KR20240003121A - Real time PCR primer set for detecting Enterococcus species and uses thereof - Google Patents

Abstract

The present invention relates to a real-time PCR primer set for detecting Enterococcus strains and its use. The present invention relates to four types of Enterococcus ( E. durans, E. hirae, E. lactis, E. mundtii ) accurately and quickly. To identify new species-specific target genes using pan-genome analysis, primers and real-time PCR methods that can detect specific genes were developed. It was confirmed that the new primers were able to specifically detect each species, and that they could be detected more accurately than the 16S rRNA gene analysis method using various isolates. In addition, this method is effective against Enterococcus in various fermented foods. It was successfully applied in an experiment to identify clusters. Accordingly, the present invention can quickly, efficiently and accurately identify four types of Enterococcus strains at the species level, so it is expected to be used in research to identify Enterococcus communities contained in various food and environmental samples, including fermented foods. .

Description

Real time PCR primer set for detecting Enterococcus species and uses thereof}

The present invention relates to a real-time PCR primer set for detecting Enterococcus strains and its use.

Enterococcus is a genus that lives in a variety of environments. Some species are involved in the fermentation of fermented foods, and some species are attracting attention for their probiotic effects. In addition, E. durans and E. hirae mainly produce useful secondary metabolites and are of interest for industrial applications. However, some species have pathogenic factors, so Enterococcus There is a need for accurate and rapid classification of species. The technique mainly used to identify and detect Enterococcus to the species level is the method using the 16S rRNA gene. Enterococcus For species, 16S rRNA gene sequences had high similarity (89.2% to 99.2%), E. durans and E. lactis , E, and mundtii And some Enterococcus species, such as E. hirae , have more than 99% similarity in 16S rRNA gene sequence, making it difficult to distinguish between Enterococcus and E. hirae. There is a need for new target genes for species detection.

Korean Patent No. 10-1869832 (registered on June 15, 2018)

The object of the present invention is a biomarker composition for detecting Enterococcus strains, a composition for detecting Enterococcus strains containing as an active ingredient an agent capable of measuring the expression level of the biomarker, and an Enterococcus strain containing the composition. The aim is to provide a detection kit and a method for detecting Enterococcus strains using the same.

In order to achieve the above object, the present invention includes fructose 1,6-bisphosphatase (NCBI accession no. AKX85242.1), sodium/glutamate symport protein; NCBI accession no. AFM70771.1), helix-turn-helix transcriptional regulator (NCBI accession no. QXX72259.1) and wxL domain surface protein (NCBI accession no. BAO06098) Provided is a biomarker composition for detecting Enterococcus strains, comprising as an active ingredient one or more proteins selected from the group consisting of .1) or a gene encoding the same.

In addition, the present invention relates to fructose 1,6-bisphosphatase (NCBI accession no. AKX85242.1), sodium/glutamate symport protein (NCBI accession no. AFM70771) .1), consisting of a helix-turn-helix transcriptional regulator (NCBI accession no. QXX72259.1) and a wxL domain surface protein (NCBI accession no. BAO06098.1) Provided is a composition for detecting Enterococcus strains, comprising as an active ingredient an agent capable of measuring the expression level of one or more proteins selected from the group or the genes encoding them.

Additionally, the present invention provides a kit for detecting Enterococcus strains comprising the composition.

In addition, the present invention includes the steps of (1) isolating DNA from a sample; (2) Using the isolated DNA as a template, a primer set represented by SEQ ID NO: 1 and SEQ ID NO: 2, a primer set represented by SEQ ID NO: 3 and SEQ ID NO: 4, and a primer set represented by SEQ ID NO: 5 and SEQ ID NO: 6 and; Performing PCR amplification using one or more primer sets selected from the group consisting of primer sets represented by SEQ ID NO: 7 and SEQ ID NO: 8; and (3) analyzing the PCR amplification product.

The present invention relates to a real-time PCR primer set for detecting Enterococcus strains and its use. The present invention relates to four types of Enterococcus ( E. durans , E. hirae, E. lactis, E. mundtii ) accurately and quickly. To identify new species-specific target genes using pan-genome analysis, primers and real-time PCR methods that can detect specific genes were developed. It was confirmed that the new primers were able to specifically detect each species, and that they could be detected more accurately than the 16S rRNA gene analysis method using various isolates. In addition, this method is effective against Enterococcus in various fermented foods. It was successfully applied in an experiment to identify clusters. Accordingly, the present invention can quickly, efficiently and accurately identify four types of Enterococcus strains at the species level, so it is expected to be used in research to identify Enterococcus communities contained in various food and environmental samples, including fermented foods. .

Accordingly, in the present invention, in order to accurately distinguish four types of Enterococcus strains to the species level, we performed a pan-genome analysis based on whole-genome sequence, discovered specific genes for each species, and used this to identify Enterococcus strains. A real-time PCR method capable of detecting was developed and the present invention was completed.

The present invention relates to fructose 1,6-bisphosphatase (NCBI accession no. AKX85242.1), sodium/glutamate symport protein (NCBI accession no. AFM70771.1) ), helix-turn-helix transcriptional regulator (NCBI accession no. QXX72259.1) and wxL domain surface protein (wxL domain surface protein; NCBI accession no. BAO06098.1). Provided is a biomarker composition for detecting Enterococcus strains comprising one or more selected proteins or genes encoding the same as an active ingredient.

Preferably, the Enterococcus strain isEnterococcus durans, Enterococcus hirae , Enterococcus lactis or Enterococcus mundtiiIt may be, but is not limited to this.

In the present invention, “marker” refers to a base sequence used as a reference point when identifying genetically unspecified related loci. The term also applies to nucleic acid sequences complementary to a marker sequence, such as nucleic acids used as primer sets capable of amplifying the marker sequence. The location of a molecular marker on a genetic map is called a genetic locus.

In addition, the present invention relates to fructose 1,6-bisphosphatase (NCBI accession no. AKX85242.1), sodium/glutamate symport protein (NCBI accession no. AFM70771) .1), consisting of a helix-turn-helix transcriptional regulator (NCBI accession no. QXX72259.1) and a wxL domain surface protein (NCBI accession no. BAO06098.1) Provided is a composition for detecting Enterococcus strains, comprising as an active ingredient an agent capable of measuring the expression level of one or more proteins selected from the group or the genes encoding them.

Preferably, the agent capable of measuring the expression level of the gene is fructose 1,6-bisphosphatase (NCBI accession no. AKX85242.1), sodium/glutamate symport protein. (sodium/glutamate symport protein; NCBI accession no. AFM70771.1), helix-turn-helix transcriptional regulator (NCBI accession no. QXX72259.1), and wxL domain surface protein (wxL domain surface protein). It may be a primer or probe that specifically binds to one or more proteins selected from the group consisting of (protein; NCBI accession no. BAO06098.1) or the gene encoding them, but is not limited thereto.

More preferably, the primers include a primer set represented by SEQ ID NO: 1 and SEQ ID NO: 2, a primer set represented by SEQ ID NO: 3 and SEQ ID NO: 4, a primer set represented by SEQ ID NO: 5 and SEQ ID NO: 6, and; It may be one or more primer sets selected from the group consisting of primer sets represented by SEQ ID NO: 7 and SEQ ID NO: 8, but is not limited thereto.

Preferably, the Enterococcus strain may be Enterococcus durans, Enterococcus hirae, Enterococcus lactis, or Enterococcus mundtii , but is not limited thereto.

In the present invention, "probe" refers to a nucleic acid fragment, such as DNA, of as short as a few bases or as long as several hundred bases, that can specifically bind to DNA, and is labeled to determine the presence or absence of a specific DNA and its expression level. can confirm. Probes may be manufactured in the form of oligonucleotide probes, single strand DNA probes, double strand DNA probes, RNA probes, etc. Selection of appropriate probes and hybridization conditions can be appropriately selected according to techniques known in the art.

In the present invention, “primer” refers to a single-stranded oligonucleotide sequence complementary to the nucleic acid strand to be amplified, and can serve as a starting point for the synthesis of a primer extension product. The length and sequence of the primers should allow for initiation of synthesis of the extension product. The specific length and sequence of the primer will depend on the complexity of the DNA or RNA target desired as well as the conditions under which the primer is used, such as temperature and ionic strength. In the present invention, the oligonucleotide used as a primer may also include a nucleotide analogue, such as phosphorothioate, alkylphosphorothioate or peptide nucleic acid, or May contain an intercalating agent.

Additionally, the present invention provides a kit for detecting Enterococcus strains comprising the composition.

In addition to the primer set of the present invention, conventional components included in a PCR kit may be included. Typical components included in the kit may include reaction buffer, polymerase, dNTPs (dATP, dCTP, dGTP, and dTTP), and cofactors such as ^{Mg 2+} . A variety of DNA polymerases can be used in the amplification step of the present invention, including the Klenow fragment of E. coli DNA polymerase I, thermostable DNA polymerase, and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from various bacterial species. Most of the above polymerases can be isolated from the bacteria themselves or can be purchased commercially.

In addition, the present invention includes the steps of (1) isolating DNA from a sample; (2) Using the isolated DNA as a template, a primer set represented by SEQ ID NO: 1 and SEQ ID NO: 2, a primer set represented by SEQ ID NO: 3 and SEQ ID NO: 4, and a primer set represented by SEQ ID NO: 5 and SEQ ID NO: 6 and; Performing PCR amplification using one or more primer sets selected from the group consisting of primer sets represented by SEQ ID NO: 7 and SEQ ID NO: 8; and (3) analyzing the PCR amplification product.

Preferably, the sample may be probiotics or fermented food, but is not limited thereto.

Preferably, the Enterococcus strain may be Enterococcus durans, Enterococcus hirae, Enterococcus lactis, or Enterococcus mundtii , but is not limited thereto.

In the present invention, “probiotics” refer to live bacteria that enter the body and provide good health effects. Most probiotics known to date have been consumed as fermented milk products made using lactic acid bacteria such as Lactobacillus, but recently, fermented milk, granules, and powders containing some strains such as Enterococcus, Weissella , and Bifidobacterium in addition to Lactobacillus have been consumed. It is sold in this form.

Methods known in the art can be used to isolate DNA from a sample. In addition, PCR is a method of amplifying a target nucleic acid from a set of primers that specifically bind to the target nucleic acid using polymerase. These PCR methods are well known in the art, and commercially available kits can also be used.

Below, the present invention will be described in detail according to examples that do not limit the present invention. Of course, the following examples of the present invention are only intended to embody the present invention and do not limit or limit the scope of the present invention. Accordingly, what can be easily inferred by an expert in the technical field to which the present invention belongs from the detailed description and examples of the present invention is interpreted to fall within the scope of the rights of the present invention.

< Experiment example >

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

One. Enterococcus inside standard strain and DNA extraction

A total of 119 standard strains were used to establish the real-time PCR method (Table 1). Strains were distributed from the National Academy of Agricultural Sciences Microbial Bank (KACC), Korea Biological Resources Center (KCTC), National Pathogen Resource Bank (NCCP), and Korea Microbial Conservation Center (KCCM). All received strains were cultured under anaerobic conditions at 37°C for 48 hours in MRS liquid medium (Difco, Becton & Dickinson, Sparks, MD). To extract the DNA of all standard strains, the strains were cultured and then centrifuged at 16,200 g for 3 minutes, and genomic DNA was extracted from the pellet from which the supernatant was removed. Genomic DNA of standard strains and DNA of food were extracted using the DNeasy®Blood & Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The concentration and purity of the extracted DNA were measured at 260 nm and 280 nm using a MaestroNano® spectrophotometer (Maestrogen, Las Vegas, NV, USA).

Species Strain no. Target species Enterococcus durans KCTC 13289 Enterococcus durans KACC 10787 Enterococcus hirae KACC 16328 Enterococcus hirae KACC 10779 Enterococcus hirae KACC 10782 Enterococcus lactis KACC 15681 Enterococcus lactis KACC 14552 Enterococcus lactis KACC 21015 Enterococcus mundtii KCTC 3630 Enterococcus mundtii KACC 13824 Non-target Enterococcus species Enterococcus avium NCCP 10761 Enterococcus avium KACC 10788 Enterococcus casseliflavus KCTC 3552 Enterococcus casseliflavus KCTC 3638 Enterococcus cecorum KACC 13884 Enterococcus devriesei KACC 14590 Enterococcus faecalis KACC 11859 Enterococcus faecalis KCTC 5290 Enterococcus faecalis KCTC 3206 Enterococcus faecium KACC 11954 Enterococcus faecium KCTC 13225 Enterococcus gallinarum NCCP 11518 Enterococcus gilvus KACC 13847 Enterococcus malodoratus KACC 13883 Enterococcus pseudoavium KACC 13781 Enterococcus raffinosus KACC 13782 Enterococcus saccharolyticus KACC 10783 Enterococcus thailandicus KCTC 13134 Non-target species Apilactobacillus kunkei KACC 19371 Bacillus cereus ATCC 11778 Companilactobacillus crustorum KACC 16344 Companilactobacillus farciminis KACC 12423 Companilactobacillus heilongjiangensis KACC 18741 Escherichia coli ATCC 25922 Escherichia coli ATCC 23763 Fructilactobacillus lindneri KACC 12445 Lacticaseibacillus casei KCTC 3109 Lacticaseibacillus casei KCTC 13086 Lacticaseibacillus casei KCTC 3110 Lacticaseibacillus paracasei KCTC 3165 Lacticaseibacillus paracasei KACC 12427 Lacticaseibacillus rhamnosus KCTC 3237 Lacticaseibacillus rhamnosus KCTC 13088 Lactiplantibacillus paraplantarum KACC 12373 Lactiplantibacillus plantarum KACC 11451 Lactiplantibacillus plantarum KACC 12404 Lactobacillus gasseri KACC 12424 Lactobacillus acetotolerans KACC 12447 Lactobacillus acidipiscis KACC 12394 Lactobacillus acidophilus KACC 12419 Lactobacillus acidophilus KCTC 3164 Lactobacillus agilis KACC 12433 Lactobacillus amylolyticus KACC 12374 Lactobacillus amylophilus KACC 11430 Lactobacillus amylovorus KACC 12435 Lactobacillus delbrueckii KACC 12420 Lactobacillus delbrueckii KACC 13439 Lactobacillus delbrueckii KACC 12417 Lactobacillus gallinarum KACC 12370 Lactobacillus gasseri KCTC 3163 Lactobacillus helveticus KACC 12418 Lactobacillus jesenii KCTC 5194 Lactobacillus johnsonii KCTC 3801 Lactobacillus pentosus KACC 12428 Lactobacillus salivarius KCTC 3600 Lactococcus lactis KCTC 3769 Lactococcus lactis KCTC 2013 Latilactobacillus curvatus KACC 12415 Latilactobacillus sakei KCTC 3603 Lentilactobacillus buchneri KACC 12416 Lentilactobacillus parabuchneri KACC 12363 Levilactobacillus brevis KCTC 3498 Levilactobacillus zymae KACC 16349 Ligilactobacillus ruminis KACC 12429 Limosilactobacillus fermentum KACC 11441 Limosilactobacillus fermentum KCTC 3112 Limosilactobacillus fermentum KCTC 5049 Limosilactobacillus mucosae KACC 12381 Limosilactobacillus reuteri KCTC 3594 Listeria ivanovii ATCC 19119 Listeria monocytogenes ATCC 19115 Listeria monocytogenes KCTC 3569 Loigolactobacillus coryniformis KACC 12411 Staphylococcus aureus NCCP 14560 Staphylococcus aureus ATCC 25923 Staphylococcus aureus ATCC 29213 Staphylococcus aureus KCTC 1928 Staphylococcus auricularis KACC 13252 Staphylococcus capitis NCCP 14663 Staphylococcus caprae KACC 13242 Staphylococcus caprae KCTC 3583 Staphylococcus carnosus KACC 13190 Staphylococcus cohnii KACC 13237 Staphylococcus cohnii KCTC 3574 Staphylococcus delphini KACC 13258 Staphylococcus epidermidis NCCP 14723 Staphylococcus epidermidis KACC 13234 Staphylococcus equiorum KACC 13255 Staphylococcus fleurettii KACC 13199 Staphylococcus gallinarum KACC 13253 Staphylococcus haemolyticus NCCP 14691 Staphylococcus hominis NCCP 10748 Staphylococcus hominis KACC 13409 Staphylococcus kloosii KACC 13256 Staphylococcus lentus KCCM 41469 Staphylococcus lugdunensis NCCP 15630 Staphylococcus lugdunensis KACC 11270 Staphylococcus pasteuri KCTC 13167 Staphylococcus saprophyticus NCCP 14670 Staphylococcus saprophyticus KCTC 3345 Staphylococcus saprophyticus KACC 15799 Staphylococcus schleiferi KCCM 41634 Staphylococcus sciuri KACC 13217 Staphylococcus sciuri KCCM 41468 Staphylococcus warneri KCTC 3340 Staphylococcus warneri KACC 10785 Staphylococcus xylosus NCCP 10937 Staphylococcus xylosus KACC 13239 Streptococcus thermophilus KACC 11857

2. Enterococcus Discovering species-specific genes and primer design

To develop specific primers that can detect Enterococcus at the species level, 87 Enterococcus genome sequences were downloaded from the National Center for Biotechnology Information (NCBI) in the United States. Genes specific to each species were obtained by performing pan-genome analysis. By comparing genes possessed by all of the same target species and the full-length gene base sequences of Enterococcus species excluding the target species, specific genes present only in the target species were selected. The final specific genes were selected from genes present in all target species but absent in non-target species using BLASTN (https://blast.ncbi.nlm.nih.gov) (Table 2). Primers were developed from the specific genes selected in this way, and the developed four-pair primer set was designed to have an amplification product size of 200 bp or less to increase amplification efficiency and be suitable for application to processed foods (Table 3). Primers were designed using Primer Designer (Scientific and Education Software, Durham, NC, USA).

Species Unique gene (accession no.) E. durans fructose 1,6-bisphosphatase (accession no. AKX85242.1) E. hirae sodium/glutamate symport protein (AFM70771.1) E. lactis helix-turn-helix transcriptional regulator (QXX72259.1) E. mundtii wxL domain surface protein (BAO06098.1)

Species Primer name Sequence (5'-3') Size (bp) E. durans ED_F CGA GGA ATG GTT CAG CAA GA (SEQ ID NO: 1) 123 ED_R CCA TTG GCT CTG ATC GGT AA (SEQ ID NO: 2) E. hirae EH_F GAC CGT TGC TGC GAC AGT TA (SEQ ID NO: 3) 126 EH_R GAG CCT GCG ACG TTC AAC AT (SEQ ID NO: 4) E. lactis EL_F AGT CCG GTT AAA CCG ATC AA (SEQ ID NO: 5) 101 EL_R ACA CCT ATC ACA CCG GCT AA (SEQ ID NO: 6) E. mundtii EM_F TAG GAA TGC GCT CTC GAT GA (SEQ ID NO: 7) 133 EM_R ACC TGA TGC CAC ATC GAT TG (SEQ ID NO: 8)

3. Real-time PCR method Specificity and accuracy assessment

To confirm the specificity of each developed species-specific primer, real-time PCR was performed using the CFX96 Deep Well Real-time System (Bio-Rad, Hercules, CA, USA). Real-time PCR identified 28 Enterococcus Standard strains and 91 other lactic acid bacteria and pathogenic bacteria mainly isolated from fermented foods were used as controls (Table 1). The reaction solution composition consisted of 10 μL of 2X Thunderbird SYBR®qPCR Mix (Toyobo, Osaka, Japan), 20 ng of DNA template, 0.5 μM primer set, and DW to fill a total volume of 20 μL. Each target was amplified under the following conditions: denaturation was performed at 95°C for 5 minutes, and then the reaction was repeated 35 times for 5 seconds at 95°C and 30 seconds at 60°C. Melt curve was performed by increasing the temperature from 65℃ to 95℃. To evaluate the accuracy of real-time PCR, a standard curve experiment was performed with serial dilution (20~0.00002 ng) of Enterococcus standard strain DNA. The standard curve for the four pairs of primers developed in this study was tested according to the CODEX guidelines to satisfy the slope value of -3.1 to -3.6, the R ² value of 0.98 or more, and the efficiency of 90-110%. .

4. Fermented food application experiment

The developed real-time PCR method was applied to isolate identification and food monitoring. To secure Enterococcus isolates from food, samples were spread on MRS solid medium, colonies with different shapes were selected, and DNA was extracted. The extracted DNA was identified using real-time PCR and 16S rRNA gene sequencing. For food monitoring, total genomic DNA from food purchased from the market was extracted according to the above method, and real-time PCR was performed according to the above method to determine Enterococcus in fermented foods. Clusters were confirmed.

< Example >

One. Enterococcus Acquisition of species-specific genes

87 Enterococcus genome sequences were downloaded from NCBI, and pan-genome and core-genome analyzes were performed depending on the species. As a result of confirming all species-specific genes obtained through pan-genome analysis using BLAST, it was confirmed that these genes were present in all species of the target strain, but were not present in any non-target species or other genera (Table 2). From the identified genes, primers specific to the species or subspecies were designed as shown in Table 3.

2. Real-time developed PCR's Specificity and Accuracy

To confirm the specificity and accuracy of the real-time PCR experiment, specificity was confirmed using four pairs of Enterococcus species-specific primers. When each species-specific primer set was reacted with the DNA of 28 Enterococcus standard strains, amplification occurred within the Ct value range of 12.45 to 16.26 for the corresponding target strains, and no amplification occurred for non-target strains. Therefore, all four pairs of species-specific primer sets developed in this study were judged to be specific (Table 4). To evaluate the accuracy of the developed species-specific primers, a standard curve was obtained by serial dilution (20~0.00002 ng) of the Enterococcus standard strain. The standard curve for the four pairs of primers developed in this study satisfied the slope value of -3.1 to -3.6, R ² value of 0.98 or more, and efficiency of 90-110% according to CODEX guidelines. Real-time PCR can be judged to be accurate and reliable.

Primers
Specificity Standard curve Detected strains CT value
Strains Slope R ² Efficiency (%) Enterococcus durans KCTC 13289, KACC 10787 16.26, 15.5 KACC 10787 -3.137 0.998 108.3 Enterococcus hirae KACC 16328, KACC 10779, KACC 10782 13.09, 13.23, 13.01 KACC 16328 -3.467 0.998 94.3 Enterococcus lactis KACC 15681, KACC 14552, KACC 21015 12.54, 12.56, 12.59 KACC 14552 -3.565 0.999 90 Enterococcus mundtii KCTC 3630, KACC 13824 12.45, 13.21 KCTC 3630 -3.433 0.999 95.6

3. Food application experiment

An experiment was designed to detect four Enterococcus in one 96-well plate using real-time PCR with proven specificity, and was applied to monitoring isolates isolated from food and food colonies.

A total of 80 Enterococcus isolates were isolated, and all 80 isolates showed one amplification curve: 2 E. durans , 16 E. hirae , 60 E. lactis , and 2 E. mundtii . All isolates were re-identified by 16S rRNA gene sequencing and compared with real-time PCR results. Most isolates were identified identically to the real-time PCR results. However, unlike real-time PCR, some isolates were identified as two candidate species by 16S rRNA gene sequencing (Table 5). These are all species with more than 99% similarity in the 16S rRNA gene, and although it was judged difficult to distinguish using the 16S rRNA gene, they could be identified as exactly one species using real-time PCR.

The results of monitoring Enterococcus colonies in food using the developed method are shown in Table 6. All foods contained one or more species of Enterococcus , and E. hirae was present in most fermented foods. As a result, Enterococcus species present in fermented foods can be accurately detected using the real-time PCR developed in this study.

Source Strains qPCR 16S rRNA sequencing (no. of isolates) E. durans E. hirae E. lactis E. mundtii Description Accession no. (identity) Beef S1-S7 (7) - - + - E. lactis /
E. faecium OM760842.1/
CP089092.2
(100%) Cabbage S8-S14 (7) - - + - E. lactis /
E. faecium OM760842.1/
CP089092.2
(100%) Sikhae S15-S22 (8) - - + - E. lactis /
E. faecium JQ446562.1/
MG098997.1
(100%) Raw milk S23-S36 (14) - + - - E. hirae /
E. faecium CP055232.1/
MN826473.1
(100%) Soy Sauce S37-S38 (2) + - - - E. durans /
E. faecium MT545097.1/
MT196421.1
(100%) Soy Sauce S39-S40 (2) - + - - E. hirae CP055232.1/
MN826473.1
(100%) Soybean paste S41-S78 (38) - - + - E. lactis /
E. faecium OM760842.1/
CP089092.2
(100%) Soybean paste S79-S80 (2) - - - + E. mundtii OM691709.1
(100%)

Sample type No. of sample Number of positive results by qPCR analysis E. durans E. hirae E. lactis E. mundtii Pork 4 4 3 0 0 Fish 4 0 4 0 0 Lettuce 4 0 3 0 0 Raw milk 6 0 5 One 0 Soybean paste 8 3 One 7 3 Soy Sauce 2 One One 2 One Gajami-sikhae 4 0 0 One 0

<110> University-Industry Cooperation Group of Kyung Hee University <120> Real time PCR primer set for detecting Enterococcus species and uses it <130> ADP-2022-0221 <160> 8 <170>CopatentIn 2.0 <210> 1 <211> 20 <212> DNA <213> Enterococcus durans <400> 1 cgaggaatgg ttcagcaaga 20 <210> 2 <211> 20 <212> DNA <213> Enterococcus durans <400> 2 ccattggctc tgatcggtaa 20 <210> 3 <211> 20 <212> DNA <213> Enterococcus hirae <400> 3 gaccgttgct gcgacagtta 20 <210> 4 <211> 20 <212> DNA <213> Enterococcus hirae <400> 4 gagcctgcga cgttcaacat 20 <210> 5 <211> 20 <212> DNA <213> Enterococcus lactis <400> 5 gagcctgcga cgttcaacat 20 <210> 6 <211> 20 <212> DNA <213> Enterococcus lactis <400> 6 acacctatca caccggctaa 20 <210> 7 <211> 20 <212> DNA <213> Enterococcus mundtii <400> 7 taggaatgcg ctctcgatga 20 <210> 8 <211> 20 <212> DNA <213> Enterococcus mundtii <400> 8 acctgatgcc acatcgatg 20

Claims (10)

fructose 1,6-bisphosphatase (NCBI accession no. AKX85242.1), sodium/glutamate symport protein (NCBI accession no. AFM70771.1), helix -Any one selected from the group consisting of helix-turn-helix transcriptional regulator (NCBI accession no. QXX72259.1) and wxL domain surface protein (NCBI accession no. BAO06098.1) A biomarker composition for detecting Enterococcus strains comprising the above proteins or genes encoding them as active ingredients. The method of claim 1, wherein the Enterococcus strain is Enterococcus durans , Enterococcus hirae , Enterococcus lactis or Enterococcus A biomarker composition for detecting Enterococcus strains, characterized in that mundtii . fructose 1,6-bisphosphatase (NCBI accession no. AKX85242.1), sodium/glutamate symport protein (NCBI accession no. AFM70771.1), helix -Any one selected from the group consisting of helix-turn-helix transcriptional regulator (NCBI accession no. QXX72259.1) and wxL domain surface protein (NCBI accession no. BAO06098.1) A composition for detecting Enterococcus strains, comprising as an active ingredient an agent capable of measuring the expression level of the above proteins or genes encoding them. The method of claim 3, wherein the agent capable of measuring the expression level of the gene is fructose 1,6-bisphosphatase (NCBI accession no. AKX85242.1), sodium/glutamate Port protein (sodium/glutamate symport protein; NCBI accession no. AFM70771.1), helix-turn-helix transcriptional regulator (NCBI accession no. QXX72259.1) and wxL domain surface protein (wxL). A composition for detecting Enterococcus strains, characterized in that it is a primer or probe that specifically binds to one or more proteins selected from the group consisting of domain surface protein; NCBI accession no. BAO06098.1) or a gene encoding the same. The method of claim 4, wherein the primers include a primer set represented by SEQ ID NO: 1 and SEQ ID NO: 2, a primer set represented by SEQ ID NO: 3 and SEQ ID NO: 4, a primer set represented by SEQ ID NO: 5 and SEQ ID NO: 6, and; A composition for detecting Enterococcus strains, characterized in that it is one or more primer sets selected from the group consisting of primer sets represented by SEQ ID NO: 7 and SEQ ID NO: 8. The composition for detecting Enterococcus strains according to claim 3, wherein the Enterococcus strains are Enterococcus durans, Enterococcus hirae, Enterococcus lactis, or Enterococcus mundtii . A kit for detecting Enterococcus strains comprising the composition of any one of claims 3 to 6. (1) separating DNA from the sample;
(2) Using the isolated DNA as a template, a primer set represented by SEQ ID NO: 1 and SEQ ID NO: 2, a primer set represented by SEQ ID NO: 3 and SEQ ID NO: 4, and a primer set represented by SEQ ID NO: 5 and SEQ ID NO: 6 and; Performing PCR amplification using one or more primer sets selected from the group consisting of primer sets represented by SEQ ID NO: 7 and SEQ ID NO: 8; and
(3) A method for detecting Enterococcus strains comprising the step of analyzing the PCR amplification product. The method of claim 8, wherein the sample is probiotics or fermented food. The method of claim 8, wherein the Enterococcus strain is Enterococcus durans, Enterococcus hirae, Enterococcus lactis, or Enterococcus mundtii .