KR20150143347A - Primer set for high sensitive real-time multiplex loop-mediated isothermal amplification reaction for determining type of shiga toxin genes of Enterohemorrhagic Escherichia coli, and method for determining type of shiga toxin genes of Enterohemorrhagic Escherichia coli using the same - Google Patents

Abstract

The present invention relates to a primer set for highly sensitive real time multiplex isothermal amplification reaction for discriminating a cigarette toxin genotype of intestinal hemorrhagic Escherichia coli, a composition and a kit for the determination of a cigarette toxin genotype of intestinal hemorrhagic Escherichia coli comprising the primer set, The present invention relates to a method for identifying a cigarette toxin genotype of Escherichia coli. When the isothermal amplification reaction is performed using the primer set according to the present invention, the intestinal hemorrhagic Escherichia coli can be specifically detected with high sensitivity before the bacterial isolation is completed, , The result can be quantified, and the reaction time can be greatly improved, so that contamination and occurrence level of intestinal hemorrhagic Escherichia coli can be confirmed quickly and accurately, and its cigar poisoning can be discriminated at the same time. When a food poisoning accident occurs It can be effectively used as an early screening system for detecting a causal agent.

Description

TECHNICAL FIELD [0001] The present invention relates to a primer set for high-sensitivity real-time multiplex isothermal amplification reaction for distinguishing a cigarette toxin genotype of intestinal hemorrhagic Escherichia coli, and a method for identifying a cigarette toxin genotype of intestinal hemorrhagic Escherichia coli using the primer set. determining type of shiga toxin genes of Enterohemorrhagic Escherichia coli, and method for determining type of shiga toxin genes of Enterohemorrhagic Escherichia coli using the same}

The present invention relates to a primer set for highly sensitive real time multiplex isothermal amplification reaction for discriminating a cigarette toxin genotype of intestinal hemorrhagic Escherichia coli, a composition and a kit for the determination of a cigarette toxin genotype of intestinal hemorrhagic Escherichia coli comprising the primer set, The present invention relates to a method for identifying a cigarette toxin genotype of Escherichia coli.

The number of food poisoning cases and the number of patients are increasing year by year and becoming larger. It is estimated that the social loss cost due to food poisoning in Korea is about 1.6 trillion won including medical expenses and loss of productivity. Among them, productivity loss cost is 74.6%, which is 1,220 billion won, and medical expenses are 24.8%, which is 420 billion won. The number of outbreaks of food poisoning in Korea was 109 cases in 2005 and 5,711 cases in 2005, but it increased every year since then to 259 cases, 10,833 cases in 2006, 510 cases in 2007, 9,686 cases in 2007, and 7,218 cases in 271 cases in 2010 The number of patients was reported. The major causes of foodborne illness in 2010 were pathogenic Escherichia coli, which is the leading cause of bacterial food poisoning in 2010, and Salmonella, Bacillus cesarethus, Staphylococcus aureus, and Campylobacter jejuni were also found to be causative agents of food poisoning. The risk of E. coli O157: H7 in pathogenic Escherichia coli is the most widely known, and the E. coli O157: H7 negative standard has been established in several foods including fresh convenience foods. Outside the country In Germany, 941 foodborne outbreaks occurred in 2011 due to pathogenic Escherichia coli in May 2011, and until July 26, the end of the official epidemic, resulting in 3,910 patients, of which 46 I have died. The causative organism associated with food poisoning was E. coli O104: H4, a serotype of Shiga toxin producing E. coli (STEC), a type of intestinal hemolytic Escherichia coli producing toxins. Thus, the problem caused by E. coli O157: H7 as well as pathogenic Escherichia coli (STEC) causing toxin production and intestinal bleeding has become serious, and attention has been focused worldwide.

Recently, gene-based detection methods such as PCR (Polymerase Chain Reaction) and real-time PCR have been applied to various fields in combination with existing standard culture methods. This method can detect the causative organism by amplifying the bacterial specific gene. It has the merit that it is possible to judge the result easily and quickly when compared with the standard culture method. However, in the case of PCR, the electrophoresis process on the agar gel (Bickley et al., 1996; Kim et al., 2000; Rossen et al., 1992), as well as the inconveniences of gene amplification by food samples such as milk and food .

On the other hand, Loop-mediated isothermal amplification (LAMP) is similar to PCR and real-time PCR, but it is fast, sensitive and accurate. (Jiang et al., 2010; Jiang et al., 2012; Techathuvanan et al., 2010; Thekisoe et al., 2010; Wang et al., 2012). Unlike conventional gene amplification methods that undergo the reaction under temperature gradient conditions, the isothermal amplification reaction proceeds by isothermal amplification using Bst DNA polymerase or the like without thermal denaturation of the template DNA duplex structure. In addition, six primers used in the isothermal amplification method recognize eight different regions of the target gene sequence and amplify the gene so that sensitivity and specificity can be improved compared to other methods (Nagamine et al., 2002; Notomi et al., 2000; Tomita et al., 2008).

As a result of continuing research to develop a novel method for detecting intestinal hemorrhagic Escherichia coli, the present inventors have found that a highly sensitive real-time multiplex isothermal amplification reaction capable of simultaneously detecting each of stigmatoxin genes stx1 and stx2 of intestinal hemorrhagic Escherichia coli It is possible to specifically detect the intestinal hemorrhagic Escherichia coli in a sample in which a variety of living organisms are mixed using the primer set and to quantify the microorganism as well as discriminate the cytokines The present invention has been completed.

Accordingly, an object of the present invention is to provide a primer set for the determination of a cigarette toxin genotype of enterohemorrhagic Escherichia coli.

Another object of the present invention is to provide a composition for identifying a cigarette toxin genotype of enterohemorrhagic Escherichia coli comprising the primer set.

It is another object of the present invention to provide a kits for the determination of a cigarette toxin genotype of enterohemorrhagic Escherichia coli comprising the above composition.

It is another object of the present invention to provide a method for determining a cigarette toxin genotype of enterohemorrhagic Escherichia coli using the primer set.

Another object of the present invention is to provide a primer set for detecting a cytotoxic gene of enterohemorrhagic Escherichia coli.

In order to solve the above problems, the present invention provides a primer set comprising a first primer set represented by the nucleotide sequence of SEQ ID NOS: 1 to 6; And a second primer set selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 7 to 12, and a set of at least one primer selected from the group consisting of SEQ ID NOS: 7 to 12.

In addition, the present invention provides a composition for identifying a cigarette toxin genotype of enterohemorrhagic Escherichia coli comprising the primer set.

In addition, the present invention provides a kits for the determination of a cigarette toxin genotype of enterohemorrhagic Escherichia coli comprising the above composition.

In addition,

(a) separating the genomic DNA (gDNA) of the sample;

(b) amplifying the target sequence by using the separated genomic DNA as a template and performing an isothermal amplification reaction using the primer set; And

 (c) detecting the amplified product, wherein the method comprises the steps of:

The present invention also relates to a first primer set represented by the nucleotide sequences of SEQ ID NOS: 1 to 6; And a second primer set selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 7 to 12, and a set of at least one primer selected from the group consisting of SEQ ID NOS: 7 to 12.

When the isothermal amplification reaction is performed using the primer set according to the present invention, the intestinal hemorrhagic Escherichia coli can be specifically detected with high sensitivity before the bacterial isolation is completed, , The result can be quantified, and the reaction time can be greatly improved, so that contamination and occurrence level of intestinal hemorrhagic Escherichia coli can be confirmed quickly and accurately, and its cigar poisoning can be discriminated at the same time. When a food poisoning accident occurs It can be effectively used as an early screening system for detecting a causal agent.

FIG. 1 is a graph showing the extent of amplification products according to reaction time when the temperature condition of the isothermal amplification reaction is different. FIG.
FIG. 2 is a diagram showing the Tm values of the stx1 and stx2 genes confirmed by the annealing curve analysis of the isothermal amplification reaction. FIG.
3 is stx1 and when in stx2 sheet with both gene of hemorrhagic E. coli strain (ATCC 43894) by the stx1 and stx2 genes to target obtained by performing the isothermal amplification reaction, a measure of the amount of fluorophore to be detected in time Tp value Fig.
Figure 4 when in Chapter hemorrhagic E. coli strain (ATCC 43894) which has both the stx1 and stx2 genes stx1 and stx2 genes to the target performing the isothermal amplification reaction, a diagram illustrating the Tm value.
Figure 5 is when the chapter hemorrhagic E. coli strain (ATCC 43894) which has both the stx1 and stx2 genes stx1 and stx2 genes to the target performing the isothermal amplification reaction, a diagram showing a standard curve.

Hereinafter, the present invention will be described in more detail.

A first primer set represented by the nucleotide sequences of SEQ ID NOS: 1 to 6; And a second primer set selected from the group consisting of nucleotide sequences of SEQ ID NOS: 7 to 12, and a set of at least one primer selected from the group consisting of SEQ ID NOS: 7 to 12.

In the present invention, " Enterohemorrhagic Escherichia coli (EHEC) " is an Escherichia coli which produces a toxic toxin such as verotoxin and Shiga toxin in the intestines of livestock, E. coli O157, E. coli O91, E. coli O111, E. coli O104, E. coli O26, E. coli O114, E. coli O79 and E. coli O178 belong to E. coli .

In the present invention, "Loop-mediated isothermal amplification" (LAMP) is a method of performing a reaction under an isothermal condition using a polymerase such as Bst or Gsp, which does not require a PCR cycle, unlike the conventional PCR method, Basically, four primers are used to synthesize both ends in a loop structure to amplify a specific part of a gene infinitely and confirm whether the amplified DNA product is amplified by using a fluorescence detection reagent or by precipitation reaction Preferably, a reagent for detecting fluorescence can be used, but the present invention is not limited thereto. In the present invention, the " real-time multiple isothermal amplification reaction " is a method for real-time confirmation of amplification, and can detect various genotypes at the same time. Preferably , the genotypes stx1 and stx2 can be detected and identified simultaneously However, the present invention is not limited thereto.

It is preferable that the primer set of the present invention is used for a Loop-mediated isothermal amplification (LAMP), wherein the first primer set is stx1 , a cytotoxic gene of intestinal hemorrhagic Escherichia coli, and the second primer set is enterohemorrhagic Escherichia coli Shiga toxin genes of the target stx2, each primer set consists of the inner primers, the outer primer set, the primer set and the ring. Specific information of each primer is as follows.

The FIP of SEQ ID NO: 1 constituting the first primer set includes a forward inner primer of the stx1 gene;

The BIP of SEQ ID NO: 2 is a backward inner primer of the stx1 gene;

LF of SEQ ID NO: 3 is a loop forward primer of the stx1 gene;

LB of SEQ ID NO: 4 is a loop backward primer of the stx1 gene;

F3 in SEQ ID NO: 5 is a forward outer primer of the stx1 gene;

B3 in SEQ ID NO: 6 is a backward outer primer of the stx1 gene.

The FIP of SEQ ID NO: 7 constituting the second primer set includes a forward inner primer of the stx2 gene;

The BIP of SEQ ID NO: 8 is a backward inner primer of the stx2 gene;

LF of SEQ ID NO: 9 is a loop forward primer of the stx2 gene;

LB of SEQ ID NO: 10 is a loop backward primer of the stx2 gene;

F3 in SEQ ID NO: 11 is a forward outer primer of the stx2 gene;

B3 in SEQ ID NO: 12 is a backward outer primer of the stx2 gene.

In the present invention, the term "primer" refers to a primer capable of hybridizing under appropriate conditions in a suitable buffer solution (for example, four different nucleoside triphosphates and a polymer such as DNA, RNA polymerase or reverse transcriptase) Quot; refers to single stranded oligonucleotides that can serve as a starting point for directed DNA synthesis. The appropriate length of the primer may vary depending on the intended use, but is usually 15 to 30 nucleotides. Short primer molecules generally require a lower temperature to form a stable hybrid with the template. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template. The primers of the present invention can be chemically synthesized using methods known in the art such as, for example, the phosphoramidite solid support method. Further, it can be modified by methylation, capping or the like by a known method. In addition, the primers of the present invention may, if necessary, comprise labels that are detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of labels include enzymes (e.g., horseradish peroxidase, alkaline phosphatase), radioactive isotopes (e.g., 32P), fluorescent molecules, chemical groups (e.g., biotin) have.

In the present invention, the term " inner primer " means a single stranded oligonucleotide capable of binding to template DNA and serving as a starting point for new DNA chain synthesis. The primers of SEQ ID NOS: 1 and 2 are internal primers located inside the stx1 gene to be amplified, the primers of SEQ ID NO: 1 are forward internal primers (FIP), the primers of SEQ ID NO: 2 are reverse internal primers (BIP )to be. The primers of SEQ ID NOs: 6 and 7 are internal primers located inside the stx2 gene to be amplified, the primer of SEQ ID NO: 6 is a forward internal primer (FIP), the primer of SEQ ID NO: 7 is a reverse internal primer (BIP )to be.

In the present invention, " outer primer " means a single-stranded oligonucleotide that binds to the template DNA at a position farther than the position at which the internal primer binds to the template DNA. The internal primer binds to the template DNA, After elongation, the strand displacement occurs due to the combination of the template DNA and the external primer, so that the previously formed chain is released. The primer of SEQ ID NO: 5 and 6 is an external primer located inside the stx1 gene to be amplified, the primer of SEQ ID NO: 5 is a forward external primer (F3), the primer of SEQ ID NO: 6 is a reverse external primer )to be. The primers of SEQ ID NOs: 11 and 12 are external primers located inside the stx2 gene to be amplified, the primer of SEQ ID NO: 11 is a forward external primer (F3), the primer of SEQ ID NO: 12 is a reverse external primer (B3 )to be.

In the present invention, "loop primer" means that the initial stem loop structure chain formed by binding the internal primer and the external primer to the template DNA increases the number of loop structure generation times, Quot; means a single stranded oligonucleotide that can serve as a starting point for nucleotide synthesis, which allows the reaction to be accelerated. The primers of SEQ ID NOS: 3 and 4 of the present invention are ring primers of the stx1 gene to be amplified, the primer of SEQ ID NO: 3 is a forward link primer (LF), and the primer of SEQ ID NO: 4 is a reverse link primer (LB). In addition, the primers of SEQ ID NOS: 9 and 10 are the ring primers of the stx2 gene to be amplified, the primer of SEQ ID NO: 9 is the forward link primer (LF) and the primer of SEQ ID NO: 10 is the reverse linker (LB).

According to one embodiment of the present invention, when the isothermal amplification reaction is carried out using the primer set according to the present invention, it is possible to detect the intestinal hemorrhagic Escherichia coli The result can be quantified, and the reaction time can be greatly improved. Thus, it is possible to quickly and accurately confirm contamination and occurrence level of intestinal hemorrhagic Escherichia coli, And can be effectively used as an early screening system for detecting a causal agent when a food poisoning accident occurs.

Accordingly, the present invention provides a composition for determining the cytotoxic genotype of enterohemorrhagic Escherichia coli comprising the primer set.

In addition, the present invention provides a kits for the determination of a cigarette toxin genotype of enterohemorrhagic Escherichia coli comprising the above composition.

The identifying composition or kit of the present invention may comprise one or more other component compositions, solutions or devices suitable for the analytical method. The kit of the present invention may include a conventional component included in the microorganism / gene detection kit together with a primer set for the determination of the cigarette toxin genotyping of the enterohemorrhagic Escherichia coli, and the kit for identifying a cigarette toxin genotype of the enterohemorrhagic Escherichia coli Typical components include, but are not limited to, reaction buffer, DNA polymerase, dNTP and MgCl ₂ , and the like.

In addition,

(a) separating the genomic DNA (gDNA) of the sample;

(b) amplifying the target sequence by using the separated genomic DNA as a template and performing an isothermal amplification reaction using the primer set; And

(c) detecting the amplified product, wherein the method comprises the steps of:

In the genotyping method, the sample of step (a) means an unknown substance for extracting DNA necessary for confirming the presence of a cytotoxic gene of enterohemorrhagic Escherichia coli, for example, a food contaminated with enterohemorrhagic Escherichia coli , Natural products (e.g., water), biological samples, and the like. The biological sample includes, but is not limited to, blood, sweat, urine, feces, tissue, and the like, which are separated from an individual. The subject includes all species susceptible to enterohemorrhagic Escherichia coli and includes, but is not limited to, a person, a dog, a cow, a pig, a sheep, a whale, a chicken, a duck,

In the genotyping method, the sample of step (a) may be a mixed sample. Preferably, the mixed sample is obtained by subjecting the stx1 and stx2 genes of enterohemorrhagic Escherichia coli Can be discriminated before the completion of the process, and may be a bacterium of a sample in which a mixture such as a feces or an environmental sample is mixed rather than a pure culture medium in which the microorganism has been separated.

In the genotyping method, the isothermal amplification reaction of step (b) may be performed at 60 to 67 ° C, preferably at 64 ° C.

In the genotyping method, the detection of the amplification product of step (c) may be performed by DNA chip, gel electrophoresis, radioactive measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto.

And (d) measuring the detection time of step (c) to quantify the intestinal hemorrhagic Escherichia coli.

The "quantification" may be a quantification of the fluorescence detection time according to the DNA concentration by comparing with the standard curve at the time of performing the amplification reaction using the primer set according to the present invention. Preferably, the amplification product of step (c) And measuring the fluorescence detection time by detecting using an indicator to quantify the DNA concentration.

The present invention also relates to a first primer set represented by the nucleotide sequences of SEQ ID NOS: 1 to 6; And a second primer set selected from the group consisting of nucleotide sequences of SEQ ID NOS: 7 to 12, and a set of at least one primer selected from the group consisting of SEQ ID NOS: 7 to 12.

Hereinafter, the present invention will be described more specifically based on examples and experimental examples. It is to be understood that both the present invention and the following examples are for illustrative purposes only and that the scope of the invention is not limited by these examples and experimental examples in accordance with the spirit of the present invention. It will be obvious.

Example 1: Primer design for high sensitivity real-time multiplex isothermal amplification (LAMP)

The primers for amplifying the stx1 and stx2 genes of enterohemorrhagic Escherichia coli are the stigmatoxin gene sequences of the various serotypes of enterohemorrhagic Escherichia coli registered with Genbank (the stx1 gene sequences are STEC O26, O79, O103, O111 and O157 the stx2 gene sequence referred to the gene sequences of STEC O26, O157 and O178 (Genbank accession number: FR850034, AB048240, X07865, FR850037) ) And a consensus sequence was generated. The designed primers were selected from a variety of candidate primers by means of isothermal amplification reaction conditions described later. Each of the six primers for isothermal amplification reaction contained internal primers (FIP, BIP), external primers (F3, B3) , And a set of ring primers (LF, LB). The internal primer and the external primer, essential for the reaction, generate the initial stem loop structure of the reaction product. The ring primer increases the number of times the loop structure of the reaction product is generated, thereby accelerating the overall reaction.

The specific nucleotide sequences of the primers are shown in Table 1.

Example 2: High Sensitivity Real Time Multi-Amplification (LAMP) Condition Setting

The reaction mixture was prepared by using GspSSD polymerase (OptiGene Ltd., UK), Evagreen (Solgent, Korea) and dNTPs including the primers, and finally prepared by optimizing the conditions. The concentration of dNTPs at a concentration of 10 mM, GspSSD polymerase at a concentration of 8 U / ul, MgSO ₂ .H ₂ O at a concentration of 50 mM, Betaine at a concentration of 5 M, and Evagreen at a concentration of 20 X were combined to prepare a Genie II platform (Optigene Ltd., UK) confirmed the combination with the fastest amplification product observation time. Make final concentration of 1X buffer, 1M Betaine, 4mM MgSO _4, 0.8mM of each dNTP, 0.8μM of each of FIP and BIP, 0.4μM each of the LF and LB, F3 and B3 in each of 0.2μM, 1X Eva Green (SolGent , Korea), confirming that the combination produced the amplification product in the earliest time.

In order to set the optimal temperature condition, the temperature condition was changed in the range of 60 to 67 ° C. The amount of the amplified product detected per each temperature was confirmed, and the amount of the amplified product per hour was expressed as a ratio Is shown in Fig.

As shown in Fig. 1, the isothermal amplification reaction confirmed that the amplification product was produced at the earliest time at 64 ° C.

Experimental Example 1: Measurement of inclusivity and exclusivity

In order to measure the inclusivity and exclusivity of the 12 isotope-amplification (LAMP) primers and reaction solutions prepared in Examples 1 and 2, a total of 48 species And the specific information thereof is shown in Table 2. In the case of the intestinal hemorrhagic Escherichia coli, the accession number ATCC 43894 contains all the stx1 and stx2 genes, the ATCC 43890 contains the stx1 gene, and the ATCC 43889 contains the stx2 gene. Lt; / RTI > In particular, Accession No. ATCC 43894, Enterohemorrhagic Escherichia coli, was used to measure the detection limit of the isothermal amplification reaction.

When the primers and reaction solutions for LAMP of Examples 1 and 2 were applied to 19 enterohemorrhagic Escherichia coli and 29 spleen hemorrhagic Escherichia coli strains, the inclusivity and exclusivity results were analyzed And the results are shown in Table 2.

As shown in Table 2, when an isothermal amplification reaction (LAMP) was performed using the primer and reaction solution of the present invention, the inclusivity and exclusivity were 100%, respectively.

Thus, it can be seen that various serotypes of intestinal hemorrhagic Escherichia coli can be selectively detected using the primer set for isothermal amplification reaction of the present invention, and the cigarette toxin genotype can be discriminated very accurately.

Experimental Example 2: Measurement of annealing temperature

To measure the annealing temperature of the isothermal amplification reaction (LAMP) using the primer prepared in Example 1, 100 ng / 쨉 l of DNA was extracted from three kinds of enterohemorrhagic Escherichia coli (ATCC 43894, ATCC 43890, ATCC 43889) Respectively. Then, the isothermal amplification reaction was performed using Genie II (Optigene, UK), and 1X buffer, 1M betaine, 4 mM MgSO ₄ , 0.8 mM dNTP, 0.8 μM FIP and BIP, A reaction volume of 25 μL total volume containing 0.4 μM each of LF and LB, 0.2 μM each of F3 and B3, 1 × EVA green (SolGent, Korea), 8 U Gsp polymerase (Optigene, UK) and 4 μL of template DNA (NC) (negative control) was treated with distilled water (Invitrogen, USA). An isothermal reaction was carried out at a temperature of 64 DEG C for 30 minutes to 1 hour, and an annealing curve was analyzed from 98 DEG C to 80 DEG C. The results are shown in FIG.

As shown in Figure 2, Tm values of the genes stx1 and stx2 observed in stx1 and stx2 field comprising both the gene of hemorrhagic E. coli, the accession number of ATCC 43894 strain was measured as 84.55 ± 0.32 ℃ and 87.22 ± 0.21 ℃, stx1 gene The Tm value observed in the Accession No. ATCC 43890 strain, which is the intestinal hemorrhagic Escherichia coli containing only the stx2 gene, was 87.61 +/- 0.17 [deg.] C, which was observed in Accession Number ATCC 43889, which is the enterohemorrhagic Escherichia coli containing only the stx2 gene .

As a result, stx1 and stx2 genes have different Tm values, and it was confirmed that stx1 and stx2 genes can be discriminated simultaneously by isothermal amplification reaction using the primer set of the present invention.

Experimental Example 3: Measurement of the detection limit

Field that contains all of the stx1, stx2 gene of a pure culture three different strains used in Experimental Example 2 to determine the detection limit of the isothermal amplification reaction (LAMP) using the prepared primers in Example 1, hemorrhagic E. coli strain (ATCC 43894), and the extracted DNAs were each diluted at a concentration of 1 fg / 占 퐇 to 100 ng / 占 퐇 in 10-fold steps and subjected to an isothermal amplification reaction performed under the same conditions as in Experimental Example 2 The detection limit was measured, and the results are shown in Figs. 3 to 5. Fig.

As shown in FIG. 3, when the DNA of the enterohemorrhagic Escherichia coli was diluted to a concentration of 10 fg / μl to 100 ng / μl and the detection time of the fluorescent substance expressed as the gene was amplified was measured in real time, Tp was measured from 9 minutes 6 seconds to 22 minutes 16 seconds. Thus, it can be seen that fluorescence substances are sequentially detected from a DNA having a high concentration during the equalization amplification reaction using the primer set of the present invention and can be confirmed in real time.

As shown in Fig. 4, the stx1 gene and the stx2 gene of enterohemorrhagic Escherichia coli have different Tm values of 87.24 +/- 0.25 [deg.] C, respectively. Thus, It can be identified.

As shown in FIG. 5, the quantitative equation of the isothermal amplification reaction using the primer set of the present invention was y = -1.8145x + 23.671 And the correlation coefficient was measured as R ² = 0.9313. It was confirmed that the detection time of the fluorescent substance according to the DNA concentration can be quantified by measuring the cytotoxic gene of the intestinal hemolytic Escherichia coli.

Therefore, it can be seen that stx1 and stx2 genes of intestinal hemorrhagic Escherichia coli can be rapidly identified and quantitated in real time during the isothermal amplification reaction using the isothermal amplification primer of the present invention.

Experimental Example 4: Isothermal amplification reaction of a sample obtained directly from a cow farm

In order to confirm the field applicability of the method for determining the cigarette toxin genotyping of intestinal hemorrhagic Escherichia coli according to the present invention, various serotypes of enterohemorrhagic Escherichia coli isolates isolated from environmental samples (bottom samples) of cattle feces and cattle farms were used PCR and isothermal amplification reaction were carried out in the same manner as described above.

EXPERIMENTAL EXAMPLE 4-1: Culturing of Escherichia coli outdoors

The intestinal hemorrhagic Escherichia coli outgrowth was obtained through the cultivation of 253 small cattle samples (237 cattle feces, 13 farm soil samples, 1 raw milk sample, 1 water sample, 1 feed sample) Was obtained from 15 cows farms in Gyeonggi-do from August 2012 to May 2013. Cattle stool samples were obtained directly from the bovine workplace and at least one environmental sample (bottom sample) was obtained from each farm. All samples were transferred to the laboratory at 4 ° C and immediately used in the experiment.

In order to cultivate intestinal hemorrhagic Escherichia coli outdoors, general culture method was first performed. More specifically, 20 g / L novobiocin (Oxoid, UK) was added to each 1 g sample and homogenized in 9 mL mEC broth (Becton, Dickinson and Company, USA) Lt; RTI ID = 0.0 > 37 C < / RTI > overnight. One loop of the mEC culture was then added to a MacConkey agar (Becton, Calif.) Supplemented with potassium tellurite (T-SMAC; 2.5 mg / L, Sigma-Aldrich, Canada) and sorbitol, Dickinson and Company, USA) and CHROMagr O157 (CHROM; CHROMagar Microbiology, France) agar, and then incubated overnight at 37 ° C. Then, up to six typical populations of MAC agar-pink, CHROM agar and light purple, were isolated using the E. coli O157 latex experiment kit (Oxoid, UK) and isolated populations were harvested from the MAC agar Subcultured in agar such as CHROM agar and incubated overnight at 37 < 0 > C.

E. coli O157 (Dynabeads MAX anti- E. coli O157) was used to perform another culture method, immunomagnetic separation. Suspensions of immunomagnetic beads were injected into T-SMAC and incubated overnight at 37 ° C. In the same manner as the above general culture method, the four colonies were cultured to become enterohemorrhagic Escherichia coli (STEC O157) to isolate enterohemorrhagic Escherichia coli.

In order to carry out another PCR-based culture method, first, one loop of the mEC culture solution obtained after homogenizing the intestinal hemorrhagic Escherichia coli outdoors was streaked in T-SMAC, and then incubated overnight at 37 ° C Lt; / RTI > The DNA was extracted from the strain in which the cytotoxin gene was confirmed to be present by the following PCR, and the plate in which the cytotoxin gene was present was subcultured in the MAC, followed by incubation at 37 DEG C overnight. Then, the aggregation reaction was carried out using antiserum (Joongkyeom, Korea) to determine the serotype of the community containing the cytotoxin gene.

EXPERIMENTAL EXAMPLE 4-2: Isothermal amplification reaction for the isolation of intestinal hemorrhagic Escherichia coli

Of the 253 samples of Experimental Example 4-1, 12 samples (4.7%) were cultivated in the outdoor cultures cultured by the above general cultivation method, 17 samples (6.7%) cultivated by IMS cultivation cultivated, PCR- (4.7%) were found to be intestinal hemorrhagic Escherichia coli.

The DNA extracted from the intestinal hemorrhagic Escherichia coli detected in the outdoor cultures cultured by the respective culturing methods was preliminarily denatured at 90 ° C for 5 minutes, denatured at 90 ° C for 30 seconds, annealed at 57 ° C for 30 seconds, PCR was performed by repeating the process of extending for 45 seconds 30 times and finally extending for 5 minutes at 72 ° C. Table 3 shows the primers used.

Also, the Tm value and the detection limit were measured by performing isothermal amplification of the DNA extracted by known methods in the above-mentioned outdoor isolate in the same manner as in Example 2 above. As a result, the cytotoxin gene was detected within 8 min 21 sec to 12 min 03 sec (average Tp value was 10 min 03 sec), and the stx1 gene was 85.76 ± 0.42 ° C. and the stx2 gene was 87.73 ± 0.29 ° C. Was measured.

In addition, the sensitivity of the Isopoiesis isolate separated by the general culture method, the IMS method, and the PCR-based culture method in Experimental Example 4-1 was compared with the result of performing PCR, and the results are shown in Table 4 .

As shown in Table 4, when isothermal amplification reaction was performed on the culture medium (mEC broth) of fecal and cow farm environmental samples which were confirmed to be contaminated with intestinal hemorrhagic Escherichia coli by a general culture method, sensitivity of 100% , The sensitivity was 88.2% and the PCR-based culture method was 90.0%. The sensitivities of the primer set of the present invention were 25.0%, 23.5%, and 0%, respectively.

In general, when the isothermal amplification reaction using the primer set of the present invention is carried out, it is possible to simultaneously detect the cigarette toxin gene of intestinal hemorrhagic Escherichia coli and discriminate the genotype at high sensitivity and quickly in real time through the detection of the fluorescent substance, In addition, it is possible to perform early screening of intestinal hemorrhagic Escherichia coli and its cytokines in a mEC broth of a sample mixed with a mixture such as feces and environmental samples, which is not a pure culture medium in which the microorganism has been separated. It can be seen that the field applicability of the isothermal amplification reaction is remarkably excellent.

<110> Seoul National University R & DB Foundation <120> Primer set for high sensitive real-time multiplex loop-mediated          isothermal amplification reaction for determining type of shiga          toxin genes of Enterohemorrhagic Escherichia coli, and method for          determining type of shiga toxin genes of Enterohemorrhagic          Escherichia coli using the same <130> SNU-2015-0404 <150> KR 10-2014-0072314 <151> 2014-06-13 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> stx1_FIP <400> 1 gcgatttatc tgcatccccg tatgtctggt gacagtagct at 42 <210> 2 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> stx1_BIP <400> 2 ggaacctcac tgacgcagtc cttcagctgt cacagtaaca 40 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> stx1_LF <400> 3 actgatccct gcaacacg 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> stx1_LB <400> 4 tgtggcaaga gcgatgtt 18 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> stx1_F3 <400> 5 acaacagcgg ttacattgt 19 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> stx1_B3 <400> 6 gatcatccag tgttgtacga a 21 <210> 7 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> stx2_FIP <400> 7 ggcgtcatcg tatacacagg agcgcttcag gcagatacag 40 <210> 8 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> stx2_BIP <400> 8 agacgtggac ctcactctga aactctgaca ccatcctctc 40 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> stx2_LF <400> 9 cagacagtgc ctgacgaa 18 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> stx2_LB <400> 10 ggcgaatcag caatgtgc 18 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> stx2_F3 <400> 11 gcatccagag cagttctg 18 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> stx2_B3 <400> 12 cagtataacg gccacagtc 19

Claims (13)

A first primer set represented by the nucleotide sequences of SEQ ID NOS: 1 to 6; And
And a second primer set selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 7 to 12; and a set of at least one primer selected from the group consisting of SEQ ID NOS: 7 to 12.
The primer set for the determination of the cigarette toxin genotyping of enterohemorrhagic Escherichia coli according to claim 1, wherein the primer set is a real-time multiplex loop-mediated isothermal amplification reaction.
The primer set for discriminating the cigarette toxin genotypes of enterohemorrhagic Escherichia coli according to claim 1, wherein the cytotoxin gene is stx1 or stx2 gene.
A composition for identifying a cigarette toxin genotype of enterohemorrhagic Escherichia coli comprising the primer set of any one of claims 1 to 3.
A kit for identifying a cigarette toxin genotype of enterohemorrhagic Escherichia coli comprising the composition of claim 4.
(a) separating the genomic DNA (gDNA) of the sample;
(b) performing a real-time isothermal amplification reaction using the separated genomic DNA as a template and using the primer set of any one of claims 1 to 3 to amplify the target sequence; And
(c) detecting the amplified product. &lt; Desc / Clms Page number 19 &gt;
The method according to claim 6, further comprising: (d) measuring the detection time to quantify the intestinal hemorrhagic Escherichia coli.
The method according to claim 6, wherein the sample of step (a) is a mixed sample.
[7] The method according to claim 6, wherein the sample of step (a) is at least one selected from the group consisting of food, natural products, and biological samples.
10. The method according to claim 9, wherein the biological sample is at least one species selected from the group consisting of blood, sweat, urine, feces and tissues separated from an individual.
7. The method according to claim 6, wherein the isothermal amplification reaction in step (b) is performed at 60 to 67 占 폚.
7. The method according to claim 6, wherein the cytotoxin gene is stx1 or stx2 gene.
A first primer set represented by the nucleotide sequences of SEQ ID NOS: 1 to 6; And
And a second primer set selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 7 to 12; and a set of primers selected from the group consisting of SEQ ID NOS: 7 to 12.

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