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Dual-PCR detecting method for pathogenic enterocolitis yersinia

A technology for enterocolitis and Yersinia, which is applied in the field of food safety detection, can solve the problems of instability, poor specificity, cumbersome operation, etc., and achieve the effect of strong specificity, high sensitivity, simple and fast operation

Inactive Publication Date: 2015-03-04
HEFEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The object of the present invention is to provide a kind of pathogenic Yersinia enterocolitica double PCR detection method, the gene that this method selects promptly comprises the ail gene that is located on the chromosome, comprises the virF gene that is located on the plasmid again, reaches the detection of pathogenic Yersinia enterocolitica. The rapid detection of pathogenic strains overcomes the shortcomings of traditional pathogenic Yersinia enterocolitica such as cumbersome operation, instability, and poor specificity, and also avoids the missed detection of virulent strains based on a single virulence gene

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  • Dual-PCR detecting method for pathogenic enterocolitis yersinia
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  • Dual-PCR detecting method for pathogenic enterocolitis yersinia

Examples

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Embodiment 1

[0019] Example 1 Establishment of a dual PCR detection system for pathogenic Yersinia enterocolitica

[0020] 1. Extraction of genome and plasmid DNA: Inoculate Yersinia enterocolitica isolated by GB / T4789.8-2008 method into sterilized fresh soybean tryptone broth, cultivate overnight at 25-28°C, Take 1ml of bacterial solution or different dilutions of bacterial solution (6.5×10 9 -6.5×10 0 cfu / mL) Use the Genomic DNA Extraction Kit and Plasmid Extraction Kit of Guangzhou Meiji Biological Co., Ltd. to extract the genomic DNA and plasmid DNA of the pathogenic bacteria respectively, and store them at -30°C for later use;

[0021] 2. Double PCR amplification procedure:

[0022] PCR system (25 μL): 0.5-1.0 μL primers (the original primer concentration is 10 μM), including ail gene (its nucleotide sequence is as SEQ ID NO.1) primer and virF gene (its nucleotide sequence is as SEQ ID NO. 2) primers, wherein, ail gene primer: F: 5'-taatgtgtacgctgcgag-3' (its nucleotide sequence is...

Embodiment 2

[0028] Example 2 Application test of double PCR sensitivity detection of pathogenic Yersinia enterocolitica

[0029] Add 1 gram of yogurt (negative for pathogenic Yersinia enterocolitica) into 8ml sterile water, and then dilute with different dilutions (6.5×10 6 cfu / mL, 6.5×10 5 cfu / mL, 6.5×10 4 cfu / mL, 6.5×10 3 cfu / mL, 6.5×10 2 cfu / mL, 6.5×10 1 cfu / mL, 6.5×10 0 cfu / mL) of pathogenic Yersinia enterocolitica. Aseptically pipette 1.0ml of the bacterial solution to extract the genome and plasmid DNA of the pathogenic bacteria using a genome extraction kit and a plasmid extraction kit, respectively.

[0030] PCR system (25 μL): 1.0 μL primers (original primer concentration 10 μM) (ail gene: F: 5'-taatgtgtacgctgcgag-3', R: '-gacgtcttacttgcactg-3'; virF gene: F: 5'-ggcagaaca gcagtcagacata-3 ', R: 5'-ggtgagcatagagaatacgtcg-3'), 200-250μM dNTP, Ta q DNA polymerase 4U, 10× buffer 2.5μL, 5ng DNA template, add double distilled water to make up to 25μL.

[0031] Double PCR amplifi...

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Abstract

The invention discloses a dual-PCR detecting method for pathogenic enterocolitis yersinia. The dual-PCR detecting method for the pathogenic enterocolitis yersinia comprises the following steps of treating samples, extracting nucleic acid, performing PCR amplification and detecting PCR products. The dual-PCR detecting method can be used for detecting virulence genes on chromosome, and also can be used for detecting pathogenic plasmids, has the advantages of strong specificity, simple and quick operation, high sensitivity and the like, and can be used for avoiding the missing detection results caused by single PCR detection and the defects of the conventional pathogenic detection.

Description

technical field [0001] The invention belongs to the technical field of food safety detection, and in particular relates to a double PCR detection method for pathogenic Yersinia enterocolitica. Background technique [0002] Enterocolitis is an important foodborne pathogen that causes acute gastrointestinal and bacteremia diseases in humans. Yersinia enterocolitica is the third most common intestinal infection after salmonella and campylobacteriosis in Europe. Yersinia enterocolitica can be divided into pathogenic and non-curative. The virulence factors of pathogenic strains include virulence genes on the chromosome and pathogenic plasmids, while non-pathogenic strains do not contain virulence virulence genes or virulence plasmids. At present, the traditional methods for identifying the pathogenicity of the bacteria include self-coagulation test, Congo red test, calcium-dependent growth at 37°C, etc., but these methods are time-consuming, poor in specificity, unstable, and t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/686C12Q2537/143
Inventor 叶应旺韩永佳焦芮高吉娜
Owner HEFEI UNIV OF TECH
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