CN109355404B - Primer, kit and detection method for isothermal detection of listeria monocytogenes based on polymerase helix reaction - Google Patents
Primer, kit and detection method for isothermal detection of listeria monocytogenes based on polymerase helix reaction Download PDFInfo
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Abstract
The invention discloses a primer, a kit and a detection method for constant-temperature detection of Listeria monocytogenes based on polymerase helix reaction. The primer comprises detection primers Ft and Bt; wherein the nucleotide sequence of the detection primer Ft is shown as SEQ ID NO. 1; the nucleotide sequence of the detection primer Bt is shown as SEQ ID NO. 2. The invention also provides a kit for detecting listeria monocytogenes at constant temperature based on polymerase helix reaction, which comprises the primer, Bst DNA polymerase, a mixed solution of calcein and manganese chloride and the like. The primer or the kit can be used for detecting the polymerase helix reaction of the listeria monocytogenes, can not cause time loss due to temperature change, has short time consumption, simple and convenient reaction process, short detection period and strong specificity, and can observe the detection result by naked eyes. The invention has important significance for the development of amplification of a novel constant-temperature amplification technology and the field detection of microorganisms.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a primer, a kit and a detection method for detecting listeria monocytogenes at constant temperature based on polymerase helix reaction.
Background
Listeria is a facultative anaerobic bacterium that is found primarily in soil where dead and decaying organisms are consumed. Of these, only Listeria monocytogenes (Listeria monocytogenes) and Listeria ivalis (Listeria ivanovovii) are pathogenic, while Listeria monocytogenes is the only zoonotic pathogen in this group. The listeria monocytogenes is widely existed in food, has very high detection rate in meat, poultry and fresh seafood, and has average lethality reaching 30 percent which is far more than other common food-borne pathogenic bacteria, such as salmonella enteritidis (0.38 percent), campylobacter (0.02 to 0.1 percent) and vibrio cholerae (0.005 to 0.01 percent). At present, most of the conventional detection methods in China need to be subjected to enrichment of a liquid selective culture medium and separation of a solid selective culture medium, and finally, the processes of biochemical identification, serology and the like are carried out on the strains. The experiments are complicated in operation, long in time consumption and low in detection rate, the food processing factory, the entry and exit inspection and quarantine and the food health supervision efficiency are greatly reduced, the sensory properties of fresh foods are influenced, and great loss is caused to the whole food industry. Therefore, it is necessary to establish a rapid, accurate, specific and sensitive detection and diagnosis method for listeria monocytogenes.
The current detection method for listeria monocytogenes has some emerging rapid nucleic acid detection methods and immunological detection, such as Polymerase Chain Reaction (PCR), real-time fluorescent quantitative PCR, isothermal amplification (LAMP) and Elisa enzyme-linked immunoassay kit, in addition to the traditional biochemical identification method. Although the rapid detection methods have the characteristics of high specificity, short time, high sensitivity and the like, the detection cost is high, the immunological detection needs to prepare expensive monoclonal antibodies and has high requirements on samples, nucleic acid detection based on polymerase chain reaction needs expensive constant temperature equipment, and even the constant temperature amplification technology LAMP which is most widely applied at present gradually exposes many limitations, including the problems of complicated primer design, high false positive and the like. Although these problems can be avoided to some extent, in order for rapid detection techniques to be able to gain a wider range of popularity, the above-mentioned limitations must be overcome. The Polymerase Spiral Reaction (PSR) technology is a novel isothermal amplification technology, can rapidly, efficiently and specifically amplify a target sequence under an isothermal condition, only needs a pair of primers, does not need precise temperature-changing equipment, and is simple to operate, so that the cost is low, the PCR amplification technology is very suitable for field safety detection and field care test in various factories and mechanisms, and has a very wide development prospect in the field of food-borne microorganism detection. Therefore, the establishment of an isothermal nucleic acid amplification method aiming at the listeria monocytogenes with independent intellectual property rights is of great significance.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a primer for isothermal detection of listeria monocytogenes based on polymerase helix reaction.
The invention also aims to provide a kit for isothermal detection of listeria monocytogenes based on polymerase helix reaction.
The invention also aims to provide a method for isothermal detection of listeria monocytogenes based on polymerase helix reaction.
The purpose of the invention is realized by the following technical scheme: a primer for isothermal detection of Listeria monocytogenes based on polymerase helix reaction comprises a detection primer Ft and a detection primer Bt, and the nucleotide sequence of the primer is as follows:
5'-ACACCAGGAGTTCCCATTGCAACCTCGGAGACTTA-3' (SEQ ID NO. 1);
and (3) detecting a primer Bt: 5'-TTACCCTTGAGGACCACAGTAGCCTCCAGAGTGAT-3' (SEQ ID NO. 2).
The listeria monocytogenes is preferably listeria monocytogenes ATCC19115, listeria monocytogenes ATCC19116, listeria monocytogenes 15313, listeria monocytogenes ATCC19114 or listeria monocytogenes ATCC 19113.
A kit for isothermal detection of Listeria monocytogenes based on polymerase helix reaction comprises the primer for isothermal detection of Listeria monocytogenes based on polymerase helix reaction.
The concentration of the detection primers Ft and Bt in the primer for isothermal detection of Listeria monocytogenes based on polymerase helix reaction is 50 mu M.
The kit for isothermal detection of listeria monocytogenes based on polymerase helix reaction further comprises the following components:
A. 2 × reaction buffer: 40.0mM Tris-HCl, 20.0mM ammonium sulfate, 20.0mM potassium chloride, 16.0mM magnesium sulfate, 0.2% (v/v) Tween 20, 1.4M betaine, 10.0mM dNTPs (reach);
B. bst DNA polymerase;
C. a mixed solution of calcein and manganese chloride.
The Bst DNA polymerase described in component B is preferably an aqueous solution of Bst DNA polymerase at a concentration of 8U/. mu.L.
The mixed solution of the calcein and the manganese chloride in the component C is prepared by the following method:
(i) dissolving calcein in dimethyl sulfoxide (DMSO) to obtain 50 μ M calcein solution; dissolving manganese chloride in water to prepare a 1mM manganese chloride aqueous solution;
(ii) 25 mu L of 50 mu M calcein solution and 10 mu L of 1mM manganese chloride aqueous solution are uniformly mixed to obtain a mixed solution of calcein and manganese chloride (the concentration ratio of the calcein solution to the manganese chloride solution is 1: 8).
The primer for isothermal detection of listeria monocytogenes based on polymerase chain reaction or the kit for isothermal detection of listeria monocytogenes based on polymerase chain reaction are applied to detection of listeria monocytogenes.
A method for isothermal detection of Listeria monocytogenes based on polymerase helix reaction comprises the following steps:
(1) extracting bacterial DNA of a sample to be detected as template DNA, and controlling OD of template DNA aqueous solution260/OD280The value is 1.8 to 2.0;
(2) keeping the temperature in a water bath at 65 ℃ for 60 minutes to carry out polymerase helix amplification reaction; wherein, the polymerase spiral amplification reaction system is a 26 μ L reaction system: 2 times reaction buffer solution 12.5U L, 50U M detection primer Ft and 50U M detection primer Bt each 0.8U L, DNA template 2.0U L, 8U/U L Bst DNA polymerase 1.0U L, deionized water make up to 25U L; finally, adding 1 mu L of mixed solution of calcein and manganese chloride;
(3) after the reaction is finished, preserving the heat in a water bath at 80 ℃ for 2 minutes to terminate the reaction, and then observing the color change by naked eyes, wherein if the color is yellow, the sample to be detected does not contain Listeria monocytogenes; if the color is changed to green, the sample to be detected contains the listeria monocytogenes.
The nucleotide sequence of the detection primer Ft in the step (2) is shown as SEQ ID NO.1, and the nucleotide sequence of the detection primer Bt is shown as SEQ ID NO. 2.
The mixed solution of the calcein and the manganese chloride in the step (2) is prepared by the following method:
(i) dissolving calcein in dimethyl sulfoxide (DMSO) to obtain 50 μ M calcein solution; dissolving manganese chloride in water to prepare a 1mM manganese chloride aqueous solution;
(ii) 25 mu L of 50 mu M calcein solution and 10 mu L of 1mM manganese chloride aqueous solution are uniformly mixed to obtain a mixed solution of calcein and manganese chloride (the concentration ratio of the calcein solution to the manganese chloride solution is 1: 8).
Compared with the prior art, the invention has the following advantages and effects:
(1) the polymerase helix detection and identification system designed aiming at the specific target sequence hylA of the Listeria monocytogenes provided by the invention solves the defects of long required period, low sensitivity, high cost, difficult field application and the like of the method in the prior art.
(2) The method can reduce the detection time to 60 minutes, and compared with the traditional loop-mediated isothermal amplification technology, the method can quickly break the detection period, and has important significance for the development of amplification of a novel isothermal amplification technology and the field detection of microorganisms. Meanwhile, the invention also discloses a pair of detection primers designed aiming at the specific region of the specific target sequence hylA conserved region of the listeria monocytogenes, thereby ensuring the reliability of the detection result. Secondly, the method can amplify under the isothermal condition, cannot cause time loss due to temperature change, is short in time consumption, and can finish result interpretation within 60 minutes. In addition, the technology does not need special and expensive instruments and reagents, the amplification product does not need gel electrophoresis, the result can be judged by naked eyes by directly using fluorescent dye for color development, the operation is simple, convenient and quick, and the detection cost is lower. The kit and the method are particularly suitable for small and medium-sized units and field detection.
Drawings
FIG. 1 is a diagram showing the results of electrophoresis in an hlyA primer screening experiment; wherein, the graph A is the amplification result of the first set of primers (hlyA-1) and the second set of designed primers (hlyA-2), and the graph B is the amplification result of the third set of primers (hlyA-3); in the figure, lane M is DNA Marker, lane 1 is listeria monocytogenes ATCC19115, lane 2 is listeria monocytogenes ATCC19116, lane 3 is listeria monocytogenes ATCC15313, lane 4 is listeria monocytogenes ATCC19114, lane 5 is listeria monocytogenes ATCC19113, NG is blank control.
FIG. 2 is a diagram showing the results of detecting Listeria monocytogenes by polymerase helix reaction; wherein, 1 is Listeria monocytogenes ATCC19115, 2 is Listeria monocytogenes ATCC19116, 3 is Listeria monocytogenes ATCC15313, 4 is Listeria monocytogenes ATCC19114, 5 is Listeria monocytogenes ATCC19113, and NG is blank control.
FIG. 3 is a graph showing the results of a specificity detection experiment; wherein, lane M: DNA Marker, lane 1: listeria monocytogenes ATCC 19115; lane 2: listeria monocytogenes ATCC 19116; lane 3: listeria monocytogenes ATCC 15313; lane 4: listeria monocytogenes ATCC 19114; lane 5: listeria monocytogenes ATCC 19113; lane 6: salmonella ATCC 29629; lane 7: salmonella ATCC 19585; lane 8: salmonella ATCC 14028; lane 9: salmonella ATCC 13076; lane 10: e019 of escherichia coli; lane 11: e020 of Escherichia coli; lane 12: e043 of Escherichia coli; lane 13: escherichia coli ATCC 43894; lane 14: escherichia coli ATCC 43895; lane 15: pseudomonas aeruginosa ATCC 27853; lane 16: pseudomonas aeruginosa ATCC 10145; lane 17: pseudomonas aeruginosa ATCC 9027; lane 18: pseudomonas aeruginosa ATCC 15442; lane 19: pseudomonas aeruginosa ATCC 19429; lane 20: staphylococcus aureus ATCC 23235; lane 21: staphylococcus aureus ATCC 6358; lane 22: staphylococcus aureus ATCC 12600; lane 23: staphylococcus aureus ATCC 27664; lane 24: staphylococcus aureus ATCC 13565; lane 25: vibrio parahaemolyticus ATCC 17802; lane 26: vibrio parahaemolyticus ATCC 27969.
FIG. 4 is an electrophoretogram of a sensitivity detection experiment; wherein lane M is DNA Marker, lane 1 is 41 ng/. mu.L, lane 2 is 4.1 ng/. mu.L, lane 3 is 410 pg/. mu.L, lane 4 is 41 pg/. mu.L, lane 5 is 4.1 pg/. mu.L, lane 6 is 410 fg/. mu.L, lane 7 is 41 fg/. mu.L, and lane 8 is a negative control.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise specified, reagents, materials and strains used in the present invention are commercially available.
Example 1 primer screening for polymerase helix reaction detection of Listeria monocytogenes
1. Design of primers
Sets of primers as shown in table 1 were designed for hlyA target using PrimerPremier software according to PSR amplification reaction principles.
TABLE 1
| Primer name | Sequence (5 '-3') |
| Ft-hlyA-1 | CCATTCCCAAGCTAAACCGCCAGGTAACGCAAGAAA |
| Bt-hlyA-1 | CCAAATCGAACCCTTACCCGGATAAAGCGTAGTGCC |
| Ft-hlyA-2 | TTACGGCTTTGAAGGAAGATTTGACGCTGCCGTAAGTG |
| Bt-hlyA-2 | AGAAGGAAGTTTCGGCATTGGTTACCGTCGATGATTTG |
| Ft-hlyA-3 | ACACCAGGAGTTCCCATTGCAACCTCGGAGACTTA |
| Bt-hlyA-3 | TTACCCTTGAGGACCACAGTAGCCTCCAGAGTGAT |
| Accelerated primer name | Sequence (5 '-3') |
| IF-hlyA-1 | CATTCTTTGGCGTAAAC |
| IB-hlyA-1 | ATGACCGGAACTTACCA |
| IF-hlyA-2 | GTTCTACATCACCTGAG |
| IB-hlyA-2 | CTCCGCAAAAGATGAAG |
2. Method for establishing polymerase helix reaction detection
(1) Reaction system
Combinations of primer pairs at concentrations of 50. mu.M each in Table 1.
2 × reaction stock solution: consists of a mixture of Tris-HCl 40.0mM, ammonium sulfate 20.0mM, potassium chloride 20.0mM, magnesium sulfate 16.0mM, Tween 20 0.2% (v/v), betaine 1.4M, dNTPs (research) 10.0 mM.
③ an aqueous solution of Bst DNA Polymerase (Bst DNA Polymerase, Large Fragment, available from NEB) at a concentration of 8U/. mu.L;
(2) detection method
Firstly, extracting bacterial DNA of a sample to be detected as template DNA: the primers designed by the listeria monocytogenes ATCC19115, ATCC19116, ATCC15313, ATCC19114 and ATCC19113 are selected as the study objects. Extracting the bacterial DNA of each group by using a DNA extraction kit (purchased from Guangdong Sheng Biotech Co., Ltd., product number N1152), and performing operation according to the kit specification to obtain the OD of the bacterial DNA aqueous solution of the experimental group260/OD280The value of (ratio of absorbance at 260nm to 280 nm) was 1.8; the blank was prepared with enucleated acid water.
Polymerase helical amplification reaction of listeria monocytogenes: aiming at a target point hlyA, respectively configuring a polymerase spiral amplification reaction system with the total volume of 25 mu L in a reaction tube; add 2X reaction stock 12.5. mu.L, corresponding Ft and Bt (Ft-hlyA-1 and Bt-hlyA-1, Ft-hlyA-2 and Bt-hlyA-2, or Ft-hlyA-3 and Bt-hlyA-3) equal volume of mixed primer mix 1.6. mu.L (IF accelerating primer, equal volume of mix 1.6uL corresponding to accelerating primer IF and IB), Bst DNA polymerase 1. mu.L, DNA template 2.0. mu.L, make up volume to 25. mu.L with deionized water. The concentrations of the substances are as follows: Tris-HCl 20.0mM, ammonium sulfate 10.0mM, potassium chloride 10.0mM, magnesium sulfate 8.0mM, Tween 200.1% (v/v), betaine 0.7M, dNTPs (each)1.4mM, Bst DNA polymerase 8U, primers Ft, Bt each 1.6. mu.M (IF accelerating primers, accelerating primers IF, IB each 1.6. mu.M). The reaction tube is placed in a water bath at 65 ℃ for heat preservation reaction for 60 minutes, and then is placed in a water bath at 80 ℃ for heat preservation for 2 minutes to terminate the reaction.
And thirdly, carrying out 2% agarose gel electrophoresis on the product after the amplification is finished. The results are shown in FIG. 1, which is an electrophoretogram of target hlyA primer screening, wherein the target hlyA corresponds to that no amplification band appears in only the fifth lane of the first set of primers (Ft-hlyA-1 and Bt-hlyA-1; hlyA-1), and only the third lane of the second set of primers (Ft-hlyA-2 and Bt-hlyA-2; hlyA-2). The third set of primers (Ft-hlyA-3 and Bt-hlyA-3; hlyA-3)5 lanes are designed to have amplification bands, and no band appears in the lane NG added with enucleated acid water, which indicates that the effect of the primers is optimal. Therefore, the third set of primers is selected as the optimal primer for detecting the hlyA target. The primer disclosed by the invention can be used for quickly and accurately detecting five different Listeria monocytogenes, so that the primer is good in applicability.
Example 2 microbial method for detecting Listeria monocytogenes (L.monocytogenes) ATCC19113 based on polymerase helix reaction isothermal amplification technology
1. The present example takes listeria monocytogenes (l.monocytogenes) ATCC19113 as an example, and uses the following reagents:
a. the primer sequences of the detection primer Ft aqueous solution and the Bt aqueous solution at concentrations of 50. mu.M each were as follows (5 '-3'):
ACACCAGGAGTTCCCATTGCAACCTCGGAGACTTA (SEQ ID NO. 1);
and (3) detecting a primer Bt: TTACCCTTGAGGACCACAGTAGCCTCCAGAGTGAT (SEQ ID NO. 2);
b.2 × reaction stock: consists of a mixture of Tris-HCl 40.0mM, ammonium sulfate 20.0mM, potassium chloride 20.0mM, magnesium sulfate 16.0mM, Tween 20 0.2% (v/v), betaine 1.4M, dNTPs (research) 10.0 mM;
c. bst DNA polymerase (Large fragment, NEB) in water at a concentration of 8U/. mu.l;
d. mixed solution of calcein and manganese chloride: firstly, preparing a calcein solution (dissolved by dimethyl sulfoxide) with the concentration of 50 mu M; then, 25. mu.L of 50. mu.M calcein solution was mixed with 10. mu.L of 1mM manganese chloride aqueous solution (concentration ratio of calcein solution to manganese chloride solution was 1: 8).
2. The detection of the listeria monocytogenes by using the reagent and utilizing a polymerase helix reaction amplification technology comprises the following steps:
(1) extracting bacterial DNA of a sample to be detected as template DNA:
this example simultaneously set up an experimental group and a blank control group, wherein the experimental group was five strains of listeria monocytogenes ATCC19115 (american type culture collection), ATCC19116, ATCC15313, ATCC19114, ATCC 19113; extracting the bacterial DNA of each group by using a DNA extraction kit (Guangdong Sheng Biotech Co., Ltd.), and performing the operation according to the kit specification to obtain the OD of the bacterial DNA aqueous solution of the experimental group260/OD280The value of (absorbance ratio at 260nm and 280 nm) was 1.8.
(2) Polymerase helix amplification reaction of listeria monocytogenes: configuring a polymerase spiral amplification reaction system with the total volume of 26 mu L in a reaction tube; adding 12.5 mu L of 2 multiplied reaction stock solution, 1.6 mu L of mixed primer mixed solution with equal volume of detection primer Ft and Bt, 1 mu L of Bst DNA polymerase and 2.0 mu L of DNA template, and supplementing the volume to 25 mu L by deionized water; and finally, adding 1 mu L of the mixed solution of the calcein and the manganese chloride with the concentration, and uniformly mixing. The concentrations of the substances are as follows: Tris-HCl 20.0mM, ammonium sulfate 10.0mM, potassium chloride 10.0mM, magnesium sulfate 8.0mM, Tween 200.1% (v/v), betaine 0.7M, dNTPs (ideal) 1.4mM, Bst DNA polymerase 8U, and detection primers Ft and Bt each 1.6. mu.M. The reaction tube is placed in a water bath at 65 ℃ for heat preservation reaction for 60 minutes, and then is placed in a water bath at 80 ℃ for heat preservation for 2 minutes to terminate the reaction.
(3) And (3) color development detection: after the reaction was completed, the color change was observed with the naked eye
The results are shown in FIG. 2, which shows: the color of the blank control group is yellow, which indicates that the detection strain does not contain the listeria monocytogenes gene; the colors of the experimental groups are changed into green, which indicates that the genes contain the listeria monocytogenes, and then 2 percent of agarose gel electrophoresis is carried out on the amplification products, the positive group presents a trapezoidal strip, and the negative group has no amplification strip, which is consistent with the expected result.
Example 3 polymerase helix reaction assay for detecting Listeria monocytogenes specificity
The specific test was carried out by establishing a helical reaction between Listeria monocytogenes (ATCC19115, ATCC19116, ATCC15313, ATCC19114, ATCC19113) and the genomic DNA of other strains (Salmonella ATCC 29629; Salmonella ATCC 19585; Salmonella ATCC 14028; Salmonella ATCC 13076; Escherichia coli E019; Escherichia coli E020; Escherichia coli E043; Escherichia coli ATCC 43894; Escherichia coli ATCC 43895; Pseudomonas aeruginosa ATCC 27853; Pseudomonas aeruginosa ATCC 10145; Pseudomonas aeruginosa ATCC 9027; Pseudomonas aeruginosa ATCC 15442; Pseudomonas aeruginosa ATCC 19429; Staphylococcus aureus ATCC 23235; Staphylococcus aureus ATCC 6358; Staphylococcus aureus ATCC 12600; Staphylococcus aureus ATCC 27664; Staphylococcus aureus ATCC 13565; Vibrio parahemolyticus ATCC 17802; Vibrio parahemolyticus ATCC27969) in accordance with the reaction system of example 1 (primers Ft-hl-yA-3 and Bt-yA-3, i.e.detection primers Ft and Btl) and conditions. The listeria monocytogenes-containing genome was set as a positive control and ultrapure water was set as a negative control (the negative control gave the same results as the blank control in fig. 1). Wherein, the Escherichia coli E019, Escherichia coli E020 and Escherichia coli E043 can be obtained by reference documents (cistanche deserticola low-temperature storage researches on the induction of the VBNC state of the Escherichia coli with intestinal hemorrhage and the influence of toxin expression amount [ D ]. southern China university 2015.).
The results are shown in FIG. 3. The results show that only the genome containing the listeria monocytogenes has positive reactions, and the rest of the genome has negative reactions.
Example 4 susceptibility assay for PSR detection of Listeria monocytogenes
The genome of Listeria monocytogenes was diluted with 10-fold concentration gradient to 41 ng/. mu.L, 4.1 ng/. mu.L, 410 pg/. mu.L, 41 pg/. mu.L, 4.1 pg/. mu.L, 410 fg/. mu.L, 41 fg/. mu.L, while negative control (deionized water) was set, and the PCR amplification method was constructed according to the reaction system described in example 1 above (primers Ft-hlyA-3 and Bt-hlyA-3 in the reaction system, i.e., primers Ft and Bt), to determine the sensitivity of the detection method.
The results are shown in FIG. 4. The results show that: the established Listeria monocytogenes polymerase helix reaction method can detect the Listeria monocytogenes DNA of 41 pg/mu L in a sample.
And (4) conclusion: as can be seen from the above experimental results, the PCR amplification method has the following advantages compared with the conventional PCR and the fluorescence PCR:
the operation and the identification are simple and quick: the result can be obtained in 2-4 hours in the whole process of conventional PCR, the fluorescent quantitative PCR needs 1-1.5 hours, and the detection method provided by the invention can obtain a positive result in 60 minutes. And secondly, the requirement on the instrument is low, only one common water bath is needed, and the detection result can be directly observed through fluorescent dye, so that the traditional electrophoresis detection step is omitted. Has wide application prospect in the practice of rapid detection and field detection.
The specificity is strong: the presence or absence of the target gene can be judged only by whether amplification has occurred, thereby completing qualitative detection of the bacteria.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
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Claims (5)
1. A kit for isothermal detection of Listeria monocytogenes based on polymerase helix reaction is characterized in that: the kit comprises a primer for detecting the listeria monocytogenes, 2X reaction buffer solution,BstDNA polymerase, and a mixed solution prepared from calcein and manganese chloride; wherein the content of the first and second substances,
the primer for detecting the listeria monocytogenes consists of a detection primer Ft and a detection primer Bt, and the nucleotide sequence of the primer is as follows:
5'-ACACCAGGAGTTCCCATTGCAACCTCGGAGACTTA-3' is used for detecting the primer Ft;
and (3) detecting a primer Bt: 5'-TTACCCTTGAGGACCACAGTAGCCTCCAGAGTGAT-3', respectively;
the 2 × reaction buffer consists of the following components: 40.0mM Tris-HCl, 20.0mM ammonium sulfate, 20.0mM potassium chloride, 16.0mM magnesium sulfate, 0.2% Tween 20 by volume, 1.4M betaine, 10.0mM dNTPs;
the mixed solution is prepared by the following method:
i) dissolving calcein in dimethyl sulfoxide to obtain 50 μ M calcein solution; dissolving manganese chloride in water to prepare a 1mM manganese chloride aqueous solution;
ii) taking 25 mu L of 50 mu M calcein solution and mixing with 10 mu L of 1mM manganese chloride aqueous solution uniformly to obtain a mixed solution of calcein and manganese chloride.
2. The kit of claim 1, wherein: the concentration of the detection primers Ft and Bt in the primer for isothermal detection of Listeria monocytogenes based on polymerase helix reaction is 50 mu M.
3. The kit of claim 1, wherein: saidBstThe concentration of DNA polymerase was 8U/. mu.L.
4. Use of the kit of any one of claims 1 to 3 for the detection of listeria monocytogenes for non-disease diagnostic purposes.
5. A method for isothermal detection of Listeria monocytogenes based on polymerase helix reaction for non-disease diagnostic purposes, comprising the steps of:
1) extracting bacterial DNA of bacterial sample as template DNA, and controlling OD of template DNA aqueous solution260/OD280The value is 1.8 to 2.0;
2) keeping the temperature in a water bath at 65 ℃ for 60 minutes to carry out polymerase helix amplification reaction; wherein, the polymerase helix amplification reaction system is as follows: 2 Xreaction buffer 12.5. mu.L, 50. mu.M detection primer Ft and 50. mu.M detection primer Bt each 0.8. mu.L, DNA template 2.0. mu.l, 8U/. mu.LBstDNA polymerase 1.0 μ L, deionized water make up to 25 μ L; finally, adding 1 mu L of mixed solution of calcein and manganese chloride;
3) after the reaction is finished, preserving the heat in a water bath at 80 ℃ for 2 minutes to terminate the reaction, and observing the color change by naked eyes, wherein if the color is yellow, the bacteria sample does not contain the Listeria monocytogenes; if the color is changed to green, the bacterial sample contains the listeria monocytogenes;
the nucleotide sequence of the detection primer Ft in the step 2) is shown as SEQ ID NO.1, and the nucleotide sequence of the detection primer Bt is shown as SEQ ID NO. 2.
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