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Multi-PCR (polymerase chain reaction) detection method of four diarrhoeic Escherichia coli and primer group thereof

A technology that causes Escherichia coli and diarrhea, applied in the field of biological detection, can solve the problems of single detection target, long detection time, complex and cumbersome operation, etc.

Inactive Publication Date: 2013-01-02
哈尔滨海关技术中心
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Problems solved by technology

At present, the detection methods for diarrhea-causing Escherichia coli in my country are mainly based on traditional detection methods. The specific operation process is: samples to be tested→enrichment→isolation and culture→Gram staining microscopy / biochemical and colony observation / biochemical reaction test / intestinal Toxin inspection, etc., the operation is complicated and cumbersome, and the detection time is long. It takes 5-10 days to complete the entire detection procedure, and the detection target is single.

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  • Multi-PCR (polymerase chain reaction) detection method of four diarrhoeic Escherichia coli and primer group thereof
  • Multi-PCR (polymerase chain reaction) detection method of four diarrhoeic Escherichia coli and primer group thereof
  • Multi-PCR (polymerase chain reaction) detection method of four diarrhoeic Escherichia coli and primer group thereof

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Embodiment Construction

[0083] The present invention will be further described below in conjunction with specific embodiment:

[0084] 1. Preparation of PCR template

[0085] Extract and purify bacterial genomic DNA according to the instructions of TIANamp Bacteria DNA Kit, and add the corresponding reagents in order:

[0086] 1.1 Take 1.5mL overnight enrichment solution of bacteria in the sample to be tested, centrifuge at 10000r / min for 1min, discard the supernatant, add 200μL buffer GA, and suspend the bacteria thoroughly;

[0087] 1.2 Add 20 μL of proteinase K (20mg / mL), and add 220 μL of buffer solution GB, mix thoroughly, and act in a 70°C water bath for 10 minutes;

[0088] 1.3 Add 220 μL of absolute ethanol, mix thoroughly for 15 seconds, transfer the obtained solution (including flocculent precipitate) to the adsorption column CB3 after brief centrifugation, centrifuge at 12000 r / min for 30 seconds, and discard the liquid in the collection tube;

[0089] 1.4 Add 500μL buffer GD (containin...

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Abstract

The invention relates to a multi-PCR (polymerase chain reaction)-based method for quickly detecting four diarrhoeic Escherichia coli and special primers thereof. The method comprises the following steps: respectively selecting enterohemorrhagic Escherichia coli O157:H7O-antigen gene, enterotoxigenic Echerichia coli LT gene, enteropathogenic Echerichia coli bfpA gene and enteroinvasive Echerichia coli invasion plasmid gene as target genes, carrying out comparative analysis on the gene sequences, selecting the conserved region of the target gene sequence to design and synthesize the primers which are disclosed as SEQ ID NO.1-8 in the sequence table, and establishing a multi-PCR detection method to carry out qualitative detection on the four diarrhoeic Escherichia coli. The method provided by the invention has strong detection specificity. The invention also relates to a kit for detection. The invention is quick and simple and has the advantages of high accuracy and high sensitivity.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for rapidly detecting four kinds of diarrhea-causing Escherichia coli based on multiplex PCR technology and special primers thereof. Background technique [0002] Food safety is a major global public health problem. There are more than 250 pathogenic factors of foodborne diseases reported, among which enteropathogenic bacteria are the most common biological pathogenic factors in foodborne diseases. According to the World Health Organization (WHO), there are hundreds of millions of cases of diarrhea caused by food-borne microbial contamination every year in the world, and about 1.7 million children aged 0-15 die. At present, diarrhea-causing Escherichia coli is still one of the important pathogens causing diarrhea, especially infantile diarrhea. The detection rate, composition ratio, dominant type and serotype of diarrhea-causing Escherichia coli...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10C12N15/11
Inventor 徐义刚李丹丹刘忠梅吴岩刘新亮李苏龙
Owner 哈尔滨海关技术中心
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