Establishment Of Duplex Fluorescence Quantitative PCR Method For Detection Of Bovine ETEC And Separation And Identification

Enterotoxigenic Escherichia coli(ETEC)is a clinically important intestinal pathogens cause diarrhea in infants and young,In order to establish a rapid,simple,accurate and sensitive ETEC detection method,the test according to GenBank of ETEC no resistance to good heat-labile enterotoxin(LT)and heat-labile enterotoxin(ST)gene sequence primers and probe design synthesis,A dual fluorescence quantitative PCR method for detecting ST and LT was established.The positive samples were isolated and identified by ETEC and the isolated were tested for drug sensitivity.The lowest copy number of ST and LT genes in ETEC was 1 ×101copies/?L,which showed high sensitivity.Only LT and ST genes were detected from the reference strains of enterotoxigenic LT and ST,which proved high specificity.The coefficient of variation of intra-group and inter-group tests was less than 2%,indicating good repeatability.Three ST positive and one LT positive ETEC were detected from 60 fecal samples by the established duplex PCR method.The 4 positive samples were further subjected to bacterial isolation,staining microscopy,biochemical analysis and 16S rRNA identification,which proved to be E.coli.The 16S rRNA homology analysis of 4 strains showed that the homology of Hohhot ETEC-3 with E.coli E4 and U12 strains was 96.0%,and that with E.coli Q1 was 99.4%.The highest homology between E.coli Xuyi-382-2 and Hohhot ETEC-4 was 89.7%;The highest homology between isolates Hohhot ETEC-2 and E.coli Q1 was 99.8%,and the genetic analysis showed that isolates Hohhot ETEC-2 had close genetic relationship with E.coli IRQBAS57 strain,which was an evolutionary group.The isolate Hohhot ETEC-1 was closely related to E.coli 42L and E.coli E84-1,and belonged to an evolutionary group.The results of drug sensitivity test showed that the four isolates were sensitive to ciprofloxacin and ofloxacin,sensitive to amicacin and had strong resistance to amoxicillin,penicillin and vancomycin.This study provides a rapid,sensitive and specific method for the detection of ETEC in farms,and provides guidance for the prevention and control of E.coli diseases caused by ETEC.