Visetsripong Apirak, Pattaragulwanit Kobchai, Thaniyavarn Jiraporn, Matsuura Ryosuke, Kuroda Akio, Sutheinkul Orasa
Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
Southeast Asian J Trop Med Public Health. 2007 Jan;38(1):82-90.
A rapid method for detection of Escherichia coli O157: H7 using multiplex PCR was developed. Two oligonucleotide primer pairs were used for simultaneously detection of vt encoding verotoxin genes for virulence factor and rfb(O157) encoding the O-antigen specific for E. coli O157: H7. Multiplex PCR generated two products of 215 bp and 420 bp for vt and rfb(O157), respectively. Multiplex PCR detected reference strain O157: H7 (NF-7777) with a sensitivity of 10(5) CFU per ml with no amplification of other 15 pathogenic bacteria. After incubation of 10(2) CFU/25 gram raw meat in tryptic soy broth at 37 degrees C for 8 hours, multiplex PCR conducted with the addition of 100 mg bovine serum albumin produced the two specific PCR products for E. coli O157: H7. This modified multiplex PCR is a rapid, sensitive, and specific technique for detecting and differentiating E. coli O157: H7 and has the potential to be used as an alternative to conventional methods for the screening of O157: H7 strains isolated from raw meat.
建立了一种使用多重聚合酶链反应（PCR）检测大肠杆菌O157:H7的快速方法。使用两对寡核苷酸引物同时检测编码毒素的vt基因（一种毒力因子）和编码大肠杆菌O157:H7特异性O抗原的rfb(O157)。多重PCR分别产生了215 bp和420 bp的vt和rfb(O157)两种产物。多重PCR检测参考菌株O157:H7（NF-7777）的灵敏度为每毫升10(5) CFU，其他15种病原菌无扩增。将10(2) CFU/25克生肉在胰蛋白胨大豆肉汤中于37℃孵育8小时后，加入100毫克牛血清白蛋白进行多重PCR，产生了大肠杆菌O157:H7的两种特异性PCR产物。这种改良的多重PCR是一种快速、灵敏且特异的检测和区分大肠杆菌O157:H7的技术，有潜力作为从生肉中分离的O157:H7菌株常规筛选方法的替代方法。
