Establishment And Application Of Multiplex Fluorescence Quantitative PCR Detection Method Of The Main Virulence Factors Of Escherichia Coli 0157:h7 And Listeria Monocytogenes

0157:H7 is the main pathogenic strain of enterohemorrhage Escherichia coli,and also it is the most important serum type of hemorrhagic Escherichia coli associated with public health.After Escherichia coli 0157:H7 infected the host,mainly lead to serious complications of infectious diarrhea,hemorrhagic colitis,thrombotic thrombocytopenic purpura,hemolytic uremic syndrome,kidney,seriously leading to death.As one of the four major food borne pathogens as the same with E.scherichia coli 0157:H7,Listeria monocytogenes infected host mainly manifested as gastroenteritis,meningitis,septicemia,damage the nervous system,abortion and mononuclear cells increased.This study established several real-time fluorescence quantitative PCR reaction system and made a primary application to practice by researching for the virulence factors of Escherichia coli 0157:H7 and Listeria monocytogenes,so as to lay the foundation for further research and monitoring of these two kinds of bacteria.Tests were divided into the following four parts:Test Ⅰ Establishment of multiplex fluorescence quantitative PCR detection method of Escherichia coli 0157:H7In this study,madding a homology comparison analysis on the sequences of Escherichia coli 0157:H7 O-antigen rfbE gene,H-antigen fliC gene,(hemolysin gene)hlyA,intimin gene(eaeA),and shiga-like toxin 1,toxin 2 gene published on GenBank,and six pairs of specific primers and corresponding Taqman probes were designed by selecting conserved sequences by software Primer Express 5.0.The 5’-end of probes of rfbE/eaeA,Stxl/hlyA,fliC/Stx2 were individually labeled with FAM,HEX,CY5 fluorescence reporting group,and 3’-end of probes were all labeled with BHQ1 fluorescence quenching group.By optimizing of reaction system and procedures,three testing attributes(detection time,specificity,and sensitivity)were obtained to establish two triple real-time fluorescence quantitative PCR method were developed for rapid and specific detection of Escherichia coli 0157:H7 and four virulence genes.The results showed that the sensitivity of this method was high,the length of target genes Stx1,rfbE,fliC,eaeA,hlyA and Stx2 were 108 bp,87 bp,86 bp,125 bp,141 bp and 118 bp,and the detection limits of recombinant plasmids Stxl,rfbE,fliC,eaeA,hlyA,Stx2 respectively were 20,30,20,20,30,40 copies/μL;Specificity test confirmed that there was no cross reaction with other strains;It had good reproducibility,the coefficient of variations in reproducibility test were all less than 2%;The detection process took about one hour.All the results showed that,the sensitivity,reproducibility and specificity of the triple real-time fluorescence quantitative PCR established in this research were good,which could be used as a powerful method for the rapid detection of Escherichia coli 0157:H7 and virulence genes.Test Ⅱ Establishment of single fluorescence quantitative PCR detection method of Listeria monocytogenesAccording to the sequence of Listeria monocytogenes virulence gene hlyA published on GenBank,and one pair of specific primer and corresponding Taqman probe were designed by selecting conserved sequences.The 5,-end of probe was labeled with FAM fluorescence reporting group,and 3’-end was labeled with BHQ1 fluorescence quenching group.Construction of positive recombinant plasmid hlyA for fluorescence quantitative PCR detection.The reaction system and procedure were optimized to establish a single real-time fluorescent quantitative PCR method and analysis the sensitivity.The results showed that the sensitivity of this method was high,the detection limit was 70 copies/μL;Specificity test confirmed that there was no cross reaction with other strains;It had good reproducibility,the coefficient of variations in reproducibility test were all less than 2%;The detection process took about one hour.The correlation coefficient of the standard curve established by quantitative amplification of the positive recombinant plasmid was 0.998.The results showed that the method had strong specificity and high sensitivity,which can lay the foundation for the rapid and accurate quantitative detection of Listeria bacteria in meat and meat products.Test Ⅲ Establishment of multliplex fluorescence quantitative PCR detection method of Listeria monocytogenes and Escherichia coli 0157:H7The primers and corresponding TaqMan probes designed according to gene hlyA of Listerzamonocytogenes and genes rfbE,Stxl,fliC,Stx2,eaeA,hlyA of Escherichi coli 0157.H7,a multiplex real-time fluorescent quantitative PCR detection method of Escherichia coli 0157:H7 and Listeria monocytogenes was established with recombinant plasmids of two strains as template.The results showed that the specific genes of the two strains could be detected in the same reaction system by fluorescence quantitative PCR as long as the selected channels were different,and the test successfully established a simultaneous detection of Escherichia coli 0157:H7 and Listeria monocytogenes TaqMan fluorescence quantitative PCR detection method.Test IV Detection of pork samples artificially contaminated with Escherichia coli 0157:117 and Listeria monocytogenesAccording to the primers and corresponding TaqMan probes designed of Listeria monocytogenes and Escherichia coli 0157:H7 in this study,and the multiplex real-time fluorescent quantitative PCR detection method of Escherichia coli 0157:H7 and a single real-time fluorescent quantitative PCR detection method of Listeria monoaytogenes were established,and then be applied to the detection of simulated pork samples,the results showed that the lowest detection limits of Escherichia coli 0157:H7 without enrichment or 8h enrichment respectively were 200 CFU/mL and 1 CFU/mL,and Listeria monocytogenes were respectively 200 CFU/mL and 5CFU/mL.