Quantitative Detection Of Three Virulence Genes Of Pathogenic Escherichia Coli In Municipal Wastewater By Real-time PCR

Urban sewage reuse is one of the effective way that can improve the integratedwater resources utilization and alleviate the shortage of water resources. Howerver,there are a large amount of pathogenic bacterials in wastewater, which posed a threat onhuman health. Escherichia coli is the most common kind of gut germs. The mainpathogenic of E.coli is related to virulence factor and proteins. These virulence factorscorrespond coding genes, namely virulence genes. Gene eaeA codes out-membraneprotein―inminate. It is a necessary factors that EHEC and EPEC in intestinal mucosasettled and cause adhesion and effacing. Gene rfbE is specific gene of EHEC O157: H7strain. Lipopolysaccharide coded by rfbE, the main of poison factor can cause diarrhea,hemorrhagic colitis and dangerous hemolytic uremic syndrome. Gene EAgg is specificgene of EAEC strain, which can cause acute and long-term performance diarrhea.About the outbreak case of EHEC, EPEC and EAEC have occurred and at home andabroad.For pathogenic Escherichia coli，the quantitative detection method targeted tovirulence genes eaeA, rfbE and EAgg was established by real-time PCR technology,respectively. Recombinant plasmid was constructed by cloning purified DNA fragmentfrom positivewater sample into vector. By BLAST analysis, the specificity ofamplification was proved. Utilizing the recombinant plasmid as targeted gene standard,there was good linear relationship between the Ct values and the logarithm of inputcopy numbers over a range from8.77×10~0copy to8.77×10~5copy for eaeAgene(R~2=0.997),4.98×10~0copy to4.98×10~5copy for rfbE gene(R~2=1.000),and5.34×10~1copy to5.34×106copy for EAgg gene(R~2=0.996). Combined withconcentration procedure, the detection limit was1.75×10~2copy per10~0mL water sample for eaeA,9.96×10~1copy/10~0mL for rfbE and1.07×10~3copy/10~0mL for EAgg,respectively. Through practical amplication, it showed that municipal wastewatertreatment process can effectively remove these virulence genes.Municipal waster was detected by real-time PCR. It made a simple analysis aboutdistribution and tracking sources of virulence factors of Escherichia coli in secondaryeffluent. It showed that there are chickens, pigs and dog in the pollution source of eaeAand pigs, dogs, sheep and duck in the pollution source of EAgg. The pollution source ofrfbE is not clear.