CN113584197A - PSR primer, detection kit and detection method for detecting enterotoxin SEA - Google Patents
PSR primer, detection kit and detection method for detecting enterotoxin SEA Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses a PSR primer, a detection kit and a detection method for detecting enterotoxin SEA, wherein the primer comprises a detection primer Ft and a detection primer Bt, and the nucleotide sequences of the primers are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2. The PSR detection primer and the method designed aiming at the enterotoxin SEA solve the problems of long required period, low sensitivity, high cost, difficult field application and the like of the prior art. A pair of detection primers Ft and Bt are designed to construct a PSR reaction system by selecting a conserved region of the SEA enterotoxin specific sequence of the target strain, and a detection result is obtained in about 60 minutes, so that the detection period is greatly shortened compared with the traditional MRSA enterotoxin detection method.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to a PSR primer, a detection kit and a detection method for detecting enterotoxin SEA.
Background
Staphylococcus aureus is a common infectious bacterium in hospitals and communities, and in recent years, with the wide use of antibiotics, the separation rate of methicillin-resistant staphylococcus aureus (MRSA) is increasing. Staphylococcus aureus under certain conditions can secrete an enterotoxin SEs. Enterotoxins of the type SEA, SEB, SEC, SED, SEE, etc. have been found. Such enterotoxins can cause food poisoning, causing vomiting symptoms. Several studies have shown a higher detection rate of SEA enterotoxin in MRSA. Therefore, the SEA enterotoxin detection technology of MRSA in food becomes the key for preventing and controlling food-borne diseases. Therefore, it is very important to develop an SEA toxin kit and a method for rapidly detecting MRSA with high efficiency and sensitivity.
At present, the detection and identification methods of microorganisms are mainly divided into the traditional culture method, the immunological detection method and the molecular biological technology. The traditional culture method has the disadvantages of complicated operation steps, low detection sensitivity and long experimental period. The immunological detection method is widely applied to the detection of the enterotoxin SEA, but has high requirements on the professional level of experimenters, higher cost and lower accuracy. Fluorescence quantitative PCR in molecular biology is also used for detecting the enterotoxin SEA, but the defects of high requirement on operator level and high cost exist.
Disclosure of Invention
The invention aims to provide a PSR primer, a detection kit and a detection method for detecting enterotoxin SEA, which improve the sensitivity (up to 630 pg/mu L) by optimizing the primer, and have the characteristics of simple and convenient operation, good specificity, 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 PSR primer for detecting enterotoxin SEA comprises a detection primer Ft and a detection primer Bt, and the nucleotide sequences are respectively shown as follows:
detection primer Ft: 5'-gtctagccataaattgattgg gtggtacaccaaacaa aaca-3' (SEQ ID NO. 1);
and (3) detecting a primer Bt: 5'-ggttagttaaataccgatctggcttgaaga tccaa ctcctg-3' (SEQ ID NO. 2);
a kit for detecting enterotoxin SEA comprises the PSR primer;
in the kit, the concentration of each PSR primer is preferably 50 mu M;
the kit also 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 concentration of the Bst DNA polymerase is preferably 8U/. mu.L;
the preparation method of the mixed solution of the calcein and the manganese chloride comprises the following steps:
(i) dissolving calcein in dimethyl sulfoxide (DMSO) to obtain 50 μ M calcein solution; dissolving manganese chloride in water to prepare 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 mass ratio of calcein to manganese chloride in the solution is 1: 8).
The kit can be used for detecting the enterotoxin SEA.
A PSR method for detecting enterotoxin SEA comprising the steps of:
(1) extracting bacterial DNA of a sample to be detected as template DNA;
the template DNA, OD260/OD280The value is 1.8 to 2.0;
(2) establishing a PSR amplification reaction system for detecting SEA toxin, and carrying out PSR reaction in a water bath at 60-68 ℃ for at least 60 minutes;
wherein, the PSR amplification reaction system is as follows: 2 Xreaction buffer solution 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, Bst DNA polymerase 1.0. mu.L of 8U/. mu.L, water to make up to 25. mu.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 the temperature of 75-85 ℃ for 2 minutes to terminate the reaction, and then observing the color by naked eyes, wherein if the color is yellow, the sample to be detected does not contain the enterotoxin SEA; if the color is changed to green, the enterotoxin SEA is contained in the sample to be detected;
preferably, after the reaction is terminated in the step (3), performing agarose gel electrophoresis on the amplification product, and showing a trapezoidal band to indicate that the sample to be detected contains enterotoxin SEA; the absence of amplified bands indicates that the sample to be tested does not contain enterotoxin SEA.
Compared with the prior art, the invention has the following advantages and effects:
(1) the PSR detection primer and the method designed aiming at the enterotoxin SEA solve the problems of long required period, low sensitivity, high cost, difficult field application and the like of the prior art. A pair of detection primers Ft and Bt are designed to construct a PSR reaction system by selecting a conserved region of the SEA enterotoxin specific sequence of the target strain, and a detection result is obtained in about 60 minutes, so that the detection period is greatly shortened compared with the traditional MRSA enterotoxin detection method.
(2) Compared with the ordinary PCR, the PSR technology has remarkable superiority in sensitivity. The invention can reduce the probability of false positive result, and ensure the reliability, specificity and high sensitivity of detection. The method has very important significance for early disease diagnosis and improvement of the diagnosis rate of diseases of a large group.
(3) The method disclosed by the invention can be used for amplification under a constant temperature condition, a large amount of time is not needed for changing the temperature of a reaction system, and the time consumption is short. In addition, the technology does not need expensive instruments and reagents, amplification products do not need to undergo gel electrophoresis, fluorescent dyes are directly used, results can be judged by naked eyes through color reaction, the operation is simple and convenient, and the cost is low. Therefore, the kit and the method can be well applied to field detection, and bring convenience to small and medium-sized units.
Drawings
FIG. 1 shows the color development of the detection of enterotoxin SEA by PSR reaction technique;
FIG. 2 shows the result of gel electrophoresis of the detection of enterotoxin SEA by PSR technique;
wherein, 1 is MRSA132115, 2 is MRS0315022822, 3 is MRSA0613120003, 4 is MRSA0314020129, 5 is Lactobacillus casei BM-LC14617, and 6 is Salmonella ATCC 29629; 7 is salmonella ATCC 19585; 8 is salmonella ATCC 14028; 9 is salmonella ATCC 29629; 10 is listeria monocytogenes ATCC 19116; 11 is listeria monocytogenes ATCC19114, 12 is listeria monocytogenes ATCC 19115; 13 is listeria monocytogenes ATCC 15313; 14 is listeria monocytogenes ATCC 19113; 15 pseudomonas aeruginosa ATCC 27853; 16 is Escherichia coli ATCC 43895; 17 is Escherichia coli E019; 18 is Escherichia coli E20; 19 is Escherichia coli E43; 20 is Escherichia coli E44; 21 is Vibrio parahaemolyticus ATCC 17802; 22 is Vibrio parahaemolyticus ATCC27969, and 23 is blank control.
FIG. 3 is a graph showing the results of an experiment for detecting the sensitivity of enterotoxin SEA, in which the graph A shows the result of electrophoresis and the graph B shows the result of color development;
wherein 1 is 63 ng/. mu.L; 2 is 6.3 ng/. mu.L; 3 is 630pg/μ L; 4 is 63pg/μ L; 5 is 6.3pg/μ L; 6 is 630 fg/. mu.L; 7 was 63 fg/. mu.L, NG-negative control.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
The method for detecting the enterotoxin SEA based on the PSR reaction technology comprises the following steps:
1. the present embodiment takes MRSA bacteria as an example, and the reagents used in the method are as follows:
a. ft and Bt primers with the concentration of 50 mu M respectively, and the sequences are shown in SEQ ID NO.1-SEQ ID NO. 2;
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, 0.2% (v/v) Tween 20, betaine 1.4M, dNTPs (research) 10.0 mM.
b. Bst DNA polymerase (Large fragment, NEB Corp.) in water at a concentration of 8U/. mu.L;
c. 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 (the mass ratio of calcein to manganese chloride was 1: 8).
2. The MRSA enterotoxin is detected by using the reagent and utilizing a PSR reaction technology, and the method comprises the following steps:
(1) extracting bacterial DNA of a sample to be detected as template DNA:
in this example, the experimental group and the control group were set simultaneously, wherein the experimental group of enterotoxin SEA was MRSA132115, MRSA 0315022822, MRSA0613120003, and MRSA 0314020129.
The control group is non-MRSA bacteria, Lactobacillus casei BM-LC14617, Salmonella ATCC 29629; salmonella ATCC 19585; salmonella ATCC 14028; salmonella ATCC 29629; listeria monocytogenes ATCC 19116; listeria monocytogenes ATCC19114, listeria monocytogenes ATCC 19115; listeria monocytogenes ATCC 15313; listeria monocytogenes ATCC 19113; pseudomonas aeruginosa ATCC 27853; escherichia coli ATCC 43895; e019 of escherichia coli; e.coli E20; e.coli E43; e.coli E44; vibrio parahaemolyticus ATCC 17802; vibrio parahaemolyticus ATCC 27969.
All strains are publicly available;
lactobacillus casei BM-LC14617 is disclosed in the literature (Junyan Liu, Lin Li, Bing Li, Brian M.Peters, Yang Deng, Zhenbo Xu, Mark E.Shirtliff. the visible but non-functional state and genetic analysis of Lactobacillus casei BM-LC14617, abeer-spoilage bacterium [ J ]. microbiology, 2017,6(5): e 00506);
coli E019, E020, E043 and E044 have been disclosed in the literature (cistanche tubulosa. cryo-storage effects on induction of VBNC status of enterohemorrhagic escherichia coli and toxin expression amount study [ D ]. southern china university 2015.);
extracting the bacterial DNA of each group by using a DNA extraction kit (Guangdong Sheng Biotech Co., Ltd.), and measuring the OD of the bacterial DNA aqueous solution obtained by the experimental group according to the operation of the kit specification260/OD280The value of (absorbance ratio at 260nm and 280 nm) was 1.8.
(2) Establishing a PSR amplification reaction for detecting enterotoxin:
a PSR amplification reaction system was prepared in a reaction tube in a total volume of 26 μ L: adding 12.5 μ L of 2 × reaction stock solution, 1.6 μ L of corresponding primer mixed solution of equal volume of Ft and Bt, 1 μ L of Bst DNA polymerase and 2.0 μ L of DNA template, supplementing the volume to 25 μ L with deionized water, finally adding 1 μ L of calcein and manganese chloride mixed solution water solution with the concentration, and mixing uniformly. At this time, the concentration of each substance in the reaction system is 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, primers Ft, 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. 1 and show that: the control group was yellow in color, indicating no enterotoxin SEA; the color of the experimental group turned green, indicating the presence of enterotoxin SEA.
The amplification products were subjected to 2% agarose gel electrophoresis, and the results are shown in FIG. 2, in which the positive group showed a trapezoidal band and the negative group showed no amplification band.
The bacteria reaction tube containing the SEA toxin has ladder-shaped strips and color development change, but the non-MRSA bacteria do not have non-specific amplification, so that the primer for detecting the MRSA enterotoxin SEA based on the PSR reaction has higher specificity.
Example 2
A sensitivity-contrast assay for detecting enterotoxin SEA in PSR response comprising the steps of:
performing 10-fold concentration gradient dilution on genome of MRSA132115 enterotoxin SEA, wherein the concentration is 63 ng/mu L respectively; 6.3 ng/. mu.L; 630pg/μ L; 63 pg/. mu.L; 6.3 pg/. mu.L; 630 fg/. mu.L; 63 fg/. mu.L. Meanwhile, a negative control (core-removed acid water) was set, the PSR amplification method was constructed according to the reaction system in example 1 and the amplification product was subjected to 2% agarose gel electrophoresis to determine the sensitivity of the detection method.
As shown in FIG. 3, it can be seen from FIG. A that trapezoidal bands were observed at DNA concentrations higher than 630 pg/. mu.L, and a positive result was obtained. As can be seen from panel B, the tubes with DNA concentration higher than 630 pg/. mu.L in the sample all turned from orange to green with a positive result.
The results show that: the established PSR reaction method of MRSA enterotoxin SEA can detect the reacted MRSA enterotoxin SEA with the concentration of 630 pg/mu L in a sample.
And (4) conclusion: as can be seen from the above experimental results, the PSR reaction amplification method has the following advantages compared with the conventional PCR and the fluorescent 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, 2-3 hours are needed for the fluorescent quantitative PCR, and the positive result can be obtained in 60 minutes by the detection method provided by the invention. Expensive instruments are not needed, only a common water bath is needed, and the detection result can be directly observed through the color reaction of the fluorescent dye, so that the traditional agarose gel electrophoresis step is omitted. Has wide application prospect in the fields of rapid detection and field detection.
The specificity is strong: the existence of the target gene can be judged only by the existence of amplification, thereby completing the qualitative detection of the bacteria.
The sensitivity is high: the detection limit of the MRSA enterotoxin SEA is 630 pg/mu L, which is about 10-1000 times of that of the conventional PCR reaction, and the sensitivity is higher.
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
<110> university of southern China's science
<120> PSR primer, detection kit and detection method for detecting enterotoxin SEA
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> detection primer Ft
<400> 1
gtctagccat aaattgattg ggtggtacac caaacaaaac a 41
<210> 2
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> detection primer Bt
<400> 2
ggttagttaa ataccgatct ggcttgaaga tccaactcct g 41
Claims (10)
1. The PSR primer for detecting the enterotoxin SEA is characterized by comprising a detection primer Ft and a detection primer Bt, wherein the nucleotide sequences of the primers are respectively shown as follows:
detection primer Ft: 5'-gtctagccataaattgattgg gtggtacaccaaacaa aaca-3' (SEQ ID NO. 1);
and (3) detecting a primer Bt: 5'-ggttagttaaataccgatctggcttgaaga tccaa ctcctg-3' (SEQ ID NO. 2).
2. A kit for detecting enterotoxin SEA comprising the PSR primer of claim 1.
3. The kit of claim 2, wherein: the concentration of the PSR primer is 50 μ M.
4. The kit according to claim 2, characterized by further comprising 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.
5. The kit of claim 4, wherein: the concentration of Bst DNA polymerase is 8U/. mu.L.
6. The kit of claim 4, wherein: the preparation method of the mixed solution of the calcein and the manganese chloride comprises the following steps:
(i) dissolving calcein in dimethyl sulfoxide to obtain 50 μ M calcein solution; dissolving manganese chloride in water to prepare 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.
7. Use of a kit according to any of claims 2 to 6 for the detection of enterotoxin SEA.
8. A PSR method for detecting enterotoxin SEA comprising the steps of:
(1) extracting bacterial DNA of a sample to be detected as template DNA;
(2) establishing a PSR amplification reaction system for detecting SEA toxin, and carrying out PSR reaction in a water bath at 65 ℃ for at least 60 minutes;
(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 by naked eyes, wherein if the color is yellow, the sample to be detected does not contain the enterotoxin SEA; if the color is changed to green, the enterotoxin SEA is contained in the sample to be detected.
9. The method of claim 8, wherein: after the reaction is terminated in the step (3), carrying out agarose gel electrophoresis on the amplification product to show a trapezoidal band to indicate that the sample to be detected contains enterotoxin SEA; the absence of amplified bands indicates that the sample to be tested does not contain enterotoxin SEA.
10. The method of claim 8, wherein:
the template DNA, OD of step (1)260/OD280The value is 1.8 to 2.0;
the PSR amplification reaction system in the step (2) is as follows: 2 Xreaction buffer solution 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, Bst DNA polymerase 1.0. mu.L of 8U/. mu.L, water to make up to 25. mu.L; finally, 1 μ L of a mixed solution of calcein and manganese chloride was added.
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