JPH10234377A - Oligonucleotide for detecting lactobacillus and detection of the bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject oligonucleotide containing a part of a nucleotide sequence encoding a specific 16S ribosome RNA gene and capable of selectively detecting and identifying Lactobacillus s.p. AB No. 434 which is a beer- contaminating bacterium. SOLUTION: This oligonucleotide used for detecting lactobacillus contains a part of a nucleotide sequence encoding the 16S ribosome RNA gene of Lactobacillus s.p. AB No. 434. The oligonucleotide contains a part or the whole part of the No. 50-300 sequence among the oligonucleotide sequence of the formula or the corresponding complementary sequence. For example, 5'- CACGGAGACCGCATGGTCTC-3'. The oligonucleotide may be a chemically synthesized oligonucleotide or a natural olignucleotide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a lactobacillus sp. Lactobacillus sp.
The present invention relates to an oligonucleotide for detecting BNo.434 and a method for detecting the same using the oligonucleotide.

[0002]

2. Description of the Related Art At present, in microbial testing in beer production, bacteria must be isolated through growth culture in order to identify bacterial species.
It takes at least seven days. Thereafter, the isolated bacteria are grown, and identification is performed by performing many property tests such as morphological observation, Gram staining, catalase test, and sugar utilization. In addition to these commonly used identification tests, DNA is extracted from the isolated bacteria, immobilized on a membrane or other support, and a hybridization test is performed using the DNA of the standard bacteria 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.

A more rapid detection method has recently been described in J.
Am. Soc. Brew. Chem .: 52 (1) 19-23, 1994. There is a method of detecting live bacteria by the ATP luminescence method using bioluminescence, but detection is possible regardless of the bacterial species. Therefore, it cannot be used for identification. Also, some lactic acid bacteria harmful to beer, such as Lactobacillus brevis, Lactobac
illus casei, Lactobacillus plantarum, Lactobacillu
As described in JP-A-6-46811 and JP-A-6-31894, s coryniformis and the like can be identified relatively early by using an antibody specific to lactic acid bacteria. However, in order to apply this method, it is necessary to perform an operation until the bacteria are isolated, so that it takes days, and it is difficult to obtain sufficient detection sensitivity and selectivity.

[0004] Therefore, a quicker detection method has been studied.
Recently, Japanese Patent Application Laid-Open Nos.
99, JP-A-7-289295, or J. Am. Soc. Brew.
Chem .: 52 (3) 95-99, 1994, the DNA of a lactic acid bacterium to be sampled was extracted, and a P was prepared by using an oligonucleotide complementary to this DNA as a primer.
There is a method for detecting lactic acid bacteria using the CR method.

Japanese Patent Application Laid-Open Nos. 5-15400 and 6-141899 disclose Lactobacillus brevis 5
This is a method for detecting lactic acid bacteria using an oligonucleotide which targets the S ribosomal RNA gene and is chemically synthesized so as to be complementary to the nucleotide sequence thereof. Japanese Patent Application Laid-Open No. 7-289295 discloses that Lactobacillus sp.D deposited at the National Institute of Advanced Industrial Science and Technology
The purpose of the present invention is to detect lactic acid bacteria that cannot be detected by known primers using the gene sequence of lactic acid bacteria A1. These are different from lactic acid bacteria whose specificity of detection is to be examined in the present invention.

[0006]

SUMMARY OF THE INVENTION The present invention provides a nucleotide sequence that selectively hybridizes with a 16S rRNA gene derived from a lactic acid bacterium, Lactobacillus sp., Or a nucleotide sequence having a sequence complementary to the nucleotide sequence. More specifically, Lactobacillus species belonging to Lactobacillus sp.
illus sp.) A method for selectively detecting and simultaneously identifying ABNo.434 bacteria is provided.

[0007] Lactobacillus species AB
No. 434 is a lactobacillus isolated from a brewery in Japan, and is the lactic acid bacterium described above or Lactobacillus sp. D. described in JP-A-7-289295.
A1) Lactobacillus species AB No. 7 entitled "Oligonucleotide for detecting lactic acid bacteria and method for detecting said bacteria" filed on Jan. 29, 1997 by the present applicant.
No. 4 (Lactobacillus sp. AB No. 74), and has the characteristic of being able to grow using beer as a medium.

[0008] Techniques for gene amplification are already known, and Polymerase Chain Reaction method (hereinafter abbreviated as PCR method) developed by Saiki et al .; Science 230, 13-50, 1985.
Can be performed based on This method is a reaction that amplifies a specific gene sequence and has rapid, high sensitivity, high specificity, and simplicity. Also, applications have been attempted for rapid detection of harmful bacteria in the food field. By performing the PCR method, even if only a small amount of the target sequence is present in the sample, the target nucleotide sequence sandwiched between the two primers is amplified by a million times, and the target nucleotide sequence is increased in a large amount before detection is possible. A copy is produced. In addition, in order to perform the PCR method, it is necessary to release nucleic acid components from bacteria present in the sample, but in the PCR method, the amplification reaction proceeds if the target sequence is present in several molecules or more. The test can be sufficiently performed only by carrying out a simple pretreatment using. Therefore, the utility value is extremely high as compared with the conventional bacteria detection method.

To take advantage of these facts, the present invention provides:
Lactobacillus sp. Belonging to the genus Lactobacillus sp.
An oligonucleotide prepared so as to specifically hybridize with the 6S ribosomal gene is used as a primer with P
By performing the CR method and detecting the sequence to be recognized, the presence or absence of the bacterium in the sample can be determined quickly, with high sensitivity,
A method to determine specifically was developed.

The sample may be beer or a semi-finished product during beer production, or a sample collected from an anaerobic environment such as sewage. Oligonucleotides used as primers can be either chemically synthesized or natural. It is sufficient that the DNA polymerase used in the PCR method has heat resistance at a temperature of 90 ° C. or higher. P
The temperature conditions in the CR method are 90-98 ° C. in a heat denaturation reaction for converting a double-stranded DNA into a single strand, and a primer is used as a template DNA.
37-65 by annealing reaction to hybridize to
C., 50-
The target sequence can be amplified by performing the reaction at 75 ° C., and performing this as one cycle for several tens of cycles.

After the PCR, the reaction product is separated by electrophoresis, stained with nucleic acid with ethidium bromide or the like. If the base length of the amplified nucleotide sequence is equal to the base length of the target sequence described above, the reaction product is ligated into the sample. Bacterial species (Lactobacillus sp.) ABNo. 434 can be determined. For detection of the amplified nucleotide sequence,
Chromatography is also effective.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION

1. Lactobacillus s
p.) Determination of base sequence of 16S rRNA gene of AB No. 434 bacteria Extraction of DNA from Lactobacillus sp. AB No. 434 bacteria First, DNA was extracted from Lactobacillus sp. AB No. 434 bacteria. DNA extraction was performed as follows. After 1 ml of the bacterial solution of the test bacterium was centrifuged at 4 ° C. for 5 minutes at 15 K rpm, the supernatant was discarded. Suspend by adding 1 ml of sterile water, again at 15 Krpm for 5 minutes,
After centrifugation at 4 ° C. and discarding the supernatant, 200 μl of cell wall lysate (10 mM Tris-HCl, 1 mM EDTA, 0.35 M Sucrose pH 8.0)
1 mg / ml Lysozyme and 50 μg / m N-Acetylmuramidase
l) and incubated at 37 ° C for 30 minutes. 400 μl of proteolysis solution (100 mM Tris-HCl, 1.5 MN
aCl, 20mM EDTA, 2% CTAB, 2% 2-Mercaptoethanol pH
8.0, and then add ProteinK to a concentration of 120 μg / ml), incubate at 50 ° C for 60 minutes, and then add 400 μl of phenol
Add chloroform, stir and then 15Krpm for 5 minutes at 25 ° C
And 400 μl of the supernatant was transferred to a new Eppendorf tube. After adding 300 μl of 2-propanol and stirring, the mixture was allowed to stand at −80 ° C. for 10 minutes, and centrifuged at 4 ° C. for 15 minutes at 15 K rpm to obtain a precipitate. 200 μl of cold 70% ethanol was added, centrifuged at 4 ° C. for 5 minutes at 15 K rpm, and the supernatant was discarded.
The residue was suspended in 50 μl of sterilized water to prepare a DNA solution.

Amplification of 16S rRNA of Lactobacillus sp. AB No. 434 Bacteria Next, 16S rR of Lactobacillus sp.
NA amplification was performed. DNA obtained from the above DNA solution
About 20 ng as a template, 16S rRNA shown in Table 1
Using gene sequencing primers 1 and 11, P
Performed CR. For PCR, 5 μl of the test DNA solution, 10 × PCR buffer (100 mM Tris-HCl (pH 9.0), 500 mM KCl, 1% Triton
X-100) 5 μl, 25 mM MgCl _{2 4} μl, 20 mM dNTP mixture
0.5 μl, primer 1 0.5 μl, primer 110.
5 μl, Taq DNA polymerase (Toyobo; TAP-101) 0.25 μ
and 34.25 μl of sterile distilled water.
90-98 ° C for 1 minute, annealing at 37-60 ° C for 2 minutes, elongation
30 cycles were performed at 70-75 ° C for 2 minutes. Add 5 μl of the reaction solution to 1 × T
The sample was subjected to 1.2% agarose gel electrophoresis using a BE buffer, stained with ethidium bromide for about 20 minutes, and then irradiated with ultraviolet rays to observe the PCR product.

After agarose electrophoresis, the resultant was stained with ethidium bromide, and an agarose gel containing a target DNA molecule of about 1.5 Kbp was cut out with a razor or the like. After transferring to Takara Shuzo's SUPRECTM-01 (a centrifuge tube with a filter for collecting DNA), the gel was finely crushed using a microspatula or the like, allowed to stand at -80 ° C for 10 minutes, and frozen. 300 μl of sterile water was added, and the mixture was centrifuged at 5 krpm for 10 minutes to collect the DNA solution collected at the bottom of the centrifuge tube. The resulting solution 3
An equal volume of phenol / chloroform was added to 00 μl, and the mixture was centrifuged at 15 Krpm for 5 minutes at 25 ° C., and 200 μl of the supernatant was transferred to a new Eppendorf tube. About 400 μl of cold ethanol was added, and the mixture was allowed to stand at −80 ° C. for 10 minutes, then centrifuged at 15 K rpm for 5 minutes at 4 ° C., and the supernatant was discarded. After adding about 150 μl of cold 70% ethanol, the mixture was centrifuged at 4K at 15K rpm for 5 minutes. After discarding the supernatant, it was dried under reduced pressure and suspended in 50 μl of sterile distilled water to obtain a 16S rDNA solution. Table 1 shows 16SrRN
2 shows the A gene sequencing primer.

[0015]

[Table 1] However, M in the table indicates the sequence of the forward (−29) primer of M13: (T) CACGACGTTGTAAAACG AC.

Preparation of Template for Sequence Reaction Using about 20 ng of the above 16S rDNA as a template, various 16S rRNA gene S sequencing primers shown in Table 1 were used for 2 and 3, 1 and 4, 6 and 7, 5 and 8, 10 and 11, 9 and 12, 2 and 7, 1 and
Used in a total of 10 combinations of 8, 6 and 13, and 5 and 14, PCR conditions were 90-98 ° C for 1 minute, 37-60 ° C for 2 minutes, and 70-75 ° C for 2 minutes.
Performed 0 cycles. The amplified DNA molecule of the desired length was purified in the same manner as described above and subjected to a sequencing reaction.

Sequencing Using the above DNA as a template, IRD41-labeled M13 Forward (-
29) A sequencing reaction was performed using primers according to LI-COR's DNA sequencer instruction manual 2). Electrophoresis and analysis were performed according to the same instructions. DNA
Sequence editing and data analysis were performed using DNAsis (Hitachi Software).

2. Determination of specific primers Lactobacillus sp.
ABNo. Based on the sequence obtained in the above, a base sequence of about 20 bp considered to be specific was selected and prepared as an upstream primer.

The downstream primer, which is common to all bacteria, is prepared by using a sequence near the 907th position of the 16S rRNA gene, and a universal primer (hereinafter referred to as UN
V1). The primers prepared above are shown in Table 2. Oligonucleotides as various primers were chemically synthesized by a known method. Primer 1 with sterile water
It was adjusted to 00 μM and used for PCR.

[0020]

[Table 2]

3. Confirmation test for detection specificity Next, the reaction of the selected primers against the corresponding various preserving bacteria was examined. The standard strain of each lactic acid bacterium stored, L.
brevis (JCM 1059), L.casei (ATCC334), L.plantar
um (JCM1149), L.coryniformis (JCM1164), L.lind
neri (DSM20690) and L.sp. ABNo.
Lactobacillus species AB No. 74) described in “Oligonucleotides for detecting lactic acid bacteria and method for detecting the bacteria” filed on Jan. 29, 2013, and L. sp. AB No. 43
DNA was extracted from four bacteria, and the reaction of the corresponding specific primer when each bacterial DNA was used as a template was investigated. The results are shown in Tables 3 and 4, where the primer L434P3 is L.sp.AB
It was found that it reacted specifically with No. 434 bacteria and did not react with bacteria of other genera.

[0022]

[Table 3]

[0023]

[Table 4]

[0024]

According to the conventional method, it took about 10 days to identify the strain of lactic acid bacterium. However, by using the present invention, it is possible to quickly and clearly identify Lactic acid in the specimen within 1 to 3 days.
ABNo. 74 bacteria can be specifically detected and simultaneously identified. Moreover, the PCR method is easy to operate, including the pretreatment of the sample, and uses the chemicals, instruments,
Since the apparatus is also inexpensive, low-cost and highly-reliable inspection can be performed without skill of an operator.

[0025]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1555 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Lactobacillus sp. Strain name: ABNo. 434 Sequence characteristics Features how to determine the: E sequence: AGAGTTTGAT CCTGGCTCAG GACGAACGCT GGCGGCGTGC CTAATACATG CAAGTCGAAC 60 GAGGTTTGGT CAGTTTGCGG TGGTGCTTGC ATCACCAATT ACCGATCAAA CCGAGTGGCG 120 GACGGGTGAG TAACACGTGG GTAACCTGCC CTTCAGCAGG GGATAACATT TGGAAACAGA 180 TGCTAATACC GTATAACCAC GGAGACCGCA TGGTCTCCGG GTAAAAGATG GCGCAAGTAT 240 CACTGAAGGA TGGACCCGCG GCGTATTAGC CAGTTGGTGG GGTAACGGCC TACCAAAGCG 300 ATGATACGTA GCCGACCTGA GAGGGTAATC GGCCACATTG GGACTGAGAC ACGGCCCAAA 360 CTCCTACGGG AGGCAGCAGT AGGGAATCTT CCACAATGGA CGCAAGTCTG ATGGAGCAAC 420 GCCGCGTGAG TGAAGAAGGC TTTCGGGTCG TAAAACTCTG TTATTGAAGA AGAACGTGTG 480 TGACAGTAAC TGGTCATGCA GTGACGTTAG GTAG GTTCTCAGTCGGTCAG GTTCTCAGTCGTGCAG GTTCTCAGTCGTGCAGGTTCTCAGGTGCAGGTTCTCAGGTGCAGGTTCTCAGGGTCAGCTAG CTGATGTG AAACTTCGGC TTAACCGAAG AAGGGCATCG 660 GAAACTGGGA GACTTGAGTG CAGAAGAGGA GAGTGGAACT CCATGTGTAG CGGTGAAATG 720 CGTAGATATA TGGAAGAACA CCAGTGGCGA AGGCGTGCTC TCTGGTCTGT AACTGACGCT 780 GAGGCTCGAA GCGTGGGTAG CAAACAGATT AGATACCCTG TAGTCCACGC GGTAACGATG 840 AATACTAAGT GTTGGAGGGT TCCGCCCTTC AGTGCTGCGC TAACGCATTA AGTATTCCGC 900 CTGGGAGTAC GACCGCAAGG TTGAAACTCA AAGGAATTGA CGGGGGCCCG CACAAGCGGT 960 GGAGCATGTG GTTTAATTCG AAGCAACGCG AAGAACCTTA CCAGGTCTTG ACATCTTCTG 1020 CCAGGCTGAG AGATCAGCTG TTCTTCGGGG ACAGAATGAC AGGTGGTGCA TGGTTGTCGT 1080 CAGCTCGTGT CGTGAGATGT TGGGTTAAGT CCCGCAACGA GCGCAACCCT TATGATCAGT 1140 TGCCAGCATT CAGTTGGGCA CTCTGGTCAG ACTGCCGGTG ACAAACCGGA GGAAGGCGGG 1200 GATGACGTCA AATCATCATG CCCCTTATGA CCTGGGCTAC ACACGTGCTA CAATGGGTGG 1260 TACAACGAGC AGCGAGACCG CGAGGTCAAG CGAATCTCTA AAAACCATCC TCAGTTCGGA 1320 TTGCAGGCTG CAACTCGCCT GCATGAAGCT GGAATCGCTA GTAATCGCGG ATCAGCACGC 1380 CGCGGTGAAT ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGA GAGTATGTAA 1440 CACCCAAAGC CGGTGAGACA ACCGCAAGGA GTC AGCCGTC TAAGGTGGGA CAAATGATTA 1550 GGGTGAAGTC GTAACAAGGT AGCCGTAGGA GAACCTGCGG CTGGATCACC TCCTT 1555

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:01)

Claims (10)

[Claims] 1. Lactobacillus sp. Seeds belonging to the genus Lactobacillus present in a sample.
s sp.) Lactobacillus sp. A (Lactobacillus sp.) A to selectively detect ABNo.
An oligonucleotide which targets a nucleotide sequence encoding the 16S ribosomal RNA gene of BNo. 434 and comprises a part of the nucleotide sequence, wherein the oligonucleotide is an oligonucleotide sequence described in SEQ ID NO: 1, An oligonucleotide for detecting a lactic acid bacterium, which has a part or all of the sequence described from the 50th to the 300th, or has a corresponding complementary sequence. 2. Lactobacillus sp. Seeds belonging to the genus Lactobacillus present in a sample.
s sp.) Lactobacillus sp. A (Lactobacillus sp.) A to selectively detect ABNo.
An oligonucleotide which targets a nucleotide sequence encoding the 16S ribosomal RNA gene of BNo. 434 and comprises a part of the nucleotide sequence, wherein the oligonucleotide is an oligonucleotide sequence described in SEQ ID NO: 1, An oligonucleotide for detecting a lactic acid bacterium, which has a partial or entire sequence of the first to 500th sequences or has a corresponding complementary sequence. 3. Lactobacillus sp. Seeds belonging to the genus Lactobacillus present in a sample.
s sp.) Lactobacillus sp. A (Lactobacillus sp.) A to selectively detect ABNo.
An oligonucleotide which targets a nucleotide sequence encoding the 16S ribosomal RNA gene of BNo. 434 and comprises a part of the nucleotide sequence, wherein the oligonucleotide is a part of the oligonucleotide sequence described in SEQ ID NO: 1. Or an oligonucleotide for detecting a lactic acid bacterium, having an entire sequence or a corresponding complementary sequence. 4. Lactobacillus sp. Seeds belonging to the genus Lactobacillus present in a sample.
s sp.) ABNo, 434 bacteria for selective detection, Lactobacillus sp.
An oligonucleotide targeting a nucleotide sequence encoding the 16S ribosomal RNA gene of BNo. 434 and comprising a portion of the nucleotide sequence, wherein said oligonucleotide has the following sequence 5'-CACGGAGACCGCATGGTCTC-3 ', Alternatively, an oligonucleotide for detecting a lactic acid bacterium having a corresponding complementary sequence. 5. An oligonucleotide comprising at least 10 consecutive nucleotides or more in the oligonucleotide sequence according to claim 1, 2, 3, or 4. 6. A method for amplifying a target nucleotide sequence by using the oligonucleotide sequence according to claim 1, 2, 3 or 4 as a primer for a chain length reaction. 7. The nucleotide sequence amplified by the method according to claim 6 is separated by electrophoresis or chromatography, and as a result, it is determined whether or not a sequence to be recognized is present. A method for detecting Lactobacillus sp. In a sample. 8. A method for detecting the nucleotide sequence amplified by the method according to claim 6 by gel electrophoresis and nucleic acid staining. 9. A method for causing the oligonucleotide according to claim 1, 2, 3, 4 or 5 to function as a DNA detection probe. 10. An oligonucleotide wherein the oligonucleotide sequence according to claim 1, 2, 3, 4 or 5 is modified with a label.