WO2021003878A1 - Cpa primer and kit for detecting methicillin-resistant staphylococcus aureus, and detection method - Google Patents

Abstract

Disclosed are a CPA primer for detecting methicillin-resistant Staphylococcus aureus, a detection kit therefor and a detection method therefor. The CPA primer is designed for two targets, femA and mecA, and comprises stripping primers 4s and 5a, cross amplification primer 2a1s, and specific primers 2a and 3a, wherein the sequences of the primers are shown in SEQ ID NO.1-SEQ ID NO.10. The detection method therefor includes: establishing a cross-primer constant-temperature amplification reaction system for detecting femA and mecA, performing a cross-primer constant-temperature amplification reaction, and observing the colour change of two reaction systems. If the colours of the two reaction systems both change to green, this indicates that a sample to be tested contains methicillin-resistant Staphylococcus aureus; otherwise, this indicates that the tested sample does not contain methicillin-resistant Staphylococcus aureus. The detection time of the primer and method is fast, and a detection result can be obtained in about 60 minutes; in addition, the detection sensitivity is high, reaching the level of fg/mL.

Description

CPA primer, kit and detection method for detecting methicillin-resistant Staphylococcus aureus Technical field

The invention belongs to the field of biotechnology detection, and specifically relates to a CPA primer for detecting methicillin-resistant Staphylococcus aureus, a detection kit and a detection method thereof.

Background technique

At present, microorganism detection and identification methods are mainly divided into culture identification method, immunoassay method and nucleic acid detection. The cultivation identification method and the immunoassay method are cumbersome to operate, the experiment cycle is long, and the professional level of the experiment personnel is high.

Compared with other nucleic acid amplification technologies, Crossing Priming Amplification (CPA) technology can quickly, efficiently, and specifically amplify target sequences under isothermal conditions. It is easy to operate, does not require precise temperature changing equipment, and costs Low, showing broad development prospects in the field of food-borne microbial detection. However, the design of primers for this reaction is more difficult. It is necessary to design five primers in a limited product length, and to avoid non-specific amplification of the five primers themselves and affect the results, so the design of primers is particularly important.

Staphylococcus aureus (Staphylococcus aureus) is ubiquitous in nature, in air, water, dust, and excreta of animals and humans. This situation leads to a high chance of contamination of food. Staphylococcus aureus is a common conditional pathogenic food-borne microorganism. It is an invasive bacteria. The staphylococcal enterotoxin secreted by it can cause food poisoning in humans. The toxins produced by it are highly destructive to the intestines, causing acute onset of enteritis and severe poisoning symptoms, mainly manifested as vomiting, fever and diarrhea. There are many types of poisoned foods caused by Staphylococcus aureus infection, such as milk, meat, eggs, fish and their products. At the same time, the use of antibiotics in the treatment process has also led to the discovery of a large number of methicillin-resistant Staphylococcus aureus in food-borne foods, which has brought huge challenges to human health and clinical treatment. At present, food safety accidents caused by methicillin-resistant Staphylococcus aureus have become a worldwide health problem.

Chinese patent application CN109355403A discloses a primer, kit and method for PSR to detect methicillin-resistant Staphylococcus aureus. However, the detection limit of this patent only reaches the pg/μL level, and the sensitivity is not high enough, which limits the scope of application.

Summary of the invention

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a CPA primer for detecting methicillin-resistant Staphylococcus aureus.

Another object of the present invention is to provide a methicillin-resistant Staphylococcus aureus detection kit containing the above-mentioned CPA primer.

Another object of the present invention is to provide a CPA detection method for methicillin-resistant Staphylococcus aureus, which has high sensitivity (the detection limit reaches fg/μL level), good specificity, simple and fast operation, accurate and reliable results, and detection Low cost, suitable for on-site inspection applications and other characteristics.

The purpose of the present invention is achieved through the following technical solutions:

A set of CPA primers for the detection of methicillin-resistant Staphylococcus aureus, designed for the two targets femA and mecA, including stripping primers 4s and 5a, cross-amplification primers 2a1s, and specific primers 2a, 3a; its nucleosides The acid sequence is as follows:

Target femA stripping primer 4s: 5’-tcaaatcgcggtccagtg-3’ (SEQ ID NO.1)

Target femA stripping primer 5a: 5’-aaccaatcattaccagca-3’ (SEQ ID NO.2)

Target femA cross primer 2a1s: 5’-tacctgtaatctcgccataacatcgttgtctatacct-3’ (SEQ ID NO.3)

Target femA specific primer 2a: 5’-tacctgtaatctcgccat-3’ (SEQ ID NO.4)

Target femA specific primer 3a: 5’-ggtaaatatggatcgatatg-3’ (SEQ ID NO.5)

Target mecA stripping primer 4s: 5’-gcgataatggtgaagtag-3’ (SEQ ID NO.6)

Target mecA stripping primer 5a: 5’-gatcaatgttaccgtagtt-3’ (SEQ ID NO.7)

Target mecA cross primer 2a1s: 5’-ttacgatcctgaatgtttatgactgaacgtccgata-3’ (SEQ ID NO.8)

Target mecA specific primer 2a: 5’-ttacgatcctgaatgttt-3’ (SEQ ID NO.9)

Target mecA specific primer 3a: 5'-tctttaacgcctaaacta-3' (SEQ ID NO. 10).

A kit for detecting methicillin-resistant Staphylococcus aureus, comprising the above-mentioned CPA primer;

In the kit, the concentration of each CPA primer is preferably 10 μM;

The kit also includes 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; preferably a Bst DNA polymerase aqueous solution with a concentration of 8 U/μL;

C. Mixed solution of calcein and manganese chloride;

Component C is prepared by the following method:

(i) Dissolve calcein in dimethyl sulfoxide (DMSO) to prepare a 50μM calcein solution; dissolve manganese chloride in water to prepare a 1 mM manganese chloride aqueous solution;

(ii) Take 25 μL of 50 μM calcein solution and 10 μL of 1 mM manganese chloride aqueous solution and mix well to obtain a mixed solution of calcein and manganese chloride (the concentration ratio of calcein to manganese chloride is 1:8).

A method for detecting methicillin-resistant Staphylococcus aureus based on the above kit includes the following steps:

(1) Extract the DNA of the sample to be tested as template DNA, and control the OD ₂₆₀ /OD ₂₈₀ value of the template DNA aqueous solution to 1.8～2.0;

(2) Establish a cross-primer thermostatic amplification reaction system for detecting femA and mecA respectively, and heat it in a 63℃ water bath for at least 60 minutes to carry out the cross-primer thermostatic amplification reaction. After the reaction is completed, keep it in a water bath higher than 80℃ for at least 2 Stop the reaction in minutes;

Among them, the cross primer constant temperature amplification reaction system is: 2× reaction buffer 12.5 μL, 10 μM stripping primer 4s and 10 μM stripping primer 5a each 1.5 μL, 10 μM cross primer 2a1s 2.5 μL, 10 μM specific primer 2a and 10 μM Specific primers 3a are each 1.25μL, DNA template 1.0μL, 8U/μL Bst DNA polymerase 1.0μL, add nucleic acid water to make up to 25μL; finally add 1μL of a mixed solution of calcein and manganese chloride;

(3) Observe the color changes of the two reaction systems. If the colors of the two reaction systems both turn green, it means that the sample to be tested contains methicillin-resistant Staphylococcus aureus; otherwise, it means that the test sample does not contain methicillin-resistant golden yellow staphylococcus.

The femA gene inherent in Staphylococcus aureus is the MRSA resistance auxiliary gene, and mecA is the MRSA resistance determining gene. Therefore, the combined use of mecA and femA genes can not only quickly screen drug-resistant strains, but also eliminate tedious biochemical identification.

Compared with the prior art, the present invention has the following advantages and effects:

(1) The present invention provides a set of CPA primers for detecting methicillin-resistant Staphylococcus aureus. The primers can achieve rapid and accurate detection of different MRSA strains, have good applicability and high sensitivity (detection limit). Reach the fg/μL level).

(2) The present invention is amplified under constant temperature conditions without time loss due to temperature changes, and it takes a short time. In addition, the technology does not require special and expensive instruments and reagents, and the amplified products do not require gel electrophoresis. Direct color development with fluorescent dyes can judge the result by naked eyes, the operation is simple and fast, and the detection cost is low. The kit and method of the present invention are particularly suitable for small and medium-sized units and on-site detection.

(3) The cross-primer isothermal amplification reaction detection and identification system designed for the specific target sequences of methicillin-resistant Staphylococcus aureus femA and mecA provided in the present invention solves the problem of long cycle and low sensitivity required by the prior art method , High cost, difficult field application and other defects. By selecting the conserved regions of the specific sequence femA and mecA of the target strain, design a pair of stripping primers, cross primers and specific primers to construct a cross primer constant temperature amplification reaction system, and obtain the test results in about 60 minutes to shorten the traditional methicillin resistance The cycle of Staphylococcus aureus detection.

Description of the drawings

Figure 1 shows the gel electrophoresis results and color results of femA and mecA targets detected by cross-primer isothermal amplification reaction technology; Figure A shows the gel electrophoresis results of femA and mecA targets detected by cross-primer isothermal amplification (lane 1 is Methicillin-resistant Staphylococcus aureus NCTC10442, lane 2 is methicillin-resistant Staphylococcus aureus NCTC10442, lane 3 is methicillin-resistant Staphylococcus aureus NCTC10442, NG is a blank control); Figure B is cross primer constant temperature amplification Reaction technology detects the color development results of mecA target (1 is methicillin-resistant Staphylococcus aureus NCTC1044, 2 is methicillin-resistant Staphylococcus aureus N315, 3 is methicillin-resistant Staphylococcus aureus N315, NG is Blank control).

Figure 2 is a graph showing the results of specific experiments for detecting femA and mecA targets. Figure A is a graph showing the results of a sensitivity test for detecting femA on a cross constant temperature amplification reaction. Figure B is a sensitivity experiment for detecting the target mecA. Among them, 1: Methicillin-resistant Staphylococcus aureus NCTC10442, 2: Methicillin-resistant Staphylococcus aureus NCTC10442, 3: Methicillin-resistant Staphylococcus aureus NCTC10442, 4: Escherichia coli O157: H7 E019; 5: Large intestine Bacillus O157: H7 E020; 6: E. coli E043; 7: E. coli E044; 8: E. coli ATCC43895; 9: Salmonella ATCC29629; 10: Salmonella ATCC19585; 11: Salmonella ATCC14028; 12: Salmonella ATCC13076; 13: Listeria monocytogenes Bacteria ATCC19116; 14: Listeria monocytogenes ATCC19114; 15: Listeria monocytogenes ATCC19115; 16: Listeria monocytogenes ATCC15313; 17: Listeria monocytogenes ATCC19113; 18: Pseudomonas aeruginosa ATCC27853; 19: Vibrio parahaemolyticus ATCC17802; 20: Vibrio parahaemolyticus ATCC27969; 21: Lactobacillus casei BM-LC14617; 22: negative control.

Figure 3 shows the results of the sensitivity test for detecting the target femA and mecA. Figure A shows the result of the sensitivity test for detecting the target femA by the cross constant temperature amplification reaction. Figure B shows the sensitivity test for detecting the target mecA. 1 is 3.0ng/ μL, 2 is 300pg/μL, 3 is 30pg/μL, 4 is 3pg/μL, 5 is 300fg/μL, 6 is 30fg/μL, 7 is 3fg/μL, 8 is 300ag/μL, NG is a negative control.

Detailed ways

The present invention will be further described in detail below in conjunction with the examples and drawings, but the implementation of the present invention is not limited thereto.

Example 1

The method for detecting methicillin-resistant Staphylococcus aureus based on the cross-primer constant temperature amplification (CPA) reaction technology includes the following steps:

(1) Use reagents:

a. Stripping primers 4s, 5a, cross-amplification primers 2a1s, and specific primers 2a, 3a, each with a concentration of 10μM. The primer sequence is shown in SEQ ID NO.1-SEQ ID NO.10;

b.2×Reaction stock solution: composed of 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) composition;

c. Bst DNA polymerase (large fragment, NEB company) aqueous solution with a concentration of 8U/μL;

d. Mixed solution of calcein and manganese chloride: first prepare a 50μM calcein solution (dissolved in dimethyl sulfoxide); then take 25μL of 50μM calcein solution and mix it with 10μL of 1mM manganese chloride aqueous solution ( The concentration ratio of calcein to manganese chloride solution is 1:8).

(2) Extract the DNA of the sample to be tested as template DNA:

In this example, an experimental group and a blank control group are set at the same time. The experimental group is three strains of methicillin-resistant Staphylococcus aureus, namely methicillin-resistant Staphylococcus aureus NCTC10442, methicillin-resistant Staphylococcus aureus NCTC10442, and armour-resistant Oxycillin Staphylococcus aureus NCTC10442; all strains can be obtained from public sources;

The DNA extraction kit (Guangdong Dongsheng Biotechnology Co., Ltd.) was used to extract the bacterial DNA of each group, and operate according to the kit instructions. The OD ₂₆₀ /OD ₂₈₀ value of the bacterial DNA aqueous solution obtained in the experimental group (the ratio of absorbance at 260 nm and 280 nm) is 1.8.

(2) Establish a constant temperature amplification reaction with cross primers to detect femA and mecA targets:

Prepare a cross-primer thermostatic amplification reaction system with a total volume of 26μL in the reaction tube: add 2× reaction stock solution 12.5μL, 4s and 5a equal volume mixed primer mixture 3.0μL, cross primer 2a1s 2.5μL, specific primers 2a and 3a An equal volume of 2.5μL of the mixed solution, 1μL of Bst DNA polymerase, 1.0μL of DNA template, replenish the volume to 25μL with de-nucleic acid water, and finally add 1μL of calcein and manganese chloride mixed solution with the above concentration and mix well. At this time, the concentration of each substance is: Tris-HCl 20.0mM, ammonium sulfate 10.0mM, potassium chloride 10.0mM, magnesium sulfate 8.0mM, Tween 20 0.1% (v/v), betaine 0.7M, dNTPs(each) 1.4mM , Bst DNA polymerase 8U, stripping primer 4s, 5a each 0.6μM, cross primer 2a1s 1.0μM, specific primer 2a and 3a each 0.5μM. The reaction tube was kept in a water bath at 63°C for 60 minutes, and then kept in a water bath at 80°C for 2 minutes to terminate the reaction.

(3) Color detection:

After the reaction is over, observe the color change with naked eyes.

The results are shown in Figure 1. The results show that the color of the experimental group (1-3) corresponding to femA and mecA targets changed to green, indicating that it contains methicillin-resistant Staphylococcus aureus, and the color of the blank control group (NG) is yellow , Indicating that it does not contain methicillin-resistant Staphylococcus aureus; then the amplified products were subjected to 2% agarose gel electrophoresis, the positive group showed a trapezoidal band, and the negative group had no amplified band, which was consistent with the expected result.

Example 2

The cross constant temperature amplification reaction (CPA) to detect the specificity of methicillin-resistant Staphylococcus aureus includes the following steps:

The genomic DNA of methicillin-resistant Staphylococcus aureus NCTC10442 and non-methicillin-resistant Staphylococcus aureus was established in accordance with the reaction system and conditions in Example 1 to establish a cross-isothermal amplification reaction detection method to conduct a specific test;

Among them, non-methicillin-resistant Staphylococcus aureus are: Escherichia coli O157:H7 E019; Escherichia coli O157:H7 E020; Escherichia coli E043; Escherichia coli E044; Escherichia coli ATCC43895; Salmonella ATCC29629; Salmonella ATCC19585; Salmonella ATCC14028; Salmonella ATCC13076; Listeria monocytogenes ATCC19116; Listeria monocytogenes ATCC19114; Listeria monocytogenes ATCC19115; Listeria monocytogenes ATCC15313; Listeria monocytogenes ATCC19113; Pseudomonas aeruginosa ATCC27853; Vibrio parahaemolyticus ATCC17802; parahaemolytic Vibrio ATCC27969; Lactobacillus casei BM-LC14617.

The genome of methicillin-resistant Staphylococcus aureus NCTC10442 was set as a positive control, and nucleic acid-free water was set as a negative control. The amplified products were subjected to 2% agarose gel electrophoresis. The results are shown in Figure 2. Figure A shows the specific detection result of femA, and Figure B shows the specific detection result of mecA. The results showed that only NCTC10442 containing methicillin-resistant Staphylococcus aureus showed trapezoidal bands (lanes 1 to 3), while non-methicillin-resistant Staphylococcus aureus did not. This shows that the primers for detecting methicillin-resistant Staphylococcus aureus based on cross-primer isothermal amplification reaction have high specificity.

Example 3

The comparative test of sensitivity of cross constant temperature amplification reaction (CPA) for detecting methicillin-resistant Staphylococcus aureus includes the following steps:

The genome of methicillin-resistant Staphylococcus aureus NCTC10442 was diluted in 10-fold concentration, respectively, 3.0ng/μL, 300pg/μL, 30pg/μL, 3pg/μL, 300fg/μL, 30fg/μL, 3fg/μL, 300ag /μL, and set a negative control (de-nucleic acid water) at the same time, construct a cross constant temperature amplification method according to the reaction system in Example 1, and perform 2% agarose gel electrophoresis on the amplified products to determine the sensitivity of the detection method.

The results are shown in Fig. 3, Fig. A is the result of the sensitivity test for detecting femA target by cross isothermal amplification reaction, and Fig. B is the sensitivity test for detecting mecA target.

The results show that the established methicillin-resistant Staphylococcus aureus femA and mecA target cross-primer constant temperature amplification reaction method can detect 30fg/μL (femA), 300fg/μL (mecA) methicillin-resistant golden yellow grapes in the sample Coccus DNA.

Conclusion: It can be seen from the above experimental results that the cross-isothermal amplification reaction amplification method has the following advantages compared with conventional PCR and fluorescent PCR:

Operation and identification are simple and quick: the whole process of conventional PCR takes 2 to 4 hours to produce results, and the fluorescent quantitative PCR takes 2 to 3 hours. The detection method provided by the present invention can produce positive results in 60 minutes. Secondly, the requirements for the instrument are low, only an ordinary water bath is needed, and the detection results can be directly observed through fluorescent dyes, eliminating the need for traditional electrophoresis detection steps. It has broad application prospects in the practice of rapid detection and on-site detection.

Strong specificity: The presence or absence of the target gene can be judged only by whether or not it is amplified, thus completing the qualitative detection of bacteria.

High sensitivity: The detection limit for fema and mecA of oxycillin-resistant Staphylococcus aureus is 30fg/μL, 300fg/μL, which is about 10-100 times that of conventional PCR, and has high sensitivity.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, etc. made without departing from the spirit and principle of the present invention Simplified, all should be equivalent replacement methods, and they are all included in the protection scope of the present invention.

Claims (8)

1. A set of CPA primers for detecting methicillin-resistant Staphylococcus aureus, which are characterized by including stripping primers 4s and 5a, cross-amplification primer 2a1s, and specific primers 2a and 3a; their nucleotide sequences are as follows:

   Target femA stripping primer 4s: 5’-tcaaatcgcggtccagtg-3’ (SEQ ID NO.1)

   Target femA stripping primer 5a: 5’-aaccaatcattaccagca-3’ (SEQ ID NO.2)

   Target femA cross primer 2a1s: 5’-tacctgtaatctcgccataacatcgttgtctatacct-3’ (SEQ ID NO.3)

   Target femA specific primer 2a: 5’-tacctgtaatctcgccat-3’ (SEQ ID NO.4)

   Target femA specific primer 3a: 5’-ggtaaatatggatcgatatg-3’ (SEQ ID NO.5)

   Target mecA stripping primer 4s: 5’-gcgataatggtgaagtag-3’ (SEQ ID NO.6)

   Target mecA stripping primer 5a: 5’-gatcaatgttaccgtagtt-3’ (SEQ ID NO.7)

   Target mecA cross primer 2a1s: 5’-ttacgatcctgaatgtttatgactgaacgtccgata-3’ (SEQ ID NO.8)

   Target mecA specific primer 2a: 5’-ttacgatcctgaatgttt-3’ (SEQ ID NO.9)

   Target mecA specific primer 3a: 5'-tctttaacgcctaaacta-3' (SEQ ID NO. 10).
2. A kit for detecting methicillin-resistant Staphylococcus aureus, characterized by comprising the CPA primer of claim 1.
3. The kit according to claim 2, wherein the concentration of each CPA primer is 10 μM.
4. The kit according to claim 2, characterized in that it 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. Mixed solution of calcein and manganese chloride.
5. The kit according to claim 4, wherein the component B is an aqueous solution of Bst DNA polymerase with a concentration of 8 U/μL.
6. The kit according to claim 4, wherein in the component C, the concentration ratio of calcein to manganese chloride is 1:8.
7. A method for detecting methicillin-resistant Staphylococcus aureus based on the above kit is characterized in that it comprises the following steps:

   (1) Extract the DNA of the sample to be tested as template DNA, and control the OD ₂₆₀ /OD ₂₈₀ value of the template DNA aqueous solution to 1.8～2.0;

   (2) Establish a cross-primer thermostatic amplification reaction system for detecting femA and mecA respectively, and heat it in a 63℃ water bath for at least 60 minutes to carry out the cross-primer thermostatic amplification reaction. After the reaction is completed, keep it in a water bath higher than 80℃ for at least 2 Stop the reaction in minutes;

   (3) Observe the color changes of the two reaction systems. If the colors of the two reaction systems both turn green, it means that the sample to be tested contains methicillin-resistant Staphylococcus aureus; otherwise, it means that the test sample does not contain methicillin-resistant golden yellow staphylococcus.
8. The method according to claim 7, characterized in that: in step (2), the cross-primer constant-temperature amplification reaction system is: 2 x reaction buffer 12.5 μL, 10 μM stripping primer 4s and 10 μM stripping primer 5a 1.5μL each, 10μM cross primer 2a1s 2.5μL, 10μM specific primer 2a and 10μM specific primer 3a 1.25μL each, DNA template 1.0μL, 8U/μL Bst DNA polymerase 1.0μL, add denucleic acid water to make up to 25μL ; Finally add 1μL of calcein and manganese chloride mixed solution.

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