摘要
目的:建立一种同时快速检测肠出血性大肠杆菌(EHEC)O157∶H7和霍乱弧菌O139的基因芯片,并验证该芯片的特异性和敏感性。方法:选择EHEC O157∶H7的产志贺样毒素基因stx1、stx2和β-葡糖醛酸糖苷酶基因(u idA);霍乱弧菌O139的肠毒素A亚单位(ctxA)、毒力协调菌毛A亚单位(tcpA)、糖基转移酶(glycosotransferaseLPSgt)基因序列分别设计引物和探针,反向引物用荧光素Cy3标记,探针在3′端氨基修饰。在优化的PCR和杂交反应条件下,分别进行三重PCR扩增,产物混合后与芯片进行杂交,产生特异性荧光信号,进一步筛选探针。随后将筛选出的探针制备芯片用于检测临床样本。结果:PCR产物在相应探针处均产生特异性杂交信号,临床样本检测结果表明,此芯片比常规细菌学检测方法灵敏。结论:所研制的同时定性检测EHEC O157∶H7和霍乱弧菌O139的基因芯片是特异、灵敏而且快速的,为这两种肠道致病菌感染的快速诊断提供了新的方法。
Objective： To develop a rapid, specific and sensitive method of genechip assay for simultaneous identification of enterohemorrhagic Escherichia coli（EHEC） O157：H7 and Vibrio cholerae O139. Methods： Three pairs of primers were designed for EHEC O157：H7 multiplex PCR, corresponding to stxl, stx2 （Shiga-like toxins gene） and uidA （β-glucuronidase gene） respectively,and three pairs of primers for O139 corresponding to toxin sub-unit A （ctxA）, toxin co-regulated pili subunit A （tcpA） and glycosotransferase （LPSgt）. Sixty spare oligonucleotide probes were designed to capture the reverse strand of the PCR product. Consequently, only the reverse primers required fluorescein （Cy3） modification at 5＇-terminal, and all probes were synthesized with 3＇ amino-group modification. Then the multiplex PCR system was optimized, two sets of multiplexed PCR amplified product were mixed and then hybridized on the surface of the microarray containing sixty oligonucleotide probes, and specific signal were detected. The best probes were chosen according to the six reference gene segments and were used to prepare the microarray which contained positive control and negative control. The microarray was used to detect clinical samples. Results： The results showed that the sensitivity of oligonucleotide microarray was better than that of the routine bacteriology method. Conclusion： It is proved that the microarrs,y is a reliable method for detection and identification of EHEC O157：H7 and V. eholerae O139 simultaneously.
出处
《军事医学科学院院刊》
CSCD
北大核心
2006年第2期122-126,130,共6页
Bulletin of the Academy of Military Medical Sciences
基金
国家科技攻关计划项目(2003BA712A03-09)
