Double PCR (polymerase chain reaction) rapid detection method for escherichia coli O157:H7 and kit

Abstract

The invention discloses a double PCR (polymerase chain reaction)) rapid detection method for escherichia coli O157:H7 and a kit; the method comprises the following steps of: selecting a 0517 specific antigen gene rfbE and a coding H7 flagellum gene Flic of escherichia coli as amplified target genes, and simultaneously taking homology of different serotype nucleotides into consideration, and designing two pairs of specific primers which are used for rapidly detecting escherichia coli O157:H7 by PCR (polymerase chain reaction). The method is simple and convenient in operation, saves time, has high specificity and good sensitivity, only can detect the escherichia coli O157:H7 and is not affected by non-F7 serotype and other related bacterium of the escherichia coli. The kit which is researched based on the double PCR (polymerase chain reaction)) rapid detection method is suitable for bulk detection of various samples and provides a new technical means for rapidly detecting the escherichia coli O157:H7.

Description

technical field [0001] The invention relates to a detection technology for Escherichia coli O157:H7, in particular to a double PCR rapid detection method and a kit for Escherichia coli O157:H7. Background technique [0002] Escherichia coli O157:H7 (E.coli O157:H7) is a zoonotic food-borne pathogen with wide prevalence and high pathogenicity to humans, and its infectivity is strong (100-200 bacteria coli can cause infection, while other pathogenic Escherichia coli require more than a million bacteria to cause disease), can infect humans through various livestock and poultry host animals such as cattle, pigs, and chickens, and become popular among humans. Infecting humans can cause Diarrhea, hemorrhagic colitis, hemolytic uremic syndrome and thrombotic thrombocytopenia, etc., can lead to death in severe cases. Therefore, rapid and accurate detection of Escherichia coli O157:H7 is the key to control the outbreak and prevalence of food poisoning or infection caused by it. [...

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Problems solved by technology

[0003] At present, the laboratory diagnosis methods of E. coli O157:H7 are mostly selective medium-Sorbitol MacConkey medium isolation method and antiserum identification method, but both of them have the problems of time-consuming and false positive

Studies have shown that Escherichia coli O157:H7 and non-H7 serotypes are white translucent colonies on Sorbitol MacConkey medium, with similar biochemical characteristics and cannot be distinguished by selective medium; in addition, Escherichia coli O157:H7 and non-H7 The sequence comparison of the flagellar gene (Flic) of H7 serotype bacteria shows that the nucleotide homology is about 70%, and there is antigenic crossover, which is not easy to distinguish by H7 single-factor serum agglutination reaction

Li Li and others studied the multiplex PCR detection of Escherichia coli O157:H7 (Li Li. Multiplex PCR detection of Escherichia coli O157:H7 and the expression of hemolysin protein and its immunogenicity analysis[D]. Nanjing Agricultural University 2009: 21 -32), and established a double PCR detection method with O157 antigen gene rfbE and H7 marker gene Flic as target genes, however, due to the need to extract bacterial DNA and the amplified fragments are too long (812bp and 609bp), processing and detection It takes a long time and the operation is time-consuming; moreover, this study did not cover E. coli O157 non-H7 serotypes, and the possibility of false positives caused by the aforementioned nucleotide homology cannot be ruled out, that is, the specificity of this method is questionable

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