KR20170030190A - Primers used for LAMP reaction for the detection of Clostridium perfringens and its use - Google Patents

Abstract

The present invention discloses: a primer set capable of specifically detecting Clostridium perfringens, which is food poisoning bacteria, by an isothermal amplification method; a composition or a kit comprising the same; and a method using the same. The primer set according to the present invention can rapidly detect infection or contamination of a specimen or pathogens in a food with high sensitivity and high specificity, and also can be detected immediately without any additional treatment such as electrophoresis after ending reaction, if necessary, thereby having improved convenience.

Description

[0001] The present invention relates to a primer for detecting Clostridium perfringens using LAMP, and a primer for detecting Clostridium perfringens and its use.

The present invention relates to a primer for accurately and rapidly detecting Clostridium perfringens causing food poisoning using the LAMP method and its use.

Food poisoning refers to symptoms caused by ingestion of food contaminated with pathogenic bacteria, toxins, viruses, parasites, chemicals, natural poisons, etc. Main symptoms are abdominal pain, nausea, vomiting, diarrhea, fever, headache, fatigue, (Heymann DL, Control of Communicable Diseases Manual, pp 232-242, The American Public Health Association, Washington, 2004.). Foodborne diseases caused by microorganisms are classified into bacterial food poisoning and viral food poisoning. Bacterial food poisoning is classified into poisonous and infectious types. Staphylococcus aureus, Bacillus cereus, and Clostridium are the causative organisms of poisonous food poisoning. Pathogenic Escherichia coli, enteritis Vibrio bacteria, Salmonella, and Shigella are the causative organisms of infectious food poisoning.

Clostridium perfringens , a major causative agent of poisonous food poisoning, is a gram-positive, non-motile, anaerobic, apo-forming bacterium that occurs in natural environments such as soil, rivers, sewage, and dust, Food and the like. Especially, apo is heat resistance which is not killed even when heated at 100 ° C for 1 to 4 hours. (Czeczulin JR1, Collie RE, McClane BA. Regulated expression of Clostridium perfringens enterotoxin in naturally cpe-negative type A, B, and C isolates of C. perfringens. Infect Immun 1996 Aug; 64 (8): 3301-9; Miyamoto K1, Li J, McClane BA. Enterotoxigenic Clostridium perfringens: detection and identification Microbes Environ. 2012; 27 (4): 343-9. Approved August 17, 2015).

Clostridium perfringens enterotoxin (CPE), which is produced during the process of producing spores by the presence of 10 ⁵ cfu / g or more of bacteria in suspect foods or 10 ⁶ cfu / (Miyamoto ibid : Guide to the pathogenic microorganism. 2013. http: //www.nifds.go.kr/ Approached on August 17, 2015). Fungi are classified into six types of A to F according to secreted toxins (alpha, beta, epsilon, and iota), among which A (alpha toxin) (Czeczulin, ibid ; Miyamoto K1, ibid ) have also been reported.

The seasonal biases that occur throughout the year are hard to find and are characterized by massive outbreaks in a short time due to the short incubation period compared to infectious food poisoning (see page 2013. http: //www.nifds.go. Approach on August 17, 2015).

The total number of cases caused by Clostridium perfringens is 4,891, which is 324 in 2011, and 516 in 2013. The number of cases of food poisoning by the causative organism (2002 ~ 2014) And 1,689 in 2014, and is on the increase.

Thus, the rate of food poisoning is increasing due to the increase of group meal due to social change such as school meal, nuclear family, dual income and aging, increase of eating out due to improvement of living standard, increase of demand for instant food.

Korean Patent Publication No. 1544050 relates to a kit and a method for detecting bacteria, and discloses a method of detecting Clostridia using an antibody.

Recently, however, food poisoning has occurred regardless of the season, and the size of the food has been gradually increased due to the spread of the food, so that the monitoring and rapid monitoring based on the new technology with high sensitivity and specificity for quickly and accurately checking the pathogen for reduction and inhibition of food poisoning Development of test methods is necessary.

The present invention aims to provide a method for rapidly and easily detecting infection with Clostridium perfringens while exhibiting high sensitivity and specificity.

In one embodiment, the present invention provides a primer set for LAMP analysis for detection of Clostridium perfringens comprising an F3 primer, a B3 primer, a FIP primer, and a BIP primer, wherein the F3 primer comprises SEQ ID NO: 1 or 7 ≪ / RTI > The B3 primer is selected from SEQ ID NO: 2 or 8; Wherein said FIP primer is selected from SEQ ID NO: 3 or 9; Wherein said BIP primer is selected from the group consisting of Clostridium < RTI ID = 0.0 > provides a primer set for LAMP Analysis for perfringens) detected.

The F3 primer, the B3 primer, the FIP primer, and the BIP primer included in each primer set according to the present invention may be included in various combinations. In one embodiment according to the present invention, the primer set of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: A primer set comprising 4; A primer set comprising SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10; A primer set comprising SEQ ID NO: 7, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4; Or a combination of primers set forth in SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 3 and SEQ ID NO: 4.

The primer set according to the present invention may further comprise an LF primer and an LB primer for rapid reaction, wherein said LF primer is selected from SEQ ID NO: 5 or SEQ ID NO: 11 and said LB primer is selected from SEQ ID NO: 6 or SEQ ID NO: 12 Is selected.

The F3 primer, the B3 primer, the FIP primer, the BIP primer, the LF and the LB primer included in each primer set according to the present invention may be included in various combinations, and in one embodiment according to the present invention, SEQ ID NO: 1, SEQ ID NO: A primer set comprising SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; A primer set comprising SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12; A primer set comprising SEQ ID NO: 7, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; A primer set comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11 and SEQ ID NO: 6; A primer set comprising SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; Or a set of primers including SEQ ID NO: 7, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11 and SEQ ID NO:

When a primer set according to the present invention is required for real time or detection of an amplification product at the termination of the reaction, one or more primers contained in each set may be labeled with a detectable labeling substance.

In another aspect, the disclosure also provides a kit or composition for detecting Clostridium perfringens via a LAMP method comprising a primer set according to the present invention.

In another aspect, the present invention is also directed to a method for detecting Clostridium perfringens in Invitro via the LAMP method using the primer sets, compositions and / or kits disclosed herein, wherein Clostridium perfringens infection or contamination Providing the suspected sample; Providing a primer set according to the present invention; Performing a LAMP reaction using the sample and the primer set; And analyzing the results during or after the LAMP reaction and comparing the results to a control sample, if there is a difference, determining the sample as Clostridium perfringens infection or contamination.

The primer sets, kits and / or compositions according to the present invention may be used in a variety of foodstuffs, including food, drinking water, preserved food, foodstuffs and foodstuffs, which require the detection of clostridia; feed; Blood, urine, feces, or body fluids; But is not limited to, environmental samples, including, but not limited to, tap water, ground water, water in public facilities, cooking utensils including chopping boards, knives, kitchen cloths, or various equipment or devices used in hospitals.

In the method according to the present invention, the analysis of the LAMP reaction results is possible both in real time or after the termination of the reaction, and the amplification is determined, for example, by measurement using one or more of color change measurement or turbidity, Or when the turbidity increases, it can be judged to be positive for clostridium by amplification.

A primer set, a composition or kit containing the same, and a method using the primer set, which can specifically detect Clostridium perfringens according to the present invention by means of isothermal amplification method, can be used not only for high sensitivity and specificity, And if necessary, detection can be performed without any additional treatment such as electrophoresis after completion of the reaction, thereby increasing convenience.

FIG. 1 shows the results of detection of sensitivity by color change and agarose gel electrophoresis using Clostridium perfringens-specific LAMP primer set of combination shown in Table 1 according to one embodiment of the present invention.
FIG. 2 shows the results of detection of sensitivity by color change and agarose gel electrophoresis using a Clostridium perfringance-specific LAMP primer set of the combination shown in Table 2 according to another embodiment of the present invention.
FIG. 3 shows the results of detection of sensitivity by color change and agarose gel electrophoresis using Clostridium perfringens-specific LAMP primer set of the combination shown in Table 3 according to another embodiment of the present invention.
FIG. 4 shows the results of detecting the sensitivity of the Clostridium perfringance-specific LAMP primer set of the combination shown in Table 4 according to another embodiment of the present invention by color change and agarose gel electrophoresis.
FIG. 5 shows the results of detection of sensitivity by color change and agarose gel electrophoresis using Clostridium perfringens-specific LAMP primer set of combination shown in Table 5 according to another embodiment of the present invention.
Figure 6 shows the results of colorimetry and agarose gel electrophoresis of the specificity and sensitivity of the Clostridium perfringance-specific LAMP primer set of the combination described in Table 6 according to another embodiment of the present invention.
Sensitivity is shown in red in each of the above figures. Reactions were performed in a total of 7 different concentrations and negative controls for each set and the concentrations were as follows: 1: 100 pg / [mu] l; 2: 10 pg / l; 3: 1 pg / l; 4: 500 fg / l; 5: 100 fg / l; 6: 50 fg / l; 7: 10 fg / 占 퐇 and 8: Negative control group.

The present invention is based on the discovery that Clostridium perfringens can be easily detected with high accuracy and sensitivity at a nucleic acid level by using the LAMP method. In the present invention, Clostridium perfringens is expressed by LAMP (Loop Mediated Isothermal Amplification, Mediated isothermal amplification method), a composition or kit comprising the same, and a method for detecting Clostridium perfringens using the primer set or the composition or kit.

In one embodiment, the present invention relates to a primer set for LAMP analysis capable of specifically detecting Clostridium perfringens.

The term " primer " as used herein refers to a nucleic acid molecule in which a nucleotide is extended by covalent bonding at its 3 'end in a nucleic acid amplification or synthesis reaction using a polymerase as a single strand oligonucleotide. The primer set according to the present invention is used for amplification of nucleic acid, i.e., LAMP analysis. In this application, a primer which can be composed in various combinations by designing a binding site so as to specifically bind to a toxin gene or its periphery is designed in consideration of sensitivity, specificity, promptness and convenience of detection.

As used herein, the term " nucleic acid " refers to a single or double stranded oligonucleotide or polynucleotide, which refers to an RNA or DNA molecule or analogs and derivatives thereof comprising one or more nucleotides. The Clostridium perfringens nucleic acid in which the primer set according to the present invention is used includes all or a part of the genome, and in one embodiment, DNA.

As used herein, the terms " oligonucleotide " and " polynucleotide " are used interchangeably, and both encompass both. The term " primer " can also be used in combination with " oligonucleotide " and " polynucleotide ", including nucleic acid (RNA or DNA), aptamer and the like.

As used herein, the term " detection " is intended to analyze the presence or absence of nucleic acid contamination or infection of Clostridium perfringens, particularly the LAMP assay described below.

As used herein, the term " target " or " target nucleic acid " refers to a nucleic acid sequence which is specifically amplified by a set of primers according to the present invention, wherein the primer set herein is combined and comprises RNA or DNA.

The primer according to the present invention specifically binds to the target nucleic acid through complementation and amplifies the target nucleic acid by the LAMP method. LAMP was amplified by strand amplification using a nucleic acid amplification method (Notomi, T. et al. 2000. Loop-Mediated Isothermal Amplification of DNA, Nucleic Acids Res 28, E63) capable of amplifying a target nucleic acid with high sensitivity and specificity under isothermal conditions (Nagamine, K < / RTI > (K)) using a set of at least four primers specially designed at various sites of the target nucleic acid (see Korean Patent No. 612551 and the like) et al., 2002. Accelerated reaction by Loop mediated isothermal amplification using loop primers, see Mol Cell Probes 16, 223-9).

As used herein, the term " complementary " or " complementarity " refers to a condition in which a primer or oligonucleotide under sequence hybridization, annealing, or annealing conditions is capable of sequence-specific binding to a target nucleic acid through pairing of Watson- Quot; includes both partial complementarity, substantially complementary and perfectly complementary. Substantially complementarity means that the two strands of the nucleic acid sequence are not completely complementary to each other, but are complementary enough to bind to the target nucleic acid so as to amplify the effect according to the present invention, that is, to amplify the target sequence through the LAMP method.

As used herein, the term " hybridization " or " hybridization " refers to a reaction in which two strands of complementary nucleic acid molecules form a sequence-specific complex through hydrogen bonding. Hybridization can constitute the step of binding the primer to the target nucleic acid or template in the LAMP reaction in which the primer according to the present invention is used. The degree of hybridization is affected by various factors such as the degree of complementation of any two nucleic acid molecules involved, their melting temperature (Tm), or the stringency of hybridization. The hybridization reaction conditions can also be determined in view of this. The stringency of the hybridization reaction is a condition that determines how easily any two nucleic acid molecules that are to be hybridized with each other can be easily conjugated, and the complementary, reaction temperature, ionic strength and / or concentration And can be determined according to the kind, for example, J. Sambrook and DW Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th Ed., 2012; and F. M. Ausubel, Ed., Short Protocols in Molecular Biology, Wiley; 5th Ed, 2002 can be referred to.

Specific binding or hybridization herein means that a particular nucleic acid molecule or primer binds only to the target nucleic acid without substantial binding to a nucleic acid other than the non-target nucleic acid. The primers according to the present invention can specifically bind to the target Clostridium perfringance nucleic acid according to the present invention or its complementary sequence under high stringency conditions, and can be used in primer-based DNA synthesis for primer sequence length, buffer, pH, magnesium concentration and reaction temperature.

The primer according to the present invention is designed to specifically detect Clostridium perfringens through the LAMP method, and specifically binds to a target nucleic acid or its complementary sequence. The primers according to the present invention are designed to detect bacteria with high sensitivity by specifically recognizing the Clostridium perfringens alpha toxin gene site in various combinations.

As described above, at least four types of primers are used in the standard LAMP reaction: FIP (forward inner primer), BIP (backward inner primer), F3 (forward outer primer) and B3 (backward outer primer). For accelerated LAMP, a total of six primers can be used including LF (Loop F) and LB (Loop B). The above-mentioned references to features and functions of each F3 primer, B3 primer, FIP primer, BIP primer, Loop F primer (LF), and Loop B (LB) primer are described in Notomi et al., 2000; And Nagamine et al., 2002.

A primer specifically binding to a target nucleic acid or a complementary sequence thereof according to the present invention has a length of at least 10 nucleotides, a minimum of 15 nucleotides for F3 and B3, a minimum of 30 nucleotides for FIP and BIP, and a minimum of 30 nucleotides for LF and LB It is 15 nucleotides. With at least 70% complementarity, particularly 80% complementarity, even 90% complementarity, even more particularly 95%, 96%, 97%, 98%, 99% or 100% complementarity with the corresponding portion of the target nucleic acid.

The LAMP primer according to the present invention specifically binds to the target Clostridium perfringens nucleic acid and detects it with high sensitivity. In one embodiment, the F3 primer included in the primer set according to the present invention is selected from SEQ ID NO: 1 or 7, the B3 primer is selected from SEQ ID NO: 2 or 8, the FIP primer is selected from SEQ ID NO: 3 or 9, Is selected from SEQ ID NO: 4 or 10, and each of the sequences comprises a primer having substantially the same sequence. In another embodiment, the primer set comprises six primer sets, for example, each primer set comprises an LF primer of SEQ ID NO: 5 or SEQ ID NO: 11 and an LB primer of SEQ ID NO: 6 or 12, or a sequence of substantially the same sequence Further comprising a primer. Each F3 primer, B3 primer, FIP primer, and BIP primer disclosed in the present application may be used in various combinations, and the LB and LF may be additionally included in various combinations.

A primer according to the present invention includes substantially the same primer and specifically binds to a target nucleic acid or a complementary sequence thereof. The primers of substantially the same sequence have 70% complementarity, particularly 80% complementarity, more particularly 90% complementarity, even more particularly 95%, 96%, 97%, 98%, 99% 100% complementarity. In the case of FIP and BIP, two sequences, each of which binds to the target nucleic acid and its complementary sequence, can be linked by a linker that does not bind to the target nucleic acid, in which case the complementarity means complementarity with the portion excluding the linker portion.

In one embodiment, a set of primers comprising six primers is used, including, but not limited to, combinations of, for example, Tables 1 to 6.

The primer according to the present invention and the composition or kit or method comprising the same can be used for screening for contamination or infection in various samples suspected of infection or contamination of a bacterium for detection of infection or contamination, It can be useful for analysis.

Samples in which a primer set, method, or kit according to the present invention can be used include, for example, foodstuffs, drinking water, preserved foods, foodstuffs, various foods including foodstuffs; feed; Blood, urine, feces, or body fluids; Or environmental samples including water, ground water, water in public facilities, cooking utensils including chopping boards, knives, kitchen cloths, or various equipment or devices used in hospitals. Methods of sampling are known in the art. For example, samples suspected of contamination with food poisoning bacteria were collected, suspended in sterilized water or 0.85% physiological saline, treated with Proteinase K, recovered, and subjected to LAMP reaction by hot water extraction Can be performed.

These biological samples may also be those obtained directly by the usual method of obtaining the samples immediately before the test or stored separately in advance.

In another embodiment, the sample according to the present invention can be used as a sample in which a nucleic acid separated from the above-mentioned sample is used. Separation is separation from the sample containing the nucleic acid, including separation into various purity.

The primers according to the present application and compositions or kits and methods comprising them are used in the LAMP method for the detection of bacteria in any sample in which there is a possibility that the bacteria are present.

For the reaction for LAMP analysis, primer sets, dNTPs, buffers, magnesium and DNA polymerases and samples according to the present invention are used. It may further comprise a substance such as betaine or DMSO to enhance the reaction. The primer set included in the reaction product is as described above, and four or six primer sets can be used.

Magnesium is used in the form of a salt such as magnesium acetate, magnesium chloride or magnesium sulfate.

The buffer may be, for example, sodium phosphate buffer, potassium phosphate buffer, Tris-HCl buffer or Tricine buffer.

The DNA polymerase that can be used in the reaction is a polymerase derived from a thermophilic microorganism, in particular a polymerase lacking 5 '->3' exonuclease function. Non-limiting examples include Bacillus stearothermophilus , Bst, DNA polymerase; Thermus , thermophilus , Tth, DNA polymerase; Thermus aquaticus , Taq, DNA polymerase; Thermococcus litoralis DNA polymerase; Pyrococcus furiosus , Pfu, DNA polymerase; And Bacillus caldotenax DNA polymerase.

Also, instead of dNTP, a nucleotide analog may be used as the reactant. Nucleotide analogues can be modified or co-polymerized with natural nucleotides in a template-induced DNA synthesis process that is not altered or found in nature. Examples of nucleotide-based analogues that can be polymerized through such Wasson Creek-based pairing include, for example, substituted purines or pyrimidines, deazapurines, methylpurines, methylpyrimidines, aminopurines, aminopyrimidines, But are not limited to, purine, thiopyrimidine, indole, pyrrole, 7-deazaguanine, 7-methylguanine, hypoxanthine, shodocytosine, shido isocytosine, isocytosine, isoguanine, , 6-thioguanine, nitropyrrol, nitroindole, and 4-methylindole. Nucleotides comprising a substituted deoxyribose analog include substituted or unsubstituted arabinose, substituted or unsubstituted xylose, and substituted or unsubstituted pyranose. Nucleotides containing phosphate ester analogs include, but are not limited to, phosphorothioate, phosphorodithioate, phosphoroamidate, phosphorocellinoate, phosphoroanilothioate, phosphoroanilide, phosphoroamidate, boron Phosphate, phosphotriester, and alkylphosphonates such as methylphosphonate.

In one embodiment, the reactants were added to a total of 25 μl with 0.2 μM of each F3 and B3 primer, 1.6 μM of each FIP and BIP primer, 0.8 μM of each LF and LB primer, 0.4 M betaine, 10 mM MgSO4, 1.4 mM dNTPs, 1 × ThermoPol reaction buffer (New England Biolabs), 16 U Bst DNA polymerase (New England Biolabs) and samples requiring analysis of 1.0 to 8.5 μl.

The reaction is then allowed to react at a temperature suitable for DNA polymerase activity. The reaction temperature can be determined without difficulty in view of the nucleotide sequence of the target enzyme and the enzyme used in the reaction. The reaction is then allowed to react at a time appropriate for amplification of the target nucleic acid. Those skilled in the art will be able to determine the reaction time in consideration of the reaction conditions, and can be determined, for example, at a time of 15 minutes to 60 minutes, depending on the amount of the target nucleic acid contained in the sample, and can be determined. For example, the reaction time can be increased to increase the sensitivity.

In one embodiment according to the present application, a sample can be directly used without isolating the nucleic acid in particular.

LAMP assays according to the present invention can be analyzed in a variety of analytical formats, for example, in a liquid phase reaction or in a state where some components are adsorbed to a solid substrate.

The primers according to the present invention and the compositions or kits and methods comprising them are used for LAMP analysis using any biological sample for which there is a possibility that the bacterium is present and the amplified product has a sequence corresponding to the molecule used as the template, Can be analyzed by a variety of methods known in the art.

The amplification reaction product after amplification can be detected in various ways, for example, by color change of reaction solution, turbidity change due to DNA synthesis, fluorescence and / or electrophoresis, and the detected product is positive and / or Is compared with the product of the negative control sample and used for quantitative or qualitative analysis.

In one embodiment, the amplification product according to the present invention can be detected by color change of the reaction solution according to DNA synthesis. In this case, the reaction solution may contain an appropriate indicator, and HNB (hydroxy naphthol blue) may be used as a dye whose color changes in response to the magnesium ion concentration in the reaction solution to produce a product of the positive and / or negative control sample , Clostridium perfringens can be quantitatively or qualitatively analyzed. Especially, detection is possible in the inside, and convenience is enhanced.

In other embodiments, the amplification product can be detected by a direct or indirect method. In a direct method, a detectably labeled primer can be used, and the nucleic acid amplification is detected through a signal emitted through a primer labeled with a specific binding to a nucleic acid, and real-time detection is possible. In indirect detection methods, labeled probes capable of binding to amplified nucleic acids can be used. Means a substance capable of emitting a detectable signal using any suitable method such as spectroscopic, optical, photochemical, biochemical, enzymatic, electrical and / or immunochemical methods. Means a labeled substance. Such materials include, for example, fluorescent moieties, chemiluminescent moieties, bioluminescent moieties, magnetic particles, enzymes, substrates, radioactivity and chromophore materials.

In one embodiment, the label for detection includes, but is not limited to, a compound capable of generating or erasing a detectable fluorescence, chemiluminescent, or bioluminescent signal such as light emission, light scattering, light absorbing material, For example, Garman A., Non-Radioactive Labeling, Academic Press 1997. Fluorescent materials include, but are not limited to, fluorescein (e.g., U.S. Patent 6,020,481), rhodamine (e.g., U.S. Patent 6,191,278), benzophenoxaphone (e.g., U.S. Patent 6,140,500), donors and receptors Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 (for example, US Pat. No. 5,945,526) and cyanine (for example, WO1997-45539) , FluorX (Amersham), Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY- TRX, Cascade Blue, 6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA, Tetramethylrhodamine, and / Lt; RTI ID = 0.0 > fluorescence < / RTI > The fluorescent dye is 6-carboxyfluorescein; 2 ', 4', 1,4, -tetrachlorofluorescein; And 2 ', 4', 5 ', 7', 1, 4-hexachlorofluorescein. In one embodiment, SYBR-Green, 6-carboxyfluorescein ("FAM"), TET, ROX, VIC, or JOE are used as fluorescent labels. In one embodiment, a probe labeled with two fluorescent materials, a reporter fluorescent substance and an erasing fluorescent substance, is used. In this case, a fluorescent substance is used in which a fluorescent substance emits a spectrum having a distinguishable wavelength. Also, markers may include compounds capable of enhancing, stabilizing, or affecting the binding of nucleic acids, such as intercalators, minor groove associations and crosslinkable functional groups, including epidymium bromide and SYBR-Green, , But are not limited to, Blackburn et al., Eds. &Quot; DNA and RNA Structure " in Nucleic Acids in Chemistry and Biology (1996).

In addition, when it is necessary to detect non-specific amplification by non-specific priming of a primer, a non-specific nucleic acid binding agent such as acidium bromide or picogreen is used in a control reaction not containing a template to generate nonspecific total amplification The amount of product can be determined.

The primer set according to the present invention can be usefully used in various analyzes for detecting contamination or screening for the presence or absence of infection in various samples suspected of being infected or contaminated for detection of infection or contamination.

Accordingly, in another aspect, the present invention is also directed to a composition or kit for detecting Clostridium perfringens comprising a primer or primer set according to the present invention as described above.

The composition according to the present invention may comprise the components used in the LAMP reaction as described above except for the template and the four or six primer sets described above.

The kit according to the present invention can provide the components used in the LAMP reaction as described above except the template and the above described four or six primer sets separately or in one tube. The kit according to the present invention further comprises a positive control, a negative control, and instructions for use. As a negative control, a sample containing no nucleic acid, a positive control group may include one or more detection target nucleic acids.

The primers, kits and compositions according to the present invention can be prepared by detecting Clostridium perfringens at the nucleic acid level in a sample and comparing the presence or absence of the nucleic acid with the control or reference group to determine the presence of Clostridium perfrin The effects of treatment can be predicted if you are receiving Zens infection, contamination, or antibiotic treatment.

In this regard, the disclosure also relates to a method for detecting Clostridium perfringens in a biological sample to provide information necessary for the detection, contamination, infection or prognosis of Clostridium perfringens.

The method according to the present invention may be carried out using a primer set, composition or kit as described above, in one embodiment providing a sample suspected of contamination or infection with Clostridium perfringens; Amplifying the target nucleic acid by performing LAMP using the primer set according to the present invention from the sample; Comparing the result of amplification with a sample of the control group during or after the reaction, and when there is a change in the amount of the amplified product as compared with the sample, judging it as Clostridium perfringens contamination or infection . The method according to the present invention can determine the infection by qualitative or quantitative analysis, for example, when the amount thereof is increased as compared with the negative control, or when the positive reaction is not detected in the negative control, If it is detected more than a certain amount, it can be judged as contamination or infection. The judgment or judgment criterion can be easily determined in consideration of the standard in the art.

Samples, reagents, amplification methods and reaction conditions used in the method of the present invention can be referred to the above.

Hereinafter, the present invention will be described in detail with reference to the following examples, which are intended to be illustrative and not limit the scope of the present invention in any way.

The present invention may be practiced using conventional techniques within the skill of those skilled in the art of cell biology, cell culture, molecular biology, gene transformation techniques, microbiology, DNA recombinant techniques, unless otherwise indicated. Further, a more detailed description of common techniques can be found in Molecular Biotechnology: (Bernard et al., ASM press 2014); Molecular Cloning: A Laboratory Manual, 4th Ed. (Sambrook et al., Cold Spring Harbor Laboratory Press 2012); Short Protocols in Molecular Biology, 5th Ed. (Ausubel F. et al. Eds., John Wiley & Sons 2002).

Example 1. Obtaining a standard strain related to food poisoning induction

Representative strains known to cause food poisoning worldwide include Bacillus cereus , Campylobacter jejuni , Clostridium perfringens , Escherichia coli O157: H7 O157: H7 ), Listeria monocytogenes , Staphylococcus aureus , Salmonella spp. , Shigella spp. , Vibrio parahaemoliticus Vibrio parahaemolyticus , Vibrio vulnifcus , and Yersinia enterocolitica were obtained from the Korean Microorganism Conservation Center.

Example 2. Production of primers specific to food poisoning pathogens

For accurate detection of Clostridium perfringens strains, toxin genes were obtained from NCBI and GenBank, and the final primers specific for the strains were selected. Specifically, two kinds of primers were prepared for each primer of F3, B3, FIP, BIP, LF and LB based on the gene of cpa (GenBank accession number, L43547.1) as a target of Clostridium perfringens , And these were subjected to various combinations to complete six types of LAMP amplification primer sets (Tables 1 to 6). Each primer sequence was verified using NCBI (National Center for Biotechnology Information), Genbank, and Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi website). As a result, Did not exist. Each primer set was designed and combined to be optimized for the LAMP reaction and showed excellent effects in terms of specificity and sensitivity. Primers were synthesized by request from outside.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

Example 3. Specificity and Sensitivity of LAMP Reaction

First, the specificity was confirmed by the color change of the reaction product and the electrophoresis result through the cross reaction between the strains causing food poisoning using one combination of six primer combinations (see Table 6) (FIG. 6A).

For this purpose, Clostridium perfringens obtained in Example 1 and 11 strains induced by food poisoning were cultured according to standard methods, and the DNAs were prepared with 20 ng / ul of each DNA. For LAMP analysis, 0.2 μM of each F3 and B3 primer, 1.6 μM of each FIP and BIP primer, 0.8 μM of each LF and LB primer, 0.4 M betaine, 10 mM MgSO ₄ , 1.4 mM dNTPs, 1 × ThermoPol reaction buffer (New England Biolabs), 16 U Bst DNA polymerase (New England Biolabs), 120 μM HNB (Sigma) and 2 μl of the separated DNA. Isothermal amplification was carried out at 63 ° C for 30 minutes in the above composition, followed by reaction at 80 ° C for 5 minutes to terminate the reaction.

The results show that the primer set according to the present invention does not cross-react to other similar strains and specifically detects only Clostridium perfringens as shown in Fig. 6A. The above results can sufficiently show the specificity with other combinations of primer sets (Tables 1 to 5).

Next, the sensitivity of each primer set (Table 1 to Table 6) to Clostridium perfringens was confirmed. For this, isothermal amplification was carried out at 63 ° C. for 30 minutes in the same composition using the Clostridium perfringens genome prepared as described above at a concentration of 100 pg, 10 pg, 1 pg, 500 fg, 100 fg, 50 fg and 10 fg in the reaction solution The reaction was carried out at 80 ° C for 5 minutes, and the sensitivity was confirmed by the color change of the reaction product and electrophoresis (FIGS. 1 to 5 and 6b).

Color analysis was performed by adding HNB (hydroxy naphthol blue) dye to the reaction solution. HNB acts as an indicator of Mg ²⁺ ion concentration. When there is no Mg ²⁺ ion, it appears to be light blue. When Mg ²⁺ ion is present, it is purple. Therefore, before the reaction, it is present in purple, and as the DNA synthesis reaction occurs, the concentration of Mg ²⁺ ions decreases and thus the color changes from purple to blue. The change in color is a positive reaction indicating that the nucleic acid has been amplified and qualitative and quantitative analysis is possible by measuring the absorbance at a wavelength of about 650 nm. The method according to the present invention is advantageous in qualitative and quantitative analysis when absorbance is measured at a wavelength of 650 nm using a spectrophotometer.

The detection of bacteria using the primer set according to the present invention as described in Figures 1 to 5 and 6b was found to be very high, with sensitivity ranging from 10 pg to 500 fg using genomic DNA.

<110> Mmonitor Inc. <120> Primers used for LAMP reaction for the detection of Clostridium          perfringens and its use <130> DP201508007P <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of C. perfringens <400> 1 tgtaaggcgc ttatttgtgc 20 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of C. perfringens <400> 2 catgcatgtt ctcttttaaa atctc 25 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of C. perfringens <400> 3 caatctttcc atcccaagcg tgcgctagca actagcctat 40 <210> 4 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of C. perfringens <400> 4 ggaacaggaa ctcatgctat gaggttcatt tttggacaga tcat 44 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of C. perfringens <400> 5 actttagttg atgccccagc 20 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of C. perfringens <400> 6 gtaactcaag gggtttcaat cttag 25 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of C. perfringens <400> 7 gatttgtaag gcgcttattt gt 22 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of C. perfringens <400> 8 gctcatgcat gttctctttt aaaa 24 <210> 9 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of C. perfringens <400> 9 ctttccatcc caagcgtaga ccgcgctagc aactagccta t 41 <210> 10 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of C. perfringens <400> 10 attgatggaa caggaactca tgctttctgg ttcatttttg gacagatc 48 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of C. perfringens <400> 11 tttagttgat gccccagcc 19 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of C. perfringens <400> 12 ctatgattgt aactcaaggg gtttca 26

Claims (10)

As a primer set for LAMP analysis for detection of Clostridium perfringens including F3 primer, B3 primer, FIP primer and BIP primer,
The F3 primer is selected from SEQ ID NO: 1 or 7;
The B3 primer is selected from SEQ ID NO: 2 or 8;
Wherein said FIP primer is selected from SEQ ID NO: 3 or 9;
Wherein said BIP primer is selected from the group consisting of Clostridium &lt; RTI ID = 0.0 &gt; A primer set for LAMP analysis for detection of perfringens .
The method according to claim 1,
Wherein the primer set further comprises an LF primer and an LB primer,
Wherein said LF primer is selected from SEQ ID NO: 5 or SEQ ID NO: 11,
Wherein the LB primer is selected from SEQ ID NO: 6 or SEQ ID NO: 12, and a primer set for LAMP analysis for detection of Clostridium perfringens.
The method according to claim 1,
A primer set for LAMP analysis for detection of Clostridium perfringens, wherein the primer set is selected from the group consisting of:
A primer set comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4;
A primer set comprising SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10;
A primer set comprising SEQ ID NO: 7, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4; or
SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 3 and SEQ ID NO: 4.
3. The method of claim 2,
A primer set for LAMP analysis for detection of Clostridium perfringens, wherein the primer set is selected from the group consisting of:
A primer set comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6;
A primer set comprising SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12;
A primer set comprising SEQ ID NO: 7, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6;
A primer set comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11 and SEQ ID NO: 6;
A primer set comprising SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; or
A primer set comprising SEQ ID NO: 7, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11 and SEQ ID NO:
5. The method according to any one of claims 1 to 4,
A primer set for LAMP analysis for detection of Clostridium perfringens, wherein each set of at least one primer is labeled with a detectable labeling substance.
A method for detecting Clostridium perfringens by the LAMP method in Invitro using a primer set according to any one of claims 1 to 4, the method comprising the steps of:
Providing a sample suspected of having Clostridium perfringens infection or contamination;
Providing a primer set according to any one of claims 1 to 4;
Performing a LAMP reaction using the sample and the primer set; And
Analyzing the reaction product during or after the LAMP reaction and comparing the result to a control sample, if there is a difference, determining the sample as Clostridium perfringens infection or contamination.
The method according to claim 6,
Wherein the sample is an environmental sample comprising a sample, a food material, a food, a feed, a food water, or a drinking water, or a public facility water, cooking utensil, or hospital apparatus including blood, feces, urine or fish.
The method according to claim 6,
Wherein the analysis of the LAMP reaction result is determined by measurement using at least one of a color change measurement of the reaction result or turbidity.
A composition for detection of Clostridium perfringens using a LAMP system comprising a primer set according to any one of claims 1 to 4.
A kit for detecting Clostridium perfringens using a LAMP system comprising a primer set according to any one of claims 1 to 4.

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