应用多重引物PCR技术检测O157∶H7毒力基因

Using multiplex PCR for the detection of virulence genes in Escherichia coli O157∶H7

目的 对江苏省 6个不同地区不同宿主动物中分离的O15 7∶H7菌株进行毒力基因的检测分析。方法 应用肠出血性大肠杆菌 (EHEC)的多重引物聚合酶链反应 (PCR)方法 ,以志贺样毒素 (SLT2 和SLT1)基因、“粘附抹平”因子eaeA基因和溶血素 (hly)基因为靶基因进行检测。结果 江苏省分离的O15 7∶H7菌株毒力基因携带率为 5 6 .5 % ,不同地区的分离株携带率有所不同 ,个别地区高达 90 %以上 ,有的地区则未检测到带毒力基因的菌株 ,这一结果与不同地区发病率的高低有平行的关系。疾病高发地区 ,菌株毒力基因携带率达 85 .7% (36 /4 2 ) ,低发或散发地区为 5 2 .6 % (10 /19) ,非流行地区为 8.3 % (2 /2 4)。不同的宿主动物分离株其毒力基因携带阳性率从高到低依次为羊 >牛 >猪 >鸡。仅有的一份兔粪便标本分离株也检出毒力基因。菌株毒力基因图谱以SLT2 +eaeA +hly为主 ,占 79.2 % ,其次为SLT2 +SLT1+eaeA +hly和SLT2 +hly ,分别占 16 .6 %和 4.2 % ;有毒力基因的菌株均有hly和SLT2 ,绝大多数菌株有eaeA基因 ,携带SLT1的菌株则较少 ,这与国外一些报道有所不同。结论 O15 7∶H7毒力基因图谱是一个重要的分子流行病学标志 ,应用多重引物PCR方法检测O15 7∶H7毒力基因 ,简便、快速、特异、敏感 。

Objective To detect and characterize the virulence genes in E.coli O157∶H7 isolated from various reservoir in six areas of Jiangsu province. Method The virulence genes of Shiga like toxin(SLT 1 and SLT 2), intimin(eaeA) and hemolysin(hlyA) were chosen as the target genes and amplified in multiplex PCR assays. Results Of the eighty five E. coli O157∶H7 strains, the overall virulence gene prevalence was found to be 56.5 %(48/85). The prevalence rates viralence genes of isolates from various areas were different from 0% up to 90.5 %. It seemed to exist a relationship between the virulence gene prevalence and the level of incidence. In the areas where rates of incidence were divided into high, low, sporadic or zero, the prevalence rates were 85.7 %(36/42), 52.6 %(10/19) and 8.3 %(2/24), respectively. The prevalence rates of isolates were also different from various reservoirs, decrcasing by sheep, cattle, pig and poultry. One isolate from a rabbit was positive for SLT 2, eaeA and hly genes. Of forty eight isolates carrying virulence genes, 38( 79.2 %) had SLT 2, eaeA and hly genes, taking the dominate virulence gene pattern, 8( 16.6 %) had all of the four virulence genes 2( 4.2 %)had both SLT 2 and hly genes respectively. In addition, SLT 1 gene showed a lower prevalence, which was different from some findings abroad. Conclusion Since virulence gene pattern of E.coli O157∶H7 is an important molecular epidemiological marker, it can provide an useful information for epidemiologic studies, and helpful to the design of prevention and control strategies. For virulence gene detection, multiplex PCR seems to be a simple, rapid, specific and sensitive method.