.

[0008] "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 are determined from the base composition of the probe / target double-stranded nucleic acid, and the degree of mismatch between the nucleic acid sequences, and the concentration and type of ionic species present in the hybridization solution, denaturation. Expressed as a function of hybridization reaction parameters such as the nature and concentration of the agent, or 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.

[0009] "Homologous" means to indicate the degree of similarity between two or more nucleic acid sequences, but does not mean the taxonomic degree of similarity between organisms. The degree of similarity is expressed as a percentage, for example, 2
90% homologous between two sequences means that the base sequence of the first sequence
90% means match identically to a base in the second sequence. A “probe” has hybridization specificity under defined conditions, and in the case of the present invention, rRNA,
Or a nucleotide fragment that forms a hybridization complex with a target nucleic acid having a nucleotide sequence contained in rDNA, for example, a nucleotide fragment containing 5 to 100 nucleotide units, particularly 8 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, chromogenic, fluorescent or luminescent compounds, It can be labeled with a marker selected from nucleotide base analogs and ligands such as biotin.

A "primer" comprises, for example, 8 to 100 nucleotide units and comprises a defined condition for the initiation of enzymatic polymerization, for example in an amplification method such as PCR (polymerase chain reaction), a sequencing process, a reverse transcription method, etc. Oligonucleotides with hybridization specificity below. Oligonucleotides according to the present invention can be used to test all known hybridization techniques, especially "dot blot"
Technology for spot attachment on filters called MANIATIS
Et al., Molecular Cloning, Cold Spring Harbor, 1982),
It can be used according to a technique of DNA separation and transcription called "Southern blot" (SOUTHERN EM, Mol. Biol., 98, 503 (1975)), and a technique of RNA separation and transcription called "Northern blot".

[0011]

According to the present invention, there is provided an oligonucleotide that hybridizes preferentially with Lactobacillus and Pediococcus. The oligonucleotides of the present invention are useful for detecting the presence of organisms that cause beer turbidity. One aspect of the invention is for oligonucleotides that are complementary to or hybridize to a region of 23S rRNA or rDNA of Beer turbidity. Specifically, the oligonucleotide is homologous to at least 90% of the sequence containing any eight consecutive oligonucleotides in the sequence group shown in SEQ ID NOs: 1 to 81. Another aspect of the present invention is the gene sequence of 23S rRNA of Lactobacillus genus and Pediococcus genus, specifically, the DNA sequence of SEQ ID NOS: 82 to 86.

[0012] Yet another aspect of the present invention is a method for detecting the presence of beer turbid bacteria. The method comprises at least one sample suspected of containing the target bacteria.
Contacting with a type of nucleotide fragment. This nucleotide fragment contains lactic acid bacteria that cause beer turbidity.
About 8 to 10 that hybridizes preferentially with rRNA or rDNA
Contains 0 bases. This method involves applying hybridization conditions to the sample such that the nucleotide probe preferentially binds to the target rRNA or rDNA to form a complex, and as an indicator of the presence of the target organism (s). Detecting the complex. Preferably, the nucleotide probe is homologous to at least 90% of the sequence comprising any eight consecutive oligonucleotides in a sequence selected from the group of sequences set forth in SEQ ID NOs: 1-81. The nucleotide fragments of the present invention provide the basis for the development of hybridization assays for the specific detection of beer turbid organisms in beer and semi-finished products during beer production, or in samples taken from environments such as breweries and sewage. provide. The nucleotide fragments of the present invention also provide a basis for confirming the presence of beer turbidity.

The first step taken in the development of the probes of the present invention was to identify a unique region within the 23S rRNA of the target beer turbidity bacterium for a specific nucleic acid probe. This means that any target region in the 23S rRNA
It was to find out whether it is specific for Lactobacillus and Pediococcus, which cause beer turbidity. Therefore, to achieve these, Lactobacillus
Lactobacillusbrevis, Lactobacillus
Species ABBC no.74 (Lactobacillus speciesABBC n
o.74), Lactobacillus lndneri
indneri), Lactobacillus plantarum (Lactobaci)
llus plantarum), Pediococcus damnosus (Ped
sequence of 23S rRNA of iococcus damnosus (SEQ ID NOS: 82 to 8)
6) was determined by a known experimental protocol and compared with the sequence of 23S rRNA of other bacteria. Based on the results, probes specific to the Lactobacillus and Pediococcus were designed as shown in SEQ ID NOs: 1 to 81.

Specifically, (1) a probe specific to Lactobacillus brevis is SEQ ID NO: 1 to 14, and (2)
A probe specific for Lactobacillus brevis and Lactobacillus species ABBC no.74 is SEQ ID NO: 15.
(3) Lactobacillus species ABBC
No. 74-specific probes are SEQ ID NOS: 18-31, (4) Lactobacillus lindneri-specific probes are SEQ ID NOs: 32-48, and (5) Lactobacillus plantarum-specific probes. Numbers 49-64,
(6) A probe specific to Lactobacillus plantarum and Pediococcus damnosus is SEQ ID NO: 65, and (7) a probe specific to Pediococcus damnosus is SEQ ID NO: 66 to 81.

[0015] The probe of the present invention is fluorescence in situ.
It can be used for hybridization (hereinafter abbreviated as FISH). That is, beer and a semi-finished product during beer production or a sample collected from an environment such as a brewery or sewage may be centrifuged or treated with a membrane filter capable of capturing beer turbid bacteria, and may be present in the sample. The potential target beer turbid bacteria and the detection probe are contacted under appropriate hybridization conditions. The detection probe penetrates into the cytoplasm of the target bacterium, where the 23S rR
The target site in the NA 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, the assay is performed by a method such as autoradiography, and when the detection probe is labeled with a fluorescent substance, the assay is performed using a fluorescence microscope or laser scanning technology. However, if the target organism is 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 the target organism.

The oligonucleotide defined above is used as a specific primer for amplifying and synthesizing 23S rDNA or 23S rRNA of Lactobacillus and Pediococcus in the presence of a thermostable polymerase by a known method. (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.

[0017]

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 turbid 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 50 ml of the test sample is placed in a centrifuge tube, and centrifuged (1
The supernatant was discarded and the bacterial pellet was collected. The bacterial pellet was further suspended in 30 ml of a PBS solution, and centrifuged under the same conditions for washing. After washing, remove 1 bacterial pellet
Resuspended in ml of fixative (PBS containing 50% ethanol). Place a drop of these bacterial solutions on a slide glass coated with gelatin, air-dry, then add 50% ethanol solution, 80%
Ethanol solution followed by 2% each in 99% ethanol solution
The sample was left to stand for 5 minutes to perform a dehydration treatment, and used as a test specimen to be subjected to FISH. As a probe used for FISH, a fluorescent probe labeled with FITC at the 5 ′ end was used. FISH is performed using a hybridization solution containing 50 pmol fluorescent probe (0.9 M NaCl, 0.0
5% SDS, 20mM Tris, 5mM EDTA, 10% formamide, pH
7.6) 20 μl was brought into contact with the test specimen described above, and the test was performed at 44 ° C. for 1 hour in a chamber maintained at high humidity. Washing was performed as follows. The test specimen was allowed to stand in the above-mentioned hybridization solution at 46 ° 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 an anti-fading agent (ProLong (registered trademark); Molecular Probe) was added dropwise and observed with a fluorescence microscope.

(2) Collection by Membrane Filter 50 ml of the test sample was suction-filtered through a polycarbonate membrane filter (diameter: 47 mm, pore size: 0.4 μm), and then washed twice with 50 ml of PBS. Subsequently, 10 ml of hybridization buffer (0.9 M NaCl, 0.05% SD
S, 20mM Tris, 5mM EDTA, 10% formamide, pH 7.6)
And subjected to FISH. FISH is performed using a hybridization solution containing 50 pmol fluorescent probe (0.9 M NaCl, 0.0
5% SDS, 20mM Tris, 5mM EDTA, 10% formamide, pH
7.6) 200 μl was dropped on a plastic petri dish, and a filter was brought into contact with the plastic petri dish so that the surface on which the cells were captured faced up. The operation was performed at 44 ° C. for 1 hour in a chamber maintained at high humidity. Washing was performed as follows. Set the filter on which the hybridization has been completed in the suction filtration device, and set the hybridization buffer 100m at 46 ° C.
l, followed by suction washing with 50 ml of sterile water filtered at room temperature. After the hybridization and washing, the membrane filter is aseptically air-dried, placed on a non-fluorescent slide glass with the bacteria capturing surface facing up, and an anti-fading agent (ProLong (registered trademark); Molecular Probe) is added dropwise. The filter was covered with a glass and observed with a fluorescence microscope. The observed image is shown in FIG.

FISH using FITC-labeled specific probe
When the analysis is performed and observed under a fluorescence microscope, it can be seen that the entire target bacterium emits fluorescence. For example, FITC
In situ using labeled complementary probe of SEQ ID NO: 1
When hybridization is carried out, as shown in FIG. 1, Lactobacillus brevis is detected by fluorescent labeling, but Escherichia coli and the like are not detected. The results of the two methods described above are completely consistent, and the specificity of the probe in the FISH method is shown in Table 1.
And as shown in Table 2.

[0021]

[Table 1]

[0022]

[Table 2]

There is also a technique for simultaneously using a plurality of probes. For example, probe 1 and probe 2
When used for analysis, probe 1 and probe 2
Since the target region in the 23S 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 Determination of Lactobacillus and Pediococcus by FISH The determination of Lactobacillus and Pediococcus in a sample was performed as follows. Immediately before collecting the bacteria in the sample as in Example 1, control bacteria directly counted and controlled using a bactometer were added to the sample, and the results of FISH analysis were analyzed. For example, beer contaminated with Lactobacillus brevis is mixed with E. coli (C) controlled by a direct counting method and performed using a fluorescently labeled probe shown in SEQ ID NO: 3 specific to 23S rRNA of Lactobacillus brevis. After FISH analysis was performed as in Example 1, all bacteria were stained with an anti-fading agent (ProLong (registered trademark); Molecular Probe) containing 10 μg / ml of DAPI. From the obtained fluorescent images, the number of bacteria (A) stained with DAPI and the number of bacteria (B) labeled with FITC can be determined as shown in FIG. 2, and the number of Lactobacillus brevis present in the beer can be estimated. That is, the number (N) of Lactobacillus brevis present in the beer subjected to the test can be represented by the following relational expression.

N = B × C / (AB) The genus Lactobacillus and Pediococcus can be determined from the ratio of cells that do not emit fluorescence to cells that emit fluorescence by fluorescence microscopy. For example, when a sample contaminated with Lactobacillus brevis alone and 10 ⁷ E. coli cells were added thereto and collected and subjected to FISH analysis using a fluorescent-labeled complementary sequence probe of SEQ ID NO: 3, the sample was not fluorescently labeled. Fluorescently labeled cells (Lactobacillus
When brevis) is observed 10, Lactobacillus brevis will be the presence of 10 ⁵ cells in the original sample.

Example 3 Identification of Lactobacillus and Pediococcus spp. 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 Roche Diagnostics and used for hybridization. Detection is carried out using the DIG luminescence detection kit of Roche Diagnostics Inc. according to the procedure manual.The filter is finally exposed to an X-ray film for an appropriate time, and based on the presence or absence of a hybridization signal, Lactobacillus sp. And Pediococcus spp. Were identified. Tables 3 and 4 show the results of the dot blot.
Shown in

[0027]

[Table 3]

[0028]

[Table 4]

[0029]

[Sequence List] <110> Asahi Breweries Ltd. <120> Nucleotide sequences for detecting Lactobacillus and Pediococcus bacteria and the method for detecting these bacteria. <130> 2000-25P <160> 82 <210> 1 <211> 29 <212 > DNA <213> Lactobacillus brevis <400> 1 tcaagttgta atcatcggta aagatgatt 29 <210> 2 <211> 20 <212> DNA <213> Lactobacillus brevis <400> 2 tctgcctcaa cttgactatg 20 <210> 3 <211> 35 <212> DNA <213> Lactobacillus brevis <400> 3 gggggtatca ccctctatgc cgagccttcc cagac 35 <210> 4 <211> 20 <212> DNA <213> Lactobacillus brevis <400> 4 ctccggtctt tccaccttaa 20 <210> 5 <211> 20 <212> DNA <213> Lactobacillus brevis <400> 5 tctacatcaa tttactgaaa 20 <210> 6 <211> 36 <212> DNA <213> Lactobacillus brevis <400> 6 aacacacatt tccaatcgtg tgcacaccat agcctc 36 <210> 7 <211> 33 <212> DNA <213> Lactobacillus brevis <400> 7 tgtccttagc gataagcatt tgactcatca cca 33 <210> 8 <211> 20 <212> DNA <213> Lactobacillus brevis <400> 8 attcggtcct cgtcacgcct 20 <210> 9 <211> 21 <212> DNA <213> Lactobacillus brevi s <400> 9 cttcggttat catcaacttt g 21 <210> 10 <211> 22 <212> DNA <213> Lactobacillus brevis <400> 10 ctgagggttg acgatttcac c 21 <210> 11 <211> 20 <212> DNA <213> Lactobacillus brevis <400> 11 ccttgcaaat taacgtaact 20 <210> 12 <211> 23 <212> DNA <213> Lactobacillus brevis <400> 12 atcactcgac cttacggtcg aat 23 <210> 13 <211> 24 <212> DNA <213> Lactobacillus brevis <400> 13 aagtcctttgc gatcttgctt cgtt 24 <210> 14 <211> 23 <212> DNA <213> Lactobacillus brevis <400> 14 cgttcaaaca tgattaacta gta 23 <210> 15 <211> 20 <212> DNA <213> Lactobacillus brevis , Lactobacillus species ABBC no.74 <400> 15 tgcggctgca cttgcgtgca 20 <210> 16 <211> 20 <212> DNA <213> Lactobacillus brevis, Lactobacillus species ABBC no.74 <400> 16 cggatataaa tctgtggcat 20 <210> 17 < 211> 20 <212> DNA <213> Lactobacillus brevis, Lactobacillus species ABBC no.74 <400> 17 ggcttcattt ctgggcttcg 20 <210> 18 <211> 29 <212> DNA <213> Lactobacillus species ABBC no.74 <400> 18 ctaagcaata accatcggtt aagatggct 29 <210> 19 <211> 20 <212 > DNA <213> Lactobacillus species ABBC no.74 <400> 19 tctgccttta cttagctatg 20 <210> 20 <211> 35 <212> DNA <213> Lactobacillus species ABBC no.74 <400> 20 ggggctatca ccctctttgg ccaggcttcc caacc 35 <210 > 21 <211> 20 <212> DNA <213> Lactobacillus species ABBC no.74 <400> 21 ctccgtcttt tcaacttaac 20 <210> 22 <211> 20 <212> DNA <213> Lactobacillus species ABBC no.74 <400> 22 tctacaccaa catactgaaa 20 <210> 23 <211> 36 <212> DNA <213> Lactobacillus species ABBC no.74 <400> 23 aacgcacatt tccaatcgtg cgcacggctt agcctc 36 <210> 24 <211> 31 <212> DNA <213> Lactobacillus species ABBC no.74 <400> 24 aacccggctg ccagcattta actggtaacc t 31 <210> 25 <211> 20 <212> DNA <213> Lactobacillus species ABBC no.74 <400> 25 tgcggctgca cttgcgtgca 20 <210> 26 <211> 21 <212> DNA <213> Lactobacillus species ABBC no.74 <400> 26 cttcggttac caacaacttt g 21 <210> 27 <211> 21 <212> DNA <213> Lactobacillus species ABBC no.74 <400> 27 ctgagggtta ttggtttcgt t 21 <210> 28 <211> 20 <212> DNA <213> Lactobacillus species ABBC no.74 <400> 28 ccttgcatgt tagcgtaact 20 <210> 29 <211> 23 <212> DNA <213> Lactobacillus species ABBC no.74 <400> 29 atcacgtgac ctaatggtca cat 23 <210> 30 <211> 24 <212> DNA <213> Lactobacillus species ABBC no.74 <400> 30 tagtccttac gatctagctt cacc 24 <210> 31 <211> 23 <212> DNA <213> Lactobacillus species ABBC no.74 <400> 31 cgttcaaaca cactttacta gta 23 <210> 32 <211> 29 <212> DNA <213> Lactobacillus lindneri <400> 32 ctacgcagtg accagtcatg acactggtc 29 <210> 33 <211> 20 <212> DNA <213> Lactobacillus lindneri <400> 33 tctgcctctg cgcagctag 20 <210> 34 <211> 32 <212> DNA <213> Lactobacillus lindneri <400> 34 gagactatca ctctttggtg cagcttccca gc 32 <210> 35 <211> 20 <212> DNA <213> Lactobacillus lindneri <400> 35 ctccgctttt tcggcttaac 20 <210> 36 <211> 20 <212> DNA <213> Lactobacillus lindneri <400> 36 tctacggcaa catactaaaa 20 <210> 37 <211> 36 <212> DNA <213> Lactobacillus lindneri <400> 37 aacatacatt tccatcagta tgcataactt agcctt 36 <210> 38 <211> 20 <212> DNA <213> Lactobacillus lindneri <400> 38 aaccaggtta caagcattta 20 <210> 39 <211> 20 <212> DNA <213> Lactobacillus lindneri <400> 39 tgcggctggc cgtgaggcca 20 <210> 40 <211> 22 <212> DNA <213> Lactobacillus lindneri <400 > 40 gcttcaatta tcaaatactt tt 22 <210> 41 <211> 21 <212> DNA <213> Lactobacillus lindneri <400> 41 ctgagggtat tcgatttcat t 21 <210> 42 <211> 20 <212> DNA <213> Lactobacillus lindneri <400 > 42 ccttgcatgc aactgtaact 20 <210> 43 <211> 20 <212> DNA <213> Lactobacillus lindneri <400> 43 cggacataaa tcagtggtat 20 <210> 44 <211> 20 <212> DNA <213> Lactobacillus lindneri <400> 44 atcacgcaac gtagttgcgt 20 <210> 45 <211> 23 <212> DNA <213> Lactobacillus lindneri <400> 45 agtccttgcg gtctgccttc att 23 <210> 46 <211> 23 <212> DNA <213> Lactobacillus lindneri <400> 46 tgttcaaaca taattaacta gta 23 <210> 47 <211> 25 <212> DNA <213> Lactobacillus lindneri <400> 47 ttttcgatcc tcatcatcgc ttgcc 25 <210> 48 <211> 20 <212> DNA <213> Lactobacillus lindneri <400> 48 ggcttcattt cgaggctttg 20 <210> 49 <211> 29 <212> DNA <213> Lac tobacillus plantarum <400> 49 tctagtggta acagttgatt aaaactgct 29 <210> 50 <211> 21 <212> DNA <213> Lactobacillus plantarum <400> 50 tttacctcca actagactat g 21 <210> 51 <211> 30 <212> DNA <213> Lactobacillus plantarum <400> 51 ctatcaccat ctatggcgga ttttcccaaa 30 <210> 52 <211> 20 <212> DNA <213> Lactobacillus plantarum <400> 52 ctccgacttt ttagtcttaa 20 <210> 53 <211> 21 <212> DNA <213> Lactobacillus plantarum <400> 53 tctataacct cgtactcaaa a 21 <210> 54 <211> 36 <212> DNA <213> Lactobacillus plantarum <400> 54 gacattctaa tccaacagaa tgcacatctt agcctc 36 <210> 55 <211> 20 <212> DNA <213> Lactobacillus plantarum <400> 55 taaagtgaaa agcatttgac 20 <210> 56 <211> 22 <212> DNA <213> Lactobacillus plantarum <400> 56 ccttcgacta tcaagttctt tg 22 <210> 57 <211> 21 <212> DNA <213> Lactobacillus plantarum <400> 57 ctgagagaat ttgatttaat c 21 <210> 58 <211> 20 <212> DNA <213> Lactobacillus plantarum <400> 58 ccttgcacgg gatcataact 20 <210> 59 <211> 20 <212> DNA <213> Lactobacillus plantarum <400> 59 cg gatatgaa tcaatggcat 20 <210> 60 <211> 23 <212> DNA <213> Lactobacillus plantarum <400> 60 atcacgcgac ctaatggtcg cat 23 <210> 61 <211> 24 <212> DNA <213> Lactobacillus plantarum <400> 61 aagtcctcac gatcttgctt cacc 24 <210> 62 <211> 22 <212> DNA <213> Lactobacillus plantarum <400> 62 tgctcaaacg cacttactag ta 22 <210> 63 <211> 26 <212> DNA <213> Lactobacillus plantarum <400> 63 tttcgctccc cgtcacagct tgtcct 26 <210> 64 <211> 20 <212> DNA <213> Lactobacillus plantarum <400> 64 ggcttcaatt ctgagcttcg 20 <210> 65 <211> 20 <212> DNA <213> Lactobacillus plantarum, Pediococcus damnosus <400> 65 tgcggctgat cttgcgatca 20 <210> 66 <211> 29 <212> DNA <213> Pediococcus damnosus <400> 66 cgtggcggta acaacggatt aatgttgtt 29 <210> 67 <211> 21 <212> DNA <213> Pediococcus damnosus <400> 67 tctacctcca accacgttat g 21 <210> 68 <211> 28 <212> DNA <213> Pediococcus damnosus <400> 68 ggggctgtcc ccgctctggc cagtcttc 28 <210> 69 <211> 20 <212> DNA <213> Pediococcus damnosus <400> 69 gctccgtttc ttcaacttaa 20 <210> 70 <211> 20 <212> DNA <213> Pediococcus damnosus <400> 70 tctacatctg catactcagt 20 <210> 71 <211> 35 <212> DNA <213> Pediococcus damnosus <400> 71 ggcacattat ccaactgtgt gcttttctta gcctc 35 <210> 72 <211> 20 <212> DNA <213> Pediococcus damnosus <400> 72 gaaaagataa agcatttac 20 <210> 73 <211> 21 <212> DNA <213> Pediococcus damnosus <400> 73 gttcggctat gaaaatcttt g 21 <210> 74 <211> 21 <212> DNA <213> Pediococcus damnosus <400> 74 ctgagggatt ttcattttgc c 21 <210> 75 <211> 20 <212> DNA <213> Pediococcus damnosus <400> 75 cctggcatgc aaacgtaact 20 <210> 76 < 211> 20 <212> DNA <213> Pediococcus damnosus <400> 76 cggacataaa tcaatggcat 20 <210> 77 <211> 23 <212> DNA <213> Pediococcus damnosus <400> 77 atcacgccac cttacggtgg cat 23 <210> 78 <211 > 23 <212> DNA <213> Pediococcus damnosus <400> 78 agtcctcacg gtctgccttc atc 23 <210> 79 <211> 23 <212> DNA <213> Pediococcus damnosus <400> 79 tgttcaaaca agtttaacta gta 23 <210> 80 <211 > 26 <212> DNA <213> Pediococcus damnosus <400> 80 tttcgctccc catcacaac t tgtcct 26 <210> 81 <211> 20 <212> DNA <213> Pediococcus damnosus <400> 81 ggcttcattc caaagcttcg 20 <210> 82 <211> 2741 <212> DNA <213> Lactobacillus brevis <400> 82 ggcttctagg agccgatgaa ggacgggact aacaccgata tgcttcgggg agctgtacgt 60 aagctttgat ccggagattt ccaaatgggg aaacccaatc atctttaccg atgattacaa 120 cttgatgaat acatagtcaa gttgaggcag acgtggggaa ctgaaacatc taagtaccca 180 caggaagaga aagaaaaatc gattccctaa gtagcggcga gcgaacgggg aacagcccaa 240 accaggaagc ttgctttctg gggttgtagg actgaacatt tgagttacca aagttgatga 300 taaccgaaga gtctgggaag gctcggcata gagggtgata cccccgtagg tgaaatcgtc 360 aaccctcagt tcaggatcct gagtacggcc acacacgtga aacgtggtcg gaatccggga 420 ggaccatctc ccaaggctaa atactcccta gtgaccgata gtgaaccagt accgtgaggg 480 aaaggtgaaa agcaccgcgg aagcggagtg aaatagttcc tgaaaccatg tgcctacaat 540 tagttagagc ccgttaatgg gtgatagcgt gccttttgta gaatgaaccg gcgagttacg 600 ttaatttgca aggttaaggt ggaaagaccg gagccgtagc gaaagcgagt ctgaaacggg 660 cgtttcagta aattgatgta gacccgaaac caggtgacct acccatgtcc aggttgaag g 720 tgcggtaaaa cgcactggag gaccgaaccc gtgtatgttg aaaaatgctg ggatgaggtg 780 tgggtagcgg tgaaattcca aacgaacttg gagatagctg gttctctccg aaatagcttt 840 agggttagcc tcggagttaa gaatcgtgga ggtagagcca ctgtttggac taggggcccg 900 tcatgggtta ctgaattcag ataaactccg aatgccacag atttatatcc gggagtcaga 960 cgatgagtga taagatccac cgtcgaaagg ggaacagccc agaccaccaa ttaaggtccc 1020 taaatacatg ctgagtggaa aaggatgtgg agttgcatag acagctagga tgttggctca 1080 gaagcagcca ccatttaaag agtgcgtaat agctcactag ccgagtgatt ctgcgccgaa 1140 aatttaccgg ggctaagcat gttaccgaaa ttgtggattc gaccgtaagg tcgagtgata 1200 ggagagcgtt ctaagggcaa cgaagcaaga tcgcaaggac ttgtggagcg cttagaagtg 1260 agaatgccgg tatgagtagc gaaagatcag tgagaatctg atccaccgaa tgactaaggt 1320 ttcctgggga aggctcgtcc tcccagggtt agtcgggacc taagccgagg ccgcaaggcg 1380 taggcgatgg ataacaggtt gatattcctg tactagttaa tcatgtttga acgatggagg 1440 gacgcaggag gctatggtgt gcacacgatt ggaaatgtgt gttcaagcgt caagtctggt 1500 gatgagtcaa atgcttatcg ctaaggacaa ggcgtgacga ggaccgaatt ttagtaggga 1560 agcg ccagat gtcacactgc cgagaaaagc ttctagttag tgattaatta cccgtaccgc 1620 aaaccgacac aggtagtcga ggagagtatc ctaaggtgag cgagtgaact ctcgttaagg 1680 aactcggcaa aatgaccccg taacttcggg agaaggggtg ctgcacgcaa gtgcagccgc 1740 agtgaaaagg cccaggcgac tgtttatcaa aaacacaggt ttctgcaaaa tcgaaagatg 1800 acgtataggg gctgacgcct gcccggtgct ggaaggttaa gtggatgagt tagcttcggc 1860 gaagcccaga aatgaagccc cagtaaacgg cggccgtaac tataacggtc ctaaggtagc 1920 gaaattcctt gtcgggtaag ttccgacccg cacgaaaggc gtaacgatct gggcactgtc 1980 tcaacgagag actcggtgaa attatattac ctgtgaagat gcaggttacc cgcgacagga 2040 cggaaagacc ccatggagct ttactgtagc ttgatattgg gtgttgacac agcttgtaca 2100 ggataggtcg gagccgtaga actcggaacg ctagtttcga gtgaggcgct ggtgggatac 2160 gaccctcgct gtgtgaacac tctaacccgc accacttatc gtggtgggag acagtgtcag 2220 gtaggcagtt tgactggggc ggtcgcctcc taaaaagtaa cggaggcgcc caaaggttct 2280 ctcagaatgg ttggaaatca ttcgtcgagt gtaaaggcag aagagagctt gactgcgaga 2340 caggtcgagc agggacgaaa gtcgggctta gtgatccggt ggtaccgtat ggaagggcca 2400 tcgctcaac g gataaaagct accctgggga taacaggctt atctccccca agagtccaca 2460 tcgacgggga ggtttggcac ctcgatgtcg gctcatcgca tcctggggct gtagtcggtc 2520 ccaagggttg ggctgttcgc ccattaaagc ggtacgcgag ctgggttcag aacgtcgtga 2580 gacagttcgg tccctatccg tcgcgggcgt aggaaatttg agaggagctg tccttagtac 2640 gagaggaccg ggatggacat accgctggtg taccagttgt gccgccaggc gcatcgctgg 2700 gtagctatgt atggatgaga taaacgctga aagcatctaa g 2741 <210> 83 <211> 2821 < 212> DNA <213> Lactobacillus spp. ABBC74 <400> 83 aacggccgcc agtgtgctgg aattcggctt ctaggagccg atgaaggacg ggactaacac 60 cgatatgctt cggggagctg tacgtaagct ttgatccgga gatttccgaa tggggaaacc 120 cagccatctt aaccgatggt tattgcttag tgaatacata gctaagtaaa ggcagacgtg 180 gggaactgaa acatctaagt acccacagga agagaaagca attgcgattc ccatagtagc 240 ggcgagcgaa gtgggagcag cccaaaccag gaagcttgct tcctggggtt gtaggactga 300 acatttgagt taccaaagtt gttggtagtt gaacaggttg ggaagcctgg ccaaagaggg 360 tgatagcccc gtaaacgaaa ccaataaccc tcagttcagg atcctgatgagta cggccagaca 420 cgtgaaacct ggtcggaatc cgggaggacc atctcccaag gctaaatact ccctagtgac 480 cgatagtgaa ccagtaccgn gagggaaagg tgaaaagaac cccggaaggg gagtgaaata 540 gttcctgaaa ccatgtgcct acaattaggt tggagcccgt taatgggtga cagcgtgcct 600 tttgtagaat gaaccggcga gttacgctaa catgcaaggt taagttgaaa agacggagcc 660 gtagcgaaag cgagtctgaa acgggcgttt cagtatgttg gtgtagaccc gaaaccaggt 720 gacctaccca tgtccagggt gaaggtgcgg taaaacgcac tggaggcccg aacccgtgta 780 cgttgaaaag tgctgggatg aggtgtgggt agcggtgaaa ttccaaacga acttggagat 840 agctggttct ctccgaaata gctttagggc tagcctcgga attgagaatc gtggaggtag 900 agccactgtt tggactaggg gcccgtcatg ggttactgaa ttcagataaa ctccgaatgc 960 cacagattta tatccgggag tcagacggtg agtgataaga tccatcgtcg aaaggggaac 1020 agcccagacc accaattaag gtccctaagt gtatcctaag tggaaaaggc ggtgaagttg 1080 catagacagc taggatgttg gctcagaagc agccaccatt taaagagtgc gtaatagctc 1140 actagccgag tgattttgcg ccgaaaatgt accggggcta aggataccac cgaaattgtg 1200 gatgtgacca ttaggtcacg tgataggaga gcgttctaag ggcggtgaag ctagatcgta 1260 aggactagtg gagcgcttag aagtgagaat gccggtatga gtagcgaaag atc agtgaga 1320 atctgatcca ccgaatgact aaggtttcct ggggaaggct cgtcctccca gggttagtcg 1380 ggacctaagc tgaggccgag aggcgtaagc gatggctaac aggttgagat tcctgtacta 1440 gtaaagtgtg tttgaacgat ggagggacgc aggaggctaa gccgtgcgca cgattggaaa 1500 tgtgcgttca agcagtaagt caggttacca gttaaatgct ggcagccggg ttgacaagct 1560 gtgatgagga ccgaatttaa gtagggaagt ggccgacgtc acactgccga gaaaagcttc 1620 tagtgaatac tttattaccc gtaccgcaaa ccgacacagg tagtcgagga gagtatccta 1680 aggtgtgcga gtgaactctt gttaaggaac tcggcaaaat gaccccgtaa cttcgggaga 1740 aggggtgctg cacgcaagtg cagccgcagt gaaaaggctc aggcgacctg tttatcaaaa 1800 acacaggttt ctgcaaaatc gtaagatgac gtataggggc tgacgcctgc ccggtgctgg 1860 aaggttaagt ggatgagtta gcttcggcga agcccagaaa tgaagcccca gtaaacggcg 1920 gccgtaacta taacggtcct aaggtagcga aattccttgt cgggtaagtt ccgacccgca 1980 cgaaaggcgt aacgatctga gcactgtctc aacaagagac tcggtgaaat tatagtacct 2040 gtgaagatgc aggttacccg cgacaggacg gaaagacccc atggagcttt actgtagctt 2100 gatattgggt gttgacacaa cttgtacagg ataggtcgga gccgtagagg ccggaacg ct 2160 agtttcggca gaggcgctgg tgggatacga cccttgttgt gtgaacactc taacccgcac 2220 cacttagcgt ggtgggagac agtgtcaggt gggcagtttg actggggcgg tcgcctccta 2280 aaaagtaacg gaggcgccca aaggttccct cagaatggtt ggaaatcatt cgtagagtgt 2340 aaaggcagaa gggaacttga ctgcgagaca gacaggtcga gcagggacga aagtcgggct 2400 tagtgatccg gtggtaccgt atgcaagggc catcgctcaa cggataaaag ctaccctggg 2460 gataacaggc ttatctcccc caagagtcca catcgacggg gaggtttggc acctcgatgt 2520 cggctcatcg catcctgggg ctgtagtcgg tcccaagggt tgggctgttc gcccattaaa 2580 gcggtacgcg agctgggttc agaacgtcgt gagacagttc ggtccctatc cgtcgcgggc 2640 gtaggaaatt tgagaggagc tgttcctagt acgagaggac cggaatggac acaccgctgg 2700 tgtatcagtt gtgccgccag gcgcattgct gagtagctat gtgtggatga gataaacgct 2760 gaaagcatct aagtgtgaaa gccgaattct gcagatatcc atcacactgg cggccgctcg 2820 a 2821 <210> 84 <211> 2937 <212> DNA <213> Lactobacillus lindneri <400 > 84 gaattcggct taagttaata aagggcgcac ggtgaatgcc ttggtactag gagccgatga 60 aggacggaac taacaccgat atgcttcggg gagctgtacg taagctttga tccggagatt 1 20 tccgaatgag gaaactcgac cagtgtcatg actggtcact gcgtagtgaa tacatagctg 180 cgcagaggca gacgtgggga actgaaacat ctaagtaccc acaggaatat aaagaaattt 240 cgattcccaa agtagcggcg agcgaactgg gaatagccca aaccggacct ttatggtccg 300 gggttgtagg actgaacatt tgagttacaa aagtatttga taattgaagc agctgggaag 360 ctgcaccaaa gagagtgata gtctcgtaaa tgaaatcgaa taccctcagt tcaggatcct 420 gagtacggcg ccacacgtgt aacggcgtcg gaatccggga ggaccatctc ccaaggctaa 480 atactcccta gtgaccgata gtgaaccagt accgtggggg aaaggtgaaa agcaccccgg 540 aaggggagtg aaatagttcc tgcaaccatg tgcctacaag cagttagagc ccgttaacgg 600 gtgatagcgt gccttttgta gaatgaaccg gcgagttaca gttgcatgca aggttaagcc 660 gaaaaagcgg agccgtagcg aaagcaagtc ttaagagggc gttttagtat gttgccgtag 720 acccgaaacc aggtgatcta tccatgtcca ggatgaaagt gcggtaatac gcaccggagg 780 tccgaacccg tgtacgttga aaagtgctgg gatgaggtgt ggatagcggt gaaattccaa 840 acgaacttgg agatagctgg ttctctccga aatagcttta gggctagcct cggaattaga 900 atcgtggagg tagagccact gtttgagcaa ggggtccatc taggattact gagttcagat 960 aaactccgaa taccac tgat ttatgtccgg gagtcagacg atgagtgata agatccatcg 1020 tcgaaagggg aacagcccag accgccagtt aaggtcccta aatatatgct aagtggaaaa 1080 ggtagtgaag ttgcatagac aactaggatg ttggctcaga agcagccatc atttaaagag 1140 tgcgtaatag ctcactagtc gagtgattct gcgcccgaaa atttaccggg gctaagcata 1200 ttaccgagac tgcagacgca actacgttgc gtgataggag agcgttctaa gggcaatgaa 1260 ggcagaccgc aaggactgct ggagcgctta gaagtgagaa tgccggtatg agtagcgaaa 1320 gatcagtgag aatctggtcc accgaatgac taaggtttcc tggggaaggc tcgtcctccc 1380 agggttagtc gggacctaag ccgaggctga gaagcgtagg cgatggataa caggttgaga 1440 ttcctgtact agttaattat gtttgaacaa tggagggacg cagaaggcta agttatgcat 1500 actgatggaa atgtatgttc aaatcgtaag tcaggttatg agttaaatgc ttgtaacctg 1560 gttggcaagc gatgatgagg atcgaaattt aagtagagaa gtaacccatg tcacgctgcc 1620 gagaaaagct tctagttagt aattaattac ccgtaccgca aaccgacaca ggtagtcgag 1680 gagagtatcc tcaggtgagc gagagaactc tcgttaagga actcggcaaa atgaccccgt 1740 aacttcggaa gaaggggtgc tggcctcacg gccagccgca gtgaaaagac tcaaacgact 1800 gtttatcaaa aacacaggtt tatgcaaaat cgtaagatga agtatatggg ctgacgcctg 1860 cccggtgctg gaaggttaag tggaaaggtt agcttcggca aagcctcgaa atgaagcccc 1920 agtaaacggc ggccgtaact ataacggtcc taaggtagcg aaattccttg tcgggtaagt 1980 tccgacccgc acgaaaggcg taacgatttg agtactgtct caacgagaga ctcggtgaaa 2040 ttaagatacc tgtgaagaag caggttaccc gcgacaggac ggaaagaccc catggagctt 2100 tactgtagct tgatattggg tgtttacata gcttgtacag gataggtagg agccatagaa 2160 gccggaacgc tagtttcggt ggaggcgtca gtgggatact acccttgcta tgtgaacact 2220 ctaaccctga gcacttaacg tgctcggaga cagtgtctgg ttggcagttt gactggggcg 2280 gtcgcctcct aaatagtaac ggaggcgctc aaaggtttgc ttagaatggt tggaaatcat 2340 ttgtaaagtg taaaggcaaa agcaagcttg acttgcgagc caaacaggtc gagcagggac 2400 gaaagtcgga cttagtgatc cggtggtacc atatggaagg gccatcgctc aacggataaa 2460 agctaccctg gggataacag gcttatctcc cccaagagtt cacatcgacg gggaggtttg 2520 gcacctcgat gtcggctcat cgcatcctgg ggctgtagtt ggtcccaagg gttgggctgt 2580 tcgcccatta aagcggtacg cgagctgggt tcagaacgtc gtgagacagt tcggtcccta 2640 tccgtcgcgg gcgcaggaaa tttgag agga gctgtcccta gtacgagagg accgggatgg 2700 acataccgct ggtgtatcag ttgcgccgcc aggcgcattg ctgagtagct atgtatggat 2760 gagataaacg ctgaaagcat ctaagtgtga aactcgcctc aagatgagat ttcccattcc 2820 tatatggaag taagactcct gaaagatgat caggtcgata ggttagaagt ggaagcatag 2880 tgatatgtga agcggactaa tactaatagg tcgaggactt gaccaaaagc cgaattc 2937 <210> 85 <211> 2940 <212> DNA <213> Lactobacillus plantarum <400> 85 gaattcggct taagttaata aagggcgcac ggtgaatgcc ttggcactag gagccgatga 60 aggacgggac taacaccgat atgcttcggg gagctgtacg taagctatga tccggagatt 120 tccgaatggg gcaacccagc agttttaatc aactgttacc actagatgaa ttcatagtct 180 agttggaggt aaacgctgtg aactgaaaca tctcattagc agcaggaata taaagaaatt 240 tcgattccct aagtagcggc gagcgaacgg ggaacagccc aaaccaaagt gcttgcactt 300 tggggttgta ggactgaaca tttgagttac caaagaactt gatagtcgaa ggatttggga 360 aaatccgcca tagatggtga tagcccagta gattaaatca aattctctca gttcaggatc 420 ctgagtacgg cggaacacgt gaaattccgt cggaatccgg gaggaccatc tcccaaggct 480 aaatactacc tagtgaccga tagtgaacca gtaccgtgag ggaa aggtga aaagcacccc 540 gggaggggag tgaaatagtt cctgaaacca tgtgcctaca ataagtcaga gcgcgttaat 600 gcgtgatggc gtgccttttg tagaatgaac cggcgagtta tgatcccgtg caaggttaag 660 actaaaaagt cggagccgta gcgaaagcga gtctgaaatg ggcgttttga gtacgaggtt 720 atagacccga aaccaggtga cctatccatg tccaggttga aggtgcggta aaacgcactg 780 gaggaccgaa cccgtgtaag ttgaaaattg ctgggatgag gtgtggatag cggtgaaatt 840 ccaaacgaac ttggagatag ctggttctct ccgaaatagc tttagggcta gcctcggaat 900 taggatcatg gaggtagagc actatttgga ctaggggccc gtcttgggtt actgaattca 960 gataaactcc gaatgccatt gattcatatc cgggagtcag acgatgagtg ataagatcca 1020 ccgtcgaaag gggaacagcc cagaccatca gttaaggtcc ctaaatgtat gctaagtgga 1080 aaaggatgtg gagttgcata gacaactagg atgttggctc agaagcagcc accatttaaa 1140 gagtgcgtaa tagctcacta gtcgagtgat cctgcgccga aaatgtaccg gggctaagca 1200 tactaccgaa accatggatg cgaccattag gtcgcgtgat aggagagcgt tctaaggccg 1260 gtgaagcaag atcgtgagga cttgtgaagc gcttggaagt gagaatgccg gtatgagtag 1320 cgaaagatag gtgagaatcc tatccaccga atgactaagg tttcctgggg aaggctc gtc 1380 ctcccagggt tagtcgggac ctaagtcgag gccgagaggc gtagacgatg gataacaggt 1440 tgagattcct gtactagtta agtgcgtttg agcaatggag ggacgcagga ggctaagatg 1500 tgcattctgt tggattagaa tgtccaagca gtaagtcttg tgaagagtca aatgcttttc 1560 actttaagga caagctgtga cggggagcga aatttagtag cgaagcgtct gatgtcacac 1620 tgccgagaaa agcttctagt gagtacttaa ctacccgtac cgcaaaccga cacaggtagt 1680 cgaggagaga atcctaaggt gagcgagtga actctcgtta aggaactcgg caaaatgacc 1740 ccgtaacttc gggagaaggg gtgctgatcg caagatcagc cgcagtgaat aggcccaggc 1800 gactgtttat caaaaacaca ggtctctgca aaatcgcaag atgacgtata ggggctgacg 1860 cctgcccggt gctggaaggt taaaaggatg ggttagcttc ggcgaagctc agaattgaag 1920 ccccagtaaa cggcggccgt aactataacg gtcctaaggt agcgaaattc cttgtcgggt 1980 aagttccgac ccgcacgaaa ggcgtaacga tctgggcact gtctcaacga gagactcggt 2040 gaaattatat tgtccgtgaa gatgcggact acccgcgaca ggacggaaag accccatgga 2100 gctttactgt agcttgatat tgagtgtttg tacagcttgt acaggatagg taggagccat 2160 agaaaccgga acgctagttt cggtggaggc gttggtggga tactaccctc gctgtatgac 2 220 cactctaacc cgcaccacta atcgtggtgg gagacagtgt caggtgggca gtttgactgg 2280 ggcggtcgcc tcctaaaaag taacggaggc gcccaaaggt tccctcagaa tggttggaaa 2340 tcattcgcag agtgtaaagg cacaagggag cttgactgcg agacagacag gtcgagcagg 2400 gacgaaagtc gggcttagtg atccggtggt accgtatgga agggccatcg ctcaacggat 2460 aaaagctacc ctggggataa caggcttatc tcccccaaga gtccacatcg acgggaaggt 2520 ttggcacctc gatgtcggct catcgcatcc tggggctgta gtcggtccca agggttgggc 2580 tgttcgccca ttaaagcggt acgcgagctg ggttcagaac gtcgtgagac agttcggtcc 2640 ctatccgtcg cgggcgtagg aaatttgaga ggacctgtcc ttagtacgag aggaccggga 2700 tggacatacc tctggtgtac cagttgtgcc gccaggcgca tcgctgggta gctacgtatg 2760 gatgtgataa acgctgaaag catctaagtg tgaaacacac ctcgagatgg gatttcccat 2820 tcctttatgg aagtaagacc cctgaaagat gatcaggtag ataggttaga agtggcagtg 2880 cggtgacgca tgaagcggac taatactaat aggtcgagga cttgaccaaa agccgaattc 2940 <210> 86 <211> 2942 <212> DNA <213> Pediococcus damnosus <400> 86 gaattcggct taagttaata aagggcgcnc ggtgaatgcc ttggtactag gagccgatga 60 aggacggga c taacaccgat atgcttcggg gagctgtaag taagctttga tccggagatt 120 tccgaatggg gaaacccaac aacattaatc cgttgttacc gccacgtgaa tacataacgt 180 ggttggaggt agacgtgggg aactgaaaca tctcagtacc cacaggaata gaaagaaaaa 240 tcgattcccc gagtagcggc gagcgaaagg ggaacagccc aaaccaggaa gcttgcttcc 300 tggggttgta ggaccgaaca tttgagttac caaagatttt catagccgaa cagtctggga 360 agactggcca gagcgggtga cagccccgta ggcaaaatga aaatccctca gttcaggatc 420 ctgagtacgg cggaacacgt gtaactccgt cggaatccgg gaggaccatc tcccaaggct 480 aaatactccc tagtgaccga tagtgaacca gtaccgtgag ggaaaggtga aaagcacccc 540 gaaaggggag tgaaacagtt cttgaaacca tgtgcctaca agaagtcaga gcccgttaat 600 gggtgatggc gtgccttttg tagaatgaac cggcgagtta cgtttgcatg ccaggttaag 660 ttgaagaaac ggagccgtag cgaaagcgag tctgaatagg gcgactgagt atgcagatgt 720 agacccgaaa ccaagtgacc tacccatgtc caggttgaag gtgcggtaaa acgcactgga 780 ggaccgaact cgtgtacgtt gaaaagtgct gagatgaggt gtgggtagcg gtgaaattcc 840 aaacgaactt ggagatagct ggttctctcc gaaatagctt tagggctagc ctcggactta 900 ggatcatgga ggtagagcca ctgtttg gac taggggccca tctagggtta ctgaattcag 960 ataaactccg aatgccattg atttatgtcc gggagtcaga cggtgagtga taagatccat 1020 cgtcgaaagg ggaacagccc agaccaccag ttaaggtccc taaatatatg ttaagtggaa 1080 aaggatgtgg agttgcatag acaactagga tgttggctca gaagcagcca tcatttaaag 1140 agtgcgtaat agctcactag tcgagtgatt ctgcgccgaa aatgtaccgg ggcttaaaca 1200 tattaccgag actgtggatg ccaccgtaag gtggcgtgat aggagagcgt tctaagggcg 1260 atgaaggcag accgtgagga ctgttggagc gcttagaagt gagaatgccg gtatgagtag 1320 cgaaagacag gtgagaatcc tgtccaccga atgactaagg tttcctgggg aaggctcgtc 1380 cacccagggt tagtcgggac ctaagtcgag gccgagaggc gtagacgatg gataacaggt 1440 tgagattcct gtactagtta aacttgtttg aacaatggag ggacgcagga ggctaagaaa 1500 agcacacagt tggataagtg tgcccaagca acaagtctta gatagagtta aatgctttat 1560 cttttcagga caagttgtga tggggagcga aatttaagta gcgaagtttc tgatgtcaca 1620 ctgccgagaa aagcttctag ttagagttta actacccgta ccgcaaaccg acacaggtag 1680 tcgaggagag tatcctcagg tgagcgagag aactctcgtt aaggaattcg gcaaaatgac 1740 cccgtaactt cggaagaagg ggtgctgatc gc aagatcag ccgcagtgaa taggcccagg 1800 cgactgttta tcaaaaacac aggtttctgc aaaatcgtaa gatgacgtat aggggctgac 1860 acctgcccgg tgctggaagg ttaagtgaat gagttagctt cggcgaagct ttggaatgaa 1920 gccccagtaa acggcggccg taactataac ggtcctaagg tagcgaaatt ccttgtcggg 1980 taagttccga cccgcacgaa aggtgtaacg atctgggcac tgtctcaacg agagactcgg 2040 tgaaattata atacccgtga agatgcgggt tacccgcgac aggacggaaa gaccccatgg 2100 agctttactg tagcttgata ttgagtgttt gtacaacttg tacaggatag gtaggagccg 2160 tagacatcga aacgctagtt tcgatggagg cgttggtggg atactaccct cgttgtatga 2220 accctctaac ccgcgccact aagcgtggcg ggagacagtg tcaggtaggc agtttgactg 2280 gggcggtcgc ctcctaaaga gtaacggagg cgcccaaagg ttccctcaga atggttggag 2340 atcattcgca gagtgtaaag gcacaaggga gcttgactgc gagacagaca ggtcgagcag 2400 ggacgaaagt cgggcttagt gatccggtgg taccgtatgg aagggccatc gctcaacgga 2460 taaaagctac cctggggata acaggcttat ctcccccaag agttcacatc gacggggagg 2520 tttggcacct cgatgtcggc tcatcgcatc ctggggctgt agtcggtccc aagggttggg 2580 ctgctcgccc attaaagcgg tacgcgagct gggttcag aa cgtcgtgaga cagttcggtc 2640 cctatccgtc gcgggcgtag gaaatttgag aggatctgtc cttagtacga gaggaccggg 2700 atggacatac cgctggtgta ccagttgttc cgccaggagc atcgctgggt agctacgtat 2760 ggatgagata aacgctgaaa gcatctaagt gtgaaactca cctcgagatg agatttccca 2820 ttccatttat ggaagtaaga cccctgagag atgatcaggt agataggttg ggagtggaag 2880 tgtagtgata catggagcgg accaatacta ataggtcgag gacttgacca aaagccgaat 2940 tc 2942

[Brief description of the drawings]

Fig. 1 FISH (Fluorescence in situ hybridization)
The figure which shows the detection of Lactobacillus brevis by the method.

FIG. 2 is a diagram showing quantification of Lactobacillus and Pediococcus by fluorescence microscopy.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) C12R 1:25) (C12Q 1/68 A (C12Q 1/68 C12R 1: 225) C12R 1: 225) (C12Q 1 / 68 A (C12Q 1/68 C12R 1:01) C12R 1:01) C12N 15/00 ZNAA (72) Inventor Yasuro Motoyama 1-1-21 Midori, Moriyacho, Kitasoma-gun, Ibaraki Asahi Breweries Co., Ltd. F term (reference) 4B024 AA05 AA11 CA01 CA09 CA11 DA05 HA13 4B063 QA01 QQ06 QQ42 QR08 QR56 QR62 QS16 QS25 QS34 QS36 QX02

Claims (18)

[Claims] 1. A sequence targeting 23S rRNA and DNA of Lactobacillus brevis for selectively detecting Lactobacillus brevis, wherein the sequence is SEQ ID NO: 1 in the sequence listing.
Oligonucleotides having at least 8 contiguous bases or at least 90% homologous to any of -14 or corresponding complementary oligonucleotides. 2. A sequence targeting 23S rRNA and DNA for selectively detecting Lactobacillus brevis and Lactobacillus species ABBC No. 74, wherein the sequence is one of SEQ ID NOs: 15, 16, and 17 in the sequence listing. Or at least 90% homologous, or a corresponding complementary oligonucleotide. 3. Lactobacillus species for selectively detecting Lactobacillus species ABBC No. 74.
A sequence targeting 23S rRNA and DNA of ABBC No. 74, which has at least 8 consecutive bases or is at least 90% homologous to any of SEQ ID NOs: 18 to 31 in the sequence listing. Or a corresponding complementary oligonucleotide. 4. A 23S rRNA of Lactobacillus Lindneri for selectively detecting Lactobacillus Lindneri.
And oligonucleotides or corresponding complements characterized by having at least 8 contiguous bases or at least 90% homologous to any of SEQ ID NOs: 31-48 of the Sequence Listing Strand oligonucleotide. 5. Lactobacillus plantarum 23S r for selectively detecting Lactobacillus plantarum.
An oligonucleotide or corresponding sequence targeting RNA and DNA, wherein the oligonucleotide has at least 8 consecutive bases or is at least 90% homologous to any of SEQ ID NOs: 49-64 in the sequence listing Complementary oligonucleotide. 6. A method for selectively detecting Lactobacillus plantarum and Pediococcus damnosus, comprising the steps of:
An oligonucleotide targeting rRNA and DNA, wherein the oligonucleotide has at least 8 consecutive bases or is at least 90% homologous from SEQ ID NO: 65 in the sequence listing, or a corresponding complementary oligonucleotide. 7. Pediococcus damnosus 23S r for selectively detecting Pediococcus damnosus.
An oligonucleotide or a corresponding sequence targeting RNA and DNA, wherein the oligonucleotide has at least 8 consecutive bases or is at least 90% homologous to any of SEQ ID NOs: 67 to 81 in the sequence listing. Complementary oligonucleotide. 8. The oligonucleotide according to any one of claims 1 to 7, which is labeled with a tracer. 9. The oligonucleotide according to claim 1, wherein the oligonucleotide is immobilized on a solid support. 10. A method for determining the identity and presence or absence of a genus Lactobacillus or Pediococcus in a sample containing or capable of containing at least one nucleic acid of said bacterium, comprising: Contacting the oligonucleotide probe with the probe under suitable hybridization conditions, and then determining, in a manner known per se, the formation or absence of hybridization complex between the probe and the nucleic acid of the sample. Is an oligonucleotide selected from those according to any one of claims 1 to 9. 11. The method according to claim 10, wherein the quantification of the genus Lactobacillus or Pediococcus is determined in a sample containing or capable of containing at least one nucleic acid of the bacterium. The control bacterium is intentionally mixed into the sample, and no hybridization complex is formed between the probe and the nucleic acid of the control bacterium under the hybridization conditions. A method comprising the formation of a hybridization complex. 12. A method for using an oligonucleotide containing a part or all of the nucleotide sequence according to any one of claims 1 to 7 as a primer for amplifying a nucleic acid in the presence of a polymerase. 13. A method for detecting a nucleic acid amplified by the method according to claim 12 by electrophoresis and nucleic acid staining. 14. A gene sequence of a gene encoding 23S rRNA of Lactobacillus brevis comprising a part or all of the nucleotide sequence of SEQ ID NO: 82. 15. Lactobacillus species ABBC no. 74 containing a part or all of the nucleotide sequence shown in SEQ ID NO: 83.
Gene sequence of the gene encoding 23S rRNA. 16. A 23S rRNA of Lactobacillus Lindneri containing a part or the whole of the nucleotide sequence of SEQ ID NO: 84.
Gene sequence of the gene encoding 17. 23S rRN of Lactobacillus plantarum containing a part or the whole of the nucleotide sequence of SEQ ID NO: 85.
Gene sequence of the gene encoding A. 18. A Pediococcus damnosus ABBC no. Containing a part or all of the nucleotide sequence shown in SEQ ID NO: 86.
Gene sequence of the gene encoding 74 23S rRNA.