JP6917355B2 - Method for discriminating species and strains of Lactobacillus microorganisms - Google Patents

Description

The present invention relates to a method for discriminating a specific species and a specific strain of a Lactobacillus microorganism.

Conventionally, it is a general method to identify bacterial species of bacteria or fungi by using DNA or RNA sequence analysis. As a method for identifying such a general strain, the RiboPrinter, Pulsed Field Gel Electrophoresis (PFGE) method, Restration Fragment Lens Polymerase (RFLP) method, and RFLP method, which use the DNA of the viable strain of the purely cultured strain, are used. Analysis of DNA (RAPD) method, Repetitive Element Palindromic PCR (Rep-PCR) method, Multilocas queuance typing (MLST) method, Multilocus sexual analysis (MLSA) method and the like are common.
For example, in a method for determining a strain of a Lactobacillus acidophilus species, live DNA of the strain is generally used. Specifically, a PFGE typing method as described in Non-Patent Document 2, a method by Riboprinter as described in Non-Patent Documents 3 and 4, and a method described in Non-Patent Document 5. Examples include a method based on the RFLP method.
Further, as an application of such bacterial species identification, Patent Document 1 discloses a DNA probe for identifying a bacterial strain of Lactobacillus herveticui species in a dairy product, and an attempt to prepare such a probe. Has been done. Further, in Patent Document 2, a probe prepared from 16S rRNA gene or 16S rRNA of a novel microorganism belonging to the genus Thalassospira capable of promoting decomposition and removal of environmental pollutants is used, and a bacterium having an ability to promote decomposition of environmental pollutants is used. It is disclosed that detection and quantification can be performed easily and quickly, and that the polluted environment can be monitored and evaluated.
As described above, techniques for identifying bacterial species and strains of bacteria and fungi are required and developed in various situations.

Japanese Unexamined Patent Publication No. 03-236799 Japanese Unexamined Patent Publication No. 2006-230255

Devopment of a Tiered Multilocus Sequence Type Scheme for Members of the Lactobacillus complexophilus Complex, Applied and English. 7220-7228 December 2013 Volume 79 Number 23 Zhonghua Yu Fang Yi Xue Za Zhi. 2011 Dec; 45 (12): 1086-9 "Development of pulsed field gel electrophoresis for characterization for characterization and identification of Lactobacillus" Microbiol. Immunol. , 45 (4), 271-275, 2001 "Charactration of the Lactobacillus casei Group and the Lactobacillus acidofhilus Group by Automated Ribotyping" APPLIED AND ENVIRONMENTAL MICROBIOROGY, July 1998, p. 2418-2423 Vol. 64, No. 7 "Differentiation of Lactobacillus Species by Molecule Typing" Journal of Gut Bacteriology 19: 193-198, 2005, Strain Level Identification-DNA Cleavage Patterns with Various Restriction Enzymes (RFLP)-

In beverages and foods such as soft drinks mixed with dead lactic acid bacteria, which are currently sold in large numbers, the DNA of the cells and the DNA of the cells are affected by the pH and heating conditions during the manufacturing process and the pH and temperature conditions during the storage period. Since RNA is degraded, it is difficult to obtain sufficient DNA and RNA necessary for identifying the bacterial species and strain, and there is a problem that a conventionally known strain discrimination method cannot be used. Therefore, it is extremely difficult to identify the bacterial species and strains that are mixed in the manufactured products (that is, foods and beverages) that are required from the viewpoint of quality assurance.
From the above, an object of the present invention is to provide a method capable of detecting a specific species of a Lactobacillus microorganism (including dead bacteria) contained in a beverage or food, or a specific strain of the specific species.

As a result of diligent research, the present inventors have not paid much attention to detect specific species or strains of bacterial cells (including dead bacteria) contained in beverages such as foods and beverages. It was found that by using an RNA fragment of about 50 to about 450 bp of the bacterial cells in the product, the DNA fragment sequence necessary for identifying the bacterial species or strain can be unexpectedly obtained.
However, on the other hand, L. Strains of the acidofilus species, such as 5 strains in public databases, ie L. cerevisiae. Among the genes that are usually examined during typing, such as 16S rDNA, fusA, gyrA, gyrB, lepA, pyrG, and recA genes, among the acidophilus NCFM, CSI4, LA1, La14, and ATCC 53544 strains, there is polymorphism between the strains. There is a problem that it does not exist, which makes it extremely difficult to type even live bacteria. Furthermore, as described above, it is impossible to obtain DNA or RNA longer than that derived from live bacteria from foods and beverages such as soft drinks. Therefore, the present inventor does not use a sequence such as a housekeeping gene that is usually used as a target for gene analysis, but L. Focusing on the gene sequence of the orf (Open reading frame) of the acidofhilus strain, we extracted the gene sequence data with differences between the strains from 1622 data (sequence is 500-2000 bp) up to orf003-orf2010, and studied diligently. At the end, L. The present invention was completed by narrowing down the six gene sequences necessary for discriminating the strain of acidofilus to establish a primer set consisting of a plurality of primer pairs.

That is, the present invention relates to the following [1] to [9].
[1] A method for detecting a specific species of a Lactobacillus microorganism (lactic acid bacterium) or a specific strain of a specific species of a Lactobacillus microorganism in a sample as a detection target microorganism, and the following steps:
i) Step of extracting RNA from a sample,
ii) A step of synthesizing cDNA using the RNA extracted in i) as a template.
A step of performing PCR using the cDNA synthesized in iii) ii) as a template and a primer set consisting of two or more sets of primers capable of amplifying a gene region of about 50 bp to about 450 bp characteristic of the detection target microorganism. And iv) Detected in the sample when any of the sequences in the region amplified by each primer pair in the primer set is identical to each of the sequences of the detection target microorganism amplified by the same primer pair. The process of determining that the target lactic acid bacterium was present,
The method described above.
[2] The method according to [1] above, wherein the sample is a beverage.
[3] The method according to [1] above, wherein the sample is heat sterilized and stored at 25 ° C. for a period of 12 months or less, or is stored under conditions corresponding thereto.

[4] A method for determining a strain of lactic acid bacterium Lactobacillus acidophilus in a sample.
i) Step of extracting RNA from a sample,
ii) A step of synthesizing cDNA using the RNA extracted in step i) as a template.
iii) A step of performing PCR using the cDNA synthesized in step ii) as a template and at least one primer set selected from the group consisting of the following primer sets (A) to (P).
-Primer set (A) consisting of primer pairs (4), (6), (8) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (B) consisting of primer pairs (4), (6), (8) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (C) consisting of primer pairs (4), (6), (9) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (D) consisting of primer pairs (4), (6), (9) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (E) consisting of primer pairs (4), (7), (8) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (F) consisting of primer pairs (4), (7), (8) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (G) consisting of primer pairs (4), (7), (9) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (H) consisting of primer pairs (4), (7), (9) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (I) consisting of primer pairs (5), (6), (8) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

A primer set (J) consisting of a primer pair (5), (6), (8) and (11).
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (K) consisting of primer pairs (5), (6), (9) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (L) consisting of primer pairs (5), (6), (9) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (M) consisting of primer pairs (5), (7), (8) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (N) consisting of primer pairs (5), (7), (8) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (O) consisting of primer pairs (5), (7), (9) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)
And ・ Primer set (P) consisting of primer pairs (5), (7), (9) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

and,
iv): The sequence of the region amplified by the primer pair (4) (excluding the primer sequence) and the sequence of SEQ ID NO: 26 are the same, or the sequence of the region amplified by the primer pair (5) (excluding the primer sequence). The sequence of SEQ ID NO: 27 is the same as the sequence of SEQ ID NO: 27 (excluding the primer sequence), and the sequence of the region amplified by the primer pair (6) (excluding the primer sequence) and the sequence of SEQ ID NO: 28 are the same. Alternatively, the sequence of the region amplified by the primer pair (7) (excluding the primer sequence) and the sequence of SEQ ID NO: 29 are the same, and the sequence of the region amplified by the primer pair (8) (primer). (Excluding the sequence) and the sequence of SEQ ID NO: 30 are the same, or the sequence of the region amplified by the primer pair (9) (excluding the primer sequence) and the sequence of SEQ ID NO: 31 are the same and , The sequence of the region amplified by the primer pair (10) (excluding only the reverse primer sequence) and the sequence of SEQ ID NO: 32 are the same, or the sequence of the region amplified by the primer pair (11) ( The method comprising the step of determining a strain in L. acidophilus when the sequence of SEQ ID NO: 33 is the same as that of (excluding only the reverse primer sequence).

[5] A method for detecting the Lactobacillus acidophilus L-92 strain in a sample.
i) Step of extracting RNA from a sample,
ii) A step of synthesizing cDNA using the RNA extracted in step i) as a template.
iii) A step of performing PCR using the cDNA synthesized in step ii) as a template and at least one primer set selected from the group consisting of the following primer sets (Q) to (V).
-Primer set (Q) consisting of primer pairs (1) and (10)
Primer pair for amplifying a part of the orf466 gene sequence (1):
Forward primer GGGGACCCCAAACAGAAAGA (SEQ ID NO: 1)
Reverse primer GCGTCTTCGATTGCTTCACC (SEQ ID NO: 2);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (R) consisting of primer pairs (1) and (11)
Primer pair for amplifying a part of the orf466 gene sequence (1):
Forward primer GGGGACCCCAAACAGAAAGA (SEQ ID NO: 1)
Reverse primer GCGTCTTCGATTGCTTCACC (SEQ ID NO: 2);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (S) consisting of primer pairs (2) and (10)
Primer pair for amplifying a part of the orf1410 gene sequence (2):
Forward primer CGTGGTCAAAAAATAAACCGCA (SEQ ID NO: 3)
Reverse primer TCACCGTTATGGTCGCTTGA (SEQ ID NO: 4);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (T) consisting of primer pairs (2) and (11)
Primer pair for amplifying a part of the orf1410 gene sequence (2):
Forward primer CGTGGTCAAAAAATAAACCGCA (SEQ ID NO: 3)
Reverse primer TCACCGTTATGGTCGCTTGA (SEQ ID NO: 4);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (U) consisting of primer pairs (3) and (10)
Primer pair for amplifying a part of the orf1410 gene sequence (3):
Forward Primer AAAAATAAAACCGCAATCTTATCGT (SEQ ID NO: 5)
Reverse primer ATCACCGTTATGGTCGCTTG (SEQ ID NO: 6);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)
And ・ Primer set (V) consisting of primer pairs (3) and (11)
Primer pair for amplifying a part of the orf1410 gene sequence (3):
Forward Primer AAAAATAAAACCGCAATCTTATCGT (SEQ ID NO: 5)
Reverse primer ATCACCGTTATGGTCGCTTG (SEQ ID NO: 6);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

and,
iv) The sequence of the region amplified by the primer pair (1) (excluding the primer sequence) and the sequence of SEQ ID NO: 23 are the same, or the sequence of the region amplified by the primer pair (2) (primer). (Excluding the sequence) and the sequence of SEQ ID NO: 24 are the same, or the sequence of the region amplified by the primer pair (3) (excluding the primer sequence) and the sequence of SEQ ID NO: 25 are the same and , The sequence of the region amplified by the primer pair (10) (excluding only the reverse primer sequence) and the sequence of SEQ ID NO: 32 are the same, or the sequence of the region amplified by the primer pair (11) ( When the sequence of SEQ ID NO: 33 (excluding only the reverse primer sequence) and the sequence of SEQ ID NO: 33 are the same, L. The process of determining that the acidofilus L-92 strain was present,
The method described above.

[6] The method according to [4] or [5] above, wherein PCR using each primer pair is performed in a separate container in step iii).
[7] In step iv), the sequence of the region (excluding the primer sequence) amplified by the primer pair (1), or the sequence of the region (excluding the primer sequence) amplified by the primer pair (2), or The sequence of the region amplified by the primer pair (10) (excluding only the reverse primer sequence) or the primer pair (11) downstream of the sequence of the region amplified by the primer pair (3) (excluding the primer sequence). The method according to [5] above, wherein a fusion sequence in which the sequences of the regions amplified by (excluding only the reverse primer sequence) are bound is used.

[8] L. cerevisiae, which comprises any of the following primer sets (Q) to (V). acidofilus L-92 strain detection kit:
-Primer set (Q) consisting of primer pairs (1) and (10)
Primer pair for amplifying a part of the orf466 gene sequence (1):
Forward primer GGGGACCCCAAACAGAAAGA (SEQ ID NO: 1)
Reverse primer GCGTCTTCGATTGCTTCACC (SEQ ID NO: 2); and primer pair for amplifying part of the orf1440 gene sequence (10) :.
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20).

-Primer set (R) consisting of primer pairs (1) and (11)
Primer pair for amplifying a part of the orf466 gene sequence (1):
Forward primer GGGGACCCCAAACAGAAAGA (SEQ ID NO: 1)
Reverse primer GCGTCTTCGATTGCTTCACC (SEQ ID NO: 2);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (S) consisting of primer pairs (2) and (10)
Primer pair for amplifying a part of the orf1410 gene sequence (2):
Forward primer CGTGGTCAAAAAATAAACCGCA (SEQ ID NO: 3)
Reverse primer TCACCGTTATGGTCGCTTGA (SEQ ID NO: 4);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (T) consisting of primer pairs (2) and (11)
Primer pair for amplifying a part of the orf1410 gene sequence (2):
Forward primer CGTGGTCAAAAAATAAACCGCA (SEQ ID NO: 3)
Reverse primer TCACCGTTATGGTCGCTTGA (SEQ ID NO: 4);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (U) consisting of primer pairs (3) and (10)
Primer pair for amplifying a part of the orf1410 gene sequence (3):
Forward Primer AAAAATAAAACCGCAATCTTATCGT (SEQ ID NO: 5)
Reverse primer ATCACCGTTATGGTCGCTTG (SEQ ID NO: 6);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (V) consisting of primer pairs (3) and (11)
Primer pair for amplifying a part of the orf1410 gene sequence (3):
Forward Primer AAAAATAAAACCGCAATCTTATCGT (SEQ ID NO: 5)
Reverse primer ATCACCGTTATGGTCGCTTG (SEQ ID NO: 6);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

[9] Primer set (Q) consisting of the following primer pairs (1) and (10):
Primer pair for amplifying a part of the orf466 gene sequence (1):
Forward primer GGGGACCCCAAACAGAAAGA (SEQ ID NO: 1)
Reverse primer GCGTCTTCGATTGCTTCACC (SEQ ID NO: 2); and primer pair for amplifying part of the orf1440 gene sequence (10) :.
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20).

Table 1 below summarizes the primer pairs (1) to (11) of the present invention.

According to the method of the present invention, specific species of Lactobacillus microorganisms (including killed bacteria) contained in beverages, particularly L. Strains of the acidofilus species, and specific strains of that particular species, especially L. cerevisiae. acidofilus L-92 can be detected.

Table 2 below, registered with GeneBank, based on the regions amplified by the primer pairs (4) or (5) and (6) or (7) and (8) or (9) and (10) or (11). Accession No. Created by connecting the sequences of the regions amplified by each primer pair for each strain from the sequence and analyzing the comparison sequence data created by processing into one gene sequence for each strain with MEGA6 sequence analysis software. Shows the phylogenetic tree. Created by connecting the orf1440 gene sequence after the orf466 gene sequence for each strain based on the sequence of the region amplified by the primer pairs (1) and (10), and processing it into one gene sequence for each strain. The phylogenetic tree created by analyzing the comparative sequence data with MEGA6 sequence analysis software is shown. It is a figure which shows the result of having performed the electrophoresis on the obtained PCR product with an agarose gel according to a standard method in Example 1. It is a figure which shows the result of having performed the electrophoresis on the obtained PCR product with an agarose gel according to a standard method in Example 4. It is a reference figure which clearly shows the different base part in the sequence of the region amplified by the primer pair (1) and (11) of each strain. Different base parts are marked with shadows.

The present inventors carry out RT-PCR (reverse transcription polymerase chain reaction) using RNA remaining in the sample as a template and a relatively short region as an amplification target even if the sample has undergone a process such as sterilization treatment or long-term storage. By doing so, it has been found that a specific species of the genus Lactobacillus or a specific strain of a specific species of the genus Lactobacillus can be specifically detected.
In particular, the present inventors have RT-PCR targeting a region relatively short with respect to RNA remaining in the sample, even if the sample has undergone a process such as sterilization and / or long-term storage for several months or longer. By performing L. acidofilus species or L. It was found that the acidofilus L-92 strain can be specifically detected. Long-term storage refers to storage under, for example, 1 month or longer, 3 months or longer, preferably 3 to 6 months, more preferably 3 to 12 months or equivalent conditions, and in particular, the sample is a beverage or food. In the case, "long-term storage" also includes storage for a period from manufacture to expiration date, a period from manufacture to expiration date, or a period exceeding these. Then, when the sample is placed in a storage environment of 25 ° C. or an environment equivalent thereto, there is no problem if it is within 9 months, at least 6 months, for example, and L. acidofilus species or L. The acidofilus L-92 strain can be specifically detected. The storage temperature is usually 4 to 35 ° C. However, even when the sample is stored under a harsh temperature condition of, for example, 55 ° C., if it is about 3 to 5 days, depending on the type of primer set, L. acidofilus species or L. The acidofilus L-92 strain may be specifically detected.
Therefore, according to the present invention, a specific strain or strain of the genus Lactobacillus can be specifically detected using RT-PCR. In particular, according to the present invention, L. cerevisiae using RT-PCR. acidofilus species or L. The acidofilus L-92 strain can be specifically detected.

According to the present invention, L. acidophilus species, especially L. Samples in which the acidophilus L-92 strain can be specifically detected include foods, beverages, feeds, etc. containing lactic acid bacteria (lactic acid bacteria (microorganisms belonging to the genus Lactobacillus)), particularly foods, beverages, feeds, etc. containing dead lactic acid bacteria. Is included. Beverages in the present invention include fermented milk, fermented milk (sterilized), dairy products lactic acid bacteria beverages, dairy products lactic acid bacteria beverages (sterilized), lactic acid bacteria beverages and soft drinks.
As used herein, "specifically detected" refers to other Lactobacillus species or L. cerevisiae. Other strains of acidofilus species and L. acidofilus species or L. Distinguishing from the acidofilus L-92 strain, the L. acidofilus species or L. It refers to detecting a specific strain of acidofilus. For example, "specifically detecting the L. acidophylus L-92 strain" means that L. acidophilus L-92 strain is specifically detected. Other strains of acidofilus species and L. The L-92 strain in the sample was distinguished from the acidofilus L-92 strain. It refers to detecting the acidofilus L-92 strain. Further, "specifically detecting L. acidofilus" means detecting L. acidofilus in a sample discriminatively by distinguishing other Lactobacillus species from L. acidofilus species.

In the method of the present invention, RT-PCR is performed using one or more sets of primer pairs using RNA derived from an organism that can be contained in a sample as a template to determine the sequence of the amplified sequence. If one set of primer pairs does not provide sufficient specificity, then two or more sets of primer pairs (each primer pair in each set may be a combination of primer pairs that amplify other regions of the same gene or another gene. , Each consisting of a forward primer and a reverse primer). In the present invention, in order to obtain sufficient specificity, it is preferable to combine two or more sets of primer pairs to perform PCR as a primer set, and more preferably three or more sets, particularly preferably four or more sets of primer pairs are combined. PCR is performed as a primer set. Each primer pair that constitutes a primer set identifies a specific strain (for example, a specific species of the genus Lactobacillus) or a specific strain (for example, a specific species of the genus Lactobacillus) whose sequence is amplified by the primer pair. Strain) and other strains or strains are selected to be different from at least one. For each primer pair, obtain the gene sequence of the target strain or strain from a known database, obtain the same gene sequence of another strain or strain, and align these to obtain the desired strain or strain. It can be designed by identifying characteristic sequences, preferably sequences that are as specific as possible. A computer program that outputs primer sequence candidates for a given sequence can also be used for primer design. The length of the region amplified by each primer pair is generally preferably from about 50 bp to about 450 bp, more preferably from about 80 bp to about 450 bp, more preferably from about 100 bp to about 450 bp, more preferably from about 100 bp to about 350 bp, and particularly about 100 bp. ~ About 250 bp is preferable. When the length of the region to be amplified is set to be longer than 450 bp, RNA of that length may not remain in the beverage, and it may not be possible to amplify to a desired amount.

By selecting the sequence and number of primer pairs contained in each primer set as described above, a particular species (eg, a particular species of the genus Lactobacillus), or a particular strain of a particular species (eg, a particular strain of the genus Lactobacillus) can be identified. A specific strain of the species) can be identified in the sample.
The PCR-amplified sequence is compared to the sequence of the respective gene region of the detection target strain or strain (eg, L. acidofillius L-92 strain) amplified by the same primer set. It is the target strain of interest that the sequence of two or more gene regions amplified from the sample by two or more primer pairs matches the sequence of the gene region of the detected target strain or strain amplified by the same primer pair. Or it can be an index for identifying a strain. At least one of the (two or more) sequences of two or more gene regions amplified using a set containing two or more primer pairs matches the sequence of the gene region of the detection target strain or strain amplified by the same primer pair. If not, it is not recognized that the detection target strain or strain was present in the sample.

Specifically, for example, one of the following primer sets was used for PCR in L. It is an acidofilus species, and the strains therein can be specifically detected.

-Primer set (A) consisting of primer pairs (4), (6), (8) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (B) consisting of primer pairs (4), (6), (8) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (C) consisting of primer pairs (4), (6), (9) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (D) consisting of primer pairs (4), (6), (9) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (E) consisting of primer pairs (4), (7), (8) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (F) consisting of primer pairs (4), (7), (8) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (G) consisting of primer pairs (4), (7), (9) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (H) consisting of primer pairs (4), (7), (9) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (I) consisting of primer pairs (5), (6), (8) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

A primer set (J) consisting of a primer pair (5), (6), (8) and (11).
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (K) consisting of primer pairs (5), (6), (9) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (L) consisting of primer pairs (5), (6), (9) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (M) consisting of primer pairs (5), (7), (8) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (N) consisting of primer pairs (5), (7), (8) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (O) consisting of primer pairs (5), (7), (9) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (P) consisting of primer pairs (5), (7), (9) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (Q) consisting of primer pairs (1) and (10)
Primer pair for amplifying a part of the orf466 gene sequence (1):
Forward primer GGGGACCCCAAACAGAAAGA (SEQ ID NO: 1)
Reverse primer GCGTCTTCGATTGCTTCACC (SEQ ID NO: 2);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (R) consisting of primer pairs (1) and (11)
Primer pair for amplifying a part of the orf466 gene sequence (1):
Forward primer GGGGACCCCAAACAGAAAGA (SEQ ID NO: 1)
Reverse primer GCGTCTTCGATTGCTTCACC (SEQ ID NO: 2);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (S) consisting of primer pairs (2) and (10)
Primer pair for amplifying a part of the orf1410 gene sequence (2):
Forward primer CGTGGTCAAAAAATAAACCGCA (SEQ ID NO: 3)
Reverse primer TCACCGTTATGGTCGCTTGA (SEQ ID NO: 4);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (T) consisting of primer pairs (2) and (11)
Primer pair for amplifying a part of the orf1410 gene sequence (2):
Forward primer CGTGGTCAAAAAATAAACCGCA (SEQ ID NO: 3)
Reverse primer TCACCGTTATGGTCGCTTGA (SEQ ID NO: 4);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (U) consisting of primer pairs (3) and (10)
Primer pair for amplifying a part of the orf1410 gene sequence (3):
Forward Primer AAAAATAAAACCGCAATCTTATCGT (SEQ ID NO: 5)
Reverse primer ATCACCGTTATGGTCGCTTG (SEQ ID NO: 6);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (V) consisting of primer pairs (3) and (11)
Primer pair for amplifying a part of the orf1410 gene sequence (3):
Forward Primer AAAAATAAAACCGCAATCTTATCGT (SEQ ID NO: 5)
Reverse primer ATCACCGTTATGGTCGCTTG (SEQ ID NO: 6);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

<(I) Primer sets (A) to (P)>
L. amplified by primer pair (4). The sequence of the orf1405 gene region of acidofilus L-92 is shown in SEQ ID NO: 26 and amplified by primer pair (5). The sequence of the orf1405 gene region of acidofilus L-92 is shown in SEQ ID NO: 27 and amplified by primer pair (6). The sequence of the orf174 gene region of acidofilus L-92 is shown in SEQ ID NO: 28 and amplified by primer pair (7). The sequence of the orf174 gene region of acidofilus L-92 is shown in SEQ ID NO: 29 and amplified by primer pair (8). The sequence of the orf1529 gene region of acidofilus L-92 is shown in SEQ ID NO: 30, which is amplified by primer pair (9). The sequence of the orf1529 gene region of acidofilus L-92 is shown in SEQ ID NO: 31 (neither contains the primer region). Then, L. amplified by the primer pair (10). The sequence of the orf1440 gene region of acidofilus L-92 is shown in SEQ ID NO: 32 and amplified by primer pair (11). The sequence of the orf1440 gene region of acidophylus L-92 is shown in SEQ ID NO: 33 (both SEQ ID NOs: 32 and 33 do not include only the reverse primer region).
In addition, the orf1405 gene sequence amplified by the primer pair (4) or (5), the orf174 gene sequence amplified by the primer pair (6) or (7), and the primer pair (8) or ( It is preferable to perform BLAST analysis with a combination of the orf1529 gene sequence amplified by 9) and the orf1440 gene sequence amplified by the primer pair (10) or (11).
Regarding the orf1405 gene sequence, the top 6 bacterial species with high sequence identity were L. Various strains such as DSM 20074 strain and NCFM strain of acidophilus species had a sequence identity of 99-100%, and no other strains or strains having high sequence identity were confirmed. Regarding the orf174 gene sequence, the top 6 bacterial species with high sequence identity were L. Various strains such as DSM 20074 strain and NCFM strain of acidophilus species had a sequence identity of 99-100%, and no other strains or strains having high sequence identity were confirmed. Regarding the orf1529 gene sequence, the top 6 bacterial species with high sequence identity were L. Various strains such as DSM 20074 strain and NCFM strain of acidophilus species, the sequence identity of which is 99-100%, and other than that, L. A crispatus species can be confirmed, but its sequence identity is about 98%. Regarding the orf1440 gene sequence, the top 6 bacterial species with high sequence identity were L. Various strains such as acidophilus DSM 20084 strain and NCFM strain, the sequence identity of which is 99-100%, and other than that, L. amylvorus, L. et al. gasseri, L. et al. Paragasseri and the like can be confirmed at the top, but their sequence identity is about 83-87%.
From the above, the orf1405 gene sequence amplified by the primer pair (4) or (5), the orf174 gene sequence amplified by the primer pair (6) or (7), and the amplification by the primer pair (8) or (9). In the case of BLAST analysis with the combination of the orf1529 gene sequence to be obtained and the orf1440 gene sequence amplified by the primer pair (10) or (11), L. The sequences of the PCR products obtained by any of the above primer sets with respect to the sequences of any of the six strains of the acidophilus species have 99 to 100% sequence identity, but L. It has only about 83 to 98% sequence identity with the sequences of Lactobacillus microorganisms other than the acidophylus species.

L. In order to specifically detect the acidofilus L-92 strain, it is particularly preferable to use a primer set (A) consisting of a primer pair (4), (6), (8) and (10).

For example, RT-PCR was performed on the sample using the primer set (A), and the sequence of the region amplified by the primer pair (4) (excluding the primer sequence) was the same as the sequence of SEQ ID NO: 26, and , The sequence of the region amplified by the primer pair (6) (excluding the primer sequence) is the same as the sequence of SEQ ID NO: 28, and the sequence of the region amplified by the primer pair (8) (excluding the primer sequence) is the sequence. When the sequence of the region amplified by the primer pair (10) (excluding only the reverse primer sequence) is the same as that of SEQ ID NO: 32, the lactic acid bacteria in the sample are L.I. It can be determined to be the acidofilus L-92 strain. Further, for example, the sequence of the region amplified by the primer pair (6) (excluding the primer sequence) is bound downstream of the sequence of the region amplified by the primer pair (4) (excluding the primer sequence), and further downstream thereof. Same using a fusion sequence in which the sequence of the region amplified by the primer pair (8) (excluding the primer sequence) and the sequence of the region amplified by the primer pair (10) (excluding only the reverse primer sequence) are bound. Gender may be determined.

Even when the primer sets (B) to (P) are used, the lactic acid bacteria in the sample can be depleted by the same operation as when the primer set (A) is used. It can be determined to be the acidofilus L-92 strain.
In PCR, it is preferable that each primer pair is carried out in a separate container.

For example, Accession No. of Table 2 below, which is registered in GeneBank. Download the sequence and L. The length of the gene sequence of the region amplified by the primer pairs (4) to (11) used in the present invention is combined with the length of the gene sequence of the region that can be amplified by the above primer set in each strain of acidophilus species. Can be adjusted.

In addition, for example, Accession No. of Table 2 below, which is registered in GeneBank. Download the sequence and L. In each strain of acidophilus species, the length of the gene sequence of the region that can be amplified by any of the above primer sets (A) to (P) is matched, and the length is matched after the orf1405 gene sequence for each strain. The orf174 gene sequence is connected, and the orf1529 gene sequence and the orf1440 gene sequence are connected after that, processed into one gene sequence for each strain to create comparative sequence data, and sequence analysis software such as MEGA is used. It is possible to create a phylogenetic tree by performing phylogenetic tree analysis (see FIG. 2). L. The sequences of various strains of acidophilus species can be obtained from the following accession numbers. By this phylogenetic tree analysis, L. It is shown that it is possible to identify any of the various strains of the acidofilus species.

<(Ii) Primer set (Q)-(V)>
RT-PCR was performed on the sample using any of the primer sets (Q) to (V), and the cells contained were L. It is an acidofilus species, and the strain in it is L. Whether it is acidofilus L-92 can be specifically detected.

L. amplified by primer pair (1). The sequence of the orf466 gene region of acidofilus L-92 is shown in SEQ ID NO: 23 and amplified by primer pair (2). The sequence of the orf1410 gene region of acidofilus L-92 is shown in SEQ ID NO: 24 and amplified by primer pair (3). The sequence of the orf1410 gene region of acidophilus L-92 is shown in SEQ ID NO: 25 (neither contains the primer region). Then, L. amplified by the primer pair (10). The sequence of the orf1440 gene region of acidofilus L-92 is shown in SEQ ID NO: 32 and amplified by primer pair (11). The sequence of the orf1440 gene region of acidophilus L-92 is shown in SEQ ID NO: 33 (for SEQ ID NOs: 32 and 33, only the reverse primer region is not included).
Further, according to the method described later, the orf466 gene sequence amplified by the primer pair (1) or the orf1410 gene sequence amplified by the primer pair (2) or (3) and the primer pair (10) or (11). It is preferable to perform BLAST analysis in combination with the orf1440 gene sequence to be obtained.

For example, RT-PCR was performed on the sample using the primer set (Q), and the sequence of the region amplified by the primer pair (1) (excluding the primer sequence) was the same as the sequence of SEQ ID NO: 23, and , When the sequence of the region amplified by the primer pair (10) (excluding only the reverse primer sequence) is the same as the sequence of SEQ ID NO: 32, the lactic acid bacteria in the sample are L.I. It can be determined to be the acidofilus L-92 strain.
Here, as a result of BLAST analysis on the sequence of the amplified region, the top 6 bacterial species having high sequence identity with the orf466 gene sequence obtained by the primer pair (1) were L. It is an acidofilus species, and its sequence identity is 99-100%. In addition to that, L. Although the crispatus species was confirmed, its sequence identity was about 85%. From the above, the analysis sequence is L. It is possible to discriminate that it is a gene of the acidofilus species.

In addition, as a result of BLAST analysis on the sequence of the amplified region, the top 6 bacterial species having high sequence identity with the orf1440 gene sequence obtained by the primer pair (10) were all L. It is an acidofilus species, and its sequence identity is 99-100%. From the above, the analysis sequence is L. It is possible to discriminate that it is a gene of the acidofilus species.

Even when the primer sets (R) to (V) are used, the lactic acid bacteria in the sample can be depleted by the same operation as when the primer set (Q) is used. It can be determined to be the acidofilus L-92 strain.
In PCR, it is preferable that each primer pair is carried out in a separate container.

In another aspect of the invention, a sequence of regions amplified by another set of primer pairs in the same primer set is attached downstream of the sequence of regions amplified by one set of primer pairs in each primer set. A fusion gene sequence can be prepared in silico and this sequence can be compared to a fusion gene sequence similarly prepared using the same primer pair for the detection target strain or strain. When preparing the fusion gene sequence, it is preferable to remove the primer sequence because the primer sequences are common.
For example, L. Use the primer pair (10) downstream of the length-adjusted sequence in the sequence amplified using the primer pair (1) with the acidophilus L-92 strain as the detection target strain. Using a fusion gene sequence created by in silico binding of a sequence whose length has been adjusted for comparison with other sequences in the amplified sequence, for example, using sequence analysis software such as MEGA6 with a Neibourd Joining Method. It is also possible to perform phylogenetic tree analysis. L. FIG. 2 shows a phylogenetic tree in which sequences are amplified by primer pairs (1) and (10) for each strain of acidofilus. As can be seen from the phylogenetic tree shown in FIG. 2, the sequence of the L-92 strain is different from that of the GeneBank registered strain, and it can be confirmed that the L-92 strain is the L-92 strain. It can be confirmed that the strain is concerned.
Further, as shown in FIG. 5, in the sequence of the region amplified by the primer pairs (1) and (11) of each strain, different base portions are present between the L-92 strain and the other strains. From this, it is shown that the L-92 strain can be confirmed from the sequences amplified by the primer pairs (1) and (11). In addition, SEQ ID NO: 23 is L.I. The regions (excluding the region of the primer sequence) amplified by the primer pair (1) in the acidofilus L-92 strain, and SEQ ID NOs: 34 to 38 are L. acidofilus FSI4, L .; acidofilus LA1, L .; acidofilus ATCC 53544, L. et al. acidofilus La-14 and L. It is a region (excluding the region of the primer sequence) amplified by the primer pair (1) in acidophylus NCFM. In addition, SEQ ID NO: 33 is L.I. The regions (excluding the region of the reverse primer sequence) amplified by the primer pair (11) in the acidophilus L-92 strain, and SEQ ID NOs: 39 to 43 are L. acidofilus FSI4, L .; acidofilus LA1, L .; acidofilus ATCC 53544, L. et al. acidofilus La-14 and L. It is a region (excluding the region of the reverse primer sequence) amplified by the primer pair (11) in acidophylus NCFM.

According to the primer set (Q) consisting of the primer pair (1) and (10), the cells contained in the sample are L.I. A kit consisting of the primer set is also included in the present invention because it can be clearly detected whether or not it is an acidofilus L-92 strain. The region amplified by the primer pairs (1) and (10) is very small, 100 to 200 bp, and targets the RNA portion that is likely to remain in the sample in large quantities, so RT-PCR is successful with a high probability. Can be made to. In addition, L. In the comparison between the strains of acidofilus, the region specific to the L-92 strain was targeted, and therefore, the L-92 strain was clearly targeted. It is possible to determine whether or not the acidofilus L-92 strain is contained in the sample. In addition, the kit according to any of the above primer sets (R) to (V) is also included in the present invention.

L. can be specifically detected by the present invention. The acidofilus L-92 strain was released on March 3, 1994, at the Institute of Biotechnology and Industrial Technology, Faculty of Industrial Technology, Ministry of International Trade and Industry (Address: 1-1-3 Higashi, Tsukuba City, Ibaraki Prefecture, Japan (Zip Code). It was deposited domestically in 305)) and was given the deposit number: Microtechnical Research Institute Bacterial Deposit No. P-14205, and then transferred to the international deposit at the same institution on January 25, 1995. Therefore, the accession number FERM BP-4941 was assigned. This deposited strain is currently available at the NITE Patented Biological Deposit Center (NITE-IPOD) of the National Institute of Technology and Evaluation (NITE-IPOD) (Room 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan, Room 120 (Postal code 292-0818)). It is stored at.

In the method of the present invention, RT-PCR can be performed according to methods well known to those skilled in the art. That is, RNA is extracted from a sample according to a conventional method, cDNA is synthesized using RNA as a template using an oligo dT primer or the like according to a conventional method, and PCR (polymerase chain reaction) is performed according to a conventional method using any of the above primer sets. )It can be performed. As the PCR conditions, standard conditions derived from the Tm value of the primer, the length of the region to be amplified, the sequence, and the like can be used. PCR with two sets of primer pairs included in the primer set (each primer pair consists of a forward primer and a reverse primer) can be performed in the same container, but the primers need to be sequenced individually for the amplification products. It is preferable to carry out PCR in separate containers for each pair. The resulting amplification product is electrophoresed to confirm that fragments of the expected length are amplified, the DNA fragments are extracted from the gel according to conventional methods and sequenced by methods well known in the art. do.
Various methods, reagents, kits and devices required for these operations are also well known to those skilled in the art for performing RNA extraction, cDNA synthesis, PCR, extraction of DNA fragments from gels, sequencing, and the like. , Also readily available commercially.

The sequence of the obtained amplification product and L. acidofilus species or L. Although comparisons with sequences derived from the acidophilus L-92 strain (for example, orf sequences) can be performed visually, various algorithms and programs for sequence comparison are commercially available, and tools such as BLASTN are also available at NCBI and EMBL. And it is provided by the DDBJ Center and is widely available to the public. When using such an algorithm or program, it would be advantageous to compare the above-mentioned fusion gene sequences because the sequence identity can be determined in one analysis.
In addition, L. acidofilus species or L. The acidofilus L-92 strain is another lactic acid bacterium species or L. A phylogenetic tree can be prepared using the created fusion gene sequence in order to make it easier to understand that it is different from other strains of the acidofilus species. Algorithms and specific programs for creating a phylogenetic tree are well known to those skilled in the art and are commercially available or publicly available. For example, free software such as MEGA and Clustal W for alignment and phylogenetic tree creation provided by NCBI, EMBL and DDBJ Centers are available.

When it is considered that the lactic acid bacteria in the sample are essentially killed by sterilization or the like, particularly sterilization and / or long-term storage (for example, at 4 ° C to 35 ° C for 1 month or more or 3 months or more, preferably 3 months or more). Lactic acid bacteria in the sample are killed and the nucleic acid derived from the lactic acid bacteria is considerably damaged by storage under 3 to 6 months, particularly preferably 3 to 12 months, or equivalent conditions (for example, accelerated test conditions). According to the present invention, L. acidofilus species or L. The acidofilus L-92 strain can be specifically detected. In the present invention, when the sample is a beverage or food, it is generally stored for a period from production to expiration date, a period from production to expiration date, or even when stored for a period exceeding these. acidofilus species or L. The acidofilus L-92 strain can be specifically detected.

According to the present invention, L. Other lactic acid bacteria that can be distinguished from the acidophylus L-92 strain and L. Other strains of the acidofilus species include, but are not limited to, for example: the following lactic acid bacteria:
L. acidofilus (ATCC314, ATCC4356, ATCC9224, ATCC53544, ATCC4357);
L. amylovorus (ATCC 33620, ATCC 33621, LMG18188, ATCC33622, ATCC53671, LMG18192, LMG18179, LMG18190, ATCC33198, LMG18180, LMG18186);
L. crispatus (ATCC53545, ATCC55221, JCM5810, LMG11415, LMG17753, ATCC33820, LMG11440, LMG18187, LMG1203, LMG1205, LMG18189);
L. Gallinarum (LMG14751, LMG18181, ATCC53373, LMG14752, LMG14753, LMG14754, LMG14755, ATCC33199);
L. acidofilus (ATCC 4963, ATCC 19992, LMG 11413, ATCC 29601, ATCC 9857, ATCC 4962, ATCC 33323, LMG 10771, LMG 18176);
L. jonsoni (LMG18175, LMG18184, LMG18193, NCC533, ATCC332, ATCC33200, LMG18204, LMG18195).

Specific embodiments of the present invention will be described for the purpose of making the present invention easier to understand.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto, and various changes and modifications are made without departing from the scope of the present invention. May be good.
In the following examples and comparative examples, sequence samples were obtained by the following experimental procedure.

<Experimental procedure>
1. 1. Collecting bacteria and cleaning the sample The PET bottle containing the sample beverage as a sample was turned upside down several times before opening and mixed well, and it was confirmed that no precipitate remained at the bottom of the PET bottle. Next, the PET bottle was opened, 10 mL of the sample beverage solution was collected in a 15 mL sterile disposable plastic tube, and centrifuged at 6000 rpm (4830 × g) for 10 minutes. After discarding the supernatant of the centrifuged sample beverage, 10 mL of sterile water was placed in a tube and resuspended. The resuspension was centrifuged at 6000 rpm (4830 xg) for 10 minutes and the supernatant was discarded. Since there are necessary bacterial cells on the lower side of the precipitate and impurities such as milk protein on the upper side, add 5 mL of sterilized water and add only the impurities such as milk protein on the upper side so as not to break the pellets of the bacteria. The supernatant was lightly suspended and the supernatant was discarded, and this washing operation was repeated twice. The bacterial precipitate was resuspended in 1 mL of sterile water and collected in a sterilized 1.5 mL tube. Then, the resuspension of the bacterial precipitate was centrifuged at 14,000 rpm (19,000 × g) for 2 minutes, 1 mL of sterilized water was added, and the upper part of the pellet was washed to prepare a sample for RNA extraction. ..

2. RNA extraction The sample for RNA extraction obtained in 1 above was suspended in 350 μl of Lysis Buffer (LBP) and dissolved well. Then, the DNA was removed using the DNA removal column of the kit. RNA extraction was performed according to the manual of the Nucleospin RNA plus kit to obtain an RNA solution.

3. 3. Amplification of target gene region by RT-PCR From the RNA solution prepared in 2 above, PrimeScript ^TM One Step RT-PCR Kit Ver. 2 was used to amplify the gene region of interest.
A 10 pmol / μl solution of each primer contained in each primer set used in each example was prepared. For each primer, the required sequence was synthesized by requesting synthesis (50 pmol / l) with a PCReady primer manufactured by Eurofin Genomics, and diluted 5-fold with DNase and RNase-free sterile water.

PrimeScript ^TM One Step RT-PCR Kit Ver. Using No. 2, a solution was prepared according to the following reaction composition, and the following composition was placed in a 200 μl volume tube for PCR and mixed well. In the protocol of the kit, the total amount of the PCR reaction solution composition was specified to be 50 μl, but in order to increase the Template concentration in the reaction solution, the total amount was set to 25 μl, and the total amount of Template RNA was set to 4 μl. Except for the primer and Template RNA (RNA solution prepared in 2 above), the ones included in the kit were used.

PrimeScript 1 step Enzyme Mix 1 μl
2 x 1 step Buffer 12.5 μl
Forward Primer (10 μM) 1 μl
Reverse Primer (10 μM) 1 μl
Template RNA (RNA solution adjusted in 2 above) 4 μl
RNase Free dH ₂ O Balance
Total amount 25 μl
When no amplification product was confirmed in the above composition, the amount of Template RNA was increased to 8 μl, and RT-PCR was performed again.

It was set in the thermal cycler C1000 Touch Thermal Cycler and operated as follows.
STEP1: 50 ° C, 30 minutes,
STEP2: 95 ° C, 2 minutes,
STEP3: 95 ° C, 30 seconds,
STEP4: 55 ° C, 30 seconds,
STEP5: The RT-PCR reaction was carried out under the conditions of 72 ° C. and 30 seconds with STEP3-5 as a total of 40 cycles. The obtained sample was electrophoresed on an agarose gel according to a conventional method to confirm the target band.

4-1. When only the target band is clearly amplified (1) 5 μl of the reaction solution was collected in a new 200 μl PCR tube ^{, 2 μl of ExoSAP-IT TM} PCR Product Cleanap Reagent was added, and the mixture was mixed well with a micropipettor.
(2) Incubation was carried out in a thermal cycler at 50 ° C. for 15 minutes → 85 ° C. for 15 minutes to purify the PCR product.

4-2. When Primer dimer and other amplification products of around 100 bp are confirmed in addition to the target band, purification was performed using Nucleospin Gel and PCR Clean up after electrophoresis.
(1) Electrophoresis was carried out on an agarose gel according to a conventional method, and after confirming the target band, a portion of the target band was cut out from the agarose gel and collected in a sterilized 1.5 ml tube.
(2) Add 200 μl of NTI solution and incubate at 50 ° C. for about 5-10 minutes. The gel was completely dissolved by mixing well with Vortex every 2-3 minutes during the incubation.
(3) Necleospin Gel and PCR Clean-up Volume was set in the attached 2 ml collection tube, and the entire amount of the sample solution dissolved in (2) was placed on a column and centrifuged at 11,000 × g for 30 seconds. The filtrate was discarded and set in the collection tube again.
(4) 700 ml of NT3 solution was applied to a column and centrifuged at 11,000 × g for 30 seconds. Discard the filtrate and set it in the collection tube again. This process was repeated once more.
(5) Centrifugation was carried out at 11,000 × g for 1 minute to remove the NT # solution remaining on the column.
(6) The column was set in a new sterilized 1.5 ml tube, 15 μl of NE solution was added, and the mixture was allowed to stand at room temperature for 1 minute.
(7) Centrifugation was carried out at 11,000 × g for 1 minute, and the column was discarded to obtain a purified PCR product for sequencing.

5. Preparation of Sequence Samples Sequence reactions were performed using both the Bigdy Terminator v1.1 Cycle Sequenceing Kit, the purified PCR product, and the primer pair forward and reverse primers of the primer set for each strain.
(1) 8 μl of the forward and reverse primer 10 pmol / μl solution of the primer pair used for PCR in 3 above was collected in a sterilized 1.5 ml tube, diluted with 92 μl of sterilized water, and 0.8 pmol / μl. It was made into a μl solution.
(2) Using the solution attached to the Bigdy Terminator v1.1 Cycle Sequensing Kit and the PCR product, primer solution, and sterilized water obtained in 4-1 or 4-2, prepare a solution for sequence reaction having the following composition. Prepared in a 200 μl PCR tube. Two samples, one for forward analysis and one for reverse analysis, were prepared for each PCR product.
However, for the PCR product amplified by the primer pair (10) or (11), the following primers shown in SEQ ID NO: 21 were used as the primers for forward analysis.
GTAAAACGACGGCCAGT (SEQ ID NO: 21)

Forward analysis sample purified PCR product 2 μl
Negative Control Water 8 μl
5 x Sequencing Buffer 2 μl
Sequencing RR-100 4 μl
Forward primer (0.8 pmol / μl) 4 μl
20 μl
Reverse analysis Sample purified PCR product 2 μl
Negative Control Water 8 μl
5 x Sequencing Buffer 2 μl
Sequencing RR-100 4 μl
Reverse primer (0.8 pmol / μl) 4 μl
20 μl
(3) Set it in the thermal cycler and
96 ° C, 10 seconds,
50 ° C, 5 seconds,
A total of 25 cycles of reaction were carried out under the conditions of 60 ° C. and 4 minutes.

6. Purification of sequence samples (1) A new sterilized 1.5 mL tube for the number of samples and a Bigdye Xterminator Purification Kit were prepared.
(2) 90 μl of the SAM solution and 20 μl of the Xterminator solution were collected in a tube, and 20 μl of the above sequence reaction product was added.
(3) The mixture was set in a tube shaker, the stirring speed was set to the maximum value, and the mixture was stirred for 30 minutes.
(4) Centrifugation was carried out at 750 × g for 2 minutes, and 20 μl of the supernatant was used as a sample, HiDi Formamide was mixed with 20 μl, and the mixture was subjected to a DNA sequencer to analyze the sequence.

7. Sequence analysis and sequence identity and bacterial species discrimination (1) Consensus sequences were obtained for the obtained sequence forward and reverse sequences using MEGA (free software) sequence analysis software.
(2) The obtained sequence is sequence-identical with the existing DNA data on the NCBI homepage BLASTN (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). Confirmed.
(3) Nucleotide Collection (nr / nt) was selected for the database, and sequence identity was confirmed without setting restrictions on the search target.

8. Strain discrimination by sequence analysis, sequence identity and phylogenetic tree analysis (1) Accession No. 2 shown in Table 2 above registered in GeneBank. The sequence was downloaded, the sequence of each gene was compared with the analyzed gene, and the extra parts before and after were deleted in order to match the sequence and length of the gene analyzed this time.
(2) Using gene sequences of the same length, each amplified orf gene of each strain was connected so as to correspond to each strain, and processed into one gene sequence for each strain.
(3) Regarding the gene sequence created by processing, a phylogenetic tree was analyzed using sequence analysis software such as MEGA to create a phylogenetic tree. As a result of phylogenetic tree analysis, it was found that the L-92 strain had a different sequence from all the strains registered in GeneBank. The phylogenetic trees of FIGS. 1 and 2 are as described above.

[Example 1]
As a sample beverage as a sample, an L92 beverage product (manufactured by Calpis) in a PET bottle stored at 25 ° C. for 6 months was used. For each of the primer pairs (1) to (11), PCR was performed on this sample in separate containers, and the obtained 11 types of PCR products were electrophoresed on an agarose gel according to a conventional method. In addition, the same operation was performed on the PCR products obtained by using the primer pairs (1) to (11) using the RNA of the cultured L-92 cells (positive control). The result is shown in FIG. Each lane in FIG. 3 corresponds as shown in Table 3-1 and Table 3-2 below.

[Example 2]
Sequence samples were prepared using each PCR product obtained in Example 1, and sequence analysis and BLAST analysis of the base sequence were performed.
Then, the sequence of the region amplified by the primer pair (1) (excluding the primer sequence) and the sequence of SEQ ID NO: 23 are the same, and the sequence of the region amplified by the primer pair (2) (primer sequence). (Excluded) and the sequence of SEQ ID NO: 24 are the same, the sequence of the region amplified by the primer pair (3) (excluding the primer sequence) and the sequence of SEQ ID NO: 25 are the same, and the primer pair (4) ) And the sequence of SEQ ID NO: 26 are the same, and the sequence of the region amplified by the primer pair (5) (excluding the primer sequence) and SEQ ID NO: 27 The sequence of the region amplified by the primer pair (6) (excluding the primer sequence) and the sequence of SEQ ID NO: 28 are the same and amplified by the primer pair (7). The sequence (excluding the primer sequence) and the sequence of SEQ ID NO: 29 are the same, and the sequence of the region amplified by the primer pair (8) (excluding the primer sequence) and the sequence of SEQ ID NO: 30 are the same. The sequence of the region amplified by the primer pair (9) (excluding the primer sequence) and the sequence of SEQ ID NO: 31 are the same, and the sequence of the region amplified by the primer pair (10) (only the reverse primer sequence). (Excluded) and the sequence of SEQ ID NO: 32 are the same, and it was confirmed that the sequence of the region amplified by the primer pair (11) (excluding only the reverse primer sequence) and the sequence of SEQ ID NO: 33 are the same. ..
The bacterial species having the relevant sequence included in the sample of Example 1 is L. It has become clear that it is highly likely that it is an acidofilus species.

[Example 3]
<Creation of phylogenetic tree>
L. Nucleotide sequence of PCR product by primer pair (4), base sequence of PCR product by primer pair (6), base sequence of PCR product by primer pair (8), PCR product by primer pair (10) for acidophilus L-92 strain The base sequences of the above were joined together and connected in silico to prepare a fusion gene sequence. Similarly, Accession No. of Table 2 registered in GeneBank. Download the sequence and L. In each strain of acidophilus, the lengths of the gene sequences of the regions that can be amplified by the above primer set were matched, and each base sequence of each strain was connected by in silico to prepare a fusion gene sequence. Then, each strain was processed into one gene sequence to create comparative sequence data, and phylogenetic tree analysis was performed using MEGA6 sequence analysis software to create a phylogenetic tree. The results are shown in FIG. As can be seen from FIG. 1, the DNA sequence obtained from the sample of Example 1 analyzed in this test is L.I. It was 100% consistent with the DNA-derived sequence of the acidofilus L-92 strain, and L. It was found that the sequence was different from the strains containing other strains of acidophilus species, and the strains contained in the sample were L. It was found to be the acidophylus L-92 strain.

[Example 4]
As a sample beverage as a sample, a PET bottled L92 beverage product (manufactured by Asahi Soft Drinks Co., Ltd.) stored at 25 ° C. for 6 months was used. For each of the primer pairs (1) and (10), PCR was performed on this sample in separate containers, and the obtained two types of PCR products were electrophoresed on an agarose gel according to a conventional method. In addition, the same operation was performed on the PCR products obtained by using the primer pairs (1) and (10) using the RNA of the cultured L-92 cells (positive control). This experiment was carried out at n = 2. The result is shown in FIG. Each lane in FIG. 4 corresponds as shown in Table 4-1 and Table 4-2 below.

In addition, more RNA could be extracted from the L-92 beverage product than the cultured cells. For reference, the amount of RNA is shown in Table 5.

For the obtained PCT product, the amplified chain length was 161 bp with the primer pair (1) for amplifying the orf466 gene sequence and 144 bp with the primer pair (10) for amplifying the orf1440 gene sequence.
From the above results, it was found that the DNA sequences obtained by the primer pairs (1) and (10) are relatively short, so that a PCR product can be obtained even in a sample after long-term storage at 25 ° C. ..

[Example 5]
Sequence samples were prepared using each PCR product obtained in Example 4, and sequence analysis and BLAST analysis of the base sequence were performed.
<Orf466 gene>
The top 6 bacterial species with high sequence identity were L. The sequence of acidofilus whose sequence identity could be confirmed was about 150 bp of the analyzed sequence, and the sequence identity was 99-100%. In addition to that, L. Although crispatus was confirmed, its sequence identity was about 85%. From the above, the analysis sequence is L. It was considered to be a gene of the acidofilus species.
<Orf1440 gene>
The top 6 bacterial species with high sequence identity were all L. It was acidofilus, and the sequence identity could be confirmed at 106 bp. Its sequence identity was 99-100%. From the above, the analysis sequence is L. It was considered to be a gene of the acidofilus species. As a result of the above, the strain having the relevant sequence was L. It was considered to be an acidofilus species.
From the above results, the bacterial species having the relevant sequence contained in the sample are L. It has become clear that it is highly likely that it is an acidofilus species.

[Example 6]
<Creation of phylogenetic tree>
L. The fusion gene by connecting the orf1440 gene sequence amplified using the primer pair (10) downstream of the orf466 gene sequence amplified using the primer pair (1) used in Example 4 for the acidophilus L-92 strain with in silico. I created an array. Similarly, L.A. registered in GenBank. Similarly, for each of the five strains including the acidofhilus strain, the orf1440 gene sequence amplified using the primer pair (10) is connected in silico downstream of the orf466 gene sequence amplified using the primer pair (1). The fusion gene sequence was prepared. Using the prepared fusion gene sequence group, a phylogenetic tree was prepared by the Neiberhood-joining method using MEGA6. The results are shown in FIG. As can be seen from FIG. 2, the DNA sequence obtained from the sample analyzed in this test is L.I. It was 100% consistent with the DNA-derived sequence of the acidofilus L-92 strain, and L. It was found that the sequence was different from the other 5 strains of the acidofilus species, and the strains contained in the sample were L. It was revealed that it was an acidophylus L-92 strain.

Claims (7)

A method for detecting the lactic acid bacterium Lactobacillus acidophilus species in a sample as a detection target microorganism, and the following steps:
i) Step of extracting RNA from a sample,
ii) A step of synthesizing cDNA using the RNA extracted in i) as a template.
cDNA as a template synthesized in iii) ii), a step of performing PCR using a primer set consisting of the detection target microorganisms characteristic 5 bp to 4 50 bp of gene region capable of amplifying two or more sets of primer pairs, And iv) Detected in the sample when any of the sequences in the region amplified by each primer pair in the primer set is identical to each of the sequences of the detection target microorganism amplified by the same primer pair. The process of determining that the target microorganism was present,
Only including,
The primer set
-Primer set (A) consisting of primer pairs (4), (6), (8) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (B) consisting of primer pairs (4), (6), (8) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (C) consisting of primer pairs (4), (6), (9) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (D) consisting of primer pairs (4), (6), (9) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (E) consisting of primer pairs (4), (7), (8) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (F) consisting of primer pairs (4), (7), (8) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (G) consisting of primer pairs (4), (7), (9) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (H) consisting of primer pairs (4), (7), (9) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (4):
Forward primer AGCTAAACGTAAACGTATTGAAGAT (SEQ ID NO: 7)
Reverse primer TTCGCCCGGTGCCATATTT (SEQ ID NO: 8);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (I) consisting of primer pairs (5), (6), (8) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (J) consisting of primer pairs (5), (6), (8) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (K) consisting of primer pairs (5), (6), (9) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (L) consisting of primer pairs (5), (6), (9) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (6):
Forward primer TGCGGATGTGAAAGATCGTGA (SEQ ID NO: 11)
Reverse primer ACATCGACATTTCTACGGGCT (SEQ ID NO: 12);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (M) consisting of primer pairs (5), (7), (8) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (N) consisting of primer pairs (5), (7), (8) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (8):
Forward primer GGCTGAAAACTTGGCTCACG (SEQ ID NO: 15)
Reverse primer ACGTAGTAAATAATGAACACCACG (SEQ ID NO: 16);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (O) consisting of primer pairs (5), (7), (9) and (10)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)
and
-Primer set (P) consisting of primer pairs (5), (7), (9) and (11)
Primer pair for amplifying a part of the orf1405 gene sequence (5):
Forward primer TTAGCTAAACGTAAACGTATTGAAG (SEQ ID NO: 9)
Reverse primer TCTGGATCATATTTTTCGCCCG (SEQ ID NO: 10);
Primer pair for amplifying a part of the orf174 gene sequence (7):
Forward primer ACAGATTTAGCTGCGGATGTGA (SEQ ID NO: 13)
Reverse primer ACACTCTTTGCACCACGCTC (SEQ ID NO: 14);
Primer pair for amplifying a part of the orf1529 gene sequence (9):
Forward primer CAGCCCAATCTTGAGCGTCTT (SEQ ID NO: 17)
Reverse primer CACCACGAACAGAATCCACATCA (SEQ ID NO: 18);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
At least one primer set is, before Symbol method selected from the group consisting of reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22). The method of claim 1, wherein the sample is a beverage. Sample is heat sterilization, a 12 months period conserved beverages at 25 ° C., The method of claim 1, wherein. A method for detecting the Lactobacillus acidophilus L-92 strain in a sample.
i) Step of extracting RNA from a sample,
ii) A step of synthesizing cDNA using the RNA extracted in step i) as a template.
iii) A step of performing PCR using the cDNA synthesized in step ii) as a template and at least one primer set selected from the group consisting of the following primer sets (Q) to (V).
-Primer set (Q) consisting of primer pairs (1) and (10)
Primer pair for amplifying a part of the orf466 gene sequence (1):
Forward primer GGGGACCCCAAACAGAAAGA (SEQ ID NO: 1)
Reverse primer GCGTCTTCGATTGCTTCACC (SEQ ID NO: 2);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (R) consisting of primer pairs (1) and (11)
Primer pair for amplifying a part of the orf466 gene sequence (1):
Forward primer GGGGACCCCAAACAGAAAGA (SEQ ID NO: 1)
Reverse primer GCGTCTTCGATTGCTTCACC (SEQ ID NO: 2);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (S) consisting of primer pairs (2) and (10)
Primer pair for amplifying a part of the orf1410 gene sequence (2):
Forward primer CGTGGTCAAAAAATAAACCGCA (SEQ ID NO: 3)
Reverse primer TCACCGTTATGGTCGCTTGA (SEQ ID NO: 4);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (T) consisting of primer pairs (2) and (11)
Primer pair for amplifying a part of the orf1410 gene sequence (2):
Forward primer CGTGGTCAAAAAATAAACCGCA (SEQ ID NO: 3)
Reverse primer TCACCGTTATGGTCGCTTGA (SEQ ID NO: 4);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (U) consisting of primer pairs (3) and (10)
Primer pair for amplifying a part of the orf1410 gene sequence (3):
Forward Primer AAAAATAAAACCGCAATCTTATCGT (SEQ ID NO: 5)
Reverse primer ATCACCGTTATGGTCGCTTG (SEQ ID NO: 6);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)
And ・ Primer set (V) consisting of primer pairs (3) and (11)
Primer pair for amplifying a part of the orf1410 gene sequence (3):
Forward Primer AAAAATAAAACCGCAATCTTATCGT (SEQ ID NO: 5)
Reverse primer ATCACCGTTATGGTCGCTTG (SEQ ID NO: 6);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

and,
iv) The sequence of the region amplified by the primer pair (1) (excluding the primer sequence) and the sequence of SEQ ID NO: 23 are the same, or the sequence of the region amplified by the primer pair (2) (primer). (Excluding the sequence) and the sequence of SEQ ID NO: 24 are the same, or the sequence of the region amplified by the primer pair (3) (excluding the primer sequence) and the sequence of SEQ ID NO: 25 are the same and , The sequence of the region amplified by the primer pair (10) (excluding only the reverse primer sequence) and the sequence of SEQ ID NO: 32 are the same, or the sequence of the region amplified by the primer pair (11) ( When the sequence of SEQ ID NO: 33 (excluding only the reverse primer sequence) and the sequence of SEQ ID NO: 33 are the same, L. The process of determining that the acidofilus L-92 strain was present,
The method described above. In step iii), PCR using the primer pair is respectively carried out in a separate vessel, according to claim 4 Symbol mounting method. L.A., which comprises any of the following primer sets (Q) to (V). acidofilus L-92 strain detection kit:
-Primer set (Q) consisting of primer pairs (1) and (10)
Primer pair for amplifying a part of the orf466 gene sequence (1):
Forward primer GGGGACCCCAAACAGAAAGA (SEQ ID NO: 1)
Reverse primer GCGTCTTCGATTGCTTCACC (SEQ ID NO: 2); and primer pair for amplifying part of the orf1440 gene sequence (10) :.
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20).

-Primer set (R) consisting of primer pairs (1) and (11)
Primer pair for amplifying a part of the orf466 gene sequence (1):
Forward primer GGGGACCCCAAACAGAAAGA (SEQ ID NO: 1)
Reverse primer GCGTCTTCGATTGCTTCACC (SEQ ID NO: 2);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (S) consisting of primer pairs (2) and (10)
Primer pair for amplifying a part of the orf1410 gene sequence (2):
Forward primer CGTGGTCAAAAAATAAACCGCA (SEQ ID NO: 3)
Reverse primer TCACCGTTATGGTCGCTTGA (SEQ ID NO: 4);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (T) consisting of primer pairs (2) and (11)
Primer pair for amplifying a part of the orf1410 gene sequence (2):
Forward primer CGTGGTCAAAAAATAAACCGCA (SEQ ID NO: 3)
Reverse primer TCACCGTTATGGTCGCTTGA (SEQ ID NO: 4);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22)

-Primer set (U) consisting of primer pairs (3) and (10)
Primer pair for amplifying a part of the orf1410 gene sequence (3):
Forward Primer AAAAATAAAACCGCAATCTTATCGT (SEQ ID NO: 5)
Reverse primer ATCACCGTTATGGTCGCTTG (SEQ ID NO: 6);
Primer pair for amplifying a part of the orf1440 gene sequence (10):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20)

-Primer set (V) consisting of primer pairs (3) and (11)
Primer pair for amplifying a part of the orf1410 gene sequence (3):
Forward Primer AAAAATAAAACCGCAATCTTATCGT (SEQ ID NO: 5)
Reverse primer ATCACCGTTATGGTCGCTTG (SEQ ID NO: 6);
Primer pair for amplifying a part of the orf1440 gene sequence (11):
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer TGCCATACCCGATGCAACTAC (SEQ ID NO: 22) Primer set (Q) consisting of the following primer pairs (1) and (10):
Primer pair for amplifying a part of the orf466 gene sequence (1):
Forward primer GGGGACCCCAAACAGAAAGA (SEQ ID NO: 1)
Reverse primer GCGTCTTCGATTGCTTCACC (SEQ ID NO: 2); and primer pair for amplifying part of the orf1440 gene sequence (10) :.
Forward primer GTAAAACGACGGCCAGTATGTCAGCAAT (SEQ ID NO: 19)
Reverse primer ATTTAAAGGATGAACTACCTG (SEQ ID NO: 20).

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