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Primer group and kit prepared by same and application

A technology of primer sets and kits, applied in the field of biological detection, can solve laborious and time-consuming problems, achieve broad market application prospects, serious cross-reaction, and simple operation

Inactive Publication Date: 2017-10-24
SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional hemagglutination test for the O antigen is the main method for detecting serotypes. This method is time-consuming and laborious. PCR detection technology has been developed and applied in the detection process of E. coli serotypes. Currently, multiplex PCR for serotypes O103, O111, and O145 The detection method has not been reported yet. Therefore, a multiplex PCR detection method suitable for different detection platforms is studied and applied to the detection method of O103, O111, O145 serotypes and assembled into a kit, which is specific, sensitive, time-saving and labor-saving. The advantage is the technical problem that the present invention needs to solve

Method used

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  • Primer group and kit prepared by same and application
  • Primer group and kit prepared by same and application
  • Primer group and kit prepared by same and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Design of Primer Sets for Detection of Escherichia coli O103, O111, O145 Serotypes

[0038] A primer set, the nucleotide sequences of which are respectively:

[0039] Upstream of O103: 5'-TGATTGGAGCGTTAACTGGACCT-3',

[0040]Downstream of O103: 5'-AAAGCTCCCGAGCACGTATAAAG-3';

[0041] Upstream of O111: 5'-ATTTGTTTCTTCGATGTTGCGAG-3',

[0042] Downstream of O111: 5'-AAGGCAAGGGACATAAGAAGCCA-3';

[0043] Upstream of O145: 5'-ATTTTCATTGTTTTGCTTGCTCG-3',

[0044] Downstream of O145: 5'-CAAGGCAAGCTTTGGAAATGAAA-3';

[0045] Three pairs of serotype-specific primers were designed. The 327bp, 444bp and 756bp serotype-specific target fragments of O103, O111 and O145 were respectively amplified according to the designed primer sets.

Embodiment 2

[0047] Preparation of Escherichia coli O103, O111, O145 serotype detection kit

[0048] (1) Preparation of E. coli template

[0049] Take a single colony of Escherichia coli and inoculate it into LB liquid medium, and enrich the bacteria overnight at 37°C. Take 1mL of bacterial liquid each to make a template for later use.

[0050] (2) Preparation of specific primers

[0051] The sizes of target fragments amplified by specific primers for Escherichia coli O103, O111 and O145 detected by this kit are 327bp (O103), 444bp (O111) and 756bp (O145), respectively. The concentration of each primer is 5 μM, and the primer dilution is stored at -20°C for future use, avoiding repeated freezing and thawing; it can be artificially synthesized according to the following primer sequence.

[0052] Upstream of O103: 5'-TGATTGGAGCGTTAACTGGACCT-3',

[0053] Downstream of O103: 5'-AAAGCTCCCGAGCACGTATAAAG-3';

[0054] Upstream of O111: 5'-ATTTGTTTCTTCGATGTTGCGAG-3',

[0055] Downstream of O1...

Embodiment 3

[0064] Specificity and Sensitivity Evaluation of Escherichia coli O103, O111, O145 Serotype Detection Kit

[0065] In this example, the main characteristics such as specificity, sensitivity and repeatability of the Escherichia coli O103, O111, O145 serotype detection kits prepared in Example 2 were evaluated respectively.

[0066] (1) Kit specificity test

[0067] Select Escherichia coli O26, O45, O121, O103, O145, O113, O111, O157, O78, O101, Salmonella typhimurium (CVCC3384), Salmonella enteritidis (CVCC1805), Salmonella pullorum (CVCC519), Salmonella duck (CAU0118), poultry Pasteurella (CVCC493), Mycoplasma gallisepticum (CVCC1651), Mycoplasma avium (CVCC274), Staphylococcus aureus (CVCC543), Bordetella avium (ATCC strain IPDH591-77), Streptococcus suis type 2. PCR was performed according to the instructions of the kit, and the product was identified by electrophoresis on 1.2% agarose gel. Escherichia coli O103, O111, O145 can be amplified to specific bands, and the PCR r...

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Abstract

The invention discloses a primer group and a kit prepared by the same and an application. The kit is capable of carrying out effective amplification on the primer group in a same reaction system. An experiment proves that the detection kit containing the primer group has the characteristics of being specific, sensitive, fast and simple in operation, is capable of relatively well enriching an existing escherichia coli O103, O111 and O145 serotyping detection technology, can meet fast detection requirements of existing escherichia coli O103, O111 and O145 serotyping, is easy to popularize and apply and suitable for screening of three serum escherichia coli in the fields of foods and biology, and has a wide market application prospect.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a primer set and a prepared kit and application thereof for detecting Escherichia coli O103, O111 and O145 serotypes. Background technique [0002] Escherichia coli, also known as Escherichia coli (Escherichia coli), was first discovered in 1885. For a long period of time, it has been regarded as a component of normal intestinal flora and considered to be a non-pathogenic bacterium. It was not until the middle of the 20th century that some specific serotypes of E. coli were recognized as pathogenic to humans and animals. Pathogenic Escherichia coli (Pathogenicescherichia coli) mainly causes acute diarrhea, so it is also called diarrhea-causing Escherichia coli. According to the O antigen type, there are about 60 serotypes of pathogenic E. coli, which can be divided into 5 categories according to the pathogenesis: ① Enterotoxigenic E. coli (ETEC); ② Enteropathogenic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12N15/11C12R1/19
CPCC12Q1/689C12Q2600/16
Inventor 王金良沈志强陈金龙董林孙翠平
Owner SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY