霍乱弧菌和大肠杆菌O157∶H7检测芯片的制备

Preparation of the DNA microchip for detection of Vibrio cholerae and enterohemorrhagic E.coli O157∶H7

目的设计和制备快速检测霍乱弧菌和大肠杆菌O157∶H7基因芯片。方法选择霍乱弧菌特异的编码外膜蛋白的ompW基因和毒素ctxA基因以及大肠杆菌O157∶H7特异的编码菌体抗原rfbE、鞭毛抗原fliC和毒素SLT1、SLT2基因,设计引物和探针,并制备检测芯片,通过两次PCR扩增,制备荧光标记的靶序列,并与芯片进行杂交,检测霍乱弧菌和大肠杆菌O157∶H7。结果所有霍乱弧菌和大肠杆菌O157:H7菌株在采用单一和多重PCR两种方法制备的荧光标记靶序列与芯片杂交,均在芯片相应探针处出现阳性信号,非霍乱弧菌和非O157:H7菌株杂交结果均为阴性。结论基因芯片可以快速检测霍乱弧菌的O157∶H7。

A rapid, specific and sensitive DNA microchip for detection of Vibrio cholerae and enterohemorrhagic E. coli O157 ： H7 was prepared, in which primers and specific probes were designed according to the genetic sequences of the ompW gene encoding the outer membrane protein of V. cholerae, ctxA gene encoding the toxin subunit A, rfbE gene encoding an enzyme involved in the biosynthesis of the somatic antigen O157, fliC gene encoding the flagellar antigen H7, and Shiga-like toxin（SLT）-1 and 2 genes encoding the virulence factors of E. coli O157 ： H7. The fluorescence-labeled PCR products were incorporated to Cy3qabeled primer during PCR amplification and were hybridized to the microchips for the detection of V. cholerae and E. coli O157 ： H7 as well as the non-V, cholerae and E. coli O157 ： H7 pathogens. It was found that positive signals were all displayed on the microchips in strains of V. cholearae and E. coli O157：H7, when the fluorescence-labeled target sequence was hybridized to the microchips prepared with single or multiple PCR amplification, but the non-V, cholerae or E. coli O157： H7 pathogens failed to yield any signals under comparable conditions. It is concluded that mieroassay with microchips prepared in the present study is proved to be a rapid, specific and efficient method for the detection of V. cholerae and E. coli O157：H7.