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CPA detection primer for escherichia coli shiga toxin type II, kit and method thereof

A detection kit and technology for Shiga toxin, applied in the field of biotechnology detection, can solve the problem of high difficulty in designing reaction primers, and achieve the effects of good applicability, short time-consuming, simple and quick operation

Pending Publication Date: 2020-04-03
SOUTH CHINA UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the design of primers for this reaction is very difficult, and 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

Method used

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  • CPA detection primer for escherichia coli shiga toxin type II, kit and method thereof
  • CPA detection primer for escherichia coli shiga toxin type II, kit and method thereof
  • CPA detection primer for escherichia coli shiga toxin type II, kit and method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0047] 1. Design primers

[0048] According to the amplification reaction principle of cross-primer constant temperature amplification (CPA), the following primers were designed for the target stx2 using Primer Premier software, including stripping primers 4s and 5a, cross-amplification primers 2a1s, and specific primers 2a and 3a; The nucleotide sequence is as follows:

[0049] Target stx2 stripping primer 4s: 5'-GTTACGGGAAGGAATCAGG-3' (SEQ ID NO.1);

[0050] Target stx2 stripping primer 5a: 5'-AAATCAGCCACCCACAGC-3' (SEQ ID NO.2);

[0051] Target stx2 cross primer 2a1s: 5'-CGAACTGACGGTTTACGCATGGGACTT GCCGGTGTT-3' (SEQID NO.3);

[0052] Target stx2 specific primer 2a: 5'-CGAACTGACGGTTTACGC-3' (SEQ ID NO.4);

[0053] Target stx2 specific primer 3a: 5'-TGGTCGTACGGACCTTTT-3' (SEQ ID NO.5).

Embodiment 2

[0055] The method for detecting Escherichia coli Shiga toxin type II based on cross-primer constant temperature amplification (CPA) reaction technology may further comprise the steps:

[0056] (1) Reagents used:

[0057] a. The stripping primers 4s and 5a, the cross-amplification primers 2a1s, and the specific primers 2a and 3a with a concentration of 10 μM, the primer sequences are as shown in SEQ ID NO.1-5 in Example 1;

[0058] b. Prepare 2× reaction stock solution:

[0059] ① 20× reaction stock solution: containing 0.2M potassium chloride, 0.2M ammonium sulfate, 160mM magnesium sulfate and 2% (v / v) Tween 20 (both final concentrations, prepared with nucleic acid-free water);

[0060] ②2× reaction stock solution: 100 μL 20× reaction stock solution, 500 μL 3.2M betaine, 280 μL 10mM dNTPs, 27 μL 1.5M Tris-HCl, 93 μL nucleic acid-free water;

[0061] c. Bst DNA polymerase (large fragment, NEB company) aqueous solution with a concentration of 8U / μL;

[0062] d. Mixed solution...

Embodiment 3

[0071] Cross isothermal amplification reaction (CPA) detection Shiga toxin type II Escherichia coli specific test, comprising the following steps:

[0072] The genomic DNA of Shiga toxin type II Escherichia coli and other bacterial strains were established according to the reaction system and conditions in Example 2 to establish a cross-isothermal amplification reaction detection method, and a specificity test was carried out; wherein,

[0073] Non-Shiga toxin type II Escherichia coli are: methicillin-resistant Staphylococcus aureus NCTC10442 (purchased from the British NCTC Culture Collection); Staphylococcus aureus ATCC23235; Staphylococcus aureus ATCC25923; Salmonella ATCC29629; Salmonella ATCC19585; Salmonella ATCC14028 ; Salmonella ATCC13076; Listeria monocytogenes ATCC19116; Listeria monocytogenes ATCC19114; Listeria monocytogenes ATCC19115; bacteria ATCC10145; Pseudomonas aeruginosa ATCC15442; Vibrio parahaemolyticus ATCC17802; Vibrio parahaemolyticus ATCC27969; Lactoba...

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Abstract

The invention discloses a CPA detection primer for escherichia coli shiga toxin type II, a kit and a method thereof. The CPA detection primer is designed for the specific target sequence stx2 of the escherichia coli shiga toxin type II, a primer sequence is shown as SEQ ID NO.1-5, and the primer ensures the reliability of the detection result. The invention further provides a CPA detection kit forthe escherichia coli shiga toxin II type. The CPA detection kit has the advantages of high sensitivity, good applicability, good specificity, simplicity, convenience and rapidness in operation, accurate and reliable result, low detection cost and the like. The CPA detection primer or the CPA detection kit is used for carrying out a cross primer isothermal amplification reaction on a to-be-detected sample, a result can be directly judged and read by naked eyes through fluorescent dye, whether the to-be-detected sample contains the escherichia coli shiga toxin type II or not can be rapidly andaccurately detected, and the kit is particularly suitable for small and medium-sized units and field detection.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and in particular relates to a CPA detection primer, kit and method for Escherichia coli Shiga toxin type II. Background technique [0002] Shiga toxin-producing Escherichia coli is an important zoonotic pathogen, which can cause mild intestinal discomfort such as watery diarrhea and hemorrhagic enteritis in humans, and lead to hemolytic uremic syndrome, end-stage renal disease and even death in severe cases. The main virulence factors of Shiga toxin-producing Escherichia coli are located on the stx1 and stx2 genes on the phage, and these two genes encode Shiga toxin stx1 and stx2 respectively. Shiga toxins stx1 and stx2 cause infection in patients at very low doses, which can cause severe abdominal pain and diarrhea in patients, and acute renal failure and death in severe cases. [0003] At present, the traditional methods for detection and identification of Shiga toxin-producing E. coli ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6853C12Q1/04C12N15/11C12R1/19
CPCC12Q1/689C12Q1/6853C12Q2527/101C12Q2537/137
Inventor 徐振波骆玉婷陈玲刘君彦梁毅毛雨竹陈雁妮石帆彭瑞欣陈锦璇
Owner SOUTH CHINA UNIV OF TECH
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