CN109355405B - Primer, kit and method for detecting vibrio parahaemolyticus by PSR isothermal amplification reaction - Google Patents
Primer, kit and method for detecting vibrio parahaemolyticus by PSR isothermal amplification reaction Download PDFInfo
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Abstract
The invention discloses a primer, a kit and a method for detecting vibrio parahaemolyticus by PSR isothermal amplification reaction. The invention designs a pair of detection primers Ft/Bt and a pair of accelerating primers IF/IB aiming at a specific target sequence tlh of vibrio parahaemolyticus, the nucleotide sequences of the primers are shown in SEQ ID NO. 1-4, and the primers ensure the reliability of the detection result. The invention also provides a kit for detecting vibrio parahaemolyticus by PSR isothermal amplification reaction, which has the characteristics of high sensitivity, good specificity, simple, convenient and quick operation, accurate and reliable result, low detection cost and suitability for small and medium-sized units and field detection application. The invention utilizes the kit to detect the polymerase helix reaction of the listeria monocytogenes, the method does not cause time loss due to the change of temperature, has short time consumption, does not need gel electrophoresis of the amplification product, and can judge the result by naked eyes after directly developing the color by fluorescent dye.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a primer, a kit and a method for detecting vibrio parahaemolyticus by PSR isothermal amplification reaction.
Background
Vibrio parahaemolyticus is a halophilic bacterium, and is mainly present in offshore seawater, submarine sediments, and marine products such as fish, shrimps, shellfish and oysters. Food poisoning is caused by eating uncooked marine products polluted by vibrio parahaemolyticus, and the bacterial food poisoning has high proportion and great harm. At present, most of the conventional detection methods in China need to be subjected to reaction processes such as selective culture, biochemical identification, serology and the like. The experiments are complicated in operation, the time is required to be 5-6 days (d), the detection rate is low, and difficulties are brought to marine product production, entry and exit inspection and quarantine, food health supervision and the like. Food poisoning caused by vibrio parahaemolyticus is reported every year in China, and the incidence rate is on the rise in recent years. Therefore, it is necessary to establish a rapid, accurate, specific and sensitive detection and diagnosis method for vibrio parahaemolyticus.
At present, the detection methods for vibrio parahaemolyticus mainly comprise a traditional biochemical identification method, a PCR (polymerase chain reaction) method of molecular biotechnology, an enzyme linked immunosorbent assay kit mainly based on immunology, a detection method applying various biochemical automatic identification instruments and a latest gene chip detection means. The gene chip detection and PCR amplification method developed in recent years has strong specificity, rapidness and sensitivity, but has higher cost. The immunological detection methods, such as ELISA, immunochromatography and the like, have high sensitivity, but have complex operation process and high cost, and are not suitable for detecting samples on a large scale. At present, LAMP, which is the most widely applied isothermal amplification technology, has limitations such as complex primer design, high false positive rate and high reagent price. Compared with other nucleic acid amplification technologies, the Polymerase Spiral Reaction (PSR) technology can rapidly, efficiently and specifically amplify a target sequence under an isothermal condition, is simple and convenient to operate, does not need precise temperature-changing equipment, is low in cost, and shows a wide development prospect in the field of food-borne microorganism detection. Therefore, it is of great significance to establish a novel isothermal nucleic acid amplification method for vibrio parahaemolyticus with independent intellectual property rights.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a primer for detecting vibrio parahaemolyticus by PSR isothermal amplification reaction.
Another objective of the invention is to provide a kit for detecting Vibrio parahaemolyticus by PSR isothermal amplification reaction.
Another objective of the invention is to provide a method for detecting Vibrio parahaemolyticus by PSR isothermal amplification reaction. The method has the characteristics of high sensitivity, good specificity, simple, convenient and quick operation, accurate and reliable result, low detection cost and suitability for field detection application.
The purpose of the invention is realized by the following technical scheme: a primer for detecting vibrio parahaemolyticus by PSR isothermal amplification reaction comprises a detection primer Ft and a detection primer Bt, an acceleration primer IF and an acceleration primer IB, and the nucleotide sequences are shown as follows:
5'-CGTGATGTTGTAACCTTGCGTGCGAAAGTGCTTGAGA T-3' (SEQ ID No. 1);
and (3) detecting a primer Bt:5'-GCGTTCCAATGTTGTAGTGCGATGAGCGGTTGATGTC C-3' (SEQ ID NO. 2);
an accelerating primer IF:5'-TGTGCCTTGATGAACTCGT-3' (SEQ ID NO. 3);
accelerating primer IB:5'-CTAACTTCTGCGCCCGA-3' (SEQ ID NO. 4).
The vibrio parahaemolyticus is preferably vibrio parahaemolyticus ATCC17802 or vibrio parahaemolyticus ATCC27969.
A kit for detecting vibrio parahaemolyticus by PSR isothermal amplification reaction, comprises the primer for detecting the vibrio parahaemolyticus by the PSR isothermal amplification reaction.
The concentration of each primer (detection primers Ft and Bt, and accelerated primers IF and IB) in the primers for detecting the vibrio parahaemolyticus in the PSR isothermal amplification reaction is 50 mu M.
The kit for detecting the vibrio parahaemolyticus by the PSR isothermal amplification 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 (each);
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 concentration ratio (molar ratio) of calcein to manganese chloride of the mixed solution of calcein and manganese chloride described in component C is 1:8.
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 primer for detecting the vibrio parahaemolyticus by the PSR isothermal amplification reaction or the kit for detecting the vibrio parahaemolyticus by the PSR isothermal amplification reaction are applied to the detection of the vibrio parahaemolyticus.
A method for detecting vibrio parahaemolyticus by PSR isothermal amplification 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 solution 260 /OD 280 The value is 1.8 to 2.0;
(2) Keeping the temperature in a water bath at 65 ℃ for 45-60 minutes to carry out polymerase helix amplification reaction; wherein, the polymerase spiral amplification reaction system is a 26 μ L reaction system: 2 x reaction buffer 12.5U L, 50U M detection primer Ft and 50U M detection primer Bt each 0.8U L, 50U M accelerating primer IF and IB each 0.8uL, DNA template 2.0U L, 8U/. Mu.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 vibrio parahaemolyticus; if the color is changed to green, the sample to be detected contains the vibrio parahaemolyticus.
The nucleotide sequence of the detection primer Ft in the step (2) is shown as SEQ ID NO.1, the nucleotide sequence of the detection primer Bt is shown as SEQ ID NO.2, the nucleotide sequence of the acceleration primer IF is shown as SEQ ID NO.3, and the nucleotide sequence of the acceleration primer IB is shown as SEQ ID NO. 4.
The time for the polymerase helix amplification reaction in step (2) is preferably 45 minutes.
The mixed solution of calcein and 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 vibrio parahaemolyticus specific target sequence tlh 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 for the specific region of the specific target sequence tlh conserved region of the vibrio parahaemolyticus, so that the reliability of the detection result is ensured. 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 a tlh primer screening experiment; wherein lane M is DNA Marker, lane 1 is Vibrio parahaemolyticus ATCC17802, lane 2 is Vibrio parahaemolyticus ATCC27969, NG is blank control.
FIG. 2 is a graph showing the results of detection of Vibrio parahaemolyticus by the polymerase chain reaction technique; wherein NG is blank control, 1 is Vibrio parahaemolyticus ATCC17802, and 2 is Vibrio parahaemolyticus ATCC27969.
FIG. 3 is a graph showing the effect of primers IF and IB accelerated by tlh primers on the interpretation of the color development result and the electrophoresis result at amplification times of 5min,10min,15min,20min,25min,30min,35min,40min,45min,50min,55min and 60min, respectively; wherein, the graph A is an electrophoresis chart under different reaction time, and lanes 1-12: the reaction time is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60min respectively, and the Lane M is DS 2000Marker; FIG. B shows the results of the color reaction at different reaction times.
FIG. 4 is a graph showing the results of a specificity detection experiment; wherein lane M is a DNA Marker, lane 1: vibrio parahaemolyticus ATCC17802; lane 2: vibrio parahaemolyticus ATCC27969; lane 3: e019 of escherichia coli; lane 4: e020 of Escherichia coli; lane 5: escherichia coli ATCC43894; lane 6: escherichia coli ATCC43895; lane 7: salmonella ATCC29629; lane 8: salmonella ATCC19585; lane 9: salmonella ATCC14028; lane 10: salmonella ATCC13076; lane 11: listeria monocytogenes ATCC19116; lane 12: listeria monocytogenes ATCC15313; lane 13: listeria monocytogenes ATCC19115; lane 14: listeria monocytogenes ATCC19113; lane 15: pseudomonas aeruginosa ATCC27853; lane 16: pseudomonas aeruginosa ATCC10145; lane 17: pseudomonas aeruginosa ATCC9027; lane 18: pseudomonas aeruginosa ATCC15442; lane 19: staphylococcus aureus ATCC23235; lane 20: staphylococcus aureus ATCC6358; lane 21: staphylococcus aureus ATCC12600; lane 22: staphylococcus aureus ATCC27664.
FIG. 5 is a graph showing the results of a sensitivity test; wherein, lane M is DNA Marker, lane 1 is 64 ng/. Mu.L, lane 2 is 6.4 ng/. Mu.L, lane 3 is 640 pg/. Mu.L, lane 4 is 64 pg/. Mu.L, lane 5 is 6.4 pg/. Mu.L, lane 6 is 640 fg/. Mu.L, lane 7 is 64 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 detecting Vibrio parahaemolyticus by polymerase helix reaction
1. Design of primers
Sets of primers as shown in table 1 were designed for tlh target using PrimerPremier software according to PSR amplification reaction principles.
TABLE 1
| Primer name | Sequence (5 '-3') |
| Ft-tlh-1 | ACGCCACGGTTGTAGTTCATTACAAACCAGCAAACACC |
| Bt-tlh-1 | TACTTGATGTTGGCACCGCAGTCCGTCAAACGAATCAG |
| Ft-tlh-2 | AGGTTTGGTTTTCTTGCGTGGAAGAGCCAACCTTATCAC |
| Bt-tlh-2 | GTGCGTTCTTTTGGTTTGGACCACCAGTAGCCGTCAAT |
| Ft-tlh-3 | CGTGATGTTGTAACCTTGCG-TGCGAAAGTGCTTGAGAT |
| Bt-tlh-3 | GCGTTCCAATGTTGTAGTGC-GATGAGCGGTTGATGTCC |
| Accelerated primer name | Sequence (5 '-3') |
| IF-tlh-1 | CCAAACTCAAGCGTAA |
| IB-tlh-1 | AGTGAAAGCGGATTATG |
| IF-tlh-2 | ATCACTTCAGACGCTG |
| IB-tlh-2 | CTATGTTCGCTGTTGG |
| IF-tlh-3 | TGTGCCTTGATGAACTCGT |
| IB-tlh-3 | CTAACTTCTGCGCCCGA |
2. Method for establishing polymerase helix reaction detection
(1) Reaction system
(1) The primer pair combinations in Table 1 were each at a concentration of 50. Mu.M.
(2) 2 × reaction stock solution: the reagent was composed of a mixture of Tris-HCl 40.0mM, ammonium sulfate 20.0mM, potassium chloride 20.0mM, magnesium sulfate 16.0mM, betaine 0.2% (v/v) Tween 20,1.4M, dNTPs (each) 10.0 mM.
(3) Bst DNA Polymerase (Bst DNA Polymerase, large Fragment, available from NEB) in an aqueous solution at a concentration of 8U/. Mu.L;
(2) Detection method
(1) Extracting bacterial DNA of a sample to be detected as template DNA:
the designed primers are screened by taking vibrio parahaemolyticus ATCC17802 and vibrio parahaemolyticus ATCC27969 as research objects. Extracting the bacterial DNA of each group by using a DNA extraction kit (purchased from Guangdong Dongsheng Biotech Co., ltd., product number N1152), and performing the operation according to the kit specification to obtain the OD of the bacterial DNA aqueous solution of the experimental group 260 /OD 280 The value (ratio of absorptivities at 260nm and 280 nm) of (2.0); the blank was prepared with enucleated acid water.
2. O. Parahemolytic Vibrio polymerase helix amplification reaction: aiming at a target point tlh, respectively configuring a polymerase spiral amplification reaction system with the total volume of 25 mu L in a reaction tube; add 2 Xreaction stock solution 12.5. Mu.L, corresponding Ft and Bt equal volume mixed primer mixture 1.6. Mu.L, corresponding accelerated primer IF and IB equal volume mixed 1.6. Mu.L, 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-HCl20.0mM, ammonium sulfate 10.0mM, potassium chloride 10.0mM, magnesium sulfate 8.0mM, tween 20.1% (v/v), betaine 0.7M, dNTPs (each) 1.4mM, bst DNA polymerase 8U, primers Ft, bt each 1.6. Mu.M, and 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.
(3) The product after the end of amplification was subjected to 2% agarose gel electrophoresis. The results are shown in FIG. 1, in which FIG. 1 shows the electrophoretogram of target tlh primer screening, and target tlh corresponds to the first set (Ft-tlh-1, bt-tlh-1, IF-tlh-1 and
IB-tlh-1; tlh 1) and a second set (Ft-tlh-2, bt-tlh-2, IF-tlh-2 and IB-tlh-2; tlh 2), the primers designed by the method have no amplification band, the third set of primer lanes 1 and 2 have amplification bands, and the lane NG added with the enucleated acid water has no band, so that the effect of the primers is optimal. Therefore, a third set of primers (Ft-tlh-3, bt-tlh-3, IF-tlh-3 and IB-tlh-3) are selected as the optimal primers for detecting the tlh target spot. The primers can be used for quickly and accurately detecting two different vibrio parahaemolyticus, and the applicability of the primers is good.
Example 2 microbial method for detecting Vibrio parahaemolyticus (V.parahaemolyticus) ATCC27969 based on polymerase helix reaction isothermal amplification technology
1. The present example takes vibrio parahaemolyticus (v. Parahaemolyticus) ATCC27969 as an example, and uses the following reagents:
a. the detection primer Ft aqueous solution and Bt aqueous solution primer, and the acceleration primer IF aqueous solution and the acceleration primer IB aqueous solution with the concentration of 50 μ M respectively have the following sequences (5 '-3'):
detection primer Ft:
CGTGATGTTGTAACCTTGCGTGCGAAAGTGCTTGAGAT(SEQ ID NO.1)；
and (3) detecting a primer Bt:
GCGTTCCAATGTTGTAGTGCGATGAGCGGTTGATGTCC(SEQ ID NO.2)；
an accelerating primer IF: TGTGCCTTGATGAACTCGT (SEQ ID NO. 3);
accelerating primer IB: CTAACTTCTGCGCCCGA (SEQ ID NO. 4).
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, betaine 0.2% (v/v) Tween 20,1.4M and dNTPs (each) 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 is 1:8).
2. The reagent is used for detecting the vibrio parahaemolyticus by utilizing a polymerase helix reaction amplification technology, and comprises the following steps:
(1) Extracting bacterial DNA of a sample to be detected as template DNA:
in this example, an experimental group and a blank control group were set simultaneously, wherein the experimental group consisted of two strains of Vibrio parahaemolyticus ATCC17802 and ATCC27969 (American type culture Collection); extracting the bacterial DNA of each group by using a DNA extraction kit (Guangdong Dong Sheng Biotech Co., ltd.), and performing the OD (optical density) of the bacterial DNA aqueous solution obtained by the experimental group according to the instruction of the kit 260 /OD 280 The value of (ratio of absorptivities at 260nm and 280 nm) was 2.0.
(2) Polymerase helix amplification reaction of vibrio parahaemolyticus: configuring a polymerase spiral amplification reaction system with the total volume of 26 mul in a reaction tube; adding 12.5 mu L of 2 Xreaction stock solution, 1.6 mu L of mixed primer mixed solution with equal volumes of detection primer Ft and Bt, 1.6uL of corresponding mixed solution with equal volumes of accelerating primer IF and IB, 1 mu L of Bst DNA polymerase and 2.0 mu L of DNA template, supplementing the volume to 25 mu L by deionized water, finally adding 1 mu L of mixed solution of calcein and manganese chloride with the above concentration, and mixing uniformly. The concentrations of the substances are as follows: tris-HCl20.0mM, ammonium sulfate 10.0mM, potassium chloride 10.0mM, magnesium sulfate 8.0mM, tween 20.1% (v/v), betaine 0.7M, dNTPs (each) 1.4mM, bst DNA polymerase 8U, detection primers Ft and Bt each at 1.6. Mu.M, and acceleration primers IF and IB each at 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 vibrio parahaemolyticus genes; the color of the experimental group is changed to green, which indicates that the experimental group contains vibrio parahaemolyticus genes, and then 2% agarose gel electrophoresis is carried out on the amplification products, so that the positive group presents a trapezoidal strip, and the negative group has no amplification strip, which is consistent with the expected result.
EXAMPLE 3 Effect of reaction time on PSR amplification System
The PSR amplification reaction is an efficient and rapid gene detection and analysis means, and a detection result can be obtained within 1h by designing a corresponding accelerating primer. At present, the PSR reaction result judgment method mainly comprises a visual observation method and gel electrophoresis verification. Since gel electrophoresis can only be operated in a laboratory and cannot achieve the purpose of on-site detection, it is desirable to construct a visual reaction system to expand the application range of the PSR amplification technology. Therefore, the experiment determines the optimal time for optimal detection of the reaction through visual color change auxiliary electrophoresis verification. The TLh target of the vibrio parahaemolyticus is used for constructing a PSR amplification reaction system, and the influence of the reaction time on the PSR amplification system at the temperature of 65 ℃ is discussed.
(1) With reference to the procedure of example 1, primers Ft-tlh-3, bt-tlh-3, IF-tlh-3 and IB-tlh-3 were selected to perform PSR amplification reactions of Vibrio parahaemolyticus ATCC17802 and Vibrio parahaemolyticus ATCC27969; wherein the reaction temperature is 65 ℃, and the reaction time is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60min respectively. The product after completion of amplification was subjected to 2% agarose gel electrophoresis, and the result is shown in FIG. 3A.
(2) With reference to the procedure of example 2, PSR amplification reactions were carried out on Vibrio parahaemolyticus ATCC17802 and Vibrio parahaemolyticus ATCC27969; wherein the reaction temperature is 65 ℃, and the reaction time is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60min respectively. After the reaction was completed, the color change was observed with the naked eye, and the color development result is shown in FIG. 3B.
(3) And (4) analyzing results: as is clear from FIGS. 3A and 3B, when the PSR amplification was carried out for a period of time of 40min or more, a significant color change (gradual change of color to green) occurred, and the reaction result was positive. The amplification product is verified by 2% agarose electrophoresis results, and a ladder-shaped band can be generated after 35min, and the result is positive. Thus, the PSR amplification reaction system established by designing the corresponding accelerating primers IF and IB can obtain the interpretation of the reaction result within 60min. However, the electrophoresis result is used for judging whether the reaction is carried out or not, so that whether the strain to be detected is contained or not takes about 50min, and meanwhile, the electrophoresis result cannot be used in field detection, which is not in accordance with the purpose of establishing a rapid and convenient reaction system. Therefore, the established PSR amplification reaction system developed judges whether or not the reaction has proceeded by a color change. The optimal PSR reaction amplification time is determined to be 45min by designing a corresponding accelerating primer and adding a mixed solution of calcein and manganese chloride.
Example 4 polymerase helix reaction assay for detecting Vibrio parahaemolyticus specificity
The specific test was carried out by reacting the genomic DNA of Vibrio parahaemolyticus (Vibrio parahaemolyticus ATCC17802, vibrio parahaemolyticus ATCC 27969) with the genomic DNA of other strains (E.coli E019; E.coli E020; E.coli ATCC43894; E.coli ATCC43895; salmonella ATCC29629; salmonella ATCC19585; salmonella ATCC14028; salmonella ATCC13076; listeria monocytogenes ATCC19116; listeria monocytogenes ATCC15313; listeria monocytogenes ATCC19115; listeria monocytogenes ATCC19113; pseudomonas aeruginosa ATCC27853; pseudomonas aeruginosa ATCC10145; pseudomonas aeruginosa ATCC9027; pseudomonas aeruginosa ATCC15442; staphylococcus aureus ATCC 35; staphylococcus aureus ATCC6358; staphylococcus aureus ATCC12600; staphylococcus aureus ATCC 27664) in the reaction system of example 1 (primers: ft-tlh-3, bt-tlh-3, IF-TLh-3 and TLH-3) and detecting conditions for detection of Escherichia coli IB. The genome containing Vibrio parahaemolyticus was set as a positive control, and ultrapure water was set as a negative control (the negative control showed the same result as the blank control in FIG. 1). Wherein, the escherichia coli E019 and the escherichia coli E020 can be obtained by reference documents (cistanche. Low-temperature storage influences on the induction of the VBNC state of the enterohemorrhagic escherichia coli and toxin expression amount research [ D ]. Southern China university 2015.).
The results are shown in FIG. 4. The result shows that only the genome containing the vibrio parahaemolyticus has positive reaction, and the rest has negative reaction.
Example 5 sensitivity test for detecting Vibrio parahaemolyticus by PSR
The genome of Vibrio parahaemolyticus ATCC27969 was diluted with 10-fold concentration gradient of 64 ng/. Mu.L, 6.4 ng/. Mu.L, 640 pg/. Mu.L, 64 pg/. Mu.L, 6.4 pg/. Mu.L, 640 fg/. Mu.L, and 64 fg/. Mu.L, respectively, while negative control (deionized water) was set, and polymerase helix reaction amplification methods (primers: ft-tlh-3, bt-tlh-3, IF-tlh-3, and IB-tlh-3) were constructed according to the reaction system described in example 1 above to determine the sensitivity of the detection method, and the results are shown in FIG. 5. The results show that: the established vibrio parahaemolyticus polymerase spiral reaction method can detect the vibrio parahaemolyticus DNA of 6.4 pg/mu L in the 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 the conventional PCR, the fluorescence 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 (6)
1. A kit for detecting vibrio parahaemolyticus based on polymerase helix reaction is characterized in that: the kit comprises a primer pair for detecting vibrio parahaemolyticus, 2 multiplied reaction buffer solution,BstDNA polymerase, and a mixed solution prepared from calcein and manganese chloride; wherein,
the primer pair for detecting the vibrio parahaemolyticus consists of a detection primer Ft, a detection primer Bt, an acceleration primer IF and an acceleration primer IB, and the nucleotide sequence of the primer pair is as follows:
5'-CGTGATGTTGTAACCTTGCGTGCGAAAGTGCTTGAGAT-3' as detection primer Ft;
and (3) detecting a primer Bt:5'-GCGTTCCAATGTTGTAGTGCGATGAGCGGTTGATGTCC-3';
an accelerating primer IF:5'-TGTGCCTTGATGAACTCGT-3';
accelerating primer IB:5'-CTAACTTCTGCGCCCGA-3';
the 2 × reaction buffer consists of the following components: 40.0 Tris-HCl in mM, ammonium sulfate in 20.0mM, potassium chloride in 20.0mM, magnesium sulfate in 16.0mM, tween 20 in 0.2% volume, betaine in 1.4M, each dNTP concentration is 10.0 mM;
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 manganese chloride aqueous solution of 1 mM;
(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.
2. The kit of claim 1, wherein: the concentration of each primer in the primer pair was 50. Mu.M.
3. The kit of claim 1, wherein: the above-mentionedBstThe 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 Vibrio parahaemolyticus, characterized in that: the application is for non-diagnostic purposes.
5. A method for detecting Vibrio parahaemolyticus based on polymerase chain reaction for non-disease diagnostic purposes, comprising the steps of:
(1) Is extracted and treatedDetecting bacterial DNA of the sample as template DNA, and controlling OD of the template DNA aqueous solution 260 /OD 280 The value is 1.8 to 2.0;
(2) Keeping the temperature in a water bath at 65 ℃ for 45-60 minutes to carry out polymerase helix amplification reaction; wherein, the polymerase helix amplification reaction system is 26 μ L, and the composition of the reaction system is as follows: 2.5. Mu.L of reaction buffer, 0.8. Mu.L each of 50. Mu.M detection primer Ft and 50. Mu.M detection primer Bt, 0.8. Mu.L each of 50. Mu.M acceleration primer IF and IB, 2.0. Mu.L of DNA template, 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 then observing the color change by naked eyes, wherein if the color is yellow, the sample to be detected does not contain vibrio parahaemolyticus; if the color is changed to green, the sample to be detected contains vibrio parahaemolyticus;
the nucleotide sequence of the detection primer Ft in the step (2) is shown as SEQ ID NO.1, the nucleotide sequence of the detection primer Bt is shown as SEQ ID NO.2, the nucleotide sequence of the acceleration primer IF is shown as SEQ ID NO.3, and the nucleotide sequence of the acceleration primer IB is shown as SEQ ID NO. 4.
6. The method of claim 5, wherein: the time of the polymerase helix amplification reaction in step (2) is 45 minutes.
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