JPH0751100A - Method for detecting bacterium of genus lactobacillus - Google Patents

Abstract

PURPOSE:To rapidly carry out amplification and detection of nucleic acid of a bacterium which belongs to the genus Lactobacillus by catching bacteria of the genus Lactobacillus as pretreatment of PCR on a filler and cleaning the bacteria. CONSTITUTION:Bacteria of the genus Lactobacillus are caught on a filter as a pretreatment of PCR using DNA of bacterium of the genus Lactobacillus in a liquid sample containing bacteria (e.g. Lactobacillus hiochii bacterium) of the genus Lactobacillus as a template and the bacteria are cleaned. There, PCR inhibitor in the liquid sample is removed and amplification of the nucleic acid of the bacterium of the genus Lactobacillus is promoted.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to Lactobacillus (Lact).
For more details on the method of detecting bacteria of the genus obacillus, PC
The present invention relates to a rapid and highly sensitive method for detecting a bacterium belonging to the genus Lactobacillus such as a fire-fall bacterium using the R method.

[0002]

2. Description of the Related Art Microorganisms that cause the burning of sake (flame-dropping bacteria)
The research on the taxonomy has been conducted by Kitahara, Noshiro and Momose [The Journal of the Brewing Society of Japan, Vol. 65, No. 7].
15-803, (1970)]. Hinobacillus belongs to the genus Lactobacillus, and is classified into true fire-fall bacterium and fire-fall lactic acid bacterium according to the requirement for mevalonic acid. Lactobacillus heterohiochii can be used for eubacteria.
And Lactobacillus homohichio (Lactobacillushomoh
iochii), and lactobacillus
Examples thereof include Lactobacillus japonicus, Lactobacillus plantarum, and Lactobacillus casei. For detection of Hojira bacteria, a Hinouchi bacteria detection medium SI medium (Japan Brewery Association) is commercially available, and detection is carried out by a culture method using this medium. However, this detection method requires more than 7 days. Therefore, there is a demand for a rapid and highly sensitive detection method for fire bacterium. On the other hand, a PCR method [Methods in Enzymology, Vol. 155, Vol.
5 to 350 (1987)]. The PCR method is a method for exponentially amplifying the specific sequence of the gene of the organism to be detected, and the use of the PCR method requires genetic information of the organism to be detected.

Regarding the rRNA gene of Lactobacillus bacteria, Lactobacillus
brevis), Lactobacillus delbruecki (Lactobac
illus delbrueckii), Lactobacillus plantarum,
And Lactobacillus viridesense (Lactobacillus vi
The nucleotide sequence of the 5S rRNA gene of ridescens has been clarified [Journal of Molecular Evolution, No. 8].
Vol. 143-153 (1976), Nucleic
Nucleic Acids Research, 1st
7: 4873 (1989), 16:10
938 (1988), Vol. 8, 979-987 (1980)], Lactobacillus kandleri, Lactobacillus minor, Lactobacillus confusus, Lactobacillus confusus, Lactobacillus confusus. 16Sr of Bacillus viridense, Lactobacillus catenaforme, and Lactobacillus vitulinus
The nucleotide sequence of the RNA gene has been clarified [Journal of Bacteriol
ogy), Volume 171, 6455-6467 (198)
9), Nucleic Acids Research Vol. 18,
Pp. 3401-3402 (1990), Systematic and Applied Microbiology (System
atic and Applied Microbiology), Volume 12, 145
Pp. 149 (1989)]. Then, Tsuchiya et al. Reported a method for detecting Lactobacillus brevis in beer using PCR, in which a primer was prepared based on the nucleotide sequence of the 5S rRNA gene of Lactobacillus brevis [The Brewing Society of Japan, Vol. 86]. , P. 720 (199
1)]. However, since the nucleotide sequences of 5S rRNA are extremely similar across genus of microorganisms, microorganisms other than Lactobacillus brevis may be erroneously detected. Similarly, 16S rRNA is also extremely similar across genus of microorganisms and is not suitable for the purpose of specifically detecting Lactobacillus bacteria.

On the other hand, 16SrRNA gene and 23SrR
It is known that the spacer region gene formed between the NA genes has a nucleotide sequence specific to the microorganism species. The present inventors have determined the nucleotide sequence of the spacer region of various Lactobacillus bacteria in Japanese Patent Application No. 4-113154, and detected the Lactobacillus bacteria by amplifying the spacer region using a primer. The method of doing is disclosed.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to detect a Lactobacillus bacterium, particularly a fire bacillus, by a PCR method using a gene specific to the Lactobacillus bacterium, and to use a more rapid and highly sensitive method. It is to provide a detection method.

[0006]

The present invention is summarized as follows. The present invention relates to a method for detecting a bacterium of the genus Lactobacillus, which comprises amplifying a nucleic acid of the bacterium of the genus Lactobacillus by using a primer in a liquid sample. The method for detecting a Lactobacillus bacterium according to 1) above, which comprises a step of passing the liquid sample through a filter to capture the Lactobacillus bacterium and then washing the bacterium.

Lactobacillus (hereinafter abbreviated as L.)
The DNA encoding the rRNA of the genus bacterium is 16SrRN.
It is composed of each DNA sequence of A-spacer region-23SrRNA-spacer region-5SrRNA. The inventors of the present invention, as described in Japanese Patent Application No. 4-113154, have 13 types of L. RRNA of genus bacteria
Part of the DNA sequence coding for 16Sr
A portion of the DNA sequence encoding the RNA-spacer region-23S rRNA was revealed and then L. Common DNA sequences of genus bacteria and DNs specific to each species
The A sequence is found.

[0008]

[Table 1] Table 1 Bacteria Strain Medium Culture temperature (genuine burn-down bacterium) L. Hetero Chio IFO13118 SI 30 ℃ IFO13119 SI 30 ℃ JCM1198 SI 30 ℃ L. Homo Hachii IFO13120 SI 30 ℃ IFO13121 SI 30 ℃ JCM1199 SI 30 ℃ (flammable lactic acid bacterium) L. Japonicas IAM10068 803 37 ° C L. Plantarum IAM1216 803 37 ℃ L. Casei Subspecies Rhamnosus IFO3532 804 37 ℃ L. Casei Subspecies Casei IFO3533 804 37 ℃ L. Species IFO3954 804 37 ℃ (General lactic acid bacteria) L. Brevis IFO13110 804 30 ℃ (Hirochimi isolate) F-1 SI 30 ℃

Medium composition: SI: 5% of SI medium (Japan Brewery Association), ethanol 10
ml, distilled water 90 ml 803: 0.5% polypeptone, 0.5% yeast extract, 0.5% glucose, 0.2% lactose, 0.05% Tween 80, 0.1% MgS
_{O 4 / 7H 2 O, pH6.8~7.0} 804: 0.5% peptone, 0.5% yeast extract, 0.5% glucose, 0.1% MgSO _4/7
H ₂ O, pH6.6~7.0

Furthermore, in order to carry out the most sensitive and simple PCR method at present as a gene detection method, L. Specific region DN of DNA sequence common to genus bacteria and species-specific DNA sequence
An oligonucleotide primer for amplifying A by PCR is synthesized. The inventors proceeded with further research,
L. L. in a liquid sample containing a genus bacterium. PCR was carried out using the genus bacterial DNA as a template, and L. The PCR inhibitors in the liquid sample were removed by capturing the bacteria of the genus on a filter and then washing the bacteria, and L. The present invention has been completed by finding that a specific region of genus bacterial DNA can be efficiently amplified and detected.

The present invention will be specifically described below. 16S
Genes encoding rRNA and 23S rRNA are known to be well conserved among microorganisms (Table 2
Table 5).

[0012]

[Table 2] Table 2 Sequence of R16-1: 5′-C T T G T A C A C A C C G C C C G T C A-3 ′ L. Casey--------------------Bacillus subtilis--------------------(Bacillus subtilis) Escherichia coli- -------------------(Escherichia coli) Mycobacterium bovis--------------------(Mycobacterium bovis ) Halobacterium halobium----C---------------(Halobacterium halobium) Mycoplasma capricola------------------ --Mu (Mycoplasma capricolum) Pseudomonus aeruginosa Therma Thermophilus------------------------------------ ------(Thermus thermophilus) Halococcus molae----C---------------(Halococcus morrhuae) Streptomyces lividan----------- ---------Streptomyces lividans Heliobacterium chlora----------- ----------(Heliobacterium chlorum) Flavobacterium heparinum---------------------Nam (Flavobacterium heparinum)

[0013]

[Table 3] Table 3 Sequence of R16-2: 5′-G T G C G G C T G G A T C A C C T C C T-3 ′ L. Casey NNNNNNNNN-----------Bacillus subtilis--------------------Escherichia coli C-----T------ -------Mycobacterium bovis--------------------Halobacterium Harobi C-------------- -----Umm mycoplasma caprico------A-------------Ram Pseudomonas Aerugi C------------------- -Nosa Thermus Thermophilus--------------------Halococcus Small Ae C-------------------Streptomyces Libida- -------------------Sheliobacterium black--------------------Lamb flavobacterium hepari- NNNNNNNN-----------Nam

[0014]

[Table 4]

[0015]

[Table 5] Table 5 R23-2R sequence: 3'-CTTTGTAGATTCATGGGCC T-5 'R23-2R complementary sequence: 5'-GAAACATCTAAGTACCCGG A-3' Bacillus subtilis------------- -------Escherichia coli-----------------C--Halobacterium Harobi---G-----C-----GGCC- Um Pseudomonas Aergi-----------------T--Nosa Therma Thermophilus---------C-------A--Harococcus mallae-- -------C------G-C-

Tables 2 to 5 above show the conserved nucleotide sequences of the prokaryotic rRNA gene and the nucleotide sequences of the primers selected from them, and Tables 2 and 3 show the 16S rRNA gene, Table 4 and Table 5 shows 23SrRN
The respective conserved nucleotide sequences of the A gene and the nucleotide sequences of the primers selected from them are shown. Table 2
In Table 5, the conserved bases are indicated by −, different bases are indicated by the symbol of the base, deficient bases are indicated by *, and unidentified bases are indicated by N.

For example, R represented by SEQ ID NO: 1 in the sequence listing
16-1 primer and R23-2 represented by SEQ ID NO: 4
R primer combination, R16-represented by SEQ ID NO: 2
2 primer and the combination of R23-1R represented by SEQ ID NO: 3 The spacer region of the genus bacterium can be amplified by the PCR method.

These primers can be synthesized by a DNA synthesizer and can be used after appropriately purified by HPLC or the like.

Regarding the PCR method, a gene amplification kit containing tack DNA polymerase and an automatic gene amplification device are commercially available from Takara Shuzo. In order to determine the nucleotide sequence containing the fragment containing the spacer region amplified by the PCR method, for example, the amplified fragment can be cloned into an M13 phage vector to prepare phage DNA, and then determined by the Sanger method.

From the base sequence of the spacer region thus determined, L. Each specific region of the genus bacteria,
Alternatively, the common area can be specified. In the above specification, the inventors of the present invention have described 13 types of L. The nucleotide sequence of the spacer region of the genus bacterium was clarified, and L. The base sequence specific to the genus bacteria has been determined. Sequence number 5 in the sequence listing is L. Heterohiotic JCM 1198 and L.H. Homo Hichii JCM119
9, SEQ ID NO: 6 is L. Hetero Chio IFO1311
8, L.S. Heterohiotic IFO 13119, L. et al. Homohiotic IFO 13120, and L. Homo Hichii
IFO13121, SEQ ID NO: 7 is L. Japonicas I
AM10068, SEQ ID NO: 8 is L. Plantarum IA
M1216, SEQ ID NO: 9 is L. Casei Subspecies Rhamnosus IFO3532, SEQ ID NO: 10 is L. Casei subspecies casei IFO3533, SEQ ID NO: 11 is L. Species IFO3954, SEQ ID NO: 12 is L. Brevis IFO 13110, SEQ ID NO: 1
3 is the rR of H-1 bacterium isolated from fresh sake
This is the base sequence of the gene encoding NA.

Based on the obtained base sequence, each L. Specific sequences and common sequences of genus bacteria can be identified. As a result, the specific sequences and common sequences shown in SEQ ID NOs: 14 to 21 of the sequence listing are obtained. When these sequences are used as primers for the PCR method, each L. The genus bacterial DNA can be specifically amplified. Alternatively, with a pair of common primers, all L. Genus bacteria DN
A can be amplified. Table 6 shows the relationship between each strain, the specific sequence including the spacer region, and the specific primer.

[0022]

[Table 6]

Specifically, SEQ ID NO: 2 in the sequence listing
LAU1 represented by 0 and LAK represented by SEQ ID NO: 14
With the 4R primer pair, L. Hetero Hichii JCM
1198 and L.L. The gene of Homohiotici JCM1199 was cloned by LAU1 and the LAM3R primer pair represented by SEQ ID NO: 15. Hetero Chio IFO1311
8, 13119 and L.S. Homochio IFO1312
The genes 0, 13121 were identified by LAU1 and the LAJ3R primer pair represented by SEQ ID NO: 16 as L. Japonicas IAM10068 and L.J. Plantarum IAM1
216 genes were identified by LAU1 and the LAC4R primer pair represented by SEQ ID NO: 17 as L. Casei Subspecies Rhamnosus IFO3532 and L. Casei
The gene of Subspecies casei IFO3533 was expressed by LAU1 and the LAB4R primer pair represented by SEQ ID NO: 18. Species IFO 3954 and L.M. Brevis IFO13110 gene has LAF1 represented by SEQ ID NO: 19 and LA represented by SEQ ID NO: 21.
The gene of the isolated strain F-1 is specifically amplified by the U3R primer pair, and each bacterium can be specifically detected. In addition, the LAU1 and LAU3R primer pairs all of these L. The gene of the genus bacterium is amplified, and these bacteria can be detected. In addition, L. In a genus bacterium, a tRNA-encoding region may be inserted in the spacer region, and even in this case, amplification and detection can be performed using these primers.

After amplification of L. The genus bacterial DNA can be detected by, for example, agarose gel electrophoresis, polyacrylamide gel electrophoresis, spot method, and Southern blotting method. In the spot method and the Southern blotting method, the probe DNA may be selected within the amplification region.

The pretreatment for the nucleic acid amplification method, for example, the PCR method can be carried out by the method exemplified below. (1) The liquid sample was filtered with a membrane filter to obtain L.I.
Capture genus bacteria. (2) The captured L. Cultivate a genus bacterium as needed. (3) When step (2) was performed, the culture solution was passed through a membrane filter to grow the L. Recapture genus bacteria. (4) L. coli captured in step (1) or (3). Wash genus bacteria. (5) Treated with proteinase K and treated at 99 ° C,
Subject to R.

The liquid sample in the present invention is, for example, L.M. Alcoholic beverages containing genus bacteria, for example, sake, mirin, wine, etc. It is a medium containing a genus bacterium.

As the membrane filter used in the step (1), L. Anything can be used as long as it can capture the genus bacteria. Examples of the material of the membrane filter include polyvinylidene fluoride, nitrocellulose, and hydrophilic polyether sulfone, and polyvinylidene fluoride is preferable. The pore size of the membrane filter is L. Anything can be used as long as it can capture a genus bacterium, specifically, 0.1 to
It is about 0.8 μm. Although the diameter of the membrane filter is not particularly limited, for example, when using 300 ml of a sample, a diameter of about 13 to 47 mm may be used, and 47 mm is preferable from the viewpoint of filtration time, but when using 13 mm and 25 mm membrane filters. In the step (2), it is convenient that the membrane filter can be put directly in a microtube having a volume of 1.5 ml, and a 25 mm filter is usually used.

In the step (2), the L. This is done to increase the number of bacteria belonging to the genus or to distinguish live and dead bacteria. SI medium is typically used as the medium used in the step (2), but 10 g of yeast extract and 50 cysteine are added to 1 liter of pure rice liquor having an alcohol concentration of 15%.
When a medium containing mg (pH: 4.2 to 4.5) (hereinafter referred to as a modified medium) is used, L. The bacteria of the genus grow quickly, and the nucleic acid amplification reaction such as PCR reaction inhibition is low,
It is suitable.

As the membrane filter used in the step (3), the same one as in the step (1) can be used, but if a centrifuge tube with a filter is used, it can be simply concentrated by centrifugation.

The washing in the step (4) can be carried out as it is on the membrane filter after carrying out the steps (1) or (3). That is, an appropriate amount of washing solution may be added directly to the membrane filter after step (1) or (3), and then centrifuged to concentrate. Examples of the cleaning liquid used in the step (4) include L.I. Any substance that can remove the nucleic acid amplification reaction adhering to the genus bacterium, for example, an inhibitor of PCR method, may be used, in addition to sterilized water, various buffers such as TE buffer (10 mM Tris-HCl pH 7.5, 0). 1 mM
EDTA).

The steps (2) and (3) may be carried out as necessary and may be omitted. When omitted, step (4) can be performed, for example, as follows, in addition to the above method. That is, L. The membrane filter of the step (1) capturing the bacteria of the genus is immersed in sterilized water in a microtube and stirred to obtain L. Suspension of genus bacteria,
Washed and then L. The sterilized water containing the bacteria of the genus is passed through a membrane filter, for example, a centrifuge tube with a filter, to give L. The genus bacterium may be captured and subjected to the step (5).

Further, when PCR is performed, by adding dUTP and uracil-N-glycosylase (UNG) to the system, non-specific amplification can be suppressed and detection with higher accuracy can be performed.

The present inventors carried out the pretreatments of (1) to (5) above, and performed L. Genus bacteria were detected by PCR. As a result, one L. The samples containing genus bacteria were also detected with good reproducibility. On the other hand, in the pretreatment without the washing step (4), 100
L. It was not detectable even in samples containing genus bacteria.

The inventors of the present invention have also proposed the L. From the spacer region of the bacterium belonging to the genus L. Specific primers for spacer region of genus bacteria, LA1198, L
AF3R and LAC3R were created. Each L. Classification of bacteria belonging to the genus and each primer and LAM3R (SEQ ID NO: 15) described above
Table 7 shows the correspondence of the above.

[0035]

[Table 7]

Specifically, SEQ ID NO: 2 in the sequence listing
LAU1 represented by 0 and LA1 represented by SEQ ID NO: 22
198 primer pairs with a true H. bacilli, for example L. The genes of Heterohiotic JCM1198 type and H. bacillus isolate P-a type were combined with the primer pair of LAUA and LAM3R. Homohiotic IFO13120 type gene is LAU1 and the LAF3R primer pair shown in SEQ ID NO: 23 of the sequence listing, and pyrolytic lactic acid bacterium, for example, Hakutachi isolate isolate F-1 type gene is LAU1 and SEQ ID NO: 24 of the sequence listing. With a primer pair of LAC3R represented by Casey Subspecies Rhamnosus IFO3532 and L. Casei subspecies Casei IFO3533 gene is amplified.

Since these primers match with the nucleotide sequences of the genes in the spacer region of each Hakutobacillus, they can be detected with higher sensitivity and accuracy than the universal primer LAU3R. In addition, LA1198, LAM3
By mixing and using four kinds of primers of R, LAF3R, and LAC3R, all types of Hakuto bacteria can be detected.

[0038]

EXAMPLES The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (1) Examination of filter L. Genus bacteria (Fushimi 1-6 strain) in 10 ml of SI liquid medium containing 10% ethanol at 30 ° C, 24
Incubated for hours. After counting the number of bacterial cells in this culture solution with a hemocytometer, dilute with 15% ethanol to obtain 100
Samples were prepared by preparing four solutions each of 20/20 ml and 10/20 ml. As a filter, Millipore MF-Millipore membrane filter (made of cellulose mixed ester, pore diameter 0.45 μm, diameter 25 mm), Millipore Durapore membrane filter (made of polyvinylidene fluoride, pore diameter 0.45 μm, diameter 25 mm), Germanic Science Four types of super pore membrane filters (made of hydrophilic polysulfone, two types having pore diameters of 0.45 μm and 0.2 μm, each having a diameter of 25 mm) were selected and examined.
After filtering each sample with its own filter, the filter was immersed in 0.4 ml of modified medium in a 1.5 ml microtube and stirred for 10 seconds. Next, after plating the entire amount of the improved medium on an SI medium (ethanol 10%) containing 1% agar, overlaying the SI medium and then overlaying 1% agar, culturing at 30 ° C. for 4 days, and colony I checked the number of. Table 8 shows the number of colonies of each sample.

[0040]

[Table 8] Table 8 Membrane filter 100 pieces / 20ml 10 pieces / 20ml ─────────────────────────────────── ─ MF Millipore membrane filter 23 1 Durapore membrane filter 197 26 Superpore membrane filter 14 4 (pore size 0.45 μm) Superpore membrane filter 21 1 (pore size 0.2 μm) ───────────── ────────────────────────

That is, the Durapore membrane filter is excellent in the cell trapping efficiency, and L. It was suitable for detection of genus bacteria.

(2) P using sake, SI medium or modified medium
CR reaction inhibition As a sample, Ginjo sake with 15% alcohol concentration, 10%
An SI medium containing ethanol and an improved medium containing 10% of brewed sake were used. As a template, L. Genomic DNA of casei
(Final concentration 2 ng / 100 μl), L as primer
AU1 and LAU3R (final concentration 0.2 μM) are used, and each sample is used in an appropriate amount, that is, in the case of Ginjo sake, 0, 5 μl, 10 μl, 20 μl, 3
0 μl, 50 μl / 100 μl In case of SI medium 0, 0.5 μl, 1 μl, 2 μl, 5
μl, 10 μl / 100 μl Modified medium 0, 5 μl, 10 μl, 20 μl, 3
100 μl of PCR reaction solution containing 0 μl, 50 μl / 100 μl [10 mM Tris-H
Cl (pH 8.3), 50 mM KCl, 1.5 mM Mg
Cl ₂ , 0.001% (W / V) gelatin, 2.5 units / 100 μl tack polymerase] was prepared.
100 μl of mineral oil (Sigma) was overlaid on each solution, and DNA thermal cycler (Takara Shuzo) under the conditions of 94 ° C 0.5 minutes, 55 ° C 1 minute, 72 ° C 1 minute, 30 cycles.
Was used to perform PCR. After completion of the reaction, 10 μl of the mixture was electrophoresed on a Nusieve 3: 1 agarose (FMC) gel and stained with ethidium bromide to observe the amplified DNA. As a result, ginjo sake is 20μ
1 or more, SI medium 1 μl or more, modified medium 50 μl or more addition, PCR reaction inhibition was observed. That is, P
It was revealed that the modified medium is superior to the SI medium as the medium used before the CR.

(3) L. Detection of genus bacteria SI liquid medium 1 containing Fushimi 1-6 strain containing 10% ethanol 1
The cells were cultured in 5 ml at 30 ° C. for 2 days. After counting the number of bacterial cells in this culture solution with a hemocytometer, it was diluted with pure rice liquor with an alcohol concentration of 10% to prepare 0, 0.1, 1, 10, 1 in 300 μl.
Each sample was prepared so that the number of bacteria was 00. next,
300μl of each sample, diameter 25mm, pore size 0.45μ
m was filtered by suction using a Durapore membrane filter to capture the cells on the filter. Next, the filter was immersed in 0.4 ml of the modified medium in a 1.5 ml microtube, stirred for 30 seconds, and then allowed to stand at 30 ° C. for 2 minutes.
Cultured for 4 hours. Next, the culture solution was transferred to a centrifugal tube with filter Suprec-01 (Takara Shuzo) and centrifuged at 5000 rpm for 1 minute to capture the amplified cells on the filter.

Next, 0.4 ml of sterilized distilled water was added on the filter of SPLEK-01 and centrifuged at 5000 rpm for 1 minute to wash the cells. Next, a PC containing 0.1 mg / ml proteinase K on the filter of Suplex-01
R buffer [10 mM Tris-HCl pH 8.3, 50
mM KCl, 1.5 mM MgCl ₂ , 0.001% (W
/ V) Gelatin] 78.5 μl was added and the suspension was 0.5 ml.
Transfer to a microtube containing 60 μl of mineral oil
After layering, the mixture was treated at 65 ° C for 60 minutes and then at 99 ° C for 10 minutes.
The cells were treated for a minute to destroy the cells and expose the DNA. On the other hand, as a comparative control, a sample omitting the step of washing with 0.4 ml of sterile distilled water was also prepared.

Next, 21.5 μl of PCR was added to each sample.
Reaction solution [50 mM Tris-HCl pH 8.3, 250 mM
KCl, 7.5 mM MgCl ₂ , 0.005% (W /
V) Gelatin, 1 mM dUTP, 1 mM dATP, 1 mM
dGTP, 1 mM dCTP, 1 μM LAU-1 primer, 1 μM primer, 0.125 U / μl Amplitak (Ampri Taq), 0.005 U / μl UNG)
Was added and PCR was performed using a DNA thermal cycler.
As the primer, L shown in SEQ ID NO: 24 in the sequence listing is used.
AC3R was newly prepared and used. PCR conditions are
One cycle of 45 ° C for 10 minutes and 95 ° C for 10 minutes, then 9
40 cycles of 0.5 minutes at 4 ° C., 1 minute at 55 ° C. and 1 minute at 72 ° C. were performed. After the reaction, 10 μl of the reaction solution was electrophoresed on Nusieve 3: 1 agarose gel and stained with ethidium bromide to detect the amplified DNA. As a result, when the washing process was added, 1, 10, 100 L. An amplification product of about 170 bp was observed in each sample containing bacteria belonging to the genus Genus. The genus bacteria could be detected. On the other hand, in the control in which the washing step was omitted, no amplification product was observed in any of the samples. No genus bacteria could be detected. In another L.S. The same result was obtained for the genus bacterial strain N5.

(4) Detection of burn-down bacterium using new primer For the purpose of further detecting the burn-down bacterium with higher sensitivity and accuracy, each primer of LA1198 (SEQ ID NO: 22) and LAF3R (SEQ ID NO: 23) shown in Table 7 was prepared. did. Next, for each of the Hakutogi bacteria shown in Table 7, the corresponding primer and LA
As a result of performing PCR with 1 ng of genomic DNA of each Hakutobacillus as a template with the primer pair of U-1, LA
It was amplified more efficiently than the case of the primer pair of U-1 and LAU3R. In addition, for each of the Hakuto bacteria, the primer pair of these primers and LAU-1 was used in Example 1.
As a result of detecting the spores in the liquid sample in the same manner as in (3), it was possible to detect even one spores in 300 ml. In addition, LA1198, LAM3R, LAF
Even when a mixture of 4 types of primers, 3R and LAC3R, was used, each fire-fall bacterium could be efficiently detected in the same manner.

[0047]

According to the present invention, L. A rapid and highly sensitive method for detecting a genus bacterium, in particular a fire bacterium, has been provided.

[0048]

[Sequence list]

SEQ ID NO: 1 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Antisense: NO Sequence: CTTGTACACA CCGCCCGTCA 20

SEQ ID NO: 2 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Antisense: NO Sequence: GTGCGGCTGG ATCACCTCCT 20

SEQ ID NO: 3 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: YES Sequence: CTCCTAGTGC CAAGSCATYC 20

SEQ ID NO: 4 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: YES Sequence: TCCGGGTACT TAGATGTTTC 20

SEQ ID NO: 5 Sequence length: 540 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: genomic DNA Origin: Organism name: Lactobacillus heterohiochii (Lactobacil)
lus heterohiochii) Strain name: JCM 1198 Organism name: Lactobacillus
s homohiochii) Strain name: JCM1199 Sequence characteristics: 1-155 16S rRNA coding region 156-371 Spacer region 220 Insertion position of tRNA coding region 372-540 23S rRNA coding region Sequence: CTTGTACACA CCGCCCGTCA CACCATGATA GTTTGTAACA CCCAAAGTCG GTTAGGTAAC 60 TTTTGGAGCC TGCCGCCTAA GGTGGGACAG ATGATTAGGG TGAAGTCGTA ACAAGGTAGC 120 CGTAGGAGAA CCTGCGGCTG GATCACCTCC TTTCTAAGGA AAAATTCGAA AACCCTACAC 180 AATTAAAGTC TTGTTTAGTT TTGAGAGTTT TACTCTCAAT ACTTTGTTCT TTGAAAACTA 240 GATAATATTA TTTTCTGTAT TAATTATATT TTAATTATAA TTTTAACCGA GAAATAACCA 300 CTACGTTATT TGAGTTTTTT AAAATAGTTT AAATCGCAAA TACTCAATAA CTTACATCAC 360 GAAGTGATGC AGGTTAAGTT ATTAAGGGCG CATGGTGAAT GCCTTGGTAC TAGGAGCCGA 420 TGAAGGACGG AACTAACACC GATATGTTTC GGGGAGCTGT ACGTAAGCTT TGATCCGGAG 480 ATTTCCGAAT GGGGAAACCC AATCATCTTA GTCGATGATT GCTCGACAGT GAATTCACTG 540

SEQ ID NO: 6 Sequence length: 585 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: genomic DNA Origin: Organism name: Lactobacillus heterohiochii (Lactobacil)
lus heterohiochii) Strain name: IFO13118, IFO13119 Organism name: Lactobacillus
s homohiochii) Strain name: IFO13120, IFO13121 Sequence characteristics: 1-162 16S rRNA coding region 163-370 Spacer region 277 tRNA coding region insertion position 371-585 23S rRNA coding region Sequence: CTTGTACACA CCGCCCGTCA CACCATGAGA GTTTGTAACA CCCAAAGCCG GCCGGATAAC 60 CTAGTTTACT AGGAGTCAGC CGTCTAAGGT GGGACAAATG ATTAGGGTGA AGTCGTAACA 120 AGGTAGCCGT AGGAGAACCT GCGGCTGGAT CACCTCCTTT CTAAGGAAAA AAAGCGAACG 180 TGACGGAGAG TAGGAGACTA CTAAGAGAAG TCAGTGAAGC AAACGGAAGC ACACGAAAGA 240 GACTTTGTTT AGTTTTGAGG GTAGTACCTC AAGAAAAGTT AGTACATTGA AAACTGAATA 300 TAATCCAAAT AAAAACCGAG ACAATCATTG AAGAACAGAT TGTAGAGCGA CCGAGAAGAG 360 CGATCTTAAA GTAAGGTCAA GTAGACAAGG GCGCACGGTG AATGCCTAGG CACTAGCAGC 420 CGAAGAAGGA CGTGACGAAC TACGAAAAGC TTCGGGGAGT TGTAAGTAAA CTAAGATCCG 480 GAGATGTCCA AATGGGGAAA CCCAATGCAG TGATGCATTA TTACTAGCCG AATAGATAGG 540 CTGGTAAAGG AAGACGCAGT GAACTGAAAC ATCTAAGTAC CCGGA 585

SEQ ID NO: 7 Sequence length: 574 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: genomic DNA Origin: Organism name: Lactobacillus japonicas (Lactobacillu)
s japonicus) Strain name: IAM10688 Sequence characteristics: 1-155 16S rRNA coding region 156-358 Spacer region 224 tRNA coding region insertion position 359-574 23SrRNA coding region Sequence: CTTGTACACA CCGCCCGTCA CACCATGAGA GTTTGTAACA CCCAAAGTCG GTGGGGTAAC 60 TTTTAGGAAC CAGCCGCCTA AGGTGGGACA GATGATTAGG GTGAAGTCGT AACAAGGTAG 120 CCGTAGAGAA CCTGCGGCTG GATCACCTCC TTTCTAAGGA ATATTACGGA AACCTACACA 180 CGCGTCGAAA CTTTGTTTAG TTTTGAGAGA TTTAACTCTC AAAACTTGTT CTTTGAAAAC 240 TAGATAATAT CAAATATATT TTTTCATAAT GAAACCGAGA ACACCGCGTT TTTTGAGTTT 300 TTTATTGAAG TTTAATTATC GCTAAACTCA TTAATCGCAT TTACCGTTAG GTAAATGAGG 360 TTAAGTTAAC AAGGGCGCAT GGTGAATGCC TTGGCACTAG GAGCCGATGA AGGACGGGAC 420 TAACACCGAT ATGCTTCGGG GAGCTGTACG TAAGCTATGA TCCGGAGATT TCCGAATGGG 480 GCAACCCAGC AGTTTTAATC AACTGTTACC ACTAGATGAA TTCATAGTCT AGTTGGAGGT 540 AAACGCTGTG AACTGAAACA TCTAAGTACC CGGA 574

SEQ ID NO: 8 Sequence length: 574 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: genomic DNA Origin: Organism name: Lactobacillus plantarum (Lactobacillu)
s plantarum) Strain name: IAM1216 Sequence characteristics: 1-155 16S rRNA coding region 156-358 Spacer region 224 tRNA coding region insertion position 359-574 23S rRNA coding region Sequence: CTTGTACACA CCGCCCGTCA CACCATGAGA GTTTGTAACA CCCAAAGTCG GTGGGGTAAC 60 TTTTAGGAAC CAGCCGCCTA AGGTGGGACA GATGATTAGG GTGAAGTCGT AACAAGGTAG 120 CCGTAGAGAA CCTGCGGCTG GATCACCTCC TTTCTAAGGA ATATTACGGA AACCTACACA 180 CGCGTCGAAA CTTTGTTCAG TTTTGAGAGA TTTAACTCTC AAAACTTGTT CTTTGAAAAC 240 TAGATAATAT CAAATATATT TTTTCATAAT GAAACCGAGA ACACCGCGTT TTTTGAGTTT 300 TTTATTGAAG TTTAATTATC GCTAAACTCA TTAATCGCAT TTACCGTTAG GTAAATGAGG 360 TTAAGTTAAC AAGGGCGCAT GGTGAATGCC TTGGCACTAG GAGCCGATGA AGGACGGGAC 420 TAACACCGAT ATGCTTCGGG GAGCTGTACG TAAGCTATGA TCCGGAGATT TCCGAATGGG 480 GCAACCCAGC AGTTTTAATC AACTGTTACC ACTAGATGAA TTCATAGTCT AGTTGGAGGT 540 AAACGCTGTG AACTGAAACA TCTAAGTACC CGGA 574

SEQ ID NO: 9 Sequence length: 588 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: genomic DNA Origin: Organism name: Lactobacillus casei Subspecies
Casei (Lactobacillus casei subsp. Rhamnosus) Strain name: IFO3532 Sequence characteristics: 1-158 16S rRNA coding region 159-375 Spacer region 250 tRNA coding region insertion position 376-588 23SrRNA coding region Sequence: CTTGTACACA CCGCCCGTCA CACCATGAGA GTTTGTAACA CCCGAAGCCG GTGGCGTAAC 60 CTTTTAGGGA GCGAGCCGTC TAAGGTGGGA CAAATGATTA GGGTGAAGTC GTAACAAGGT 120 AGCCGTAGGA GAACCTGCGG CTGGATCACC TCCTTTCTAA GGAAACAGAC TGAAAGTCTG 180 ACGGAAACCT GCACACACGA AACTTTGTTT AGTTTTGAGG GGATCACCCT CAAGCACCCT 240 AACGGGTGCG ACTTTGTTCT TTGAAAACTG GATATCATTG TATTAATTGT TTTAAATTGC 300 CGAGAACACA GCGTATTTGT ATGAGTTTCT GAAAAAGAAA TTCGCATCGC ATAACCGCTG 360 ACGCAAGTCA GTACAGGTTA AGTTACAAAG GGCGCACGGT GGATGCCTTG GCACTAGGAG 420 CCGATGAAGG ACGGAACTAA TACCGATATG CTTCGGGGAG CTATAAGTAA GCTTTGATCC 480 GGAGATTTCC GAATGGGGGA ACCCAGTACA CATCAGTGTG TTGCTTGTCA GTGAATACAT 540 AGCTGGCCGG CGGCAGACGC GGGGAACTGA AACATCTAAG TACCCGGA 588

SEQ ID NO: 10 Sequence length: 588 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: genomic DNA Origin: Organism name: Lactobacillus casei Subspecies
Casei (Lactobacillus casei subsp. Casei) Strain name: IFO3533 Sequence characteristics: 1-158 16S rRNA coding region 159-375 Spacer region 250 Insertion position of tRNA coding region 376-588 23S rRNA coding region Sequence: CTTGTACACA CCGCCCGTCA CACCATGAGA GTTTGTAACA CCCGAAGCCG GTGGCGTAAC 60 CTTTTAGGGA GCGAGCCGTC TAAGGTGGGA CAAATGATTA GGGTGAAGTC GTAACAAGGT 120 AGCCGTAGGA GAACCTGCGG CTGGATCACC TCCTTTCTAA GGAAACAGAC TGAAAGTCTG 180 ACGGAAACCT GCACACACGA AACTTTGTTT AGTTTTGAGG GGATCACCCT CAAGCACCCT 240 AGCGGGTGCG ACTTTGTTCT TTGAAAACTG GATATCATTG TATTAATTGT TTTAAATTGC 300 CGAGAACACA GCGTATTTGT ATGAGTTTCT GAAAAAGAAA TTCGCATCGC ATAACCGCTG 360 ACGCAAGTCA GTACAGGTTA AGTTACAAAG GGCGCACGGT GGATGCCTTG GCACTAGGAG 420 CCGATGAAGG ACGGAACTAA TACCGATATG CCTCGGGGAG CTATAAGTAA GCTTTGATCC 480 GGAGATTTCC GAATGGGGAA ACCCAGTACA CATCAGTGTA TTGCTTGTCA GTGAATACAT 540 AGCTGGCCGG CGGCAGACGC GGGGAACTGA AACATCTAAG TACCCGGA 588

SEQ ID NO: 11 Sequence length: 414 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: genomic DNA Origin: Organism name: Lactobacillus species
s sp.) Strain name: IFO3934 Sequence characteristics: 1-156 16S rRNA coding region 157-370 Spacer region 225 tRNA coding region insertion position 371-414 23S rRNA coding region Sequence: CTTGTACACA CCGCCCGTCA CACCATGAGA GTTTGTAACA CCCAAAGCCG GTGAGATAAC 60 CTTCGGGAGT CAGCCGTCTA AGGTGGGACA GATGATTAGG GTGAAGTCGT AACAAGGTAG 120 CCGTAGGAGA ACCTGCGGCT GGATCACCTC CTTTCTAAGG AATATACGGA GGCTACACAT 180 ACTTGTTGAA ACAATGTTCA GTTTTGAGGG GCTTACCTCT CTAAACTTGT TCTTTGAAAA 240 CTAGATATTA TCAATTATTT TCCTTTAATT ATTAAGATAA TTAAACCGAG AAACAACTGC 300 GTATTTTTGA GTTTTTTAAT TAGTTTATCG CTAATACTCA ATTAATTTGA CGATCACGAA 360 GTGACCGTTA GGTTAAGTTA TGAAGGGCGC ATGGTGGAGC CTTGGTACTA GGAG 414

SEQ ID NO: 12 Sequence length: 585 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: genomic DNA Origin: Organism name: Lactobacillus brevis
evis) Strain name: IFO13110 Sequence characteristics: 1-156 16S rRNA coding region 157-370 Spacer region 225 Insertion position of tRNA coding region 371-585 23SrRNA coding region Sequence: CTTGTACACA CCGCCCGTCA CACCACGAGA GTTTGTAACA CCCAAAGCCG GTGAGATAAC 60 CTTCGGGA CAGCCGTCTA AGGTGGGACA GATGATTAGG GTGAAGTCGT AACAAGGTAG 120 CCGTAGGAGA ACCTGCGGCT GGATCACCTC CTTTCTAAGG AATATACGGA GGCTACACAT 180 ACTTGTTGAA ACAATGTTCA GTTTTGAGGG GCTTACCTCT CTAAACTTGT TCTTTGAAAA 240 CTAGATATTA TCAATTATTT TCCTTTAATT ATTAAGATAA TTAAACCGAG AAACAACTGC 300 GTATTTTTGA GTTTTTTAAT TAGTTTATCG CTAATACTCA ATTAATTTGA CGATCACGAA 360 GTGACCGTTA GGTTAAGTTA TGAAGGGCGC ATGGTGGATG CCTTGGTACT AGGAGCCGAT 420 GAAGGACGGG ACTAACACCG ATATGCTTCG GGGAGCTGTA CGTAAGCTTT GATCCGGAGA 480 TTTCCGAATG GGGAAACCCA ATCATCTTTA CCGATGATTA CAACTTGATG AATACATAGT 540 CAAGTTGAGG CAGACGTGGG GAACTGAAAC ATCTAAGTAC CCGGA 585

SEQ ID NO: 13 Sequence length: 286 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: genomic DNA Origin: Organism name: Lactobacillus Strain name: F -1 Sequence features: 1-20 16S rRNA coding region 21-241 Spacer region 86 Insertion position of tRNA coding region 242-286 23S rRNA coding region Sequence: GGCTGGATCA CCTCCTTTCT AAGGAAAATT CGGAAACCTA CACAATGTCG AAAGTTTTGT 60 TCAGTTTTGA GAGGTCTACTCTCAAACTTG GT TATTAATTTT 120 CTGTAATTTA TTGAATTGGA TATAATCCAA TTTCAACCGA GAACACCGCG TTATTTTGAG 180 TTTGTTAACT AAGTAAAAAA TCGCAAATAC TCAATTAACT AAAGTATCCG TAGGATACTT 240 AGGTTAAGTT ATCAAGGGCG CATGGTGAAT GCCTTGGCAC TAGGAG286

SEQ ID NO: 14 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: YES Sequence: CCTGCATCAC TTCGTGATGT 20

SEQ ID NO: 15 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: YES Sequence: GCTCTACAAT CTGTTCTTCA 20

SEQ ID NO: 16 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: YES Sequence: TTTACCTAAC GGTAAATGCG 20

SEQ ID NO: 17 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: YES Sequence: GTACTGACTT GCGTCAGCGG 20

SEQ ID NO: 18 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: YES Sequence: GGTCACTTCG TGATCGTCAA 20

SEQ ID NO: 19 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: NO Sequence: AATTCGGAAA CCTACACAAT 20

SEQ ID NO: 20 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: NO Sequence: ATCACCTCCT TTCTAAGGAA 20

SEQ ID NO: 21 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: YES Sequence: AAAAAACGCG GTGTTCTCGG 20

SEQ ID NO: 22 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: YES Sequence: TAACGTAGTG GTTATTTCTC GG twenty two

SEQ ID NO: 23 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: YES Sequence: CAAATACGCT GTGTTCTCGG 20

SEQ ID NO: 24 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: YES Sequence: AAATAACGCG GTGTTCTCGG 20

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kimoya Fujino 3-4-1 Seta, Otsu City, Shiga Prefecture, Central Research Laboratory, Mina Shuzo Co., Ltd. (72) Inventor Ikuno Kato, 3-chome Seta, Otsu City, Shiga Prefecture No. 1 In the Central Research Institute of Takashu Brewery

Claims (1)

[Claims] 1. A method for detecting Lactobacillus bacteria in a liquid sample by amplifying nucleic acid of Lactobacillus bacteria using a primer, comprising the step of capturing the Lactobacillus bacteria by passing the liquid sample through a filter. A method for detecting a bacterium belonging to the genus Lactobacillus, which comprises a step of washing the bacterium.