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CPA primer, kit and detection method for escherichia coli O157:H7

A technology of Escherichia coli and O157, which is applied in biochemical equipment and methods, microbial measurement/inspection, recombinant DNA technology, etc., can solve the problems of low sensitivity and low detection sensitivity, achieve low detection cost, simple and fast operation, and ensure reliability effect

Active Publication Date: 2019-04-19
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In order to overcome the problem of low detection sensitivity of PSR reaction, the primary purpose of the present invention is a kind of CPA primer of Escherichia coli O157:H7, its sensitivity has reached fg / μL, is 100~1000 times of PSR reaction sensitivity, can solve the problem of prior art The problem of low sensitivity

Method used

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  • CPA primer, kit and detection method for escherichia coli O157:H7
  • CPA primer, kit and detection method for escherichia coli O157:H7
  • CPA primer, kit and detection method for escherichia coli O157:H7

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Effect test

Embodiment 1

[0052] The method for detecting escherichia coli O157:H7 based on cross-primer constant temperature amplification reaction technology may further comprise the steps:

[0053] 1. The method for detecting pathogenic microorganisms based on cross-primer constant temperature amplification reaction technology, the present embodiment takes Escherichia coli O157:H7 as an example, and the reagents used are as follows:

[0054] 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;

[0055] 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;

[0056] c. Bst DNA polymerase (large fragment, NEB company) aqueous solution with a concentration of 8U / μL;

[...

Embodiment 2

[0068] The cross constant temperature amplification reaction detects the optimum amplification time test of Escherichia coli O157:H7, comprising the following steps:

[0069] According to the reaction system of Example 1, the cross constant temperature amplification method was constructed, and different amplification time tests of 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, and 90 minutes were carried out to determine Optimal amplification time.

[0070] The reaction system was constructed according to Example 1, sterile water was used as a blank control, and 2% agarose gel electrophoresis was performed on the amplified product.

[0071] The result is as figure 2 As shown, when the reaction time is 40 minutes to 90 minutes, a ladder-like band appears, and there is no significant difference in the ladder-like band from 60 minutes to 90 minutes, so the cross-primer constant temperature amplification reaction time can be reduc...

Embodiment 3

[0073] The cross constant temperature amplification reaction detection Escherichia coli O157:H7 specificity test comprises the following steps:

[0074] The genomic DNA of Escherichia coli O157:H7ATCC43895 and non-Escherichia coli was established according to the reaction system and conditions in Example 1. A cross-isothermal amplification reaction detection method was carried out to carry out a specificity test;

[0075] Among them, non-Escherichia coli are: Salmonella ATCC29629; Salmonella ATCC19585; Salmonella ATCC14028; Salmonella ATCC13076; Listeria monocytogenes ATCC19116; Listeria monocytogenes ATCC19114; Species ATCC19113; Pseudomonas aeruginosa ATCC27853; Staphylococcus aureus ATCC27664; MRSA NCTC10442; MRSA N315; MRSA 85 / 2082; Methicillin Staphylococcus aureus CA05; Vibrio parahaemolyticus ATCC17802; Vibrio parahaemolyticus ATCC27969; Lactobacillus casei BM-LC14617.

[0076]Set the Escherichia coli O157:H7 genome as the positive control, and the nucleic acid-free wa...

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Abstract

The invention discloses a CPA primer, kit and detection method for escherichia coli O157:H7. The CPA primer designed for a target rfbE comprises stripping primers 4s and 5a, a crossing amplification primer 2a1s and specific primers 2a and 3a; the nucleotide sequences of the primers are shown as SEQ ID NO.1-NO.5 respectively. A crossing priming amplification reaction detection and identification system designed for specific target sequences rfbE and stx1 of E.coli O157:H7 overcomes the defects of long required cycle, low sensitivity, high cost and difficult field application of methods in the prior art. By selecting a conserved region of the specific sequences rfbE and stx1 of a target strain, a pair of stripping primers, the crossing primer and the specific primers are designed to construct the crossing priming amplification reaction system, and a detection result is obtained within about 60 minutes to shorten the traditional cycle for detecting escherichia coli.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a CPA primer of Escherichia coli O157:H7, a detection kit and a detection method thereof. Background technique [0002] 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 experimenters is high. [0003] Compared with other nucleic acid amplification technologies, crossing primer constant temperature amplification (Crossing Priming Amplification, CPA) technology can quickly, efficiently and specifically amplify target sequences under isothermal conditions, and is easy to operate, does not require precise temperature-changing equipment, and costs It shows broad development prospects in the field of foodborne microbial det...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2563/107
Inventor 徐振波骆玉婷刘君彦张桂兰苗健袁牧陈玲苏健裕李冰李琳李晓玺张霞
Owner SOUTH CHINA UNIV OF TECH
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