KR20170122610A - 2 step analysis method of microorganism - Google Patents

Abstract

The present invention relates to a method of measuring the type and amount of harmful microorganisms in a beer through simultaneous analysis and cross analysis of nucleic acids by sequentially performing multiplex PCR and simultaneous PCR. Specifically, the method comprises the steps of: preparing a sample; extracting nucleic acids from the sample; performing multiplex PCR on the extracted nucleic acids to identify the type of a microorganism from which the nucleic acids are derived; and performing simultaneous PCR to confirm the amount of the microorganism.

Description

{2 STEP ANALYSIS METHOD OF MICROORGANISM}

The present invention relates to a method for rapidly quantifying microorganisms in foods.

In connection with the manufacture, distribution and sale of food, foreign matter entering the food is not only a threat to the safety of food, but also an important factor in the quality reliability of the enterprise. There are various kinds of foreign substances introduced into food such as animals, plants and minerals. For example, Korean Patent Registration No. 10-1316702 discloses a method of calculating the elapsed days from the date of death of insect foreign matter found in food.

On the other hand, each food-related enterprise focuses on the detection, analysis, tracking, and management of foreign objects. Processing of such foreign objects requires fast and accurate detection and analysis of foreign objects. However, in the case of microorganisms, it is contaminated at a low concentration, the nucleic acid content of the microorganisms is so low that it is difficult to perform qualitative and quantitative analysis, and the time and cost are considerably consumed to analyze any kind of microorganisms.

Especially, in the case of beer harmful microorganisms, the morphological and genetic characteristics are similar to each other, and therefore it is necessary to analyze the genetic identification. When the microorganisms of the same genus are mixed, they are difficult to identify because of their similar nucleotide sequences.

Therefore, the inventors of the present invention have been studying a method for detecting and analyzing microorganisms in beer rapidly, and have found that microorganisms frequently occurring in beer are selected and multiplex PCR is performed on specific primer pairs, , And cross-validation by quantitative analysis of identified microorganisms using real-time PCR with specific primer pairs and probes.

An object of the present invention is a method for rapidly qualitatively and quantitatively analyzing a microorganism in a food.

According to an aspect of the present invention,

Preparing a sample;

Extracting a nucleic acid from the sample;

Performing multiplex PCR on the extracted nucleic acid using a gene-specific first primer of a lactic acid bacterium to identify the species of the microorganism from which the nucleic acid is derived; And

And performing a real-time PCR using a second primer and a probe specific to the gene of the microorganism species to confirm the amount of the microorganism.

The analysis method of the present invention can accurately analyze the kind and amount of microorganisms in a sample in a short time.

Figure 1 shows the results of confirming the specificity of primer pairs for Lactobacillus microorganisms.
Fig. 2 shows the result of confirming the specificity of primer pairs for Pediococcus microorganisms.
Fig. 3 shows the result of performing multiplex PCR on a DNA mixture of Lactobacillus microorganisms and a DNA mixture of Pediococcus microorganisms, respectively (a: Lactobacillus b: Pediococcus).
FIG. 4 shows the result of performing multiplex PCR when only one type of lactobacillus microorganism DNA exists.
FIG. 5 shows the result of performing multiplex PCR when only DNA of various kinds of Lactobacillus microorganisms is present.
FIG. 6 shows the result of performing multiplex PCR when only one kind of Pediococcus microorganism DNA exists.
FIG. 7 shows the result of performing multiplex PCR when only DNA of various kinds of Pediococcus microorganisms is present.
Fig. 8 shows the result of performing multiplex PCR on beer that artificially caused microbial opacity.
FIGS. 9 to 16 show the result of confirming the specificity of the primer pair and the probe for Real-Time PCR.
17 to 32 show respective standard curves of Real-Time PCR for microorganisms.
33 to 35 show the result of performing the multiplex PCR of Production Examples 1 to 3 of Example 5. Fig.
36 to 38 show respective standard curves of Real-Time PCR of Production Examples 1 to 3 of Example 5. Fig.

According to the present invention,

Preparing a sample;

Extracting a nucleic acid from the sample;

Performing multiplex PCR on the extracted nucleic acid using a gene-specific first primer of a lactic acid bacterium to identify the species of the microorganism from which the nucleic acid is derived; And

And performing a real-time PCR using a second primer and a probe specific to the gene of the microorganism species to confirm the amount of the microorganism.

Hereinafter, the present invention will be described in detail.

sample

The method for analyzing a microorganism in a sample of the present invention includes a step of preparing a sample. The sample is preferably food. The food may be a general food which can be ingested or ingested by a person or an animal, and is not particularly limited. Preferably, the food is a food which is generally processed and distributed. For example, the food may be used in beverage such as meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramie noodle, dairy product including gums, ice cream, various soups, drinks, tea, drink, A powdered preparation and a vitamin complex, a processed food, a retort food, a health functional food, a health supplement food, an instant food, and the like, and preferably be a mainstream such as beer, shochu, wine, fruit wine, fermentation wine, makgeolli, More preferably beer, shochu, etc., and most preferably beer.

Nucleic acid extraction step

The method for analyzing a microorganism in a sample of the present invention includes a step of extracting a nucleic acid from a sample. The nucleic acid is DNA or RNA, preferably gDNA. The method of extracting the nucleic acid is not particularly limited. That is, a person skilled in the art can appropriately select methods known in the art to extract nucleic acids.

Microbial species identification step

The method for analyzing a microorganism in a sample of the present invention includes a step of performing multiplex PCR on the extracted nucleic acid using a gene-specific first primer of a lactic acid bacteria to identify the species of the microorganism from which the nucleic acid is derived. This allows the identification of microbial species in a single multiplex PCR even in the presence of various microorganisms in the sample.

The lactic acid bacteria are either or both selected from the group consisting of Lactobacillus and Pediococcus. Preferably the lactic acid bacteria are selected from the group consisting of L. lindneri, L. casei, L. brevis, L. plantarum, Lactobacillus koi, Lactobacillus sp., Lactobacillus sp. Is at least one selected from L. coryniformis, P. damnosus, P. claussenii, P. inopinatus, and the like. Since most of the microorganisms found in beer, that is, more than 70% belong to the above eight species, it is effective to confirm whether they correspond to the above eight microorganisms.

The first primer is preferably a primer specific to each species, more preferably a primer specific to a gene having a low homology to each species. More preferably, the pheS gene of L. lindneri, the trxB gene of L. casei, the abf2 gene of L. brevis, the L. lambda gene of Lactobacillus plantarum (L , the luxS gene of Lactobacillus plantarum, the hsp60 gene of L. coryniformis, the leuS gene of P. damnosus, the pheS gene of P. claussenii, It is a primer that is specific for the atpA gene of P. inopinatus and can amplify these genes. The first primer may comprise a primer selected from the group consisting of SEQ ID NOS: 1 to 16, preferably comprising a pair of primers selected from the following (a) to (h):

(a) a primer pair of SEQ ID NOS: 1 and 2

(b) a primer pair of SEQ ID NOS: 3 and 4

(c) a primer pair of SEQ ID NOS: 5 and 6

(d) a primer pair of SEQ ID NOS: 7 and 8

(e) a primer pair of SEQ ID NOs: 9 and 10

(f) a primer pair of SEQ ID NOs: 11 and 12

(g) a primer pair of SEQ ID NOs: 13 and 14

(h) A pair of primers of SEQ ID NOs: 15 and 16.

Preferably, the first primer comprises a pair of primers specific for Lactobacillus or a pair of primers specific for Pediococcus. It is used as a marker to distinguish between the genus of Lactobacillus sp. And Pediococcus sp. For example, a primer capable of amplifying the pheS gene specific to both Pediococcus minocus, Pediococcus clausenii, and Pediococcus inopinatus can be included as a marker, and the marker can be represented by SEQ ID NOS: 17 and 18 A pair of primers can be used.

That is, the first primer may include:

1) a primer pair specific for a gene having a low homology to each species, and

2) a pair of primers specific for the genus of Lactobacillus or a pair of primers specific for Pediococcus

.

Here, the primer pair specific for a gene having a low homology to the species of 1) may be, for example, a pair of primers selected from the primers of SEQ ID Nos. 1 to 16. And 2) a primer pair specific for Lactobacillus sp. Or a pair of primers specific for Pediococcus can be, for example, a pair of primers selected from the primers of SEQ ID NO: 17 or 18. Therefore, it is advantageous for the first primer to include two or more primer pairs to distinguish microorganism species and genus.

The multiplex PCR is performed using the primer pairs. L. lindneri, L. casei, L. brevis, L. plantarum, Lactobacillus spp., And Lactobacillus spp. Is a microorganism selected from L. coryniformis, P. damnosus, P. claussenii, P. inopinatus, or a microorganism selected from the group consisting of Lactobacillus, L. coryniformis, P. damnosus, P. claussenii, You can check at once.

Identify the amount of microorganisms

The method of analyzing a microorganism in a sample of the present invention includes a step of performing a real-time PCR using a second primer and a probe specific to a gene of the microorganism species to confirm the amount of the microorganism.

By examining the amount of microorganisms in the sample, the effect of these microorganisms on the sample, such as the change in the pH of the beer when the sample is a beer, and the degradation of the beer, can be evaluated. In addition, by checking the amount of microorganisms in the sample, it is possible to estimate the inflow point and the inflow point of microbial influx (that is, the occurrence in the sample), and it is possible to promptly process the cause of the microbial contamination.

The real-time PCR is performed using a second primer and a probe. The second primer is preferably a primer specific to each species, more preferably a primer specific to a gene having a low homology to each species. More preferably, the pheS gene of L. lindneri, the trxB gene of L. casei, the abf2 gene of L. brevis, the L. lambda gene of Lactobacillus plantarum (L , the luxS gene of Lactobacillus plantarum, the hsp60 gene of L. coryniformis, the leuS gene of P. damnosus, the pheS gene of P. claussenii, It is a primer that is specific for the atpA gene of P. inopinatus and can amplify these genes.

It is advantageous from the viewpoint of the binding ability that the probe has a GC binding of 40 to 60% with the second primer used together, and preferably, the GC binding with the second primer is 50 to 60%. The length of the probe is 15 to 30 bp, preferably 18 to 28 bp. When the length of the probe is less than 15 bp, the number of bonds that are complementary to each other between the probe and the target gene becomes too small, resulting in a low binding force. The short nucleotide sequence has a high probability of incorrectly binding to a specific site, Over-quantification can occur and accuracy can be lowered. Also, when the length of the probe exceeds 30 bp, the binding force is increased but the number of complementary bonds is excessively increased, so that the binding efficiency is lowered and the non-binding due to non-binding occurs, resulting in lower quantification.

Preferably, the second primer and the probe comprise a pair of primers selected from the following (a) to (h) and a probe:

(a) a primer pair of SEQ ID NOs: 19 and 20 and a probe of SEQ ID NO: 21

(b) a pair of primers of SEQ ID NOs: 22 and 23 and a probe of SEQ ID NO:

(c) a primer pair of SEQ ID NOs: 25 and 26 and a probe of SEQ ID NO: 27

(d) a primer pair of SEQ ID NOs: 28 and 29 and a probe of SEQ ID NO: 30

(e) a primer pair of SEQ ID NOs: 31 and 32 and a probe of SEQ ID NO: 33

(f) a primer pair of SEQ ID NOs: 34 and 35 and a probe of SEQ ID NO: 36

(g) a primer pair of SEQ ID NOs: 37 and 38 and a probe of SEQ ID NO: 39

(h) the primer pair of SEQ ID NOs: 40 and 41 and the probe of SEQ ID NO: 42.

The method for analyzing microorganisms of the present invention

The method for analyzing microorganisms in the sample of the present invention is a two-step analysis method for analyzing the amount of microorganisms identified by real-time PCR after verifying the kinds of microorganisms present in the sample through multiplex PCR. Using the method of the present invention, it is possible to quickly and accurately analyze the type and amount of microorganisms in a sample.

≪ Materials and methods >

Extraction and quantification of DNA

Genomic DNA of all microorganisms was extracted using BioFACT ™ Genomic DNA Prep Kit (Biofact, Korea). The extraction method was the same as the method provided by the manufacturer. The microorganisms before the extraction were cultured in MRS broth. In the case of the beak samples induced by turbidity, the genomic DNA was directly extracted from the precipitate by centrifugation and applied to this study. In the case of beer samples without turbidity, 100 ml of beer was filtered through a 0.25 μm filter, and the filter paper was added to the MRS broth to extract the genomic DNA by culturing the microorganism for 24 hours. Finally, distilled water was added to the silica gel membrane in the extraction column and centrifuged at 13,000 × g using a centrifuge (Micro17TR, Hanil science, Korea) to separate the genomic DNA bound to the silica gel membrane in the extraction column.

Quantitation of DNA was performed by measuring the concentration using a NanoVue (TM) Plus Spectrophotometer (GE healthcare, England).

<Example 1> Selection of harmful microorganisms causing beer pollution

More than 70% of the microorganisms that cause beer pollution around the world are known as Lactic Acid Bacteria (LAB), which can grow in low pH and anaerobic conditions in the presence of alcohol. Most of them have hop resistant genes, The most widely occurring pollution. Therefore, in consideration of this, eight kinds of representative harmful microorganisms most frequently causing beer contamination as shown in Table 1 below were selected.

The eight microorganisms were distributed from the Center for Microbial Resources, Korea Research Institute of Bioscience and Biotechnology (Table 2). The cultured microorganisms were cultured in an MRS agar plate for 3 to 5 days at 30 ° C in an anaerobic condition, and then subjected to single colony isolation, followed by an incremental culture on an MRS broth medium and used in the following experiments. Based on the National Center for Biotechnology Information (NCBI) gene bank data for eight microorganisms, specific gene information for each microorganism was obtained and used as a target gene of primer pairs and probes.

The abbreviations used herein are as follows: LL: L. lindneri, LB: L. brevis, LC: L. casei, LP: L. plantarum, LCo: L. coryniformis, PD: P. damnosus, PI: P. inopinatus, PU: Pediococcus universal primer.

&Lt; Example 2 > Preparation of primer and probe

Primers and probes were prepared based on the nucleotide sequence information registered in the National Center for Biotechnology Information (NCBI) for the detection of the eight microorganisms. The primers of the present invention have no hair-pin, loop, dimer, etc. and GC contents are between 45-60%. In addition, the forward and reverse primer pairs of the present invention can be constructed so as to be composed of 20-mer oligonucleotides to obtain a relatively short amplification product of less than 450 bp, preferably 100 bp or more and less than 450 bp, Even if the DNA is degraded, it is possible to amplify easily by binding the primer to the correct target.

Specific primers and probes are shown in Tables 3 and 4 below. First, 5 pairs of primers specific for 5 species of Lactobacillus and 3 pairs of primers specific for 3 species of Pediococcus were prepared. In addition, a pair of pediococcus universal specific to Pediococcus was constructed. Because the gene structure of Lactobacillus and Pediococcus is very similar, it is possible to distinguish between two kinds of microorganisms. To generate a PCR amplification product.

In the case of multiplex PCR, the amounts of amplified products to be expressed are different due to mutual competition among primers. Therefore, in the case of real-time PCR, both primer and probe use the same 10 pM / μl PCR was carried out. For real-time PCR primers, primer design program was used to increase the specificity of amplified products to within 100-150 bp. For real-time PCR, a probe was prepared between the forward primer, which is the starting point of the amplification product, and the reverse primer, about 20 to 25 bp. GC bonding between the primer and the probe was made between 50 and 60% to increase the binding force (Table 4). The sequence of the target genes is shown in Table 5.

Multiplex PCR primer sequence and concentration used

Real-Time PCR primers and probes

<Example 3> Multiplex PCR was performed

<3-1> Conditions for Multiplex PCR

Multiplex PCR was performed using the primer pairs shown in Table 3 above. If the number of primers is too high, the primers will compete with each other to compete with each other, resulting in degradation in amplification efficiency. Errors such as dimer formation between primers or non-specific amplification products caused by wrong primer of different target occur, , It was performed in this experiment by dividing into Lactobacillus and Pediococcus multiplex PCR.

PCR was performed with a T-Gradient thermoblock (Biometra, German) instrument. Multiplex PCR conditions were: pre-incubation at 94 ° C for 5 min, denaturation at 94 ° C for 30 sec, annealing at 60 ° C for 30 sec, and extension at 72 ° C for 30 sec. Finally, terminal elongation was carried out at 72 ° C for 10 minutes. The template DNA was quantified to be 50 ng / μl. The mixture required for PCR was 2 × Multiplex PCR Pre-Mix (Solgent, Korea) of SolGent, which was mixed with Tap polymerase, dNTP, MgCl2 and loading dye required for electrophoresis ) Products were used.

PCR amplification results were confirmed by electrophoresis. The electrophoresis equipment was Mupid-21 (Cosmo bio, Japan) and electrophoresis was carried out on 3% agarose gel at 100V for about 30 minutes.

<3-2> Identification of primer specificity

Primer specificity means that a primer binds to only a specific target to generate a PCR amplification product. In this experiment, primers were designed to produce amplification products only for specific microorganism species, and primers were designed to produce amplified products of the same size as those of target genes. For reference, in the multiplex PCR experiment of the present invention, since the size of the PCR amplification product was different for each microorganism species, it is possible to classify the microorganism species according to the size of the electrophoresis result of the amplification product.

On Lactobacillus  About Primer  Identification of specificity

PCR was carried out by treating the primer pairs of SEQ ID NOS: 1 to 10 with different types of Lactobacillus microorganisms for each lane, and as a result, i) amplification products of the same size as designed in the target gene, ii) The target specificity was confirmed by confirming whether amplification was performed only in the target gene. Therefore, the primers of the present invention were confirmed to have specificity for Lactobacillus (Fig. 1, Lane M: 100 bp DNA ladder, Lane 1: Lactobacillus brevis, Lane 2: Lactobacillus casei, Lane 3: Lane 4: Lactobacillus lindneri, Lane 5: Lactobacillus plantarum, Lane N: Negative control).

Specifically, FIG. 1 (a) is Lactobacillus brevis, and the target is abf2 gene. As a result of the multiplex PCR, an amplification product of 197 bp was generated, and it was confirmed that Lactobacillus brevis had specificity. In FIG. 1 (b), the Lactobacillus casei strain was a trxB gene, and an amplification product of 240 bp was generated. As a result, specificity was confirmed in Lactobacillus casei. Fig. 1 (c) shows that the Lactobacillus koriniformis target was the hsp60 gene, and its specificity was confirmed in Lactobacillus koriniformis, which produced an amplification product of 441 bp. Fig. 1 (d) shows that the lacto-bacillus lindneri target was a pheS gene and produced an amplified product of 120 bp, and the specificity was confirmed in Lactobacillus lindneri. Fig. 1 (e) is a lactobacillus plantarum, and the target was a luxS gene, and an amplification product of 310 bp was produced. As a result, specificity was confirmed in Lactobacillus plantarum.

Pedio Ococus  About Primer  Identification of specificity

PCR was carried out by treating the primer pairs of SEQ ID NOs: 11 to 16 with different kinds of Pediococcus microorganisms for each lane, and as a result, i) amplification products of the same size as designed in the target gene were amplified by ii ) The target specificity was confirmed by confirming whether the target gene of the microorganism was amplified only. No amplification products were produced in non-target genes. Therefore, the primers of the present invention were found to have specificity for Pediococcus (Fig. 2 (a) to (c), Lane M: 100 bp DNA ladder, Lane 1: Pediococcus clausenii, Lane 2: Lane 3: Pediococcus inopinatus, Lane N: Negative Control)

Specifically, FIG. 2 (a) shows Pediococcus clausenii, and the target is pheS gene. As a result, an amplification product of 191 bp was produced, and the specificity was confirmed in Pediococcus clausenii. Fig. 2 (b) shows that the Pediococcus sp. Strain was a leuS gene and produced an amplification product of 121 bp. Thus, the specificity was confirmed in Pediococcus sp. In Fig. 2 (c), the Pediococcus inopinatus target was an atpA gene, and an amplification product of 305 bp was produced. As a result, specificity was confirmed in Pediococcus inopinatus.

In Example 2, a pediococcus universal specific to a genus Pediococcus was prepared by using a marker between Lactobacillus and Pediococcus (SEQ ID NOS: 17 and 18), and the genome of the genus Lactobacillus and Pediococcus Multiplex PCR was performed on all of the genus Oocyte.

As a result, it was confirmed that an amplification product of 157 bp was generated only in the pediococcus in which the pheS gene was targeted, and that it could be used as a marker to differentiate between Lactobacillus and Pediococcus (Fig. 2 (d), Lane M: 100 bp DNA ladder, Lane 1: Lactobacillus brevis, Lane 2: Lactobacillus casei, Lane 3: Lactobacillus koriniformis, Lane 4: Lactobacillus lindneri, Lane 5: Lactobacillus plantarum, Lane 6: Lane 7: Pediococcus minnowsus, Lane 8: Pediococcus inopinatus, Lane N: Negative Control).

<3-3> Multiplex PCR for harmful microorganism mixture

Genomic DNAs of the five Lactobacillus microorganisms shown in Table 2 were extracted and their concentrations were measured. Lactobacillus positive control (Lactobacillus positive control) was prepared by mixing them at the same concentration (50 ng / ul). Then, multiplex PCR was carried out using the primer pairs of SEQ ID NOS: 1 to 10 at the concentrations shown in Table 3 below.

Also, the genomic DNAs of the three kinds of Pediococcus microorganisms shown in Table 2 were extracted and their concentrations were measured. Then, they were mixed at the same concentration (50 ng / ul) to prepare a pediococcus positive control. Then, multiplex PCR was carried out using the primer pairs of SEQ ID NOS: 11 to 18 at the concentrations shown in Table 3 below.

As a result, it was confirmed that amplified products of different sizes were amplified at once with similar sensitivities to 5 kinds of Lactobacillus and 3 kinds of Pediococcus (Fig. 3 (a) Multiplex PCR of Lactobacillus, (b) Multiplex PCR results of Pediococcus).

<3-4> Multiplex PCR for randomly mixed harmful microorganism mixtures

<3-4-1> When only one type of Lactobacillus microorganism DNA exists

First, multiplex PCR was performed by treating primer pairs of SEQ ID NOS: 1 to 10 with each of the five kinds of Lactobacillus microorganisms shown in Table 2. As a result, amplification products of the target were confirmed at the intended size in the production of the primer pairs. Therefore, it was possible to detect any microorganism species by confirming amplified products of different sizes (Fig. 4, Lane M: 100 bp DNA ladder, Lane 1: Lactobacillus brevis, Lane 2: Lactobacillus casei, Lane 3: Bacillus coriniformeis, Lane 4: Lactobacillus lindneri,).

&Lt; 3-4-2 > When DNA of plural kinds of Lactobacillus microorganisms exists

The DNAs of the five kinds of Lactobacillus microorganisms shown in Table 2 were mixed as shown in Table 6 below, and multiplex PCR was performed on them. At this time, the primer pairs of SEQ ID NOS: 1 to 10 were used.

As a result, amplification products of the target were increased one by one as the kinds of microorganisms to be mixed increased, and it was possible to distinguish the presence of any microorganism DNA even if the DNAs were mixed (FIG. 5).

<3-4-3> When only one kind of Pediococcus microorganism DNA exists

First, multiplex PCR was performed by treating primer pairs of SEQ ID NOS: 11 to 18 with each of the three kinds of Pediococcus microorganisms shown in Table 2. As a result, amplification products of the target were confirmed at the intended size in the production of the primer pairs. Therefore, it was possible to detect any microorganism species by confirming amplification products of different sizes (Fig. 6, Lane M: 100 bp DNA ladder, Lane 1: Pediococcus duromorphus, Lane 2: Pediococcus Clauseny, Lane 3: Pediococcus inopinatus, Lane N: Negative Control)

&Lt; 3-4-4 > When DNA of plural kinds of Pediococcus microorganisms exists

The DNAs of the three kinds of Pediococcus microorganisms in Table 2 were mixed as shown in Table 7 below, and multiplex PCR was performed on them. At this time, the primer pairs of SEQ ID NOS: 11 to 18 were used.

As a result, amplification products of the target were increased one by one as the kinds of microorganisms to be mixed increased, and it was possible to distinguish the presence of any microorganism DNA even if the DNAs were mixed (Fig. 7).

<3-4-5> Analysis of turbidity-induced beer caused by anthropogenic microbial contamination

Each of the eight microorganisms listed in Table 2 was broth-cultured to prepare a culture solution. 1 ml of the culture solution was added to 50 ml of beer, followed by culturing for 36 hours to induce turbidity. Then, turbid precipitate was obtained and DNA was extracted therefrom and analyzed by Multiplex PCR. All eight kinds of extracted DNA were applied to Lactobacillus multiplex PCR and Pediococcus multiplex PCR. Lactobacillus positive (Lactobacillus Positive) and Pediococcus Positive (Pediococcus Positive) were also prepared by adding 5 kinds of DNAs of Lactobacillus microorganisms. These were also subjected to Lactobacillus multiplex PCR and Pediococcus multi Flex PCR was performed. At this time, multiplex PCR was performed by adding all of the primer pairs of SEQ ID NOS: 1-18.

As a result, it was confirmed that a target specific microorganism gene and a specific primer were specifically bound to form an intrinsic specific amplification product. In addition, multiplex PCR of the Lactobacillus positive control group and the Pediococcus positive control group revealed that 8 kinds of microorganisms can be analyzed simultaneously (Fig. 8, Lane M: 100 bp DNA ladder, Lane LP: Lactobacillus positive, Lane PP: Pediococcus positive, Lane 1: Lactobacillus brevis, Lane 2: Lactobacillus casei, Lane 3: Lactobacillus koriniformis, Lane 4: Lactobacillus lindneri, Lane 5: Lactobacillus plantarum, Lane 6 : Pediococcus Clauseny, Lane 7: Pediococcus Minorosus, Lane 8: Pedioccus inopinatus, Lane N: Negative Control).

Example 4 Real-Time PCR Performed

<4-1> Identification of specificity of Real-Time PCR primer and probe

Using the primers and probes of the real-time PCR in Table 4, it was confirmed whether the amplification product was produced by performing general PCR or the amplification product was formed only in the target gene, and the specificity of the primer Respectively. Then, Real-Time PCR was performed by adding the probe as an additional probe, and the specificity of the probe was confirmed by whether the amplification curve was formed only in the target gene. The reproducibility of the method was verified by repeated experiments.

Specificity refers to the amplification curve by the fluorescence generated by amplifying the gene only in the microorganism corresponding to the target DNA. In the case of real-time PCR, a pair of primers and a corresponding probe are added to a mixture for PCR, which means that the amplification curve is formed as a target microorganism, and the amplification curve is not formed unless it is a target microorganism.

As a result, all of the eight primer pairs and probes of Table 4 were found to have specificity because they exhibited amplification curves only at the corresponding target in specific microorganisms (FIGS. 9 to 16).

<4-2> Real-time PCR detection limit check and standard curve creation

Serial dilution of DNA extracted from each microorganism was performed to dilute the DNA to 100, 50, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005 and 0.001 ng / A standard curve was created.

As a result, it was confirmed by real-time PCR that the analysis was possible up to a trace concentration of 0.001 ng / μl. Therefore, even when a small amount of harmful microorganisms are mixed in the beer, it is judged that the microorganism can be purified by performing DNA extraction and real-time PCR using the primers of the present invention. In addition, the amount of DNA of the microorganism incorporated through the standard curve was also quantifiable (FIGS. 17 to 32). At this time, the error rate of the method was confirmed to be less than 0.1%, and it was found that the reliability of the method was high through the error rate.

Real-Time PCR was performed using a Roche LC480 (Roche, Switzerland) instrument. Real-time PCR was performed by repeating the initial denaturation at 50 ° C for 2 minutes and 95 ° C for 10 minutes, followed by 50 cycles of amplification at 95 ° C for 15 seconds and 61 ° C for 40 seconds. Confirmation of results can be easily confirmed using LC480 program.

Example 5 Sequential Analysis of Multiplex PCR and Real-Time PCR

Specific Lactobacillus and Pediococcus were cultured in MRS broth medium to prepare a culture solution. In Production Example 1, the cultures of Lactobacillus brevis, Lactobacillus casei, and Pediococcus clauseni were mixed. In preparation example 2, the culture broth of Lactobacillus casei, Lactobacillus koriniformeus and Lactobacillus plantarum was mixed In Production Example 3, cultures of Lactobacillus brevis, Lactobacillus cell Lactobacillus koriniformis, Lactobacillus lindneri, and Pediococcus minnowusus and Pediococcus inopinatus were mixed in an equal volume, and then mixed culture (preparation Examples 1 to 3) were added to 500 ml of ordinary beer, respectively, and the bottle was wired and left at room temperature for 24 hours. After 24 hours, the beer was filtered with a 0.45 mu m filter. Then, the filtered filter was re-cultured in MRS broth medium, and the cultured medium was centrifuged to extract DNA from the precipitate and analyzed by multiplex PCR and real-time PCR. At this time, multiplex PCR was performed by adding all the primer pairs of SEQ ID NOS: 1 to 18 to the multiplex PCR.

As a result, it was confirmed that only the gene for the added microorganism was present, thus confirming the accuracy of the experiment (Fig. 33: Production Example 1, Fig. 34: Production Example 2, Fig. 35: Production Example 3).

Thereafter, real-time PCR was performed using both primer pairs and probes of SEQ ID NOS: 19 to 42 to quantitatively analyze the amount of DNA of each microorganism.

As a result, only the amplification product and the amplification curve of the microorganism added to the sample were confirmed, and it was confirmed that the qualitative analysis and the quantitative analysis were accurately performed even though the microorganisms were contained in trace amounts in the sample (FIG. 36: 37: Production Example 2, Fig. 38: Production Example 3). Specific quantitative results are shown in Table 8 below.

CP ^* : The intersection of the minimum threshold and the amplification curve that causes a reaction at the crossing point. The number of cycles at which the gene increases exponentially as the gene increases by 2 ⁿ according to the cycle repeat (n) during PCR.

<110> HITEJINRO <120> 2 STEP ANALYSIS METHOD OF MICROORGANISM <130> DNP160049 <160> 52 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for pheS of L. lindneri <400> 1 aggttcgtgt tcgtcctagt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for pheS of L. lindneri <400> 2 cttcaatcca accggatcct 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for trxB of LCtrxB <400> 3 tgctggatcg cggcgtctat 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for trxB of L. casei <400> 4 cgcctgtcgc aatgacaatc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for abf2 of L. brevis <400> 5 cgtaccgctc attcgctatc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for abf2 of L. brevis <400> 6 ttctggcgac gaagatcact 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for LuxS of L. plantarum <400> 7 ttacggtgga aaatgggcct 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Reverse Primer for LuxS of L. plantarum <400> 8 ccacagctat cgatggtcgt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for hsp60 of L. coryniformis <400> 9 gaacgtcact gccggtgcta 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for hsp60 of L. coryniformis <400> 10 tcgtcggcga tgatcaacaa 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for leuS of P. damnosus <400> 11 caatggatgc ttaagattac 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for leuS of P. damnosus <400> 12 cgttgaacgg ccaatccagt 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for pheS of P. claussenii <400> 13 tcgacgtgat acggatgatg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for pheS of P. claussenii <400> 14 cttcaactga tggttcagta 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for atpA of P. inopinatus <400> 15 ccagttggtg acgccttagt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for atpA of P. inopinatus <400> 16 ccgatcgcca catagataca 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for pheS of Pediococcus <400> 17 tcgacgtgat acggatgatg 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for pheS of Pediococcus <400> 18 tggacgaaga cgtacttcaa 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for Real-Time PCR of pheS of L. lindneri <400> 19 aggttcgtgt tcgtcctagt 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for Real-Time PCR of pheS of L. lindneri <400> 20 ccttcacagt tcatgcaagt 20 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Probe for Real-Time PCR of pheS of L. lindneri <400> 21 accgaacctt cagtagaagc agatg 25 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Real-Time PCR of trxB of L. casei <400> 22 cctatggcaa tgtcgttagc 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Real-Time PCR of trxB of L. casei <400> 23 cgcctgtcgc aatgacaatc 20 <210> 24 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Probe for Real-Time PCR of trxB of L. casei <400> 24 aactgacgag gatgagtacg aggct 25 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Real-Time PCR of abf2 of L. brevis <400> 25 tccggttgga cttggcttgg 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> Reverse primer for Real-Time PCR of abf2 of L. brevis <400> 26 cgagattgac ggccatattg 20 <210> 27 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Probe for Real-Time PCR of abf2 of L. brevis <400> 27 cttggcttgg cgggaactcg aaacc 25 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Real-Time PCR of luxS of L. plantarum <400> 28 atggatggcg tgattgactg 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Real-Time PCR of luxS of L. plantarum <400> 29 ttcaatgcct tagcaacttc 20 <210> 30 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Probe for Real-Time PCR of luxS of L. plantarum <400> 30 tcatttgatt acctggggtg aacat 25 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Real-Time PCR of 16S rRNA of L. coryniformis <400> 31 gcggctgtct ggtctgtaac 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> Reverse primer for Real-Time PCR of 16S rRNA of L. coryniformis <400> 32 ttgcgtaatt cgtaaggcgg 20 <210> 33 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Probe for Real-Time PCR of 16S rRNA of L. coryniformis <400> 33 agtaacggac aagaagatct ctaac 25 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Real-Time PCR of leuS of P. damnosus <400> 34 caatggatgc ttaagattac 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Real-Time PCR of leuS of P. damnosus <400> 35 cgttgaacgg ccaatccagt 20 <210> 36 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Probe for Real-Time PCR of leuS of P. damnosus <400> 36 ggcccgacag tatcaaggaa atgca 25 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Real-Time PCR of pheS of P. claussenii <400> 37 tcgacgtgat acggatgatg 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Real-Time PCR of pheS of P. claussenii <400> 38 cttcaactga tggttcagta 20 <210> 39 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Probe for Real-Time PCR of pheS of P. claussenii <400> 39 tgaagtacgt cttcgtccaa gttat 25 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Real-Time PCR of atpA of P. inopinatus <400> 40 actggacgga tcatggaagt 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Real-Time PCR of atpA of P. inopinatus <400> 41 ttcgttcaac tggccgtgtg 20 <210> 42 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Probe for Real-Time PCR of atpA of P. inopinatus <400> 42 tggtgacgcc ttagttggtc gagtt 25 <210> 43 <211> 455 <212> DNA <213> Lactobacillus lindneri <400> 43 aagatacttt ttatattact aaagaaattt taatgcgcac tcacacgtca cctatgcaag 60 ctcgtgcttt acaacagcat gatttcaata aaggaccatt aaaaatgatt tctcctggag 120 ttgtttttag aagagataca gatgacccaa ctcattcaca ccaatttcat caagttgagg 180 gaattgtaat cgataagcac attacgatgg ctgatttaaa aggaacctta gaggtaatgt 240 cgcataaatt atttggcgat aaattcaagg ttcgtgttcg tcctagttat tttccattta 300 ccgaaccttc agtagaagca gatgtaactt gcatgaactg tgaaggaaaa ggttgtgaag 360 tttgtaaagg atccggttgg attgaagtat taggggctgg tatggttcat cccaatgttt 420 tgaaaatggc aggcgttgac agtcaagaat atggt 455 <210> 44 <211> 954 <212> DNA <213> Lactobacillus casei <400> 44 atggcaaaaa aatatgatgt aatcgtgatc ggtgccggcc caggtggtat gacggctgca 60 ctttatgcat cgcgtgccaa tttatctgtg ctgatgctgg atcgcggcgt ctatggcggg 120 cagatgaaca acaccgctga agttgaaaac tatccgggtt tcaagtcaat cctcgggcca 180 gatcttggtc agaaaatgta tgatggtgcc acccaatttg gtgctgagta tgcctatggc 240 aatgtcgtta gcgttcaaaa ccgcggtgcc gtcaagctag tgaaaactga cgaggatgag 300 tacgaggcta aagcgattgt cattgcgaca ggcgcagagc ataagaagtt gggcgtaccg 360 ggcgaagaag cctacagtgg tcgtggcgtt tcatactgtg ctgtctgcga tggcgccttc 420 tttaaggatc gcgagttggc cgttattggt ggtggcgatt ccgcgattga agaaggcctt 480 tacctgacac aaatggccaa gaaggtcaca gttattcatc gccgtgatca attgcgtgct 540 cagcagatta tccagaagcg agcatttgcc aatgataaga tgcatttcgt ctggaatgct 600 caggttcagg aaatccaagg tgacgatatg aaagtcaccg gcgtgaaata ccgtgatcgt 660 gataaggaaa ctggcgagga acatattttg ccagtcgctg gggttttcat ttatgttggc 720 attatgccaa tgactgaagc cttccaagat ttaggtgttc tggatgagca tggttggatt 780 ccaaccgacg agcatatgcg gacaaaggtt ccaggcgttt tcgcgatcgg cgatgttcgc 840 gccaaggact tgcggcagat taccacagct gttggtgaag gtggcacagc tggacaaggc 900 gtcttcaact atattcagag tttgaatgat acgaatattg agattaaagc ttga 954 <210> 45 <211> 1506 <212> DNA <213> Lactobacillus brevis <400> 45 atgcaaggtt ctacaaagat tcaaatccaa gacgttgtcg ccccaatcga ccgccggcta 60 tatggctcct ttatcgagca attaggacgt gccgtttaca ccggtatcta tcaacctgat 120 catcccaccg ccgactcaga tggcttccgg accgatgtca ttgatgccgt aaaaactctg 180 aacgtaccgc tcattcgcta tcctggtggg aactttcttt cgcaatatcg atgggaggat 240 ggcattggtc ccaaaagcca gcgccccgtc cggttggact tggcttggcg ggaactcgaa 300 accaaccagt ttggccttca cgagtttatg cgttgggcag ataaagtcaa tgctgtcccc 360 aatatggccg tcaatctcgg cactcgggga attcaagcgg ctgctgactt agtcgaatat 420 tgtaattttc ctaaaggcac ttatttaagt gatcttcgtc gccagaatgg tgctgagcac 480 ccctttgcca ttaaaacgtg gtgtttgggt aatgaaatgg acgggccatg ggaaattggt 540 gcgaaatcgg caggcgaata tgcccacttg gctaacgaga ccgctaaggc catgcgtcgc 600 gttgatgaca ctctggaact ggtcgcctgt ggcagctcgt caatggacaa tcccaccttt 660 ggtaattggg aagaaaccgt ccttgatgct tgctacgata acgtcgatta cctgtcgctc 720 catcgttact acggctacta caatgatgac gacccaaccg aactagataa tttcttaggt 780 aaaaatcatg acttagatga ctttatcaag ggcgtcgttg ctatgtgcga tgccgttaag 840 gcccgtaaac ggtcaactaa gacgattaac ttatcctttg acgaatggaa tgtctggtat 900 cattccaacg acgctgatac ccaggttacc ccatggcaag tcggccccca tctcttagag 960 gatatctata atttcgaaga cgcgctatta attggcagtc tcctcatgac cctcttgcgc 1020 aatgctgatc gcgtcaagat cgcttgtttg gcacaactcg ttaacgtgat tgcgcccatc 1080 atgacggatg aaaatggtgg tatctggcgg caatccatct tctatccatt tatgcaggtg 1140 gccaactacg gtcaaggggt cgtcctcacc gatcacagca catcatccac ctacaattcc 1200 cgtgagttca ccgatgtggc ttatctggat accgtcacca cctatgacgc ggctacccag 1260 accgtcaccg tctttgccga aaataaacac caaacagaaa cgcttgactt tgagcttaat 1320 ttcggcgagc tgatgattga tcggctactt gatgccacgc aattttacgg ttacgatgtc 1380 aaagctgata atcgcgatgg tcatatgcag ctacaaccat tgcctgctag gactgacacc 1440 catcatgcga acactaaatt acaaccacta tcttggaacg tcttgcggtt taagctccga 1500 gattga 1506 <210> 46 <211> 477 <212> DNA <213> Lactobacillus plantarum <400> 46 atggctaaag tagaaagttt tacattagat cataccaagg ttttagcacc ttacgttcgt 60 aaaattacgg tggaaaatgg gcctaagggt gatgccatca ctaattttga tttgcggtta 120 gttcaaccta acaagaccgc tattgatacg gcgggcttac acacgattga acatatgttg 180 gctgggttat tacgtgatcg tatggatggc gtgattgact gctcaccatt tggttgtcgg 240 actggttttc atttgattac ctggggtgaa catgacaccg ttgaagttgc taaggcattg 300 aagtcctcat tagaattcat tgctggccca gctaagtggg aagacgtaca agggacgacc 360 atcgatagct gtgggaatta caaggatcat tctttattct cagctaagga atgggccaag 420 ttgattttat cgcaaggaat ttcatcggat ccatttgtac gcaaagtcgt tgaatag 477 <210> 47 <211> 506 <212> DNA <213> Lactobacillus coryniformis <400> 47 gaaggcatga agaacgtcac tgccggtgct aacccagttg gtatccgtga cgggatcgaa 60 aaagcaacta aggctgccgt tgatgaatta cacaagattt cgcataaagt taacggtaaa 120 ggtgatattg ctcaaatcgc ggctgtttct tcagctaacc aagaagttgg taaattaatt 180 gctgatgcta tggaaaaagt tggcaacgac ggtgtcatca caattgaaga atctaaagga 240 atcgatactt ctttagacgt tgttgaaggg atgcaattcg atcgtggtta tttgtcacaa 300 tacatggtta ctgacaatga caagatggaa gctgatctgg acaatccata catcttagta 360 acggacaaga agatctctaa cattcaagaa gttttaccat tactacaaaa gatcgtagaa 420 caaggtaaag cattgttgat catcgccgac gatgtagacg gtgaagcctt accaactctt 480 gtttggaaca agatccgtgg gacatt 506 <210> 48 <211> 1354 <212> DNA <213> Lactobacillus coryniformis <400> 48 ggcggcgtgc taatacatgc agtcgaacgc actgacgtcg accgaagctg cttgcagtgg 60 acgttgattg acgtgagtgg cggacgggtg agtaacacgt gggtaaccta cccttaagtg 120 ggggataaca tttggaaaca gatgctaata ccgcataacc attcagacca catggtctga 180 atgtaaaaga cggcctttgg ctgtcacttt tggacggacc cgcggcgtat tagttagttg 240 gtaaggtaac ggcttaccaa gacaatgata cgtagccgac ctgagagggt aatcggccac 300 attgggactg agacacggcc caaactccta cgggaggcag cagtagggaa tcttccacaa 360 tggacgaaag tctgatggag caacgccgcg tgagtgaaga aggttttagg atcgtaaaac 420 tctgttgttg gagaagaaca gggactagag taactgttag tcctttgacg gtatccaacc 480 agaaagccac ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt 540 ccggatttat tgggcgtaaa gcgagcgcag gcggtttttt aagtctgatg tgaaagcctt 600 cggcttaacc gaagaagtgc attagaaact gggaaacttg agtgcagaag aggacagtgg 660 aactccatgt gtagcggtga aatgcgtaga tatatggaag aacaccagtg gcgaaggcgg 720 ctgtctggtc tgtaactgac gctgaggctc gaaagtatgg ggagcgaaca ggattagata 780 ccctggtagt ccataccgta aacgatgaat gctaagtgtt ggagggtttc cgcccttcag 840 tgctgcagct aacgcattaa gcattccgcc tggggagtac gaccgcaagg ttgaaactca 900 aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 960 aagaacctta ccaggtcttg acatcctttg accactgtag agatacagct ttcccttcgg 1020 ggacaaagtg acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa 1080 gtcccgcaac gagcgcaacc cttatgacta gttgccagca tttagttggg cactctagta 1140 agactgccgg tgacaaaccg gaggaaggtg gggatgacgt caaatcagca tgccccttat 1200 gacctgggct acacacgtgc tacaatggtc ggtacaacga gttgcgaacc cgcgagggta 1260 agctaatctc ttaaagccga tctcagttcg gattgtaggc tgcaactcgc ctacatgaag 1320 ccggaatcgc tagtaatcgc ggatcagcac gccg 1354 <210> 49 <211> 677 <212> DNA <213> Pediococcus damnosus <400> 49 aaatcacttg gtttttctta tgactgggat cgcgaaatta atacaactga tcctaagtat 60 tacaagtgga cccaatggat ttttgagcaa atgtacaaaa aagggttagc ttacgaaaaa 120 gaaactttag ttaactgggc tcctgatatg atgggaggca cagtggttgc taacgaagaa 180 gtaattaatg gaaagacaga acggggtggt tacccagttt atcgggtccc catgaagcaa 240 tggatgctta agattacggc atatgctgat cgtttgctag atgatttaga cttgattgat 300 tggcccgaca gtatcaagga aatgcaacga aactggattg gccgttcaac gggggccagt 360 gttaacttta aagttgctgg gactgatgat gaaattgaga tctatacaac ccgccctgac 420 acgctgtttg gtgcaagcta catggttatc gcaccagaac actcactggt aaaaaagatt 480 acgacagacg ctcaaaaagc agctgtggaa gcttatgaga agaagattga aactaaatca 540 gattigggaac gaacagattt aaataaagat aaaactggtg tgtttaccgg agcttacgga 600 attaatccag ttaatggtga aaaattgcca atctggattg ctgattacgt cttggcttca 660 tatggaactg gcgcaat 677 <210> 50 <211> 401 <212> DNA <213> Pediococcus claussenii <400> 50 gcaagatacg ttttatatta ctaaggatat tttaatgcgt tctcaaacgt cacctgtaca 60 ggcacgaacg atggaaaagc acgatttttc taagggacca ttgaagatga tttcgccggg 120 ggttgtttat cgacgtgata cggatgatgc aacacattcg catcaatttc atcaagttga 180 gggtctcgta atcgataaaa acattactat gggtgattta aagggtaccc tagaaacact 240 tgcccaggaa ttatttggag atcgttttga agtacgtctt cgtccaagtt attttccttt 300 tactgaacca tcagttgaag cagacgtgac ctgttttaat tgtatgggaa agggatgctc 360 agtatgtaag tatactggat ggatcgaagt gttaggagcc g 401 <210> 51 <211> 1016 <212> DNA <213> Pediococcus inopinatus <400> 51 gattgcccgt gctcacggcc tggataatgc attggctggt gaacttctag agttttctaa 60 tggtgtctac ggaatggttc agaaccttga atcaaatgac gtcggaattg ttattcttgg 120 cggttacgat gatattcgtg aaggcgatac tgttaagaga actggacgga tcatggaagt 180 tccagttggt gacgccttag ttggtcgagt tgtgaataca cttggacaac cgattgatgg 240 taaaggcgaa attaaaacgg accacacacg gccagttgaa cgaaaagcac ctggcgtcat 300 ggaacgtcaa tcagttagcg aacctttaca aactgggatc aaggccattg atgccttagt 360 tccaattggt cgtggtcagc gtgagttgat cattggggac cgtaaaactg gtaaaacatc 420 tttagcaatc gatacaattt tgaatcaaaa agatcaaaat ataatttgta tctatgtggc 480 gatcggccaa aaggaatcaa ctgtgcgtac gcaggttgat accctcgaaa agtatggtgc 540 tatggactac acaatcgttt taagtgctgg cccatctgaa ccagctccaa tgttgtactt 600 ggcaccatat gctggtgcag caatgggtga ggagttcatg tttaacggca agcatgtttt 660 gattgtgtat gatgatcttt caaaacaagc agatgcttat cgtgaacttt ccttgatttt 720 gagacgtccg cctggtcgtg aagcttatcc tggtgatatc ttctatactc attcacgttt 780 actagaacgt gcagctaaat taaatgacga acttggggct ggttcaatga cagctttacc 840 attcgttgaa acaaaggctg gagatattgc cgcttatatt ccaacaaacg taatttctat 900 tactgatgga cagattttct tggatagtga tctattctat tcaggaacac gtccagcgat 960 tgatgccgga tcttctgtat cccgagttgg tggggatgcc caaatcaaag ccatga 1016 <210> 52 <211> 399 <212> DNA <213> Pediococcus <400> 52 gcaagatacg ttttatatta ctaaggatat tttaatgcgt tctcaaacgt cacctgtaca 60 ggcacgaacg atggaaaagc acgatttttc taagggacca ttgaagatga tttcgccggg 120 ggttgtttat cgacgtgata cggatgatgc aacacattcg catcaatttc atcaagttga 180 gggtctcgta atcgataaaa acattactat gggtgattta aagggtaccc tagaaacact 240 tgcccaggaa ttatttggag atcgttttga agtacgtctt cgtccaagtt attttccttt 300 tactgaacca tcagttgaag cagacgtgac ctgttttaat tgtatgggaa agggatgctc 360 agtatgtaag tatactggat ggatcgaagt gttaggagc 399

Claims (11)

Preparing a sample;
Extracting a nucleic acid from the sample;
Performing multiplex PCR on the extracted nucleic acid using a gene-specific first primer of a lactic acid bacterium to identify the species of the microorganism from which the nucleic acid is derived; And
And performing a real-time PCR using a second primer and a probe specific to the gene of the microorganism species to confirm the amount of the microorganism.
The method according to claim 1,
Wherein the sample is a beverage.
The method according to claim 1,
Wherein the lactic acid bacteria is any one selected from the group consisting of Lactobacillus and Pediococcus.
The method according to claim 1,
Wherein the first primer comprises a primer selected from the group consisting of SEQ ID NOS: 1-16.
The method according to claim 1,
Wherein the first primer comprises a primer pair selected from the following (a) to (h):
(a) a primer pair of SEQ ID NOS: 1 and 2
(b) a primer pair of SEQ ID NOS: 3 and 4
(c) a primer pair of SEQ ID NOS: 5 and 6
(d) a primer pair of SEQ ID NOS: 7 and 8
(e) a primer pair of SEQ ID NOs: 9 and 10
(f) a primer pair of SEQ ID NOs: 11 and 12
(g) a primer pair of SEQ ID NOs: 13 and 14
(h) a primer pair of SEQ ID NOs: 15 and 16
The method according to claim 1,
Wherein the first primer comprises a pair of primers specific for Lactobacillus or a pair of primers specific for Pediococcus.
Wherein the first primer is a pair of primers consisting of SEQ ID NOS: 17 and 18.
The method according to claim 1,
The first primer includes the pheS gene of L. lindneri, the trxB gene of L. casei, the abf2 gene of L. brevis, the L. lambda gene of Lactobacillus plantarum L , the luxS gene of Lactobacillus sp. plantarum, the hsp60 gene of L. coryniformis, the leuS gene of P. damnosus, the pheS gene of P. claussenii, And a primer capable of amplifying a gene selected from the group consisting of the atpA gene of P. inopinatus.
The method according to claim 1,
Wherein the first primer comprises at least two primer pairs.
The method according to claim 1,
Wherein the second primer and the probe comprise a primer or a probe selected from the group consisting of SEQ ID NOS: 19 to 42.
The method according to claim 1,
Wherein the second primer and the probe comprise a pair of primers selected from the following (a) to (h) and a probe:
(a) a primer pair of SEQ ID NOs: 19 and 20 and a probe of SEQ ID NO: 21
(b) a pair of primers of SEQ ID NOs: 22 and 23 and a probe of SEQ ID NO:
(c) a primer pair of SEQ ID NOs: 25 and 26 and a probe of SEQ ID NO: 27
(d) a primer pair of SEQ ID NOs: 28 and 29 and a probe of SEQ ID NO: 30
(e) a primer pair of SEQ ID NOs: 31 and 32 and a probe of SEQ ID NO: 33
(f) a primer pair of SEQ ID NOs: 34 and 35 and a probe of SEQ ID NO: 36
(g) a primer pair of SEQ ID NOs: 37 and 38 and a probe of SEQ ID NO: 39
(h) the primer pair of SEQ ID NOs: 40 and 41 and the probe of SEQ ID NO: 42.

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