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Multiplex PCR primer set, kit and detection method for detecting 5 pili genes of enterotoxigenic Escherichia coli

A technology of Escherichia coli and primer set, which is applied in the fields of biochemical equipment and methods, recombinant DNA technology, and the determination/inspection of microorganisms. high sex effect

Active Publication Date: 2021-08-27
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the wide application of PCR technology, there are fewer and fewer pili single-factor serum products, and the glass plate agglutination test is limited
Although there are single-PCR and multiple-PCR methods for detecting Escherichia coli pili genes in China at present, they can only detect four kinds (F4, F5, F6, F41) of pili genes at the same time at most, while for F4, F5, F6, F41, F18 The multiple PCR method of 5 kinds of pilus genes has not been reported yet, therefore, the present invention has set up a kind of multiplex PCR detection method, and this method can realize the simultaneous detection of above-mentioned 5 kinds of pilus genes, is applicable to ETEC pilus serotype identification and The diagnosis of diarrheal diseases in young animals can be popularized and applied

Method used

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  • Multiplex PCR primer set, kit and detection method for detecting 5 pili genes of enterotoxigenic Escherichia coli
  • Multiplex PCR primer set, kit and detection method for detecting 5 pili genes of enterotoxigenic Escherichia coli
  • Multiplex PCR primer set, kit and detection method for detecting 5 pili genes of enterotoxigenic Escherichia coli

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Example 1 Primer Design

[0050] The specific primer pairs designed to detect enterotoxigenic Escherichia coli F4, F5, F6, F41, and F18 pilus genes are as follows:

[0051] The primer pair used to detect the F4 pili gene of enterotoxigenic Escherichia coli is:

[0052] F1: 5'-ATTTCAATGGTTCGGTCG-3' (shown in SEQ ID NO.1)

[0053] R1: 5'-GATTGCTACGTTCAGCGGAGCG-3' (shown in SEQ ID NO.2)

[0054] The primer pair used to detect the F5 pili gene of enterotoxigenic Escherichia coli is:

[0055] F2: 5'-AAACACTGCTAGCTATTATC-3' (shown in SEQ ID NO.3)

[0056] R2: 5'-ATAAGTGACTAAGAAGGATGC-3' (shown in SEQ ID NO.4)

[0057] The primer pair used to detect the F6 pili gene of enterotoxigenic Escherichia coli is:

[0058] F3: 5'-ACTTCTAATCTGTCGCAAACC-3' (shown in SEQ ID NO.5)

[0059] R3: 5'-GAACGAATAGTCATTACTGCAC-3' (shown in SEQ ID NO.6)

[0060] The primer pair used to detect the pili gene of enterotoxigenic Escherichia coli F41 is:

[0061] F4: 5'-ATCAGCGGCAGTATCTGGTT-3' (...

Embodiment 2

[0067] The preparation of embodiment 2 kit

[0068] Described test kit comprises each component shown in following table 1:

[0069] Table 1

[0070]

[0071]

[0072] Wherein, the positive quality control standard product is prepared according to the following method: PCR method is used to amplify enterotoxigenic E. coli F4, F5, F6, F41, F18 pilus genes or partial genes respectively, and the products are respectively combined with the cloning vector pMD- 19-T connection, transforming competent Escherichia coli Trans T1 to obtain positive recombinant bacteria, and preparing positive plasmids according to the instructions of Zhongkeruitai Ordinary Plasmid Mini-prep Kit.

Embodiment 3

[0073] Example 3 Establishment of Multiplex PCR Detection Method for Enterotoxigenic Escherichia coli F4, F5, F6, F41, F18 Pili Genes

[0074] 1. Establishment of Multiplex PCR Detection Method

[0075] (1) Preparation of Escherichia coli DNA template

[0076] Coliform reference strain C83903 (F4 + ), C83199 (F5 + ), C83915 (F6 + ), C83920 (F5 + 、F41 + ), C83684 (F18 + ) inoculated into LB liquid medium, and enriched overnight at 37°C. Take 1 mL of each reference strain culture in a 1.5 mL centrifuge tube, centrifuge at 10,000×g for 5 min, remove the supernatant, add 100 μL of sterilized deionized water to resuspend, boil at 100°C for 10 min, and immediately place in ice-water mixture Cool for 5 minutes, then centrifuge at 10,000×g for 5 minutes, then take the supernatant and store it at -20°C, and use it as a DNA template for later use.

[0077] (2) Multiplex PCR amplification reaction system and composition

[0078] Prepare a multiplex PCR reaction system with a tot...

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Abstract

The invention discloses a multiplex PCR primer set, a kit and a detection method for detecting five pili genes of enterotoxigenic Escherichia coli. The enterotoxigenic Escherichia coli that causes diarrhea in young animals often carries F4, F5, F6, F18 and F41 pili and their coding genes. The present invention designs and synthesizes a set of The primer set was specifically detected, and a multiplex PCR detection method was established for detecting five pili genes of enterotoxigenic Escherichia coli, and a kit containing the specific primer set was assembled. Using the kit of the present invention to detect 5 pili genes of enterotoxigenic Escherichia coli has the advantages of strong specificity, quickness, simplicity, and good sensitivity, and is suitable for rapidly detecting the pili genes of Escherichia coli isolated from diarrhea samples of sick animals. An effective technique for screening and identifying serotypes of enterotoxigenic Escherichia coli pili.

Description

technical field [0001] The invention relates to a multiplex PCR primer set, a kit and a detection method for detecting enterotoxigenic Escherichia coli pilus genes, in particular to detection of enterotoxigenic Escherichia coli F4, F5, F6, F41, F18 pilus genes The multiplex PCR primer set, kit and detection method. The invention belongs to the technical field of biological detection. Background technique [0002] Escherichia coli (E.coli) is a Gram-negative non-spore-free brevibacterium. Since its discovery in 1885, it has been regarded as a component of normal intestinal flora and is a non-pathogenic bacterium. Until the middle of the 20th century, studies found that some special serotypes of Escherichia coli were pathogenic to humans and animals, especially infants and young animals, often causing severe diarrhea and sepsis. At present, pathogenic Escherichia coli can be divided into enterotoxigenic Escherichia coli (ETEC), Shiga toxin-producing Escherichia coli (STEC), ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/10C12N15/11
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143
Inventor 师东方孙思萌董秀梅张萍朱妍苏瑞红唐毓侯美佳冯启峥
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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