KR20040077544A - Method for identification of microorganism of Lactobacillus species - Google Patents

Abstract

PURPOSE: A method for identification of a microorganism of Lactobacillus species is provided, thereby easily, rapidly and accurately identifying the probiotic Lactobacillus species using Lactobacillus species specific primers and Lactobacillus species common primers. CONSTITUTION: The method for identification of a microorganism of Lactobacillus species comprises the steps of: (1) PCR amplifying DNA of a testing microorganism as a template using one or more of Lactobacillus species specific primers and one or more of Lactobacillus species common primers; and (2) analyzing the amplification and the size of PCR products, wherein the Lactobacillus species specific primer has the nucleotide sequence selected from SEQ ID NO:1 to SEQ ID NO:8; the Lactobacillus species common primer has the nucleotide sequence selected from SEQ ID NO: 9 and SEQ ID NO:10; and the Lactobacillus species is selected from Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus gasseri, Lactobacillus plantarum and Lactobacillus reuteri.

Description

Method for identification of microorganism of Lactobacillus species

The present invention relates to a method for identifying Lactobacillus spp., And more particularly, to detect and identify Lactobacillus spp. In a test subject using a primer specific for the Lactobacillus spp. And a primer common to the Lactobacillus spp. It is about a method.

Although the formal effects of Lactobacillus bulgaricus and L. acidophilus isolated from yogurt have been recognized, interest in lactic acid bacteria has increased and research has been continuously conducted. Research on lactic acid bacteria is still insignificant. Since long ago, various lactic acid bacteria have been separated from dairy products and cheese in various countries in Europe, and since the development of Bifidobacterium bifidum in Japan, lactic acid bacteria have been widely used as various medicines and functional foods. Recently, since the successful commercialization of Lactobacillus rhamnosus GG, which has excellent intestinal fixation and acid resistance, research on lactic acid bacteria has been actively conducted all over the world, and it has excellent anti-cancer effects by preventing adult diseases and enhancing immunity. There is a growing interest in the isolation and development of functional probiotic lactic acid bacteria. Probiotic lactobacillus is used in fermented milk, baby food, dairy products, livestock farm feed, cosmetics, dietary supplements, formal medicines, capsules, etc. Probiotic lactobacillus development technology is developed in a technology-intensive manner, and the scale of these products is continuously It is expected to increase. The domestic situation is also the same. Based on the fermented milk market, which relies on imports, most of the strains are estimated to be KRW 930 billion in 2001, with a steady growth rate of 10% annually. The marketability of products using biotic lactic acid strains is infinite (McCann et al. , J. Food.Pretect., 1996, 59, 41-45).

Lactobacillus is a bacterium that has both homo lactic fermentation and hetero lactic fermentation, and is an anaerobic fungal species found in various isolates such as animals, plants and fermented milk products. Lactobacillus has an antibacterial effect caused by the formation of organic acids such as lactic acid and acetic acid and the resulting pH drop.₂O₂It is known to inhibit the growth of food spoilage microorganisms and other pathogenic microorganisms by the production of antimicrobial metabolites such as and diacetyl and bacteriocin, a variety of proteinaceous polymers. Also widely used as. In addition, the organic acid produced by the fermentation of Lactobacillus is not only a food additive to prevent corruption, but also polylactic acid (acetealdehyde) and acrylic acid (acrylic acid), which is a raw material for surgical sewing It is used as a material of various synthetic materials such as 2,3-pentadione.et al., Appl. Environ. Microbiol.,1999, 65, 4211-4215).

There are about 90 species of genus Lactobacillus so far known, and only a few are currently commercialized as probiotic strains. Among them, L. acidophilus , L. bulgaricus ; L. delbrueckii subsp.bulgaricus , L. casei , L. gasseri , L. gasseri , L. paracasei , L. plantarum , L. plantarum , L. rhamnosus ( L. casei subsp. Rhamnosus ), L. reuteri , etc. Occupies most of it. In addition, L. helveticus , L. johnsonii ( L. acidophilus subsp.johnsonii ), and L. fermentum ( L. fermentum ) have recently been developed as functional probiotic strains. However, they belong to species similar to the existing probiotic species L. acidophilus ( L. acidophilus ), L. plantarum ( L. plantarum ).

For the phylogenetic classification of Lactobacillus, physiological characteristics tests including microscopic findings, gram staining, catalase testing, growth testing, sugar availability, and API test kits, which encompass them, have been used. However, such a classification method has a limitation of the database, has a poor reproducibility, and leads to many ambiguous identification results. Therefore, a method for specifically identifying a desired strain is urgently required.

Molecular biological identification and characterization methods have begun to overcome these problems (Tenover et al., J. Clin. Microbiol., 1995, 33 (9), 2233-2239, 1995), and recently PCR and Various electrophoretic methods have been reported (Walter et al. , Appl. Environ. Microbiol., 2001, 67 (6), 2578-2585). Among these, a lot of research is being conducted on randomly amplified polymorphic DNA (RAPD) analysis, which is a kind of fingerprinting technique using random primers consisting of about 10 sequences (Cusick et. al ., Appl. Environ.Microbiol . , 2001, 66 (5), 2227-2231). The fingerprint pattern obtained using RAPD is uniquely expressed according to genomic DNA, which allows the isolation, identification and characterization of microorganisms (Kaufman et al. , Appl. Environ. Microbiol., 1997) . , 65 (4), 1268-1273). However, studies on the reproducibility problem according to PCR conditions and the lack of suitable primers for comparison between genus, species and substrains are still underway (Wang et al. , Appl. Environ. Microbiol., 1996, 62 (4), 1241-1247). Recently, PCR using species-specific primers (Species specific PCR, abbreviated as "SS-PCR") has been used. This method uses the fact that certain regions of the genomic genes differ in species sequence. Gene amplification using species-specific primers allows the production of PCR products specific to specific species (Matsuki et al. , Appl. Environ. Microbiol., 1999, 65 (10), 4506-4512). Many of the major target genes of SS-PCR have been studied in the interspacial region between 16S, 23S rRNA and 16S-23S rRNA, and their sequencing information has been published in various web-based databases (Naimi et al. , Microbiol., 1997, 143 (6), 823-834). It has been reported that species variation in lactic acid bacteria including Enterococcus and Lactobacillus can be classified by using mutation regions within these sequences (Angeletti et al. , J. Clin. Microbiol., 2001, 39 (2), 794-797; Walter et al. , Appl. Environ.Microbiol ., 2000, 66 (1), 297-303). In addition, multiple PCR (multiplex PCR) techniques for quickly and accurately checking the presence or absence of desired strains or classifying and identifying strains by mixing SS-PCR primers of desired species in a mixed state such as intestinal environment are also used. Much has been reported recently (Lee, et al. , FEMS Microbiol. Letters., 2000, 193, 243-47).

However, even with multiple PCR methods based on 16S rRNA and 23S rRNA, which are known to date, the use of a single region is limited due to the limitation of the species-specific mutation region in the 16S rRNA, the lack of information on the 23S rRNA region, and the limitation of the sequence length. The classification and identification of many Lactobacillus species via PCR reactions is impossible (Song, et al., FEMS Microbiol. Letters. , 2000, 187, 167-173).

Thus, the inventors of the present invention, while using the PCR technique using a species-specific primer to study the probiotic Lactobacillus strains to accurately and easily, based on the sequence data of various species based on 16S rRNA, 23S rRNA or 16S Base sequences comprising the region between the rRNA and 23S rRNA were used to obtain species common and species specific sequences of the genus Probiotic Lactobacillus, and PCR products of various sizes ranging from 180 bp to 1090 bp could be generated. Multiple PCR primer sets were prepared. In the case of using this primer set, the present invention was completed by confirming that the species of the genus Probiotic Lactobacillus can be identified easily and accurately through a single PCR reaction.

It is an object of the present invention to provide a primer set forth in SEQ ID NO: 1 to SEQ ID NO: 10.

Another object of the present invention is one or more primers selected from primers having a nucleotide sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 8 and one or more primers selected from primers having a nucleotide sequence set forth in SEQ ID NO: 9 and SEQ ID NO: 10 It is to provide a method for detecting and identifying Lactobacillus spp.

Figure 1a is a diagram showing the design of the genus Lactobacillus species specific primers and common primers and the size of the expected PCR amplification products.

Reu: L. reuteri species specific primer attachment site,

Cas: Lactobacillus casi strain species specific primer attachment position,

Bul: L. delbrueckii subsp.bulgaricus species-specific primer attachment site,

Gas: L. gasseri species specific primer attachment position,

Aci: L. acidophilus species specific primer attachment site,

Par: L. paracasei species species specific primer attachment site,

Pla: Lactobacillus plantarum species specific primer attachment site,

Rha: L. casei subsp.rhamnosus species specific primer attachment site,

REV: Lactobacillus spp. Common primer attachment site as set forth in SEQ ID NO: 9, and

FOR: Lactobacillus spp. Common primer attachment site as set forth in SEQ ID NO: 10

Figure 1b is an electrophoresis picture of the chromosomal DNA of the genus Lactobacillus standard PCR using a primer combination described in SEQ ID NO: 1 to SEQ ID NO: 10.

Aci: Lactobacillus ashdophyllus, Bul: Lactobacillus bulgaricus,

Cas: Lactobacillus casei, Gas: Lactobacillus gasteria,

Hel: Lactobacillus helveticus, Pla: Lactobacillus plantarum,

Reu: Lactobacillus lusteri, Par: Lactobacillus paracazei,

Rha: Lactobacillus rhamnosus,

9-1 (0.6 kb): Production of a PCR amplification product having a size corresponding to 0.60 kb obtained by PCR using the primers set forth in SEQ ID NO: 9 and SEQ ID NO: 1,

2-10 (0.18 kb): Production of a PCR amplification product having a size corresponding to 0.18 kb obtained by PCR using the primers set forth in SEQ ID NO: 2 and SEQ ID NO: 10,

3-10 (0.71 kb): Production of a PCR amplification product having a size corresponding to 0.71 kb obtained by PCR using the primers set forth in SEQ ID NO: 3 and SEQ ID NO: 10,

9-4 (0.28 kb): Production of a PCR amplification product of a size corresponding to 0.28 kb obtained by PCR using the primers set forth in SEQ ID NO: 9 and SEQ ID NO: 4,

9-6 (0.43 kb): Production of a PCR amplification product having a size corresponding to 0.43 kb obtained by PCR using the primers set forth in SEQ ID NO: 9 and SEQ ID NO: 6,

8-10 (1.09 kb): Production of a PCR amplification product having a size corresponding to 1.09 kb obtained by PCR using the primers set forth in SEQ ID NO: 8 and SEQ ID NO: 10,

9-5 (0.65 kb): Production result of a PCR amplification product of a size corresponding to 0.65 kb obtained by PCR using the primers set forth in SEQ ID NO: 9 and SEQ ID NO: 5, and

9-7 (0.45 kb): Production of PCR amplification products having a size corresponding to 0.45 kb obtained by PCR using the primers set forth in SEQ ID NO: 9 and SEQ ID NO: 7

FIG. 2 is an electrophoretic photograph showing the results of multiple PCR reactions using chromosomal DNA of eight strains of Lactobacillus genus and primer mixtures set forth in SEQ ID NO: 1 to SEQ ID NO: 10. FIG.

Lane 1: Lactobacillus ashdophilus, lane 2: Lactobacillus bulgaricus,

Lane 3: Lactobacillus kazei, lane 4: Lactobacillus gaseri,

Lane 5: Lactobacillus plantarum, lane 6: Lactobacillus lusteri,

Lane 7: Lactobacillus rhamnosus, lane 8: Lactobacillus paracazei,

Lane M: 100 bp DNA size marker.

Figure 3 is an electrophoresis picture showing the results of multiple PCR reactions using an unevenly combined mixture of eight standard strain colonies of the genus Lactobacillus and primer mixtures described in SEQ ID NO: 1 to SEQ ID NO: 10.

Lane 1: Lactobacillus ashidophilus, Lactobacillus bulgaricus, Lactobacillus cassia, Lactobacillus gaseri, Lactobacillus plantarum and Lactobacillus luterie,

Lane M: 100 bp DNA size marker,

Lane 2: Lactobacillus casei,

Lane 3: Lactobacillus plantarum,

Lane 4: Lactobacillus ashidophilus and Lactobacillus paracazei,

Lane 5: Lactobacillus ashidophilus, Lactobacillus rhamnosus and Lactobacillus vulgaris,

Lane 6: Lactobacillus bulgaricus,

Lane 7: Lactobacillus gaseri,

Lane 8: Lactobacillus luster and Lactobacillus plantarum

Figure 4 is a schematic diagram showing the location of the species in the Phylogenetic tree of the strain isolated from the probiotic Lactobacillus strain containing products.

In order to achieve the above object, the present invention provides a primer described in SEQ ID NO: 1 to SEQ ID NO: 10.

In addition, the present invention uses one or more primers selected from primers having a nucleotide sequence represented by SEQ ID NO: 1 to SEQ ID NO: 8 and one or more primers selected from primers having a nucleotide sequence represented by SEQ ID NO: 9 and SEQ ID NO: 10 Provided is a method for detecting and identifying Lactobacillus spp. In a test subject through a PCR method.

Hereinafter, the present invention will be described in detail.

The present invention provides primers set forth in SEQ ID NO: 1 to SEQ ID NO: 10.

The primer of the present invention was prepared using a 16S rRNA, 23S rRNA or a region between 16S rRNA and 23S rRNA of the genus Lactobacillus, and is a nucleotide sequence of a specific portion of the gene or a nucleotide sequence homologous thereto. The primers described in SEQ ID NO: 1 to SEQ ID NO: 8 are genus specific primers of the genus Lactobacillus, and the primers described in SEQ ID NO: 9 and SEQ ID NO: 10 are primers common to the genus Lactobacillus.

The primer described in SEQ ID NO: 1 corresponds to the 23S rRNA gene sequence of the L. acidophilus strain, and in a preferred embodiment of the present invention, the 23S rRNA gene of the strain of the genus Lactobacillus ashidophilus. It was prepared using the nucleotide sequence (GI: 22128340, 6537241, 1771113, 1771109, etc.).

The primer set forth in SEQ ID NO: 2 corresponds to the 16S rRNA gene sequence of the L. bulgaricus strain, and in a preferred embodiment of the present invention, the Lactobacillus vulgaris 16S rRNA gene sequence (GI: 15982695, 15982694, 15982693, 2588893, 43981 and 175022, etc.).

The primer set forth in SEQ ID NO: 3 corresponds to the 16S rRNA gene sequences of the Lactobacillus casei , Lactobacillus paracasei and L. rhamnosus strains. In a preferred embodiment of the present invention, Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus 16S rRNA gene sequences (GI: 14583099, 1848057, 1848056, 1848055, 1848054, 1848053, 1848052, 1843428, 1843427, 20257220, respectively). , 12382750, 6996495, 43970, 175023, 7621525, 7621504, 1808583, 7159888, 7159887, 6996530, 7621503 and the like; GI: 6552383, 6537239 and the like; GI: 20086370, 6537244, 6815814 and the like).

The primer set forth in SEQ ID NO: 4 corresponds to the nucleotide sequence between the 16S rRNA gene and the 23S rRNA gene of the L. gasseri strain, and in a preferred embodiment of the present invention, the Lactobacillus sarycin 16S rRNA gene and It was prepared using the 23S rRNA interregion nucleotide sequence (GI: 6537236, etc.).

The primer set forth in SEQ ID NO: 5 corresponds to the 23S rRNA gene sequence of the L. paracasei strain , and in a preferred embodiment of the present invention, the Lactobacillus paracasei 23S rRNA gene sequence (GI: 6552383, 6537239, etc.).

The primer set forth in SEQ ID NO: 6 corresponds to the 23S rRNA gene sequence of the L. plantarum strain , and in a preferred embodiment of the present invention, the Lactobacillus plantarum 23S rRNA gene sequence (GI: 6537237, 20086379, 20086378, 20086376, 20086375, 20086372, 3046893, 2738840, 3420786, etc.).

The primer set forth in SEQ ID NO: 7 corresponds to the 23S rRNA gene sequence of the L. rhamnosus strain, and in a preferred embodiment of the present invention, the Lactobacillus rhamnosus 23S rRNA gene sequence (GI: 20086370, 6537244, 6815814, etc.).

The primer set forth in SEQ ID NO: 8 corresponds to the 16S rRNA gene sequence of the L. reuteri strain, and in a preferred embodiment of the present invention, the Lactobacillus luterie 16S rRNA gene sequence (GI: 3420786, 388037, etc.). Was prepared using).

The primer set forth in SEQ ID NO: 9 corresponds to the 16S rRNA gene sequence of the genus Lactobacillus, and in a preferred embodiment of the present invention, the 16S rRNA of the eight types of probiotic Lactobacillus genus described above It was prepared using the gene sequence.

The primer set forth in SEQ ID NO: 10 corresponds to the 16S rRNA gene sequence of the genus Lactobacillus, and in a preferred embodiment of the present invention, the 16S rRNA gene sequence of the eight probiotic Lactobacillus genus described above It was prepared using.

In addition, the present invention is Lactobacillus having a base sequence described in SEQ ID NO: 9 and SEQ ID NO: 10 and at least one primer selected from the group of Lactobacillus genus specific primer having a nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 8 Provided is a method for identifying a genus Lactobacillus through multiplex PCR (multiplex PCR) method using one or more primers selected from the group of common genus.

The method of identifying the genus Lactobacillus of the present invention is

1) Lactobacillus having a nucleotide sequence set forth in SEQ ID NO: 9 and SEQ ID NO: 10 and at least one primer selected from the group of Lactobacillus genus specific primers having the test sequence and the nucleotide sequence described in SEQ ID NO: 1 to SEQ ID NO: 8 Performing multiple PCR reactions using one or more primers selected from the group of genus species common primers; And

2) measuring the amplification of the PCR product and the size of the amplification product through step 1.

In step 1, the test sample may directly use a part of a sample containing a strain of the genus Lactobacillus as a test sample, using colonies obtained by culturing in a suitable medium or chromosomal DNA isolated from the colonies. . The sample may be selected from the group consisting of fermented milk, baby food, dairy products, livestock farm feed, cosmetics, dietary supplements, formal medicines, and capsules containing Lactobacillus strains, but are not necessarily limited thereto. Chromosomal DNA can be separated from colonies by known DNA extraction methods, and when used as a template for PCR reaction, it is preferable to use at a concentration of about 0.01 μg or more. In addition, in order to use as a PCR reaction template, the colony of the strain is preferably used at a concentration of 1 × 10 ⁵ cfu or more, and when the sample is directly used as a test sample, a simple treatment from a suspension diluted appropriately to contain the same colony or more as described above. Cells obtained through the use can also be used. In addition, the chromosomal DNA of the strain used as a test body or the colony of the strain can be used alone or in combination thereof. PCR reactions are carried out according to conventional methods.

In step 2, the amplification of the PCR product and the size of the amplification product can be measured by a conventional method, for example, gel electrophoresis. Since the primers prepared in the present invention are prepared to form PCR amplification products of different sizes for each Lactobacillus spp., Comparing the sizes of the products amplified by the PCR reaction, various probiotic lactobacillus contained in the test body. Genus strains can be identified simultaneously.

That is, Lactobacillus vulgaris, SEQ ID NO: 4 and SEQ ID NO using the primer pairs described in Lactobacillus ashidophilus, SEQ ID NO: 2 and SEQ ID NO: 10 using the primer pairs described in SEQ ID NO: 1 and SEQ ID NO: 9 Lactobacillus using the primer pairs described by Lactobacillus luterie, SEQ ID NO: 6 and SEQ ID NO: 9 using the primer pairs described by Lactobacillus gasser, SEQ ID NO: 8 and SEQ ID NO: 10 using the primer pair described by 10 As described in Lactobacillus rhamnosus, SEQ ID NO: 5 and SEQ ID NO: 9 or SEQ ID NO: 3 and SEQ ID NO: 10 using primer pairs described in Plantarum, SEQ ID NO: 7 and SEQ ID NO: 9 or SEQ ID NO: 3 and SEQ ID NO: 10 Using primer pairs, primer pairs described by Lactobacillus paracasei and SEQ ID NO: 3 and SEQ ID NO: 10 were prepared. Lactobacillus casei can be used to identify. In addition, by performing a multiplex PCR (multiplex PCR) reaction using one or more primer pairs corresponding to each strain, Lactobacillus ashidophilus, Lactobacillus vulgaris, Lactobacillus gaseri, Lactobacillus luteri, Lactobacillus plantarum One or more species selected from the group consisting of, Lactobacillus rhamnosus, Lactobacillus paracasei and Lactobacillus casei can be identified simultaneously.

In a preferred embodiment of the present invention, PCR was carried out using a mixture of chromosomal DNA of a single species of Lactobacillus sp. And primers described in SEQ ID NOs: 1 to 10. Was obtained (see FIG. 2 ). In addition, as a result of PCR using the Lactobacillus strain colony mixed with several strains and the primer mixture described in SEQ ID NO: 1 to SEQ ID NO: 10, even if the strains are mixed, each PCR species specifically amplified product Was obtained (see FIG. 3 ).

Lactobacillus spp. Identification method of the present invention is a fermented milk, baby food using a probiotic Lactobacillus spp. It can be used to develop raw materials for dairy products, livestock farm feed, cosmetics, dietary supplements, formal medicines and capsules.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of a Primer for Detection of Strains of the Genus Lactobacillus

Large numbers of sequences for 16S rRNA, 23S rRNA, and local genes between 16S rRNA and 23S rRNA of eight probiotic Lactobacillus spp. To prepare primers used to perform PCR methods for detecting Lactobacillus spp. Use the information to adjust the length so that the melting point (Tm) value exceeds 50 ℃ and do not generate hairpin (haipin) structure or dimer (self-dimer). The primers were prepared by searching. Specifically, using the base sequence (GI: 22128340, 6537241, 1771113 and 1771109, etc.) of the L. acidophilus 23S rRNA gene to prepare a primer described in SEQ ID NO: 1, Lactobacillus Bulgaricus ( L. bulgaricus ) A primer described in SEQ ID NO: 2 was prepared using the nucleotide sequence (GI: 15982695, 15982694, 15982693, 2588893, 43981, 175022, etc.) of the 16S rRNA gene. Base sequences of Lactobacillus casi ( L. casei ), Lactobacillus paracasei ( L. paracasei ) and Lactobacillus rhamnosus 16S rRNA genes (GI: 14583099, 1848057, 1848056, 1848055, 1848054, respectively) 1848053, 1848052, 1843428, 1843427, 20257220, 12382750, 6996495, 43970, 175023, 7621525, 7621504, 1808583, 7159888, 7159887, 6996530, 7621503, etc .; GI: 6552383, 6537239, etc .; GI: 20086370, 6537244, 6815814, etc.) To prepare a primer set forth in SEQ ID NO: 3. Using the L. gasseri 16S rRNA gene and 23S rRNA gene sequence sequences (GI: 6537236, etc.), a primer set forth in SEQ ID NO: 4 was prepared, and L. paracasei ( L. paracasei ) The primer set forth in SEQ ID NO: 5 was prepared using the nucleotide sequence of 23S rRNA gene (GI: 6552383, 6537239, etc.). Lactobacillus plantarum ( L. plantarum ) primers described in SEQ ID NO: 6 using the nucleotide sequence of the 23S rRNA gene (GI: 6537237, 20086379, 20086378, 20086376, 20086375, 20086372, 3046893, 2738840, 3420786, etc.) Using the nucleotide sequence of the L. rhamnosus 23S rRNA gene (GI: 20086370, 6537244, 6815814, etc.), a primer set forth in SEQ ID NO: 7 was prepared, and the L. reuteri 16S rRNA gene was prepared. The primer set forth in SEQ ID NO: 8 was prepared using the nucleotide sequence (GI: 3420786, 388037, etc.).

In addition, as the remaining primers for conducting PCR with the species-specific primers prepared above, a common primer of the genus Lactobacillus which detects the common sequences present in the 8 nucleotide sequences was prepared. It is described as SEQ ID NO: 9 using the 16S rRNA gene sequence of the 8 species of the genus Lactobacillus described above as a forward primer for PCR reaction in pairs with the primers described in SEQ ID NO: 1 to SEQ ID NO: 8 The primers were prepared, and primers described in SEQ ID NO: 10 were prepared using the 16S rRNA gene sequences of the 8 species of Lactobacillus genus as reverse primers.

The positions to which the primers described in SEQ ID NO: 1 to SEQ ID NO: 10 prepared above are attached, and the size of the amplification products expected in PCR amplification are shown in FIG. 1A . As shown in FIG. 1B , Lactobacillus rhamnosus (0.45 kb) is similar in size to the product amplified with Lactobacillus plantarum (0.43 kb), and Lactobacillus paracazei (0.65 kb) is Lactobacillus ashdophyllus. (0.6 kb) and the size of the product to be amplified are similar to the primers for amplifying the Lactobacillus casei as another primer for amplifying Lactobacillus paracasei, Lactobacillus rhamnosus to distinguish it The primer described as was produced.

Example 2 Detection of Standard Strains of Lactobacillus Species Using Primers for Detection of Lactobacillus Species

From the chromosomes of eight Lactobacillus standard strains using each species-specific primer and a species-specific primer pair prepared in Example 1 to determine whether the PCR amplification products using species-specific and species-specific primers have species specificity Species specific PCR (SS-PCR) analysis was performed using the obtained DNA as a template. Specifically, Lactobacillus ashidophyllus (ATCC 4356), Lactobacillus vulgaris (ATCC 11842), Lactobacillus casei (ATCC 334), Lactobacillus gasteria (ATCC 33323), which are standard strains of the probiotic Lactobacillus genus ), Lactobacillus paracasei (ATCC 25302), Lactobacillus plantarum (ATCC 14917), Lactobacillus luteri (ATCC 3594) and Lactobacillus rhamnosus (ATCC 7469) to separate chromosomes by methods such as bamanen. (Varmanen et al. , Appl. Environ. Microbiol., 1998, 64, 1831-1836). 0.25 μg of the chromosomal DNA isolated above and 125 pmol of each species and a common primer were mixed, heated at 96 ° C. for 5 minutes, and a single strand of chromosomal DNA and primers were combined at 61 ° C. for 1 minute. The polymerization was performed for 30 minutes, 35 seconds at 94 ° C, 20 seconds at 61 ° C, and 40 seconds at 68 ° C for a total of 35 times, followed by 7 minutes at 68 ° C. The reaction was performed to perform PCR reactions in the same manner as the primer pairs for amplifying the remaining strains in addition to the primer pairs for amplifying each strain. 20 μl of the final PCR reaction product was taken and electrophoresed on a 1.5% agarose gel and stained with ethidium bromide to observe the band profile of the amplified PCR product. The PCR primer pairs used to distinguish Lactobacillus species and the size of the PCR products obtained after the reaction are shown in Table 1 , and PCR results using 8 species of each species specific single primer pairs for 9 standard strains are shown in FIG. Same as 1b .

As a result, PCR products amplified from the DNA of each strain were obtained in the same size as the PCR product to be obtained after the reaction described in FIG. 1A when compared to each band of the size marker ( Table 1 and FIG. In the PCR using a strain specific primer, the amplified PCR product cannot be obtained, and thus, the strain specific lactobacillus of the genus Lactobacillus described in SEQ ID NOS: 1 to 8 prepared in the present invention, and the lactose described in SEQ ID NO: 9 and SEQ ID NO: 10. Bacillus spp. Common primer was found to be a useful primer for screening probiotic Lactobacillus spp.

Microbe detection primer Size of amplification product (kb) Forward direction Reverse Lactobacillus ashdophyllus SEQ ID NO: 9 SEQ ID NO: 1 0.60 Lactobacillus Bulgaricus SEQ ID NO: 2 SEQ ID NO: 10 0.18 Lactobacillus gasserie SEQ ID NO: 9 SEQ ID NO: 4 0.28 Lactobacillus rutheri SEQ ID NO: 8 SEQ ID NO: 10 1.09 Lactobacillus plantarum SEQ ID NO: 9 SEQ ID NO: 6 0.43 Lactobacillus rhamnosus SEQ ID NO: 9 SEQ ID NO: 7 0.45 SEQ ID NO: 3 SEQ ID NO: 10 0.71 Lactobacillus paracazei SEQ ID NO: 9 SEQ ID NO: 5 0.65 SEQ ID NO: 3 SEQ ID NO: 10 0.71 Lactobacillus casei SEQ ID NO: 3 SEQ ID NO: 10 0.71

<Example 3> Detection of Standard Strains of Probiotic Lactobacillus Using Primer Mixtures

In order to perform the identification of strains by a single PCR, multiple PCR (Multiplex PCR) methods are performed to efficiently identify species by simultaneously reacting the other eight different primers in one reaction to one strain. It was. In the present invention, the multiple PCR means performing a PCR reaction using a single species of strain using a plurality of PCR primers, or performing a PCR reaction using a variety of strains of a PCR primer. The PCR method is the same as that performed in Example 2, and the primers for performing PCR are mixed with primers ( Table 1 ) for searching all strains against the chromosomal DNA of one strain to perform multiple PCR reactions. It was.

As a result, the PCR product after performing multiple PCR when compared with each band of the size marker was the same as the expected PCR product size described in Figure 1a ( Fig. 2 ). From the above results, the PCR reaction using the species-specific multiple primer mixture prepared in the present invention specifically produces species-specific PCR amplification products for all probiotic Lactobacillus standard strains, and the species-specific primer of the present invention is species-specific multiple. It can be seen that it can be useful in performing the PCR.

Example 4 Detection of Strains of the Genus Lactobacillus from Mixed Strains

<4-1> Multiplex PCR using Colony of Strain

In order to easily search for Lactobacillus spp. By multiplex PCR method, extracting the chromosomal DNA of the strain is cumbersome. Therefore, the PCR reaction should be possible directly from the colony of the cultured strain. In order to confirm this, a suspension of colonies of eight standard strains of the probiotic Lactobacillus genus, each of which was suspended in sterile water, was used as a test for PCR reaction, and the primers described in SEQ ID NOs: 1 to 10 of the present invention Multiple PCR was performed using the mixed solution.

As a result, the PCR product containing 1 μl of the suspension (1.0 at 600 nm wavelength turbidity) in which colonies of each strain was mixed with sterile water was used as the result of PCR products containing 0.15 μg of chromosomal DNA isolated from each strain. In the PCR using a single strain colony, almost all of the 8 standard strains produced specific bands up to the concentration of 1 × 10 ⁵ cfu. As a result, it was found that the production of the same amplification product suspension having a turbidity of the strain of 0.8-1.0 and Figure 2 both in the case of using 8 kinds of strains by a factor of 10, relative to the total volume of the PCR reaction at 600 nm wavelength .

<4-2> Multiple PCR Using Mixed Strain Colonies

When using the colony of the strain as a template for PCR reaction in Example <4-1>, it showed the same result as multiple PCR with chromosomal DNA as a template. The colony of the strains were mixed and multiple PCR was performed to determine whether the strain was specifically PCR amplified. Specifically, a cell suspension (OD _{600 nm} = 1.0) in which colonies of eight probiotic Lactobacillus standard strains were clouded at an uneven ratio up to two, three, or six was used as a template for PCR reaction. Multiple PCR was performed after addition to / 10. PCR reactions were performed as described in Example 2 above.

As a result, it was found that even when various types of Lactobacillus strains were mixed when compared to each band of the size marker, the specific bands of each species were reproducibly amplified in the same manner as the expected PCR amplification products described in FIG. 1A ( FIG. 3 ). . Therefore, it can be seen that the primer of the present invention can also be usefully used in multiple PCR methods performed by mixing strains.

Example 5 Accuracy Test of Lactobacillus Species Identification Using Multiplex PCR

In Examples 1 to 4 it was confirmed that it is possible to accurately detect all 8 different Lactobacillus standard strains by using a PCR reaction using multiple primers. Next, the accuracy of the strain identification was examined by confirming whether the PCR product amplified by the multiple PCR amplified the chromosomal DNA of the species to be actually searched. Specifically, using a mixture of the primers described in SEQ ID NO: 1 to SEQ ID NO: 10 for a total of 29 distribution strains ( Table 2 ) belonging to several species of the genus Lactobacillus, which are standard strains or sold from Korean collection for type cultures (KCTC) Multiple PCR was performed, and each strain was combined with the API test (Chang, et al. , Kor. J. Appl. Microbiol. Biotechnol., 2000, 27 (1), 23-27), which is a biochemical identification method . The accuracy of the species identification of Lactobacillus was measured.

As a result of strain identification by multiple PCR, three strains of Lactobacillus ashidophilus (ATCC 1039), Lactobacillus amylovorus (ATCC 33620) and Lactobacillus john SONii ( L. johnsonii ) (JCM 1022) In other results, the average accuracy was 89.7%. In addition, as a result of strain identification by API test, L. amylophilus (DSM 20533), amyloborus (ATCC 33620), Lactobacillus casei (ATCC 334), Lactobacillus casei (ATCC 393) ), Lactobacillus gasseri (ATCC 19992), Lactobacillus gasseri (ATCC 33323), Lactobacillus gasseri (ATCC 9857), Lactobacillus helveticus ( L. helveticus ) (ATCC 15009), Lactobacillus zensenyi ( L jensenii ) (ATCC 25258 ), Lactobacillus Johnsoni (JCM 1022), Lactobacillus luteri (ATCC 23272) and Lactobacillus luteri (LMG 13090) showed different results with 58.6% accuracy. Multiple PCR identification and API assays were performed using Lactobacillus ashdophyllus (ATCC 1039) and Lactobacillus amyloborus (ATCC 33620). , Lactobacillus gasserie (ATCC 19992), Lactobacillus gasserie (ATCC 33323), Lactobacillus gasserie (ATCC 9857), Lactobacillus helveticus (ATCC 15009), Lactobacillus john sony (JCM 1022) The difference showed 75.9% (22/29) agreement ( Table 2 ). In sum, the reliability of identification was lower than that obtained by the multiplex PCR method. This is because the API test measures the activity of various enzymes contained in the culture of the strain by the change in the chromaticity of the test solution, so the determination result may be ambiguous according to the change in test chromaticity according to the reaction time. It is considered that the accuracy of the experimental results is inferior, such as the limitation of the database used for the determination. On the contrary, the multiple PCR method is based on the difference of the genes of the strains, and it can be seen that 8 strains of the probiotic Lactobacillus species can be accurately identified.

Comparison of Multiplex PCR and API Results for Lactobacillus Distribution Liquors Distribution strain name Multiplex PCR API inspection method Species Subspecies Strain ^* (strain) Identification result ^* Identification result ^* Accuracy ^* (ID;%) L. acidophilus ATCC 4356 ^T L. acidophilus L. acidophilus 99.8 L. acidophilus ATCC 832 L. acidophilus L. acidophilus 99.8 L. acidophilus ATCC 1039 L. gasseri L. acidophilus 89.2 L. amylophilus DSM 20533 ^T - L. crispatus 63.7 L. amylovorus ATCC 33620 ^T L. gasseri L. crispatus 98.9 L. casei casei ATCC 334 ^T L. casei L. paracasei 78.0 L. casei casei ATCC 393 L. casei L. paracasei 68.6 L. crispatus JCM 5808 - L. crispatus 99.6 L. crispatus JCM 5810 - L. crispatus 98.9 L. curvatus ATCC 25601 ^T - L. curvatus 95.4 L. delbrueckii bulgaricus ATCC 11842 ^T L. delbrueckii L. delbrueckii 94.3 L. delbrueckii delbrueckii ATCC 9649 ^T L. delbrueckii L. delbrueckii 99.8 L. delbrueckii lactis ATCC 21051 ^T L. delbrueckii L. delbrueckii 78.7 L. fermentum ATCC 14931 ^T - L. fermentum 99.4 L. gasseri ATCC 19992 L. gasseri L. acidophilus 99.5 L. gasseri ATCC 33323 ^T L. gasseri L. acidophilus 97.8 L. gasseri ATCC 9857 L. gasseri L.acidophilus 89.6 L. helveticus ATCC 15009 ^T - L. acidophilus 88.7 L. jensenii ATCC 25258 ^T - L. crispatus 63.2 L. johnsonii JCM 1022 L. gasseri L. acidophilus 99.3 L. paracasei paracasei ATCC 25302 ^T L. paracasei L. paracasei 77.9 L. paracasei paracasei ATCC 27216 L. paracasei L. paracasei 82.5 L. paracasei tolerans ATCC 25599 ^T L. paracasei L. paracasei 89.8 L. plantarum ATCC 10012 L. plantarum L. plantarum 75.3 L. plantarum ATCC 14917 ^T L. plantarum L. plantarum 97.3 L. rhamnosus ATCC 7469 ^T L. rhamnosus L. rhamnosus 99.8 L. reuteri ATCC 23272 ^T L. reuteri L. fermentum 92.6 L. reuteri LMG 13090 L. reuteri L. fermentum 99.2 L. salivarius salicinius ATCC 11742 ^T - L. salivarius 92.5

In the above, T means the standard strain, and-means that there is no PCR amplification product as a result of multiplex PCR. The ID is a result of how close the property is to the relationship with the microbial species in the database with the accuracy of the API result. The higher the ID%, the more likely it is to be determined to be close to the microbe.

In addition, in the above API results, because Lactobacillus casei and Lactobacillus paracasei are defined as similar strains, both are labeled as Lactobacillus paracase.

<Example 6> Identification experiment of the identified strain Lactobacillus

In order to confirm that the above-described embodiment results to the results of any amplification product is also produced to nine strains that are performing a multiplex PCR in the 5 (Table 2), the result has to do with whether to perform PCR, it is common in the probiotic Lactobacillus PCR was performed using primer pairs. Specifically, in order to simultaneously identify 29 Lactobacillus strains of Example 5, a primer having a nucleotide sequence described in SEQ ID NO: 11 and SEQ ID NO: 12, which is a probiotic Lactobacillus genus specific primer, was prepared. PCR was performed under the same conditions as in the above example using the primers described in SEQ ID NO: 11 and SEQ ID NO: 12 and 29 strains described in Table 2 . In addition, the genus Bacillus subtilis ( ATCC 6051), Bifidobacterium bifidum ATCC 29521, Bifidobacterium breve ( ATCC 15700), in addition to the 29 Lactobacillus genus a Bifidobacterium inpeon tooth (Bifidobacterium infantis, ATCC 15697), Bifidobacterium ronggeom (Bifidobacterium longum, ATCC 15707), Enterococcus faecalis (Enterococcus faecalis, ATCC 19433) and Enterococcus passive help (Enterococcus faecium ATCC 19434) Used as a control.

As a result, Lactobacillus-specific PCR amplification products were produced in all 29 strains, and controls Bacillus, Bifidobacterium and Enterococcus did not produce PCR amplification products. Therefore, it was found that the probiotic Lactobacillus strain identification method using multiple PCR specifically detects only Lactobacillus strains.

Example 7 Identification of Lactobacillus Strains from Probiotic Lactobacillus-Containing Bacterial Products

Lactobacillus sp. Strains were identified from the commercially available probiotic Lactobacillus sp. Species containing product using multiple PCR of the present invention. Specifically, the strains were isolated from the six probiotic Lactobacillus strain-containing products, and PCR was performed on the strains using multiple primers. PCR was carried out in the same manner as described in Example 2. In addition, for each strain, the accuracy of the identification of the genus Lactobacillus was determined by comparing the results of multiple PCR using a primer mixture having the nucleotide sequence described in SEQ ID NO: 1 to SEQ ID NO: 10 in parallel with the API test.

As a result, multiple PCR identification showed the same result only in the list of pre-distributed strains and 3 strains, and these results were similar to those of the API test. Considering that the API test is indicated as a nearby flexible strain even for the strains not included in the database, the multiple PCR identification method and the API test method showed an agreement of 83.3% (5/6) ( Table 3 ).

Lactobacillus strain identification results from bacterial products Lactobacillus-containing fungi Multiplex PCR API inspection method product name Display strain name (Species) Identification result Identification result ^* Accuracy ^* (ID;%) A L. acidophilus L. acidophilus L. acidophilus 98.9 B L. acidophilus L. acidophilus L. acidophilus 99.9 C L. acidophilus L. gasseri L. acidophilus 68.3 D L. acidophilus L. rhamnosus L. paracasei 78.6 E L. acidophilus L. rhamnosus L. rhamnosus 97.6 F L. rhamnosus L. rhamnosus L. rhamnosus 99.8

In the above, the ID is a result of how close the properties of the microbial species in the database are to the accuracy of the API test result.

In the above API results, because Lactobacillus casei and Lactobacillus paracasei are defined as similar strains, both were labeled as Lactobacillus paracase.

<Example 8> 16S rRNA gene sequence analysis of the strain of the genus Lactobacillus

In order to perform accurate identification on the products containing the different strain name (labeled strain name), multiple PCR identification, and API test results indicated on the probiotic Lactobacillus strain-containing product performed in Example 7, Molecular biological identification of strains isolated from C product (hereinafter abbreviated as 'C') and strains isolated from D product (hereinafter abbreviated as 'D') was performed. To this end, C and D were inoculated in MRS solid medium (Difco ^TM Lactobacilli MRS Broth, Difco and 1.5% agar), respectively, incubated at 37 ° C. for 2 days to activate, and a single colony having a diameter of 2 mm was taken and 600 nm. The colonies were suspended in sterile water to have a turbidity of about 0.8-1.0 at a wavelength and stored at −20 ° C. to be used as a template for PCR reaction. To isolate 16S rRNA genes of each strain, the samples suspended in sterile water were amplified by colony PCR method by combining primers described in SEQ ID NO: 13 and SEQ ID NO: 14 with a mixed primer mixed in a 1: 1 ratio. The amplified 16S rRNA gene fractions were separated by electrophoresis on 0.75% agarose gel using 1% TAE buffer (40 mM Tris-acetate, 1 mM EDTA). After the electrophoresis gel was stained with ethidium-bromide and UV irradiated, the gel containing the desired DNA fragment was cut to a certain size. The DNA was purified using a Gene Clean Kit (Bio101) and BamH I and Xho I Double digestion with restriction enzymes was linked to the pcDNA3.1 / Hygro (+) (5.6 kb) plasmid vector (Invitrogen). 16S rRNA genes of C and D were transformed into E. coli host cells with plasmids (7.1 kb each) linked to the pcDNA3.1 / Hygro (+) plasmid vector, and only the transformed cells were selected and cultured in large quantities to secure the desired plasmid. It was. The obtained plasmid was purified again with a gene clean kit, and 16S rRNA gene fragments were analyzed for nucleotide sequences using primers set forth in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. Sequencing reactions were performed using a cycle sequencing kit (Perkin Elmer) and dye terminator, and sequence analysis was performed using an ABI prism 310 automatical DNA sequencer. . In addition, the analyzed sequences were compared with a blast similarity search program (National Institute of Biotechnology Information) to compare the similarities with known 16S rRNA gene sequences. In addition, nine genera registered in GenBank and 85 species in the genus Lactobacillus and Lactobacillus ashidophilus and Lactobacillus crispatus, Lactobacillus gasteria, Lactobacillus gensenyi, etc. Multiple sequence alignments were performed with 16S rRNA gene sequences for 33 strains of the Ashdophilus group, followed by determination of their position in the phylogenic tree. Multiple sequence alignment program was used for ClustalX.

As a result, the 16S rRNA genes of C and D were 1,531 bp and 1,528 bp in length, respectively. As a result of blast similarity investigation, C is BLB1b (GI: 7621501), DSM 20243 (GI: 175028), KC5a (GI: 7621522), KC36a (GI: 7621508), KC26 (GI: 7621513), Higher similarities were 99.5% with BLB3 (GI: 7621499), KC29 (GI: 7621514), and TL33a (GI: 7621530), 99.1-99.7%. In contrast, the Lactobacillus ashidophilus strains ATCC 4356, NCDO 1748, BMF6Lb6 showed an average homology of 90.8%, and the strain was determined to be Lactobacillus gasery. In addition, as a result of phylogenetic position determination, the 16S rRNA gene of C was located at the position closest to the Lactobacillus gasteria ( FIG. 4 ).

In addition, the nucleotide sequence of D was 99.1% with 99.5, 99.3, and 98.4% homology with Lactobacillus rhamnosus strains F11 (GI: 7621503), ATCC 7469 (GI: 1848056), and DSM 20021 (GI: 175023), respectively. Showed homology. On the contrary, the D strain was determined to be Lactobacillus rhamnosus with an average of 82.3% homology with the Lactobacillus ashidophilus strains ATCC 4356, NCDO 1748, and BMF6Lb6. The rRNA gene was located at the position closest to the Lactobacillus rhamnosus ( FIG. 4 ).

Therefore, as a result of molecular biological identification using the 16S rRNA gene, C could be determined to be Lactobacillus gasteria and D to Lactobacillus rhamnosus, only the PCR result of the labeled strain name, API test method and multiple PCR identification method of the product were molecules. It can be seen that this is exactly the same as the biological identification. The reason for this may be due to the use of relatively old identification methods and identification criteria, and the API results may be due to data analysis errors resulting from the limitations of the database and subtle differences in culture results. In contrast, the multiplex PCR method is based on the exact difference of genes among 8 Lactobacillus species, and it is an identification method that always shows the same result regardless of the history of similar strains and the culture status and death of the strain. It was found to be much faster, more accurate and more reproducible than the identification method.

As described above, the multiple PCR method using a primer specific for the Lactobacillus spp. Of the present invention can easily and quickly and accurately identify the Lactobacillus spp. It can be used to develop raw materials for dairy products, livestock farm feed, cosmetics, dietary supplements, formal medicines and capsules.

<110> ILDONG PHARMACEUTICAL CO., LTD <120> Method for identification of microorganism of          lactobacillus species <130> 4p-02-46 <150> KR-2003-12777 <151> 2003-02-28 <160> 17 <170> KopatentIn 1.71 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> L. acidophilus reverse primer <400> 1 aactatcgct tacgctacca ctttgc 26 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> L223 bulgaricus forward primer <400> 2 ctgtgctaca cctagagata ggtgg 25 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> L. casei, L. paracasei, L. rhamnosus forward primer <400> 3 tggtcggcag agtaactgtt gtcg 24 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> L. gasseri reverse primer <400> 4 atttcaagtt gagtctctct ctcttctcgg 30 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> L paracasei reverse primer <400> 5 ccggccagct atgtattcac tgaca 25 <210> 6 <211> 31 <212> DNA <213> Artificial Sequence <220> L. plantarum reverse primer <400> 6 ctagtggtaa cagttgatta aaactgctgg g 31 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> L. rhamnosus reverse primer <400> 7 gcggttatgc gatgcgaatt tctattatt 29 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> L. reuteri forward primer <400> 8 acctgattga cgatggatca ccagt 25 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus species reverse primer <400> 9 ccaccttcct ccggtttgtc a 21 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus species forward primer <400> 10 agggtgaagt cgtaacaagg tagcc 25 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus specific forward primer <400> 11 ggaaacagrt gctaataccg 20 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus specific reverse primer <400> 12 cyctcaaaac traacaaagt ttc 23 <210> 13 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> ID8-F <400> 13 gggggatcca gagtttgatc ctggctca 28 <210> 14 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> ID8-R <400> 14 gggctcgagt accttgttac gacttcacc 29 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> T7-F <400> 15 taatacgact cactataggg 20 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> BGH-R <400> 16 tagaaggcac agtcgagg 18 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> ID13S-F <400> 17 taactacgtg ccagcagc 18

Claims (6)

Lactobacillus genus species specific primer selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 8. A common primer of the genus Lactobacillus, characterized in that it is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10. 1) performing multiplex PCR using the test sample as a template and using at least one primer selected from the primers of claim 1 and at least one primer selected from the primers of claim 2; And 2) Identification method of the genus Lactobacillus comprising the step of confirming whether the amplification of the PCR reaction product of step 1 and the size of the amplification product. 4. The method according to claim 3, wherein the test specimen is chromosomal DNA isolated from colonies or strains of strains cultured from a sample containing a strain of the probiotic Lactobacillus strain. The genus species of Probiotic Lactobacillus are Lactobacillus ashidophyllus, Lactobacillus vulgaris, Lactobacillus cascai, Lactobacillus paracaze, Lactobacillus rhamnosus, Lactobacillus gasteria, Lactobacillus fluctus. At least one selected from the group consisting of lantarum and Lactobacillus luterii. The method of claim 3, wherein the sample is selected from the group consisting of fermented milk, baby food, dairy products, livestock farm feed, cosmetics, dietary supplements, formal medicines and capsules containing the strain of the genus Probiotic Lactobacillus Way.