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Primers, kit and method for detecting MRSA enterotoxin through cross primer constant temperature technology

A cross-primer and detection method technology, applied in biochemical equipment and methods, recombinant DNA technology, and microbial assay/inspection, etc., can solve the problems of low sensitivity of PCR technology, achieve convenient operation, reduce the probability of false positive results, Detecting the effect of low cost

Pending Publication Date: 2021-10-01
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The primary purpose of the present invention is to design a CPA detection primer for MRSA enterotoxin SEA, which has a sensitivity of 300 fg / μL and can solve the problem of low sensitivity of the existing PCR technology

Method used

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  • Primers, kit and method for detecting MRSA enterotoxin through cross primer constant temperature technology
  • Primers, kit and method for detecting MRSA enterotoxin through cross primer constant temperature technology
  • Primers, kit and method for detecting MRSA enterotoxin through cross primer constant temperature technology

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Embodiment 1

[0041] The method for detecting MRSA enterotoxin SEA based on cross-primer constant temperature amplification technology comprises the following steps:

[0042] 1. The method for detecting pathogenic microorganisms based on cross-primer constant temperature amplification technology, the present embodiment takes MRSA bacteria as an example, and the reagents used are as follows:

[0043] a. The stripping primers 4s and 5a, the cross-amplification primers 2a1s, and the specific primers 2a and 3a each at a concentration of 10 μM, the primer sequences are as shown in the preceding SEQ ID NO.1-SEQ ID NO.5;

[0044] b.2×Reaction stock solution: Tris-HCl with concentration of 40.0mM, ammonium sulfate of 20.0mM, potassium chloride of 20.0mM, magnesium sulfate of 16.0mM, Tween 20 of 0.2% (v / v), 1.4M Betaine, 10.0mM dNTPs (each) composition;

[0045] c. Bst DNA polymerase (large fragment, NEB company) aqueous solution with a concentration of 8U / μL;

[0046] d. Mixed solution of calcein...

Embodiment 2

[0057] The specificity test for detecting MRSA enterotoxin by cross constant temperature amplification reaction comprises the following steps:

[0058] MRSA enterotoxin and non-MRSA enterotoxin genomic DNA were established according to the reaction system and conditions in Example 1, and a cross-isothermal amplification reaction detection method was carried out to carry out a specificity test;

[0059] Among them, non-MRSA enterotoxins are: Salmonella ATCC29629; Salmonella ATCC19585; Salmonella ATCC14028; Salmonella ATCC29629; Listeria monocytogenes ATCC19116; Listeria ATCC19113; Pseudomonas aeruginosa ATCC27853; Escherichia coli ATCC43895; Escherichia coli E019; Escherichia coli E20; Escherichia coli E43; -LC14617.

[0060] Set the MRSA enterotoxin SEA genome as a positive control, and the nucleic acid-free water as a negative control. Carry out 2% agarose gel electrophoresis to the amplified product, the result is as follows figure 2 As shown, the reaction system adding ...

Embodiment 3

[0063] The sensitivity comparison test for detecting MRSA enterotoxin SEA by cross constant temperature amplification reaction comprises the following steps:

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Abstract

The invention discloses primers, a kit and a method for detecting MRSA enterotoxin by a cross primer constant temperature technology. The CPA primer designed aiming at the MRSA enterotoxin SEA comprises stripping primers 4s and 5a, a cross amplification primer 2a1s and specific primers 2a and 3a; the nucleotide sequences of the primers are respectively as shown in SEQ ID NO.1 to SEQ ID NO.5. A constructed cross primer isothermal amplification system can judge the existence of fg / mu L-level enterotoxin SEA in about 1 hour through fluorescent dye color development of a reaction system, and the defects of long required period, low sensitivity, high cost, difficulty in field application and the like of a method in the prior art are overcome.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer, a kit and a method for detecting MRSA enterotoxin by cross-primer constant temperature technology. Background technique [0002] Staphylococcus aureus is widely distributed in nature. It is an important pathogenic bacterium that causes purulent infection in humans and animals, and is also one of the common pathogenic bacteria that cause food poisoning in humans. With the abuse of antibacterial drugs, the resistance of Staphylococcus aureus to antibacterial drugs is becoming more and more serious, especially the emergence of methicillin-resistant Staphylococcus aureus (MRSA), which makes the selection of clinical antibacterial drugs face greater pressure . Staphylococcus aureus can secrete heat-stable enterotoxins, which often cause food poisoning, extraintestinal infection, and even damage to various organs and tissues throughout the body, and eventually develo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q2531/119C12Q2537/1376C12Q2563/107
Inventor 徐振波刘子奇骆玉婷刘君彦陈玲叶燕锐李晓玺洪玮彭芳付欣户帅锋苏健裕
Owner SOUTH CHINA UNIV OF TECH
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