【摘要】 目的:对牛粪及牛肉中的非O157大肠杆菌进行分离、鉴定和毒力基因携带情况进行检测,以了解非O157大肠杆菌的污染状况。方法:参考USDA检测方法,对样品进行选择性增菌,用多重聚合酶链式反应(polymerase chain reaction,PCR)方法进行初步筛选,检测O抗原(O157、O121、O111、O103、O26),阳性样本用选择性显色培养基rainbow agar分离纯化,可疑菌株用多重PCR方法鉴定O抗原,PCR-限制性片段长度多态性鉴定H抗原,并采用血清凝集实验进行验证,确认的阳性菌株采用多重PCR方法进行毒力基因(stx1、stx2、eae、hly)检测。结果:在153份牛粪和49份牛肉样本中,共40份样品检出1个或多个O血清型阳性,牛粪检出率高于牛肉,非O157阳性率为19.3%,O157的阳性率为0.50%;经分离纯化后,共鉴定出阳性菌株30株,其中O26最多占73.3%,O121、O103、O157分别占16.7%、6.7%、3.3%。毒力基因检测结果显示,分离自牛肉的2株O26:H11,一株stx1、hly阳性,另一株hly阳性,分离自牛粪的1株O26:H11携带hly基因,因此30株菌株中带毒菌株阳性率为10.0%,非O157产志贺毒素大肠杆菌阳性率为3.4%。结论:牛粪和牛肉中非O157大肠杆菌的污染率明显高于O157,尤其是O26:H11血清型最高,且检出含志贺毒素基因的大肠杆菌,这提示零售牛肉市场存在非O157大肠杆菌污染的安全风险,我国应该加强对非O157大肠杆菌的检测和监控。更多还原

【Abstract】 Objectives: The aims of this study were to isolate and identify non-O157 Escherichia coli strains from bovine fecal and beef samples and to detect the virulence genes of the isolates, in order to understand the contamination status of non-O157 E. coli. Methods: After selective enrichment of beef and fecal samples by the USDA method with slight modifications, multiplex PCR was used as a prescreening method to determine the O-groups(O157, O121, O111, O103 and O26). The enriched positive sample was streaked for isolation and purification on chromogenic rainbow agar, and tested for the O-groups. The flagellar antigens were identified by PCR-restriction fragment length polymorphism(PCR-RFLP) and the positive isolates were further verified by means of serum agglutination test. The virulence genes(stx1, stx2, eae, and hly) of the positive isolates were detected by PCR. Results: A total of 153 fecal samples and 49 beef samples were collected.Overall, 40 samples were tested positive for one O-group, and the detection rate in feces was higher than that in beef, 19.3%of which were tested positive for non-O157, and 0.50% positive for O157. After purification, 30 positive isolates were identified. O26 was detected most frequently, accounting for 73.3% of the total isolates, followed by O26, O121, O103,and O157, accounted for 73.3%, 16.7%, 6.7% and 3.3%, respectively. The results of virulence gene analysis showed that two strains of O26:H11 were isolated from beef, one being positive for stx1 and hly and the other being positive for hly.One strain of O26:H11 harboring the hly gene was obtained from feces. Therefore, 10.0% of the 30 strains were positive for virulence genes, and 3.4% were non-O157 STEC. Conclusion: The contamination rate of non-O157 E. coli, especially O26:H11, in feces and beef was significantly higher than that of O157. Shiga toxin-producing E. coli was even detected in the beef samples, which suggested that there is a potential risk of non-O157 E. coli contamination in the retail beef market.The detection and monitoring of non-O157 E. coli should be strengthened in China.更多还原