CPA detection primers, detection kit and detection method for enterotoxin B-producing staphylococcus aureus

A technology for detection kits and detection primers, applied in biochemical equipment and methods, methods based on microorganisms, measurement/inspection of microorganisms, etc., can solve the problems of low sensitivity, achieve low detection cost, short time consumption, and reduce false positives. The effect of the probability of a positive result

Pending Publication Date: 2021-11-19
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide CPA detection primers and detection kits and methods thereof for producing enterotoxin B (SEB) Staphylococcus aureus. The detection sensitivity of the present invention reaches 4.3pg / μL, which can solve the problem of low sensitivity of existing PCR technology ; In addition, it has the characteristics of good specificity, simple and fast operation, accurate and reliable results, low detection cost, and is suitable for on-site detection applications

Method used

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  • CPA detection primers, detection kit and detection method for enterotoxin B-producing staphylococcus aureus
  • CPA detection primers, detection kit and detection method for enterotoxin B-producing staphylococcus aureus
  • CPA detection primers, detection kit and detection method for enterotoxin B-producing staphylococcus aureus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The method for detecting and detecting Staphylococcus aureus producing enterotoxin B based on cross-primer constant temperature amplification reaction technology comprises the following steps:

[0043] 1. The reagents used are as follows:

[0044] 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;

[0045] 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;

[0046] c. Bst DNA polymerase (large fragment, NEB company) aqueous solution with a concentration of 8U / μL;

[0047] d. Mixed solution of calcein and manganese chloride: first prepare a calcein solution with a concentration of 50 μM (dissolved in dimethyl sulfoxide); then take 2...

Embodiment 2

[0059] Genomic DNA of Staphylococcus aureus producing enterotoxin B and non-enterotoxin B Staphylococcus aureus was established according to the reaction system and conditions in Example 1 to establish a cross constant temperature amplification reaction detection method for specificity test;

[0060] Staphylococcus aureus that does not produce enterotoxin B is: Salmonella ATCC29629; Salmonella ATCC19585; Salmonella ATCC14028; Salmonella ATCC29629; Listeria monocytogenes ATCC19116; ; Listeria monocytogenes ATCC19113; Pseudomonas aeruginosa ATCC27853; Escherichia coli ATCC43895; Escherichia coli E019 [3] ; Escherichia coli E20 [3] ; Escherichia coli E43 [3] ; Escherichia coli E44 [3] ; Vibrio parahaemolyticus ATCC17802; Vibrio parahaemolyticus ATCC27969; Lactobacillus casei BM-LC14617 [4] ;

[0061] The enterotoxin-producing Staphylococcus aureus genome was set as a positive control, and the nucleic acid-free water was used as a negative control.

[0062] Carry out 2% agaro...

Embodiment 3

[0065] The sensitivity comparison test for the detection of enterotoxigenic B Staphylococcus aureus by cross constant temperature amplification reaction comprises the following steps:

[0066] Genomic DNA of enterotoxin-producing Staphylococcus aureus was diluted 10-fold, respectively 4.3ng / μL, 430pg / μL, 43pg / μL, 4.3pg / μL, 430fg / μL, 43fg / μL, 4.3fg / μL μL, 430ag / μL. At the same time, a negative control (nucleic acid-free water) was set, and a cross-thermothermal amplification method was constructed according to the reaction system in Example 1, and the amplified product was subjected to 2% agarose gel electrophoresis to determine the sensitivity of the detection method.

[0067] The result is as image 3 Shown is the result graph of the sensitivity test for the detection of enterotoxigenic B Staphylococcus aureus by the cross constant temperature amplification reaction. It can be seen from the figure that trapezoidal bands appear in samples with a DNA concentration higher than...

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Abstract

The invention discloses CPA detection primers, a detection kit and a detection method for enterotoxin B-producing staphylococcus aureus. The CPA primers comprise stripping primers 4s and 5a, a cross amplification primer 2a1s, and specific primers 2a and 3a, wherein the nucleotide sequences of the above primers are respectively as shown in SEQ ID NO. 1 to SEQ ID NO. 5. A cross primer isothermal amplification reaction detection and identification system designed for the gene sequence of staphylococcus aureus enterotoxin B in by the invention overcomes the defects of long period, low sensitivity, high cost, difficulty in field application and the like of methods in the prior art. By selecting a conserved region of an enterotoxin B gene sequence of a target strain, a pair of the stripping primers, the cross primer and the specific primers are designed to construct a cross primer isothermal amplification reaction system, and a detection result is obtained within about 60 minutes, so the period of detecting enterotoxin B-producing staphylococcus aureus in the prior art is shortened.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a CPA detection primer for Staphylococcus aureus producing enterotoxin B (SEB) and a detection kit and method thereof. Background technique [0002] Staphylococcus aureus is a common food-borne pathogen that widely exists in the natural environment. Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus (MRSA), has low nutritional requirements and has good tolerance and colonization ability to the hospital environment, especially the ICU. Outbreak of the epidemic, so MRSA has become one of the focus of medical attention. [0003] At present, the detection and identification methods of microorganisms are mainly divided into culture identification, immunoassay and nucleic acid detection. The culture identification method and immunoassay method are cumbersome to operate, the experiment cycle is long, and the professional level of the experimen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/14C12N15/11C12R1/445
CPCC12Q1/689C12Q1/6844C12Q2531/119C12Q2563/107
Inventor 徐振波林欣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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