KR101961652B1 - Weissella viridescens specific primer and method for detecting Weissella viridescens using thereof - Google Patents

Abstract

The present invention relates to a Weissella viridescens specific primer and a method for detecting Weissella viridescens using the same. More specifically, the present invention relates to a composition and detection kit for detecting Weissella viridescens including a Weissella viridescens specific primer set, and a method for detecting Weissella viridescens including a step of performing PCR by using the primer set. By performing conventional PCR or real-time PCR using the Weissella viridescens specific primer set according to the present invention, Weissella viridescens can be specifically detected. Therefore, the present invention can be effectively used for qualitative and quantitative analysis of Weissella viridescens present in various kinds of foods such as fermented vegetable like kimchi, fermented food, and sausage and in the digestive tract and feces of human and animal in small amounts, and for monitoring the density change of Weissella viridescens.

Description

A specific primer of Baculoviridesense and a method for detecting Bacellaviridesense using the specific primer and the method for detecting Weissella viridescens.

The present invention relates to a specific primer of Weissella viridescens and a method of detecting bicellulidisense using the primer. More particularly, the present invention relates to a composition for detecting baculovirusesense comprising a specific primer set of Baculovirus desensitization and a detection kit, and a method for detecting a baculovirus desensitization method comprising performing PCR using the primer set .

Weissella spp. Is a gram-positive spore-forming organism in lactic acid bacteria. Its typical shape is a short rod having a length of 1.5 to 2.0 μm and a width of about 0.8 to 1.0 μm. When energy is obtained by fermentation and sugar such as glucose is grown on a substrate, it undergoes heterolactic fermentation to produce carbon dioxide with lactic acid and produce acetic acid or alcohol depending on the strain. The genus Weissella was identified in 1993 by Collins et al., Leuconostoc ( Leuconostoc ), which differs from other species in Leuconostoc paramesenteroides ) and Lactobacillus ( Lactobacillus ) and proposes a new genus with six species. Since then new species have been added and currently 14 species are listed in the National Center for Biotechnology Information (NCBI). Two new species were reported in 2010 and the number will continue to increase as new species are expected to be discovered in the future. Weissella fungi are detected in a variety of natural environments such as fermented soybeans such as kimchi, fermented foods, digestive tracts of people and animals, and sausages. W. soli was detected in the soil and no specific pathogenicity was reported. In the Cellar Bar Corridor N-Sys 2002 (W. koreensis) separating kimchi, were reported by children Cellar kimchi (W. kimchii) but also reported by children Cellar kimchi (W. kimchii) are among Later Beria Cellar Bar (W. cibaria ). Although W. hanii is also listed as a Kimchi isolate in NCBI, it has not been officially recognized as a new species because it has not been reported in the literature (Lee, Gang-wook et al. , 2010). Thus, weissella fungi are detected in various fermented foods including kimchi.

On the other hand, when Weissella viridescens ) is also called Weissella viridensense , and in 1993 Collins was named Lactobacillus viridescens before the newly proposed Weissella genus.

Korean Patent No. 10-1258692 discloses that Weissella viridescens has inhibitory ability against antibiotic-resistant Staphylococcus aureus (MRSA). As such, weissella The use of viridescens is known to be effective for the fermentation of soybeans such as fermented soybeans such as kimchi, fermented foods, and digestive tracts and feces of humans and animals such as Weissella viridescens ) is required to be specifically detected.

Under these circumstances, the present inventors have completed the present invention by developing a primer set for specifically detecting only Weissella viridescens and a detection method including the primer set.

Korean Patent No. 10-1258692

An object of the present invention is to provide a composition for detecting Weissella viridescens comprising a primer set of SEQ ID NO: 1 and SEQ ID NO: 2.

Another object of the present invention is to provide a method for detecting DNA, comprising: a) extracting DNA from a sample; b) performing PCR (polymerase chain reaction) using the extracted DNA of step a) as a template and using primers set forth in SEQ ID NO: 1 and SEQ ID NO: 2; And c) detecting baculoviridesense from the amplification product resulting from the amplification by PCR in the step b). The present invention also provides a method for detecting Weissella viridisense .

Another object of the present invention is to provide a kit for detecting Weissella viridescens including a set of primers of SEQ ID NO: 1 and SEQ ID NO: 2.

In order to accomplish the above object, the present invention provides a composition for detecting Weissella viridescens including a primer set of SEQ ID NO: 1 and SEQ ID NO: 2, and a detection kit, and performing PCR using the primer set The method comprising the steps of:

Since Bacella viridense can be specifically detected through conventional PCR or real-time PCR using a specific primer set of weissella viridescens according to the present invention, It can be effectively used for the qualitative, quantitative analysis and monitoring of the density change of Bicellaviridesense present in a small amount in various foods such as fermented green vegetables such as kimchi, fermented foods, sausages, digestive tracts and feces of human and animals.

1 is by Sela of the present invention to sense irregularities (Weissella GNAT acetyl transferase family kinase (GNAT family acetyltransferase) of viridescens) (registration number: a diagram showing a blastn search (search) results in GenBank Accession No. KRN46633.1).
FIG. 2 is an electrophoresis image showing results of PCR amplification of various microbial strains shown in Table 1 of the present invention using a bispecificidense-specific primer set of the present invention. Here, M is a size marker, and the numbers displayed at the top of the electrophoresis image are numbers corresponding to the microbial strains shown in Table 1 of the present invention.
FIG. 3 is a graph showing the results of real-time PCR using Bacella viridense as a primer set for the specificity analysis of a specific primer set of Bacela viridense according to the present invention. Specifically, (A) is a cloned DNA of Baculoviridesense, which is serial diluted 10-fold in a range of 1.40 × 10 ³ to 1.40 × 10 ⁹ copies / (B) shows the standard curve derived from the amplification plot, and (C) shows the fluorescence intensity as a function of the concentration of the template sample (1 to 7) (1) to (7), and (D) shows the result of serial dilution of the bicellar viridis strain sample (1 to 7) The results of the melting peak analysis of the samples (1 to 7) of the dedense cloned DNA template are shown by the relative fluorescence unit [-d (RFU) / dT]. Here, 8 is a template-free control using distilled water and shows a low peak, and a high peak indicates an amplified product.
FIG. 4 is a graph showing a standard curve derived from real-time PCR performed using the above primer set for Biscarabydesense for the sensitivity analysis of the specific primer set of Biscarabillisense according to the present invention . Specifically, (A) shows a standard curve derived from a real-time PCR performed by step-diluting a genomic DNA obtained from Bacellaviridesense, and (B) shows a standard curve obtained from a bicellar viridense (C) shows a standard curve derived from a real-time PCR performed by inserting an amplification product into a vector and performing stepwise dilution of cloned DNA obtained from the transformed strain, and (C) shows a standard curve derived from a cell suspension of Bacella viridense strain cell Lt; RTI ID = 0.0 > of < / RTI > real-time PCR.

The present invention relates to the use of a bis- The present invention relates to a specific primer of viridescens and a method for detecting biscarabydesense using the primer. More particularly, the present invention relates to a composition for detecting bicellulidisense comprising a specific primer set of bicellulidases, , And performing PCR using the above primer set.

In one aspect, the invention provides a composition for detecting Weissella viridescens comprising a set of primers of SEQ ID NO: 1 and SEQ ID NO: 2.

The term " Weissella viridescens " in the present invention is a gram-positive sporozoite which is contained in lactic acid bacteria. It is a variety of foods such as fermented vegetable such as kimchi, fermented food, sausage, Of the digestive tract and feces.

The term " primer " in the present invention means a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming a base pair with a template of a complementary nucleic acid, Refers to a short nucleic acid sequence that serves as a starting point for strand duplication. The primer can initiate DNA synthesis in the presence of a polymerase (i.e. DNA polymerase) and a reagent for the polymerization reaction at a suitable buffer solution and temperature.

The primer set of the present invention can be used to prevent the formation of a primer dimer by the size of a primer, a melting temperature (Tm) value, a GC content of a primer, and a self-complementary sequence in a primer It has been carefully considered and designed to maximize sensitivity and specificity when detecting Weissella viridescens .

The primer set was prepared using a primer set of Weissella < RTI ID = 0.0 > viridescens ), and it has been found that when Bacellaviridesense is mixed with strains belonging to another genus other than the genus Weissella or another genus belonging to the genus Weissella , ( Weissella viridescens ) can be specifically detected.

The primer set according to the present invention is designed to amplify the GNAT family acetyltransferase gene among the genes of Weissella viridescens and can be detected with species-specific high sensitivity. When the Bacella viridense gene is amplified with the primer set according to the present invention, genes other than the GNAT family acetyl transferase gene are not amplified, and the 256 bp GNAT family acetyl transferase gene is clearly detected, so that accurate and easy detection This is possible.

The primers represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 are respectively a forward primer (WV256F) for amplifying a 256-bp nucleic acid fragment of Weissella viridescens and a reverse primer Primer (WV256R).

Specifically, the primers include a GNAT family acetyltransferase (registered trademark: GenBank Accession No. KRN46633.1 (protein id); GenBank Accession No. JQBM01000002.1, gene: 1 and a reverse primer having a nucleic acid sequence represented by SEQ. ID. NO: 2, and a reverse primer having a nucleic acid sequence shown in SEQ ID NO.

The primer set of the present invention may be a primer set amplifying a 256 bp nucleic acid fragment consisting of nucleotide sequence positions 546 to 801 of GNAT family acetyl transferase gene of Bacillus viridisense.

In one embodiment of the present invention, nucleic acid sequence information capable of specifically detecting Weissella viridescens is searched using GenBank of National Center for Biotechnology Information (NCBI) and blastn and blastx programs , A primer capable of detecting the above-mentioned specific nucleic acid sequence was prepared. As a result of carrying out conventional PCR and real-time PCR amplification reaction using the prepared primer set, it was confirmed that the primer set according to the present invention can specifically detect Baicellabidisense only (Figs. 2 to 4).

The primer set according to the present invention may include additional features that do not change the basic properties. That is, the base sequence can be modified using many means known in the art. Examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more nucleotides into nucleotides, and uncharged linkages such as phosphonates, phosphotriesters, phosphoramidates or carbamates, phosphorothioates or phosphorodithioates Or the like can be transformed into a charged nucleus. The nucleic acid may be at least one of a protein such as a nuclease, a toxin, an antibody, a signal peptide, a polylysine, an intercalating agent such as acridine or psoralen, a chelating agent such as a metal, a radioactive metal, And may have additional covalently bonded moieties.

In addition, the base sequence of the primer set according to the present invention can be modified using a label capable of directly or indirectly providing a detectable signal. The primer set can include a label that can be detected using spectroscopic, photochemical, biochemical, immunochemical or chemical means. Useful labels include 32P, 35S, fluorescent dyes, electron dense reagents, enzymes (commonly used in ELISAs), biotin or hapten, and proteins available for antisera or monoclonal antibodies.

The primer set according to the present invention can be obtained by cloning and restriction enzyme digestion of appropriate sequences and digestion with a phosphotriester method such as Narang et al . (1979, Meth . Enzymol . 68: 90-99) Can be chemically synthesized using any other well-known method including direct chemical synthesis methods such as the pamoate method (1981, Tetrahedron Lett . 22: 1859-1862), and the solid support method of U.S. Patent No. 4458066 .

In the composition of the present invention, various reagents necessary for the confirmation of the results (e.g., PCR, Southern blot, etc.) and the results necessary for detecting Bacella viridisense using the above primer set, for example, a PCR composition, a restriction enzyme, Buffer, buffer solution, and the like necessary for loss, hybridization and / or electrophoresis.

More specifically, the PCR composition may be prepared by mixing a desired DNA and a PCR primer set provided in the present invention in an appropriate amount of DNA polymerase (e.g., Thermus aquaticus ( Taq ), Thermus thermophilus ( Tth ), Thermus filiformis , Thermis flavus , Thermococcus literalis or Pyrococcus thermostable DNA polymerase from furiosus ( Pfu )), dNTP, PCR buffer solution and water (dH ₂ O).

The PCR buffer solution includes, but is not limited to, appropriate amounts of Triton X-100, dimethylsufoxide (DMSO), Tween 20, nonidet P40, PEG 6000, formamide and bovine serum albumin bovine serum albumin, BSA) may be further included.

In another aspect, the present invention provides a method for detecting Weissella viridescens comprising the steps of:

a) extracting DNA from the sample;

b) performing PCR (polymerase chain reaction) using the extracted DNA of step a) as a template and using primers set forth in SEQ ID NO: 1 and SEQ ID NO: 2; And

c) detecting the Baculovirus desensitization from the amplification product resulting from amplification by PCR in step b).

In the present invention, the sample of step a) is not limited thereto, but may be a variety of foods such as fermented vegetable such as kimchi, fermented food, sausage, etc. in which the bicellulidase is present, And feces.

The method of extracting DNA from the sample in step a) may be carried out by a conventional method known in the art, which is appropriately selected by a person skilled in the art.

The primers set forth in SEQ ID NO: 1 and SEQ ID NO: 2 in step b) are as described above, and the PCR in step b) may be performed according to real-time PCR, but the present invention is not limited thereto.

In step c), the DNA of the PCR product can be separated by size according to methods well known in the art. But not limited thereto, can be confirmed by, for example, agarose gel or polyacrylamide gel electrophoresis or an ABI prism 3100 genetic analyzer-electropherogram. In this case, in order to use the fluorescence analyzer, PCR is performed in step b) using a primer set with a fluorescent dye known in the art. The PCR amplification result can preferably be confirmed by agarose gel electrophoresis. After electrophoresis, electrophoresis results can be analyzed by ethidium bromide staining. Methods for performing conventional PCR and analyzing the results are well known in the art.

In one embodiment of the present invention, the PCR amplification may utilize a conventional PCR amplification reaction, and may be performed using conventional PCR or real-time PCR, Quantitative analysis in real time is possible when using PCR amplification reaction. The general PCR amplification reaction refers to a conventional PCR amplification reaction in which a step of amplifying a specific DNA and then confirming whether amplification of a specific DNA is additionally performed, and the step of confirming amplification of the specific DNA may be performed by electrophoresis, And then measuring the size of the amplified DNA product. Specific PCR amplification methods can be suitably selected and carried out by those skilled in the art.

The term " PCR (polymerase chain reaction) " in the present invention means a method of amplifying a target nucleic acid from a primer set that specifically binds to a target nucleic acid using a polymerase. Such PCR methods are well known in the art and commercially available kits can be used. The real-time PCR method, which is currently being used as a PCR analysis method, is a principle of measuring the amount of fluorescence by dissociation of a labeling substance from a probe during PCR amplification using a probe in which a specific primer and a labeling substance are linked, There is an advantage of quantitatively analyzing the amount of introduced genes.

The term " probe " refers to a single-stranded nucleic acid sequence that hybridizes with a complementary single-stranded target sequence to form a double-stranded molecule (hybrid).

Specifically, the real-time PCR is a technique for real-time monitoring and analysis of the increase in PCR amplification products (Levak KJ, et al. , PCR Methods Appl. , 4 (6): 357-62 (1995)). The PCR reaction can be monitored by recording the amount of fluorescence emission in each cycle during the exponential phase, during which the increase in PCR product is proportional to the initial amount of target template. The higher the starting copy number of the nucleic acid target, the faster the fluorescence increase is observed and the lower the threshold value cycle. A pronounced increase in fluorescence above the reference value measured between 3 and 15 cycles implies detection of accumulated PCR products. Compared to conventional PCR methods, real-time PCR has the following advantages: (a) conventional PCR is measured in a plateau, while real-time PCR provides data during the exponential growth phase have; (b) the increase in the reporter fluorescence signal is directly proportional to the number of amplicons generated; (c) The degraded probe provides permanent record amplification of the amplicon; (d) increase in detection range; (e) requires at least 1,000 times less nucleic acid than conventional PCR methods; (f) detection of amplified DNA without separation by electrophoresis is possible; (g) using a small amplicon size can achieve increased amplification efficiency; And (h) the risk of contamination is low.

When the amount of PCR amplified acid reaches a detectable amount by fluorescence, the amplification curve begins to occur, and the signal rises exponentially to reach the stagnation state. The higher the amount of initial DNA, the faster the amplification curve because the amount of amplified acid is less than the detectable amount of cycles. Therefore, when real-time PCR is performed using a stepwise diluted standard sample, an amplification curve is obtained in which the initial DNA amounts are arranged in the order of the same intervals. Here, if a threshold is set at an appropriate point, the CT value at which the threshold and the amplification curve intersect is calculated.

In real-time PCR, PCR amplification products are detected through fluorescence. The detection methods are largely an interchelating method (SYBR Green I method) and a method using a fluorescent label probe (TaqMan probe method). Since the intercalating method detects double stranded DNA, it is not necessary to prepare a probe for each gene, so that a reaction system can be constructed at a low cost. The method using a fluorescent label probe is costly, while the detection specificity is high, so even the similar sequence can be detected.

The amplified target sequence according to the present invention may be labeled with a detectable labeling substance, and the labeling substance may be a fluorescent, phosphorescent or radioactive substance, but is not limited thereto. When the radioactive isotope such as 32P or 35S is added to the PCR reaction solution, the amplification product is synthesized and the radioactivity is incorporated into the amplification product, so that the amplification product can be labeled as radioactive.

In one embodiment of the present invention, specific PCR conditions include denaturation at 95 ° C for 1 minute after heat treatment at 95 ° C for 5 minutes, denaturation at 95 ° C, Annealing at 63 DEG C for 30 seconds; The extension step at 72 ° C for 1 minute is repeated 35 times in total, and the reaction can be performed at 72 ° C for 10 minutes. The reaction temperature and reaction time conditions may be suitably modified by those skilled in the art.

Methods for detecting the PCR amplification products are well known in the art. The detection of the amplification product can be performed through capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto.

In addition, any method known in the art can be used as a method for analyzing the size of the amplification product. For example, the size of the amplified product can be determined by comparison with a target size marker by agarose or polyacrylamide gel electrophoresis.

In addition, as a method of analyzing the base sequence of the amplification product, there can be used a method of analyzing the base sequence known in the art. For example, as a method for directly determining the sequence part, an automatic base sequence analyzer or a sanger sequencing ), Pyrosequencing, and the like, but are not limited thereto.

The minimum amount of DNA for detection of Weissella viridescens by PCR may be 50 to 5,000,000 fg (5 ng (nanogram)) in genomic DNA, and the amount of cloned DNA And may be in the range of 5 to 5,000,000 fg (5 ng) and may be, but is not limited to, 0.85 × 10 ³ to 0.85 × 10 ⁸ CFU (colony-forming-unit) / ml in a bacterial cell suspension.

The minimum amount of DNA for detection of the GNAT family acetyltransferase gene of Baculoviridesense by real-time PCR is preferably 5 fg (femtogram) to 5 ng (nanogram), and detection is possible even with a minimum of 5 fg, Is very high.

The DNA of the GNAT family acetyltransferase gene of Baculoviridesense contains genomic DNA and cloned DNA.

The minimum amount of bacterial cell suspension for detection of the GNAT family acetyltransferase gene of Baculoviridesense by real-time PCR was 0.85 × 10 ³ CFU / ml (OD ₆₀₀ = 0.1 × 10 ^-5 unit) to ^{0.85 × 10 8 CFU / ㎖ (} OD 600 = 0.1 × 10 0 unit) is not preferred, but may detect the minimum ^{0.85 × 10 3 CFU / ㎖ (} OD 600 = 0.1 × 10 -5 unit) , because the detection sensitivity is It is very high.

In yet another aspect, the invention provides a method of screening for myocardial infarction, viridescens ) detection kit.

The primer sets of SEQ ID NO: 1 and SEQ ID NO: 2 according to the present invention are as described above.

In the present invention, the detection kit may be a real-time PCR kit, but is not limited thereto.

In the present invention, the minimum amount of DNA for detection of Weissella viridescens by the detection kit may be 50 to 5,000,000 fg (5 ng (nanogram)) in genomic DNA, may be cloned DNA (cloned DNA) 5 to 5,000,000 fg (5 ng) from, a bacterial cell suspension (bacterial cell suspension) at 0.85 × 10 ³ to ^{0.85 × 10 8 CFU (colony-} forming-Unit) may be / ㎖ but , But is not limited thereto.

In addition, the detection kit may further include reagents for performing an amplification reaction, and the reagents may be any of well-known in the art. Specifically, the reagent for performing the amplification reaction may be DNA polymerase, dNTPs, and a buffer, but is not necessarily limited thereto.

According to the present invention, WV256F (SEQ ID NO: 1) and WV256R (SEQ ID NO: 2) prepared from a specific DNA fragment of the GNAT family acetyltransferase gene (SEQ ID NO: 3) of Weissella viridescens ) Primer can be used as a PCR primer for specific detection of Baculoviridesense by generating an amplification product only in a sample containing a Baculoviridesense strain, and can be used as a PCR primer such as fermented green vegetables such as kimchi, It is confirmed that the present invention is applicable to the qualitative, quantitative analysis and monitoring of the density change of bicellulidisense present in a small amount in various foods such as sausage, digestive tract and feces of human and animal.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  One: Bissela Viridense ( Weissella viridescens ) And bacterial strain preparation and culture conditions

The term " Weissella " Other microorganisms including viridescens were distributed from Belgium's BCCM ^TM (Belgian Co-ordinated Collections of Microorganisms) and KACC (Korean Agricultural Culture Collection, Republic of Korea), and their sources are summarized in Table 1 below. The microbial strains of Table 1 were incubated with MRS medium (OXOID CM0361) for two days at 25 ° C and 30 ° C, respectively.

Example  2: Total DNA extraction of various microbial strains

Total DNA of a variety of microbial strains obtained by culturing in Example 1 was extracted by using the genomic DNA (genomic DNA) extraction kit (DNeasy Blood & Tissue Kit ^®) Inc. (Hilden, Germany) Qiagen.

Example  3: Bissela Viridense  Specific primer  Set production

Weissella For the production of a primer set for specifically detecting viridescens , the GNAT family acetyl (registered trademark) of Baicelavirisense registered at Genbank (www.ncbi.nlm.nih.gov/) of National Center for Biotechnology Information (NCBI) GenBank Accession No. KRN46633.1 (protein id); GenBank Accession No. JQBM01000002.1, gene: 392706-393572; SEQ ID NO: 3) was transfected with a blastn and blastx program Specificity was confirmed and a primer set was prepared (Fig. 1 and Table 2).

As shown in Table 2 below, WV256F (SEQ ID NO: 1) and WV256R (SEQ ID NO: 2) primer sets were amplified by amplifying a fragment of 256 bp size in Weissella viridescens .

SEQ ID NO: primer  designation primer  direction The sequence (5 '- > 3') One WV256F Forward 5'-GGC GGC AGA ACG ATA CTT GA-3 '(20mer) 2 WV256R Reverse 5'-TGC TGC CAC ATT CGT TCC TT-3 '(20mer)

The primer set was prepared by confirming the specificity of the GNAT family acetyltransferase gene of Bacella viridense registered in GenBank through blastn program (FIG. 1).

Example  4: Bissela In Viridesense  About Bissela Viridense  Specific Fry Confirmation of specific detection of mercys

Specific for the detection check, PCR amplification reactions of the cellar by irregularities having a sense-specific primer sets produced in Example 3 was performed using a ^{PTC-200 TM thermocycler (MJ research} , Watertown, mass). The PCR amplification reaction was performed in the same manner as in Example 3 except that 10 mM of Tris-HCl, 50 mM of KCl, 1.5 mM of MgCl ₂ , 0.01% of gelatin, 0.2 mM of each of dNTPs, 20 pM of each of WV256F (SEQ ID NO: 1) and WV256R (SEQ ID NO: Taq PCR mixture containing 2 units of polymerase ( Taq polymerase) (Promega, Madison, Wis.) Was performed in a total amount of 25 μl. At this time, the amount of genomic DNA obtained from the various microbial strains of Example 1 was added to the PCR mixture to about 25 ng. PCR amplification was performed at 95 ° C for 5 minutes, followed by 35 cycles (35 cycles) at 95 ° C for 60 seconds, 63 ° C for 30 seconds, and 72 ° C for 60 seconds, followed by reaction at 72 ° C for 10 minutes. After amplification reaction, 5 μl of the PCR amplification product was stained with LoadingStar (DYNEBIO, Republic of Korea), electrophoresed on a 1.5% agarose gel and confirmed in a UV transilluminator.

As shown in FIG. 2, 24 different microbial strains (Table 1) were subjected to a PCR amplification reaction using a specific primer set of Baculoviridesense. As a result, as shown in FIG. 2, It was confirmed that a nucleic acid fragment of 256 bp in size was specifically amplified only for the sense (Lane 6 in FIG. 2).

Example  5: Bissela In Viridesense  About Bissela Viridense  Specific Fry The real-time PCR PCR ) Specificity analysis

Conventional PCR was carried out on 24 different microbial strains (Table 1) using a specific primer set specific for Bicelaviridesense in Example 4, and only Bichelaviridesense was identified in various microbial strains It was confirmed that the detection was possible.

The CFX96 real-time PCR system (Bio-Rad laboratories, Inc., USA) was used to confirm whether the biosarylide sense could be detected more easily and quickly in real time Real-time PCR amplification reaction was performed using the primers. The real-time PCR amplification reaction was carried out in real-time (5 min) containing 5 pM of SYBR premix Ex Taq (2x) (TAKARA Bio, Inc., Japan) and WV256F (SEQ ID No. 1) and WV256R real-time PCR mixed solution in a total amount of 20 μl. At this time, the amount of genomic DNA obtained from Bacellaviridesense was added to the real-time PCR mixture to be about 5 ng. Real-time PCR amplification was performed at 95 ° C for 30 seconds, followed by 95 cycles of 95 ° C for 5 seconds, and 63 ° C for 30 seconds (40 cycles), followed by 95 ° C for 15 seconds. The temperature was then increased by 0.5 ° C every 5 seconds from 65 to 95 ° C to derive the melting curve and melting peak. At this time, the melting peak graph is expressed by Relative Fluorescence Units (RFU) at each temperature. That is, the relative fluorescence unit [-d (RFU) / dT] of the derivatives of the amplified product is expressed as a function of temperature.

Real-time PCR amplification was carried out to analyze the specificity of the specific primer set for Baculoviridesense specific to Baculoviridesense. As a result, as shown in FIG. 3, it was 1.40 × 10 ³ to 1.40 × 10 ⁹ copies ) / [Mu] l of 10-fold serial dilutions of Weissella A melting peak was observed at a specific temperature of 81.5 ° C only in viridescens cloned DNA samples (1 to 7). On the other hand, it was confirmed that the control group (8, distilled water) without template had no melting peak (D in FIG. 3).

Thus, (melting peak) melting peaks, as the two strands of DNA bound to the complementary temperature is increased to indicate the temperature of the moment when the DNA strands are separated from each other, by Cellar cheated to sense in a specific melting temperature of 81.5 ℃ (Weissella viridescens), it was able to specifically recognize that by Cellar cheated to sense the GNAT acetyl transferase family kinase (GNAT family acetyltransferase) can clearly detect a gene specifically.

Example  6: Bissela In Viridesense  About Bissela Viridense  Specific Fry Real-time PCR sensitivity of Mercys

In order to analyze the specificity of the specific primer set of bis-arabidide-sense of the present invention in Example 5, real-time PCR was carried out using the primer set for bis-cerarbidesense, and as a result, It was confirmed that the GNAT family acetyltransferase gene of Weissella viridescens , specifically bicellulidysense , can be specifically and clearly detected.

The CFX96 real-time PCR system (Bio-Rad Laboratories, Inc., USA) was used to analyze the sensitivity of bis-laverides-specific primer set of the present invention to Baculoviridesense Real-time PCR amplification reaction was performed using the primers. The real-time PCR amplification reaction was carried out in real-time (5 min) containing 5 pM of SYBR premix Ex Taq (2x) (TAKARA Bio, Inc., Japan) and WV256F (SEQ ID No. 1) and WV256R real-time PCR mixed solution in a total amount of 20 μl. At this time, weissella The amount of genomic DNA and cloned DNA obtained from the viridescens KACC 11850 strain was diluted 10 times in the real-time PCR mixture to be 5 ng (nanogram) to 5 fg (femtogram). The bacterial cell suspension was further diluted 10-fold to 0.85 × 10 ⁸ CFU / ml (OD ₆₀₀ = 0.1 × 10 ⁰ unit) to 0.85 × 10 ³ CFU / ml (OD ₆₀₀ = 0.1 × 10 ^-5 unit) And added to the real-time PCR mixture (Table 3). Real-time PCR amplification was performed at 95 ° C for 30 seconds, followed by 95 ° C for 5 seconds and 61 ° C for 30 seconds (40 cycles), followed by 95 ° C for 15 seconds. The temperature was then increased by 0.5 ° C every 5 seconds from 65 to 95 ° C to derive the melting curve and melting peak.

Real-time PCR amplification reaction was performed to analyze the sensitivity of bisersaviridesense-specific primer set to Baculoviridesense. As shown in FIG. 4, the R ² value of the standard curve (> 0.99) and the E value are in the appropriate range. The minimum amount of DNA for detection of bicellulidase is 50 fg in genomic DNA, 5 fg in cloned DNA, And was found to be 0.85 × 10 ³ CFU / ㎖ in a bacterial cell suspension (Table 3).

Therefore, the GNAT family acetyltransferase gene of Baicella viridense, specifically Baicella viridense, can be stably detected by quantitative detection using the Baicella viridense specific primer set of the present invention .

Through the above series of results, weissella The WV256F (SEQ ID NO: 1) and WV256R (SEQ ID NO: 2) primers prepared from the specific DNA fragment of the GNAT family acetyltransferase gene (SEQ ID NO: 3) of the viridescens are specific for Baculoviridesense Lt; RTI ID = 0.0 > PCR < / RTI > primer set for detection. Therefore, the present invention can be applied to the qualitative, quantitative analysis and monitoring of the density change of bicellar birdsense existing in various food such as fermented vegetable such as kimchi, fermented food, sausage, digestive tract and feces of human and animal .

<110> Republic of Korea <120> Weissella viridescens specific primer and method detecting          Weissella viridescens using them <130> P17R12C0508 <160> 3 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WV256F <400> 1 ggcggcagaa cgatacttga 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WV256R <400> 2 tgctgccaca ttcgttcctt 20 <210> 3 <211> 867 <212> DNA <213> Artificial Sequence <220> <223> GNAT family acetyltransferase <400> 3 atgtggataa cgaccaagac gaaactgaca gcacatgaaa aaaaacaagt acaaacatta 60 atacatcaag tgcatcaagt tgaccgcacg tttaaagcac catacttaag taaccagtat 120 aactattttc caggaatgcc gactttcatt ctggtctatg atagtgccgc gcaattagcc 180 ggttttacca tgatctatgc tgatgaagcg ccacaagatg gcattgttga tctaattgta 240 actgtgttgc ccgcacagcg ccatcaaggc attgggcgta tgatgctcaa tcgggctcag 300 tcaatccttg atgcttttgg ttataaacaa attaattaca taacggagcg tgtctttctg 360 gatgcaaatc caaacctgct gcagaagttg aatatgcgtg tagcggatag cgaatatcag 420 atggccatgg gtcaattggt agagccatcg acaaagcgag cggcaacagt aaggctgatg 480 gaagcaggtg atattgtgcc cttggtaccg atttttagta gcgcgtttga cgacatttcg 540 gacgaggcgg cagaacgata cttgacggag agtttacaag acgcatcaat tgctagttac 600 gttttaaagg tagagactac gccaattggg tattgtgcaa ttgatcttag tgattacgct 660 tatatcttta gtgtcttcat tgatgaccgt tatcgtagtc aagggtatgg tcaatattta 720 ttaacctatg caattaacca tctccaaacc caaggatgga agcggattgt gcttggcgtt 780 gaaggaacga atgtggcagc aaagcgccta tataatcgaa ttggttttca agaagaaacc 840 gaaattgtga gtttacagca aaagtaa 867

Claims (8)

Of SEQ ID NO: 3 by Cellar cheated to sense GNAT family acetyl transferase dehydratase (GNAT family acetyltransferase) containing the SEQ ID NO: 1 and a primer set of SEQ ID NO: 2 for detecting the gene specifically, by Cellar cheated to sense (Weissella viridescens ).
delete a) extracting DNA from the sample;
b) SEQ ID NO: 1 and SEQ ID NO: 2 which specifically detect the GNAT family acetyltransferase gene of Baculoviridesense of SEQ ID NO: 3, using the extracted DNA of step a) as a template, Performing a real-time polymerase chain reaction (PCR) using a primer set of the primer set; And
and c) detecting the Baculoviridesense from the amplification product obtained as a result of real-time PCR amplification in step b), wherein the Bacillaria viridisense detection method comprises the steps of:
The minimum amount of DNA for detection of the GNAT family acetyl transferase gene of Baculoviridesense by real-time PCR is 50 to 5,000,000 fg (5 ng) in genomic DNA and 5 to 5,000,000 in cloned DNA fg (5 ng) and in a bacterial cell suspension of 0.85 × 10 ³ to 0.85 × 10 ⁸ CFU / ml.
delete delete delete delete Of SEQ ID NO: 3 by Cellar cheated to sense GNAT family acetyl transferase dehydratase (GNAT family acetyltransferase) containing the SEQ ID NO: 1 and a primer set of SEQ ID NO: 2 for detecting the gene specifically, by Cellar cheated to sense (Weissella viridescens A kit for real-time PCR for detection,
The minimum amount of DNA for detection of the GNAT family acetyl transferase gene of Baculoviridesense by real-time PCR is 50 to 5,000,000 fg (5 ng) in genomic DNA and 5 to 5,000,000 in cloned DNA A kit for real-time PCR for the detection of Baculoviridesense having a fg (5 ng) of 0.85 x 10 ³ to 0.85 x 10 ⁸ CFU / ml in a bacterial cell suspension.

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