KR102407751B1 - Primers for detection of lactobacillus plantarum group, and use thereof - Google Patents

Abstract

The present invention relates to a composition and kit for detection capable of distinguishing species belonging to the Lactobacillus plantarum group, and a detection method and screening method using the same.

Description

Lactobacillus plantarum group specific primer and its use {PRIMERS FOR DETECTION OF LACTOBACILLUS PLANTARUM GROUP, AND USE THEREOF}

The present invention relates to a composition and kit for detection capable of distinguishing species belonging to the Lactobacillus plantarum group, a detection method using the same, and a screening method.

Lactobacillus plantarum ( Lactobacillus plantarum ) The group is Lactobacillus plantarum subspecies plantarum ( L. plantarum subsp . plantarum ), Lactobacillus plantarum subspecies As phylogenetic and phenotypically closely related species, including L. plantarum subsp . argentoratensis , L. paraplantarum and L. pentosus . constructed (Parente et al., 2010).

The phenotypic and genotypic heterogeneity of these species is demonstrated based on fermentation capacity and DNA-DNA hybridization data (Bringel et al., 1996; Bringel et al., 2005; Curk, Hubert, & Bringel, 1996; Torriani, Felis, & Dellaglio, 2001). For example, Lactobacillus pentosus can be distinguished from Lactobacillus plantarum by its ability to ferment xylose and glycerol (Bringel et al., 2005; Mao, Chen, & Horvath, 2015). However, not all Lactobacillus pentosus strains can ferment xylose, and some Lactobacillus plantarum strains can ferment glycerol like Lactobacillus pentosus (Bringel, Curk, & Hubert, 2005).

In the past, biochemical methods using the phenotypic characteristics of microorganisms were mainly used to identify lactic acid bacteria or to confirm the microbial community of probiotic products, but these methods consume a lot of time and have disadvantages such as fermentation patterns of lactic acid bacteria and incorrect results. there was. To improve this, molecular genetics analysis has been developed, and the most useful techniques are metagenome sequencing using 16S rRNA gene, PCR-DGGE, and 16S rRNA gene sequencing.

The 16S rRNA gene of microorganisms is a gene that is usefully used for phylogenetic studies because the sequence between the same species is highly conserved. However, the subspecies of Lactobacillus plantarum are difficult to distinguish by 16S rRNA gene sequencing. This is because the 16S rRNA gene sequence is more than 99% identical to the Lactobacillus plantarum subspecies and the other Lactobacillus species Lactobacillus paraplantarum and Lactobacillus pentosus.

Accordingly, the conventional method using the existing 16S rRNA gene has a limitation in that it is difficult to distinguish the species within the Lactobacillus plantarum group belonging to the same group.

Korean Patent Publication No. 10-2020-0004944

It is an object of the present invention to provide a composition for detection for discrimination of species belonging to the Lactobacillus plantarum group.

Another object of the present invention is to provide a kit for detecting Lactobacillus plantarum group comprising the composition for detection.

Another object of the present invention is to provide a biochip (chip) for detecting Lactobacillus plantarum group comprising the composition for detection.

Another object of the present invention is to provide a method for clearly distinguishing the Lactobacillus plantarum group present in a target sample using the composition for detection.

Another object of the present invention is to provide a method for screening a sample containing a Lactobacillus plantarum group using the composition for detection.

One aspect of the present invention is a first primer pair comprising the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2; and a second primer pair comprising the nucleotide sequence of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: 4; a primer set consisting of one or more primer pairs selected from the group consisting of; Lactobacillus plantarum , including ) relates to a composition for differential detection of species belonging to the group.

In the present invention, “primer pair” is a term that simultaneously refers to both forward and reverse primers, and “primer set” refers to one or a plurality of primer pairs, for example, from a first primer pair to a second primer pair. It was used as a term referring to a plurality of primer pair bundles composed of one or more selected ones.

The design of the primer pair is subject to various restrictions, such as the A, G, C, and T content ratio, prevention of primer dimer formation, and prohibition of repeating the same nucleotide sequence three or more times. In addition, conditions such as the amount of template DNA, primer concentration, dNTP concentration, Mg ²⁺ concentration, reaction temperature, and reaction time must be appropriate.

The primer is a short nucleic acid sequence containing a free 3' hydroxyl group at the end, and can form a base pair with a template of a complementary nucleic acid and prevent strand copying of the nucleic acid template. It can serve as a starting point for

The primer can initiate DNA synthesis in the presence of a reagent for polymerization and four different nucleoside triphosphates at an appropriate buffer solution and temperature, and hybridizes with nucleic acids present in the target material to generate a specific gene of the target material can be used to amplify.

Specifically, the primer may be single-stranded in consideration of amplification efficiency, may be deoxyribonucleotide, and may be naturally occurring dNMP (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides, or non-naturally occurring nucleotides. may contain nucleotides. In addition, the primer may also include ribonucleotides.

The hybridization means that two single-stranded nucleic acids form a duplex structure by pairing complementary nucleotide sequences.

The hybridization may occur when complementarity between single-stranded nucleic acid sequences is perfect (perfect match) or even when some mismatch (mismatch) bases exist. The degree of complementarity required for the hybridization may vary depending on the hybridization reaction conditions, and in particular may be controlled by the temperature. In general, if the hybridization temperature is high, hybridization is highly likely to occur in the case of a perfect match, and if the hybridization temperature is low, hybridization may occur even if there is some mismatch.

The primer set can hybridize to a specific gene present in a target raw material, and when a target raw material is present in the sample, the nucleotide sequence of the characteristic gene is amplified by a polymerase chain reaction, so the amplified product (amplicon) Through this, it is possible to determine the presence or absence of the raw material.

The primer may incorporate additional features that do not change the basic properties. That is, the nucleic acid sequence can be modified using a number of means known in the art. Examples of such modifications include methylation, encapsulation, substitution of a nucleotide with one or more homologues and uncharged linkages such as phosphonates, phosphotriesters, phosphoroamidates or carbamates or phosphorothioates or phosphorodithioates. Modification of nucleotides into charged linkages such as In addition, the nucleic acid may include one or more of nucleases, toxins, antibodies, signal peptides, proteins such as poly L lysine, intercalating agents such as acridine or psoralen, chelating agents such as metals, radioactive metals, iron oxidizing metals, and alkylating agents. It may have additional covalently attached residues.

In addition, the primer sequence may be modified using a label capable of directly or indirectly providing a detectable signal. The primer may include a label that can be detected using spectroscopic, photochemical, biochemical, immunochemical or chemical means. Useful labels include proteins for which 32P, fluorescent dyes, electron-dense reagents, enzymes (commonly used in ELISAs), biotin or haptens and antisera or monoclonal antibodies are available.

The primers were prepared by cloning of an appropriate sequence and digestion with restriction enzymes, and the phosphotriester method of Narang et al. (1979, Meth, Enzymol. 68:90-99), and the diethylphosphoramidite method of Beaucage et al. (1981, Tetrahedron Lett. 22: 1859-1862), and any other well known method including direct chemical synthesis such as the solid support method of US 4458066.

Specifically, the primer set includes: a first primer pair including the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2; And a second primer pair comprising the nucleotide sequence of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: 4; may include one or more selected from the group consisting of.

In one embodiment, the primer set may include a first primer pair and a second primer pair.

Also specifically, the primer set includes a third primer pair including the nucleotide sequence of SEQ ID NO: 5 and the nucleotide sequence of SEQ ID NO: 6; and a fourth primer pair comprising the base sequence of SEQ ID NO: 7 and the base sequence of SEQ ID NO: 8; may further include one or more primer pairs selected from the group consisting of.

In the present invention, “ Lactobacillus ” is a bacterium that produces a large amount of lactic acid by fermenting sugars to obtain energy. stuck Lactobacillus belongs to the lactobacilli family, and about 170 species are known so far. Lactobacillus, along with Bifidobacterium , is the most commonly used lactic acid bacterium in probiotic products. It is known to have the effect of suppressing adult diseases.

In particular, there are several groups in Lactobacillus, representative Lactobacillus acidophilus ( L. acidophilus ) Acidophilus belonging to the group ( L. acidophilus ), Helveticus ( L. helveticus ), Galinarum ( L. gallinarum ); Lactobacillus casei ( L. casei ) belonging to the group casei ( L. casei ), paracasei ( L. paracasei ), rhamnosus ( L. rhamnosus ); Lactobacillus plantarum ( L. plantarum ) Plantarum belonging to the group ( L. plantarum ), para plantarum ( L. paraplantarum ), pentosus ( L. pentosus ); Lactobacillus Sakei ( L. sakei ) belonging to the group Sakei ( L. sakei ), graminis ( L. graminis ), curvatus ( L. curvatus ) and the like correspond to this.

Specifically, the species belonging to the Lactobacillus plantarum group are Lactobacillus plantarum subspesis plantarum ( L. plantarum subsp . plantarum ), Lactobacillus plantarum subspecies. Argentora Tensis ( L. plantarum subsp . argentoratensis ), Lactobacillus paraplantarum ( L. paraplantarum ) and Lactobacillus pentosus ( L. pentosus ) It may mean one or more selected from the group consisting of.

In one embodiment, the first primer pair for detecting the gene of the Lactobacillus plantarum subsp. plantarum ( L. plantarum subsp . plantarum ) is the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2 can be configured.

In one embodiment, the Lactobacillus plantarum subspecies The second primer pair for detecting the gene of L. plantarum subsp . argentoratensis may be composed of the nucleotide sequence of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: 4.

In one embodiment, the third primer pair for detecting the gene of the Lactobacillus paraplantarum ( L. paraplantarum ) may be composed of the nucleotide sequence of SEQ ID NO: 5 and the nucleotide sequence of SEQ ID NO: 6.

In one embodiment, the fourth primer pair for detecting the gene of the Lactobacillus pentosus ( L. pentosus ) may be composed of the nucleotide sequence of SEQ ID NO: 7 and the nucleotide sequence of SEQ ID NO: 8.

In addition, the primer pair may constitute a primer set in a form including one selected primer pair or a plurality of two or more primer pairs.

In one embodiment of the present invention, it was confirmed that each primer pair of the present invention exhibits very high specificity for each species belonging to the Lactobacillus plantarum group.

Another aspect of the present invention, Lactobacillus plantarum comprising the composition Provided is a kit for differential detection of species belonging to a group.

The detection kit may have a solution, lyophilized powder, frozen solution, or strip form, and each form may be formulated in a conventional manner in the art. For example, a detection kit in the form of a solution may be formulated by separately or mixing a protein or a primer in a buffer such as sodium-phosphate, potassium-phosphate, tris-hydrochloric acid, and other various types of buffers. It can also be frozen or freeze-dried.

The detection kit may use an immunolateral flow strip method. A lateral flow assay is a method for detecting a protein or a nucleic acid based on a chromatography method. This lateral flow analysis method is widely used in various fields such as pregnancy diagnosis, cancer diagnosis, the presence of other specific proteins or genes, or detection of microorganisms. The lateral flow analysis method is based on a specific reaction between two substances, such as an antigen-antibody reaction, and has the advantages of high sensitivity and specificity, and the ability to confirm results within a short time, enabling diagnosis and rapid preventive measures. .

In the detection kit of the present invention, various reagents required for experiments (eg, PCR) and results confirmation required for detecting Lactobacillus sp. strain using the primer set of the present invention, for example, PCR composition, restriction enzymes, agarose, Buffers necessary for hybridization and electrophoresis may be additionally included. Specifically, the PCR composition comprises an appropriate amount of a DNA polymerase (eg, Thermusaquaticus (Taq), Thermusthermophilus (Tth), Thermusfiliformis, Thermisflavus, Thermococcusliteralis or thermostable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), dNTP, PCR buffer and water (dH ₂ O). The PCR buffer solution may include Tris-HCl (Tris-HCl), MgCl ₂ , KCl, and the like.

In addition, the detection kit may be a real-time-PCR (RT-PCR) kit. Specifically, the RT-PCR composition may be prepared in a freeze-dried form in a plastic tube, and the kit may further include appropriate reagents and tools used for analysis in addition to the RT-PCR composition. Such reagents and tools include suitable carriers, labels capable of producing a detectable signal, solubilizing agents, detergents, and the like. Suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyester , polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal in latex, other paper, glass, metal, agarose, and combinations thereof

Another aspect of the present invention, Lactobacillus plantarum comprising the composition Provided is a biochip for differential detection of a species belonging to a group.

In the present invention, the "biochip" is also called a DNA chip, in which very small DNA fragments are integrated on a solid surface. It may be in a form including a chamber and a channel capable of accommodating the .

Since the biochip is thin, transparent, and has a large contact area with the heater block, PCR time can be greatly shortened, and at the same time, it has the advantage of being able to search for at least hundreds of genes in a short time. In addition, by using a solid body, a very small amount of genetic material can be attached at a high density, a large number of genes can be searched at the same time, the expression level of a large amount of genes can be simultaneously measured, or the genotype of various parts of the genome can be determined can be used to

Biochips can be classified into cDNA (complementary DNA) chips and oligonucleotide chips according to the size of the attached genetic material. The cDNA chip may contain a gene (full-length open reading frame or EST) of at least 500 bp. An oligonucleotide of about 15 to 25 bases may be adhered to the oligonucleotide chip, and may be included in a solution contained in the chip, but is not limited to the above shape and may be appropriately modified as necessary.

Another aspect of the present invention provides a method for differentially detecting a species belonging to the Lactobacillus plantarum group, comprising the step of treating the composition to a target sample.

The target sample used in the detection method can be obtained from any material in which Lactobacillus genus bacteria can be found, preferably from various probiotic foods. Specifically, the target sample may be obtained through a DNA extraction method commonly used in the art, for example, may be an alkaline extraction method, hot water extraction method, column extraction method, and phenol/chloroform extraction method, but is not limited thereto.

The detection method refers to specific methods capable of detecting Lactobacillus DNA present in a target sample, and includes a DNA sequence analysis method; probe hybridization methods; structure-specific cleavage analysis; enzyme mismatch cleavage method; polymerase chain reaction (PCR); branched chain hybridization methods; rolling circle replication; Nucleic acid sequence based amplification (NASBA); molecular beacon technology; E-sensor technology (such as US Pat. No. 6,248,229); cycling probe technology; Dade Behring signal amplification method; ligase chain reaction; and a sandwich hybridization method or Southern blotting.

The detection method may be performed by amplifying a specific DNA using the composition and then confirming the amplification reaction of the specific DNA. It can be carried out by confirming whether the size of the expected DNA product is consistent with the size of the DNA product through electrophoresis or the like.

Specifically, the detection method may further include performing a polymerase chain reaction (PCR).

In the present invention, the "Polymerase Chain Reaction (PCR)" is widely known in the art as a method of exponentially amplifying a starting nucleic acid material by synthesizing a nucleic acid chain using a polymerase, It is a concept that includes both qualitative analysis PCR to check the presence or absence of a specific nucleic acid, quantitative PCR to measure the amount of a specific nucleic acid, and real-time PCR to enable qualitative and quantitative analysis by tracking the PCR process in real time.

The polymerase chain reaction may be performed according to general reaction conditions using a conventional PCR apparatus known in the art, or may be partially modified. In addition, the PCR reaction mixture may include a reaction buffer, dNTPs and a DNA polymerase, and may include instructions for performing the same.

In one embodiment of the present invention, a primer set capable of detecting one or a plurality of Lactobacillus plantarum group strains was constructed, and the primer set has high sensitivity, so that it can effectively amplify the target DNA even at a very low level of the target DNA concentration. It was confirmed that it is possible.

Specifically, the polymerase chain reaction may be a real-time polymerase chain reaction (real-time RCR). In the case of general PCR, the amount of the final product can be known after the reaction is completed, whereas real-time PCR is characterized in that it is possible to quantitatively observe the amplification process of DNA molecules during PCR.

In the present invention, the real-time PCR may use the Taqman method or SYBR Green using a fluorescent probe, and specifically, SYBR Green may be used, but is not limited thereto.

In real-time PCR using SYBR Green, the amount of DNA increases as the target sequence is amplified, and at this time, SYBR Green, a fluorescent dye having a property of being inserted into double-stranded DNA, is inserted into the double-stranded DNA. Since the SYBR exhibits a property of intervening in DNA to emit light, the amount of amplified DNA can be detected by measuring the degree of light emission. The real-time PCR method using SYBR Green has the advantage of being inexpensive because it does not need to attach fluorescent dyes to primers or probes. However, the real-time PCR method using SYBR Green requires conditions in which only one type of PCR product is amplified, and sensitive primer conditions in which non-specific products or dimers between primers that occur in conventional PCR do not occur.

Another aspect of the present invention comprises the steps of: a) treating a target sample with a composition comprising a pair of primers of SEQ ID NOs: 1 to 4; And b) Lactobacillus plantarum subspecies plantarum ( L. plantarum subsp . plantarum ) and Lactobacillus plantarum subspecies belonging to the Lactobacillus plantarum group in the sample Argentora tensis ( L. plantarum subsp . argentoratensis ) It provides a screening method of a sample containing a Lactobacillus plantarum group, comprising the step of determining whether at least one selected from the group consisting of.

Specifically, the primer set of step a) comprises a third primer pair comprising the nucleotide sequence of SEQ ID NO: 5 and the nucleotide sequence of SEQ ID NO: 6; and a fourth primer pair comprising the base sequence of SEQ ID NO: 7 and the base sequence of SEQ ID NO: 8; may further include one or more primer pairs selected from the group consisting of.

In addition, specifically, whether the sample contains Lactobacillus paraplantarum ( L. paraplantarum ) or Lactobacillus pentosus ( L. pentosus ) in the Lactobacillus plantarum group can also be checked.

In the present invention, the "sample" refers to a material to be checked whether or not the Lactobacillus sp. strain is included. Specifically, probiotic preparations, cosmetics, food, nutritional supplements, beverages, additives, substances included in various diets, etc. may be targeted, but it is not limited to the above form, and if it is intended to check whether the Lactobacillus spp. can be a sample.

The screening method may be performed through all gene screening methods performed by methods known in the art without particular limitation.

By using the composition for detecting Lactobacillus consisting of at least one selected from the first primer pair to the fourth primer pair of the present invention, it is possible to accurately determine the species belonging to the Lactobacillus plantarum group present in the target sample. have.

In addition, the first primer pair to the fourth primer pair of the present invention can accurately detect the Lactobacillus plantarum group strain through a single real-time PCR.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

Figure 1 shows the pan-genome distribution (Pan-genome distribution) of the Lactobacillus plantarum ( Lactobacillus plantarum ) group.
2 shows the results of confirming the presence/absence of species or subspecies-specific genes for 70 genomes.
3 shows the results of confirming the specificity of the Lactobacillus plantarum subspecies-specific primer (A: L. plantarum subsp. plantarum specific primer specificity, amplification curve: L. plantarum subsp. plantarum KACC 11451, KACC 15357, KCTC 3108, KCTC 3104 and KCCM 12116, B: L. plantarum subsp. argentoratensis specific primer specificity, amplification curve: L. plantarum subsp. argentoratensis KACC 12404, C: L. paraplantarum specific primer specificity, amplification curve: L. paraplantarum KACC 12373, KCTC 5045 and LI00101, D: specificity of L. pentosus -specific primers Amplification curves: L. pentosus KACC 12428, KCCM 40997, LI 00102, LI 00103, and LI 00104).
4 shows the results of constructing a standard quantitative curve for the Lactobacillus plantarum subspecies primer (A: L. plantarum subsp. plantarum standard curve, B: L. plantarum subsp. argentoratensis standard curve, C: L. paraplantarum standard curve, D: L. pentosus standard curve).

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples only illustrate the present invention, and the present invention is not limited by the following examples.

Example 1. Lactobacillus plantarum ( Lactobacillus plantarum ) group-specific primer design

Genome comparison analysis was performed to search for Lactobacillus plantarum subspecies specific genes. For genome comparative analysis, Lactobacillus plantarum subspesis plantarum ( L. plantarum subsp . plantarum ), Lactobacillus plantarum subspecies Argentora Tensis ( L. plantarum subsp . argentoratensis ), Lactobacillus paraplantarum ( L. paraplantarum ) and Lactobacillus pentosus ( L. pentosus ) A total of 70 genomes of the Bioinformation Center (NCBI: ftp://ftp) .ncbi.nlm.nih.gov/genomes/).

The analysis of pan-genomes between strains belonging to the Lactobacillus plantarum group was analyzed using Anvi'o software v6.2 (Eren et al., 2015). Anvi'o genome storage was used as the genome database for pan-genome analysis, and the phylogenetic tree based on the pan-genome was obtained using Prodigal, MCL algorithm, NCBI BLASTp and HMMER (Alneberg et al., 2018). got an estimate. Species- or subspecies-specific genes were investigated by pangenomic analysis using the Bacterial Pan Genome Analysis (BPGA) pipeline v1.3 (Chaudhari, Gupta, & Dutta, 2016).

Species-specific genes possessed by each species were selected through comparative genome analysis (identity cut-off=50%) using USEARCH 9.0 program. Specifically, the genome is compiled into two local databases, including pan- and core-genomic databases for each species- or subspecies-, and the two local databases are compared, followed by a basic local alignment search tool. (basic local alignment search tool, BLAST) was used to align with 59,344,251 sequences and searched with a cut-off value of 50% to confirm whether the specific gene was present in the bacterial genome other than the target species or subspecies. .

The results were visualized as a heat map, and species- or subspecies-specific primers were designed based on these specific genes using a primer designer (Scientific and Education Software, Durham, NC, USA) (Table 1).

set number target species primer sequence (5'-3') SEQ ID NO: One Lactobacillus plantarum subspesis plantarum ( L. plantarum subsp . plantarum ) Plantarum_F CGG CAA CAA GCC ACT AAA CT One Plantarum_R GAT AAT TAG CGG CTG CCT GA 2 2 Lactobacillus plantarum subspesis argentoratensis ( L. plantarum subsp. argentoratensis ) Argentoratensis_F TTC TTG ATG GCC CGG GTG TT 3 Argentoratensis_R GGC TGG ACC ATG GCT AAG AA 4 3 Lactobacillus paraplantarum ( L. paraplantarum ) Paraplantarum_F TTA TTC AAG CCG TCG GAG TG 5 Paraplantarum_R TCG CTG GTG CTA ATG CAA TG 6 4 Lactobacillus pentosus ( L. pentosus ) Pentosus_F GCG GTA TCG ATT CGA TTG GT 7 Pentosus_R TGA TGT CAA TCG CCT CTT GG 8

Example 2. Preparation of standard strains

In order to confirm the specificity of the primer, 55 strains of lactic acid bacteria were used. The lactic acid bacteria strains are from Korea Microbial Conservation Center (KCCM, Seoul, Korea), Korea Institute of Biotechnology and Biotechnology Biological Resources Center (KCTC, Daejeon, Korea), National Academy of Agricultural Sciences Microbial Bank (KACC, Jeonju, Korea) and laboratory isolation (LI, Yongin) , Korea) (Table 2).

number bell strain number One Lactobacillus plantarum subsp. plantarum ( Lactobacillus plantarum subsp . plantarum ) KACC ^a 11451 2 Lactobacillus plantarum subsp. plantarum ( Lactobacillus plantarum subsp . plantarum ) KACC 15357 3 Lactobacillus plantarum subsp. plantarum ( Lactobacillus plantarum subsp . plantarum ) KCTC ^b 3108 4 Lactobacillus plantarum subsp. plantarum ( Lactobacillus plantarum subsp . plantarum ) KCTC 3104 5 Lactobacillus plantarum subsp. plantarum ( Lactobacillus plantarum subsp . plantarum ) KCCM ^c 12116 6 Lactobacillus plantarum subspecies Argentoratensis ( Lactobacillus plantarum subsp . argentoratensis ) KACC 12404 7 Lactobacillus paraplantarum ( Lactobacillus paraplantarum ) KACC 12373 8 Lactobacillus paraplantarum ( Lactobacillus paraplantarum ) KCTC 5045 9 Lactobacillus paraplantarum ( Lactobacillus paraplantarum ) LI ^d 00101 10 Lactobacillus Pentosus ( Lactobacillus pentosus ) KACC 12428 11 Lactobacillus Pentosus ( Lactobacillus pentosus ) KCCM 40997 12 Lactobacillus Pentosus ( Lactobacillus pentosus ) LI 00102 13 Lactobacillus Pentosus ( Lactobacillus pentosus ) LI 00103 14 Lactobacillus Pentosus ( Lactobacillus pentosus ) LI 00104 15 Lactobacillus Aceto Tolerans ( Lactobacillus acetotolerans ) KACC 12447 16 Lactobacillus Acid pisis ( Lactobacillus acidipiscis ) KACC 12394 17 Lactobacillus Acidophilus ( Lactobacillus acidophilus ) KACC 12419 18 Lactobacillus Agilis ( Lactobacillus agilis ) KACC 12433 19 Lactobacillus Amylolyticus ( Lactobacillus amylolyticus ) KACC 12374 20 Lactobacillus Lactobacillus amylophilus ( Lactobacillus amylophilus ) KACC 11430 21 Lactobacillus Amyloborus ( Lactobacillus amylovorus ) KACC 12435 22 Lactobacillus Brevis ( Lactobacillus brevis ) KCTC 3498 23 Lactobacillus buchneri ( Lactobacillus buchneri ) KACC 12416 24 Lactobacillus casei ( Lactobacillus casei ) KACC 12413 25 Lactobacillus coryniformis ( Lactobacillus coryniformis ) KACC 12411 26 Lactobacillus crustorum ( Lactobacillus crustorum ) KACC 16344 27 Lactobacillus curvatus ( Lactobacillus curvatus ) KACC 12415 28 Lactobacillus delbrueckii ( Lactobacillus delbrueckii ) KACC 12420 29 Lactobacillus delbrueckii subsp. delbrueckii subsp. delbruecki ( Lactobacillus delbrueckii subsp . delbrueckii ) KACC 13439 30 Lactobacillus delbrueckii subsp. lactis ( Lactobacillus delbrueckii subsp . lactis ) KACC 12417 31 Lactobacillus farciminis ( Lactobacillus farciminis ) KACC 12423 32 Lactobacillus fermentum ( Lactobacillus fermentum ) KACC 11441 33 Lactobacillus gallinarum ( Lactobacillus gallinarum ) KACC 12370 34 Lactobacillus gasseri ( Lactobacillus gasseri ) KCTC 3163 35 Lactobacillus heilongjiangensis ( Lactobacillus heilongjiangensis ) KACC 18741 36 Lactobacillus helveticus ( Lactobacillus helveticus ) KACC 12418 37 Lactobacillus jensenii ( Lactobacillus jensenii ) KCTC 5194 38 Lactobacillus johnsoni ( Lactobacillus johnsoni ) i KCTC 3801 39 Lactobacillus kunkeei ( Lactobacillus kunkeei ) KACC 19371 40 Lactobacillus lindneri ( Lactobacillus lindneri ) KACC 12445 41 Lactobacillus mucosae ( Lactobacillus mucosae ) KACC 12381 42 Lactobacillus parabuchneri ( Lactobacillus parabuchneri ) KACC 12363 43 Lactobacillus paracasei ( Lactobacillus paracasei ) KCTC 3165 44 Lactobacillus reuteri ( Lactobacillus reuteri ) KCTC 3594 45 Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) KCTC 3237 46 Lactobacillus ruminis ( Lactobacillus ruminis ) KACC 12429 47 Lactobacillus sakei ( Lactobacillus sakei ) KCTC 3603 48 Lactobacillus salivarius ( Lactobacillus salivarius ) KCTC 3600 49 Lactobacillus san franciscensis ( Lactobacillus sanfranciscensis ) KACC 12431 50 Lactobacillus zymae ( Lactobacillus zymae ) KACC 16349 51 Bifidobacterium animalis lactis ( Bifidobacterium animalis subsp . lactis ) KACC 16638 52 Bifidobacterium bifidum ( Bifidobacterium bifidum ) KCTC 3418 53 Bifidobacterium breve KACC 16639 54 Bifidobacterium longum subsp. infantis ( Bifidobacterium longum subsp . infantis ) KCTC 3249 55 Bifidobacterium longum subsp. longum ( Bifidobacterium longum subsp . longum ) KCCM 11953

Example 3. DNA extraction method

To extract DNA of Lactobacillus and Bifidobacterium strains. Incubated in MRS broth (Difco, Becton & Dickinson, Sparks, MD, USA) and Bifidobacterium broth (MB cell, Seoul, Korea), respectively, at 37°C and anaerobic conditions for 48 hours. After incubation, cells were obtained by centrifugation at 13,600 g for 5 minutes. Cell pellets were used for genomic DNA extraction, and genomic DNA was extracted from lactic acid bacteria strains using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The purity and amount of DNA was confirmed using a MaestroNano® spectrophotometer (Maestrogen, Las Vegas, NV, USA).

Example 4. Real-time PCR conditions

To check the specificity and accuracy of the primers, real-time PCR was performed using a 7500 Real-time PCR system (Applied Biosystems, Foster City, CA, USA). The composition of the reaction solution for performing real-time PCR is 20 µL by mixing 10 µL of 2x Thunderbird SYBR®qPCR mix (Toyobo, Osaka, Japan), 500 nM of a species- or subspecies-primer pair, 20 ng of genomic DNA, and distilled water. prepared as much as possible.

For amplification of the target, after denaturing at 95 ^o C for 2 minutes, the reaction was repeated 30 cycles at 95 ^o C for 5 seconds and 60 ^o C for 30 seconds.

Experimental Example 1. Species or subspecies-specific gene search and specificity confirmation

A phylogenetic tree based on the pangenome was constructed to identify unique regions according to species or subspecies. Phylogenetic analysis shows that all genomes share the same lineage by subspecies or species type ( FIG. 1 ).

As a result of investigating the specific gene for Lactobacillus plantarum subspecies by the method of Example 1, 33 genomes out of 35 genomes of Lactobacillus plantarum species were Lactobacillus plantarum subspecies plantarum ( L . plantarum subsp . plantarum ) and the remaining two genomes are Lactobacillus plantarum subspesis Argentoratensis ( L. plantarum subsp . argentoratensis ) was confirmed.

Specifically, a total of 1,810 core genomes were found by comparing 15 genomes of Lactobacillus plantarum subspecies plantarum. As a result of comparing 1810 core genomes and 26,806 pangenomes, 33 genes were found in Lactobacillus It was confirmed that it was specific for Lantarum subspecies plantarum. In addition, in the case of Lactobacillus plantarum subspecies argentoratensis, 2,590 core genomes and genes encoding 86 proteins were found, of which two genes were specific for Lactobacillus plantarum subspecsis argentoratensis. It was confirmed that it is a gene. One of them encodes a transporter, which is a major promoter family protein (accession no. EFK30629.1), the other is Lactobacillus plantarum subspecies plantarum and Lactobacillus plantarum subspecies argento. It is a hypothetical protein (accession no. KRM00406.1) that shares the highest sequence identity with the genome of latensis.

The specificity of the gene was evaluated with 70 genomes, and was performed at the species level through in-silico analysis. All specific genes were present with 99-100% sequence identity in the genome of the species or subspecies. Subspecies were identified by aligning specific genes to a total of 35 genomes at the species level, and 30 Lactobacillus plantarum genomes include genes specific for Lactobacillus plantarum subspecies plantarum, and the remaining It was confirmed that five genomes ( L. plantarum SNU.Lp177, SF2A35B, Nizo1839, Nizo2264 and KMB_618) contain a specific gene for Lactobacillus subspecies argentolatensis (FIG. 2).

These results were generally consistent with the results of phylogenetic analysis based on the pangenome.

Experimental Example 2. Confirmation of specificity and sensitivity of real-time PCR

To confirm the accuracy of real-time PCR, real-time PCR was performed on a total of 55 strains, including 14 strains belonging to the Lactobacillus plantarum group and 41 strains not belonging to the Lactobacillus plantarum group.

The primers designed in Example 1 were used as the Lactobacillus plantarum subspecies plantarum and Lactobacillus plantarum subspecies argentoratensis-specific primers. In addition, primers developed in a previous study (Kim et al., 2020c) were used to detect Lactobacillus pentosus and Lactobacillus paraplantarum (Table 1).

As a result, each Lactobacillus plantarum group strain produced a detectable product (amplicon) with respect to the specific primer, while the other strains did not show an amplification signal (FIG. 3). Specifically, the Ct value corresponded to the range of 12.86 to 14.27 for the Lactobacillus plantarum group strain.

These results show that the primers of the present invention are specific for detecting species or subspecies of each Lactobacillus plantarum group strain.

In addition, in order to confirm that the specificity and sensitivity of the real-time PCR are appropriate, the accuracy of the real-time PCR was confirmed using serially diluted genomic DNA of the Lactobacillus plantarum group species or subspecies.

As a result of creating a standard graph based on the result of the fluorescence amplification curve of the real-time PCR, it was confirmed that all of the four species-specific primer pairs exhibited a linear proportional relationship over the entire range (FIG. 4).

Specifically, the slope and R ² values of the standard graph are shown in Table 3 below.

species Slope R ^{2 -} value Lactobacillus plantarum subsp. plantarum ( Lactobacillus plantarum subsp . plantarum ) -3.394 ≥0.997 Lactobacillus plantarum subspecies Argentoratensis ( Lactobacillus plantarum subsp . argentoratensis ) -3.122 ≥0.999 Lactobacillus paraplantarum ( Lactobacillus paraplantarum ) -3.335 ≥0.996 Lactobacillus Pentosus ( Lactobacillus pentosus ) -3.138 ≥0.998

In order to obtain an effective primer, the slope and R ² values of the standard curve must be -3.1 to -3.6 and ≥ 0.98 or more, respectively (Broeders et al., 2014) It was confirmed that the real-time PCR method of the present invention has high efficiency and accuracy.

In addition, the accuracy and efficiency of the PCR-based method is affected by the amplicon sizes, and it is suggested that a size of less than 150 bp is more efficient than a product of a large size (Debode, Marien, Janssen, Bragard, & Berben, 2017). ), the size of the product developed in the present invention was in the range of 120 to 145 bp, and it was confirmed that the accuracy and efficiency were excellent.

Experimental Example 3. PCR application experiment for probiotics and fermented foods

An application experiment for identification of strains belonging to the Lactobacillus plantarum group of probiotic products and kimchi products was performed using the developed real-time PCR method.

Specifically, probiotic products were purchased from the Korean, American, Canadian, British, and Italian markets, and real-time PCR analysis was performed on 32 probiotic products to confirm the presence of the bacterial species indicated on the products. The product consists of 13 powder products, 18 capsule products, and 1 chewable product.

In addition, a total of 18 kimchi samples were purchased from the market for monitoring the Lactobacillus plantarum group species. To extract DNA from the whole kimchi, a 10 g sample was blended using a blender (model: Tokebi Origin, Buwon Electronics, Seoul, Korea) and filtered using sterile gauze. The filtrate was centrifuged at 4,000 x g for 5 min, and the pellet was washed twice prior to DNA extraction. Thereafter, DNA was extracted from the probiotic and kimchi samples by the method of Example 3, and real-time PCR was performed.

Specifically, genomic DNA of probiotics and kimchi products was added to each well of a reaction plate containing species- or subspecies- primers and 2× qPCR mix (Toyobo). Species- or subspecies-specific real-time PCR was performed under the same conditions as the real-time PCR conditions of Example 4. Real-time PCR results were analyzed using 7500 software v2.0.6 (Applied Biosystems) and compared with the species of the Lactobacillus plantarum group indicated in the probiotic product.

As a result, as shown in Table 4 below, the label and the Lactobacillus plantarum species detected as a result of real-time PCR for 31 products were all identical, but the label and PCR results were different for one product.

product type Species indicated on the label Species detected as a result of PCR (detected amount (CFU/mL)) Monitoring of probiotic products product 1 Capsule, USA LPP LPP ^a (3×10 ⁷ ) product 2 Capsule, USA LPP LPP (3×10 ⁸ ) product 3 Capsule, USA LPP LPP (3×10 ⁸ ) product 4 Capsule, USA LPP LPP (3×10 ⁷ ) product 5 Capsule, USA LPP LPP (3×10 ⁷ ) product 6 Capsule, USA LPP LPP (3×10 ⁷ ) product 7 Capsule, USA LPP LPP (3×10 ⁷ ) product 8 Capsule, Canada LPP LPP (3×10 ⁷ ) product 9 Capsule, Canada LPP LPP (3×10 ⁷ ) product 10 Capsule, Canada LPP LPP (3×10 ⁸ ) product 11 Capsule, Canada LPP LPP (3×10 ⁸ ) product 12 Capsule, Canada LPP LPP (3×10 ⁸ ) product 13 Capsule, Canada LPP LPP (3×10 ⁷ ) product 14 Capsule, Canada LPP LPP (3×10 ⁶ ) product 15 capsule, UK LPP LPP (3×10 ⁸ ) product 16 capsule, italy LPP LPP (3×10 ⁷ ) product 17 capsule, Korea LPP LPP (3×10 ⁷ ) product 18 capsule, Korea LPP LPE ^b (3×10 ⁸ ) product 19 Chewable Formulation, Korea LPP LPP (3×10 ⁷ ) product 20 powder, Korea LPP LPP (3×10 ⁷ ) product 21 powder, Korea LPP LPP (3×10 ⁷ ) product 22 powder, Korea LPP LPP (3×10 ⁷ ) product 23 powder, Korea LPP LPP (3×10 ⁸ ) product 24 powder, Korea LPP LPP (3×10 ⁸ ) product 25 powder, Korea LPP LPP (3×10 ⁸ ) product 26 powder, Korea LPP LPP (3×10 ⁷ ) product 27 powder, Korea LPP LPP (3×10 ⁶ ) product 28 powder, Korea LPP LPP (3×10 ⁷ ) product 29 powder, Korea LPP LPP (3×10 ⁸ ) product 30 powder, Korea LPP LPP (3×10 ⁸ ) product 31 powder, Korea LPP LPP (3×10 ⁸ ) product 32 powder, Korea LPP LPP (3×10 ⁸ ) Fermented food monitoring product 1 Kimchi, Korea unknown LPP (3×10 ⁵ ), LPR ^c (3×10 ⁵ ) product 2 Kimchi, Korea unknown LPP (3×10 ⁵ ) product 3 Kimchi, Korea unknown LPP (3×10 ⁵ ), LPR (3×10 ⁵ ) product 4 Kimchi, Korea unknown LPP (3×10 ⁵ ), LPR (3×10 ⁵ ) product 5 Kimchi, Korea unknown LPP (3×10 ⁵ ) product 6 Kimchi, Korea unknown LPP (3×10 ⁵ ) product 7 Kimchi, Korea unknown LPP (3×10 ⁵ ), LPR (3×10 ⁵ ) product 8 Kimchi, Korea unknown LPP (3×10 ⁵ ), LPA ^d (4×10 ⁵ ), LPR (3×10 ⁵ ) product 9 Kimchi, Korea unknown LPP (3×10 ⁷ ), LPA (4×10 ⁶ ), LPR (3×10 ⁶ ) product 10 Kimchi, Korea unknown LPP (3×10 ⁸ ), LPA (4×10 ⁶ ), LPR (3×10 ⁷ ), LPE (8×10 ⁵ ) product 11 Kimchi, Korea unknown LPP (3×10 ⁷ ), LPA (4×10 ⁶ ), LPR (3×10 ⁶ ) product 12 Kimchi, Korea unknown LPP (3×10 ⁶ ), LPR (3×10 ⁵ ) product 13 Kimchi, Korea unknown LPP (3×10 ⁶ ), LPA (4×10 ⁵ ), LPR (3×10 ⁵ ) product 14 Kimchi, Korea unknown LPP (3×10 ⁵ ), LPA (4×10 ⁵ ), LPR (3×10 ⁵ ) product 15 Kimchi, Korea unknown LPP (3×10 ⁵ ), LPR (3×10 ⁵ ) product 16 Kimchi, Korea unknown LPP (3×10 ⁸ ), LPA (4×10 ⁶ ), LPR (3×10 ⁶ ) product 17 Kimchi, Korea unknown LPP (3×10 ⁷ ), LPA (4×10 ⁵ ), LPR (3×10 ⁶ ), LPE (8×10 ⁵ ) product 18 Kimchi, Korea unknown LPP (3×10 ⁸ ), LPA (4×10 ⁶ ), LPR (3×10 ⁶ ), LPE (8×10 ⁵ )

^a LPP, L. plantarum subsp. plantarum

^b LPE, L . pentosus

^c LPR, L. paraplantarum

^d LPA, L. plantarum subsp. argentoratensis

As shown in Table 4, it was confirmed that all one product with incorrect labeling was incorrectly labeling Lactobacillus pentosus as Lactobacillus plantarum. This seems to be because Lactobacillus plantarum and Lactobacillus pentosus have high genetic similarity and are closely related to each other, so it is not easy to distinguish the two strains. It was confirmed that Bacillus strains can be clearly distinguished.

The present invention provides a species-specific primer pair, a primer set, and a composition for detecting Lactobacillus including the same using comparative genomics so that the species belonging to the Lactobacillus plantarum group can be quickly and accurately identified, and Lactobacillus using them Provided are a kit, a biochip, and a detection method capable of detecting it. In addition, the detection method using the composition was applied to monitoring probiotic products and fermented food, and the ability to determine the presence of Lactobacillus reported on the product label and the presence of unreported Lactobacillus was also confirmed. This method is applicable not only to probiotic products, but also to studies that can detect various Lactobacillus from food or environmental samples at once.

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a dispersed form, and likewise components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention.

<110> University-Industry Cooperation Group of Kyung Hee University <120> PRIMERS FOR DETECTION OF LACTOBACILLUS PLANTARUM GROUP, AND USE THEREOF <130> 20PP30861 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Plantarum_F <400> 1 cggcaacaag ccactaaact 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Plantarum_R <400> 2 gataattagc ggctgcctga 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Argentoratensis_F <400> 3 ttcttgatgg cccgggtgtt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Argentoratensis_R <400> 4 ggctggacca tggctaagaa 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Paraplantarum_F <400> 5 ttattcaagc cgtcggagtg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Paraplantarum_R <400> 6 tcgctggtgc taatgcaatg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pentosus_F <400> 7 gcggtatcga ttcgattggt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pentosus_R <400> 8 tgatgtcaat cgcctcttgg 20

Claims (9)

a first primer pair comprising the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2; And a second primer pair comprising the nucleotide sequence of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: 4; a primer set consisting of; Lactobacillus plantarum comprising a; The method of claim 1,
The primer set includes a third primer pair comprising the nucleotide sequence of SEQ ID NO: 5 and the nucleotide sequence of SEQ ID NO: 6; and a fourth primer pair comprising the base sequence of SEQ ID NO: 7 and the base sequence of SEQ ID NO: 8; further comprising one or more primer pairs selected from the group consisting of, a composition for detection. 3. The method of claim 1 or 2,
Bacteria belonging to the Lactobacillus plantarum group are Lactobacillus plantarum subspecies plantarum ( L. plantarum subsp . plantarum ) and Lactobacillus plantarum subspecies Argentoratensis ( L. plantarum subsp . argentoratensis ) One or more selected from the group consisting of, the composition for detection. A kit for differential detection of a species belonging to the Lactobacillus plantarum group, comprising the composition of claim 1 or 2. A biochip for differential detection of species belonging to the Lactobacillus plantarum group, comprising the composition of claim 1 or 2. treating the subject sample with the composition of claim 1 or 2; and
A method for discriminating and detecting species belonging to the Lactobacillus plantarum group, comprising the step of performing a real time-polymerase chain reaction (PCR). delete a) treating the subject sample with the composition of claim 1 or 2;
b) performing a real time-polymerase chain reaction (PCR); and
c) The strains belonging to the Lactobacillus plantarum group in the sample are Lactobacillus plantarum subspecies plantarum ( L. plantarum subsp . plantarum ), Lactobacillus plantarum subspecies Argentora tensis ( L. plantarum subsp . argentoratensis ), Lactobacillus paraplantarum ( L. paraplantarum ) and Lactobacillus pentosus ( L. pentosus ) Including the step of confirming whether at least one selected from the group consisting of doing,
A method for screening a sample containing a Lactobacillus plantarum group. delete

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