牛源性非O157大肠杆菌的分离与鉴定

doi:10.7506/spkx1002-6630-20180103-041

食品科学 ›› 2019, Vol. 40 ›› Issue (4): 299-304.doi: 10.7506/spkx1002-6630-20180103-041

牛源性非O157大肠杆菌的分离与鉴定

禹金龙1，董?晨2，王娴静1，姬赛赛1，付文静1，胡?洁1，江?芸1,*

（1.南京师范大学金陵女子学院，江苏?南京 210097；2.江苏省疾病预防控制中心，江苏?南京 210009）

出版日期:2019-02-25 发布日期:2019-03-05
基金资助:
国家自然科学基金面上项目（31671915）；江苏省“六大人才高峰”项目（NY-080）

Isolation and Identification of Bovine-Derived Non-O157 Escherichia coli Strains

YU Jinlong1, DONG Chen2, WANG Xianjing1, JI Saisai1, FU Wenjing1, HU Jie1, JIANG Yun1,*

(1. Ginling College, Nanjing Normal University, Nanjing 210097, China; 2. Jiangsu Provincial Center for Disease Control and Prevention, Nanjing 210009, China)

Online:2019-02-25 Published:2019-03-05

摘要/Abstract

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。??

关键词: 非O157大肠杆菌, 牛粪, 牛肉, 污染状况, 毒力基因

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.

Key words: non-O157 E. coli, feces, beef, contamination status, virulence genes

中图分类号:

TS201.3

禹金龙，董晨，王娴静，姬赛赛，付文静，胡洁，江芸. 牛源性非O157大肠杆菌的分离与鉴定[J]. 食品科学, 2019, 40(4): 299-304.

YU Jinlong, DONG Chen, WANG Xianjing, JI Saisai, FU Wenjing, HU Jie, JIANG Yun. Isolation and Identification of Bovine-Derived Non-O157 Escherichia coli Strains[J]. FOOD SCIENCE, 2019, 40(4): 299-304.

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牛源性非O157大肠杆菌的分离与鉴定

Isolation and Identification of Bovine-Derived Non-O157 Escherichia coli Strains

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