大肠杆菌O116 O-抗原基因簇的破译及PCR检测方法的建立  

Characterization of Escherichia coli O116 O-antigen gene cluster and development of O-serogroup-specific PCR assay

作　　者：韩巍青[1] 王威[1] 王磊[1] 

机构地区：[1]南开大学泰达生物技术学院,天津300457

出　　处：《中国人兽共患病学报》2007年第7期667-671,共5页Chinese Journal of Zoonoses

基　　金：国家"863计划"(2002AA2Z2051);国家杰出青年科学基金资助项目(30125001)

摘　　要：目的测定大肠肝菌O116的O-抗原基因簇序列,鉴定其中的特异基因,建立PCR检测方法,替代传统的血清学检测。方法用长距离PCR方法扩增O116的O-抗原基因簇,PCR产物用鸟枪法建立测序文库进行测序,通过与GenBank中功能已知的蛋白的序列比较,进行基因功能的分析。用PCR方法证明基因簇中寡糖单位处理酶基因的特异性,以寡糖单位处理酶基因为靶基因建立PCR检测方法,并进行灵敏度测试。结果和结论基因簇全长为14323bp,共有11个基因,其中寡糖单位处理酶基因wzx和wzy具有高度特异性。所建立的PCR方法可检测2pg的基因组DNA、0.4CFU/g(猪肉样品)或4×102CFU/g(粪便样品)。可以代替传统的血清学检测。Shiga toxin-producing Escherichia coli （STEC） strains infect human populations and animals worldwide and cause severe disease in which E. coli O116 is one of important serogroups of STEC. Conventional O-serotyping is laborious and time-consuming, and often disturbed by cross-reactions and rough LPS. Rapid and reliable methods are therefore needed to overcome these difficulties for detection of E. coli O116. In the present study, O-antigen gene cluster of E. coli O116 was sequenced using shot-gun method. A sequence of 14 323 bases from galF to gnd was determined, and 11 open reading frames （ORFs） in addition to galF and gnd were identified. The functions of the 11 putative ORFs were assigned based on their amino acid identity to proteins with known functions, and genes were named accordingly. By screening against E. coli （n= 166） and Shigella （n=42） O-serogroup reference strains, O-antigen processing genes （wzx and wzy） were proved to be highly specific for E. coli O116. The sensitivity of test showed that the PCR assay can detect as low as 2 pg genomic DNA, 0.4 CFU/g in raw porck samples or 4×10^2 CFU/g in stool samples. This assay can be used as a fast, sensitive, and specific alternative to O- serotyping for the detection of E. coli O116 strains.

关 键 词：大肠肝菌O116 O-抗原基因簇 特异基因 PCR检测 

分 类 号：R378.2[医药卫生—病原生物学]