基因芯片技术直接检测常见腹泻病致病菌的应用研究

Application of microarray on the detection of pathogens of common diarrhea

目的探讨基因芯片技术在腹泻病粪便标本常见致病菌检测中的应用。方法筛选7种常见腹泻病致病菌的特异基因作为检测目的基因,分别为志贺菌ipaH基因、沙门菌hilA基因、副溶血弧菌tdh基因、空肠弯曲菌FlaA基因、肠出血性大肠埃希菌stxA2基因、肠产毒性大肠埃希菌st基因、霍乱弧菌ctxA2基因。设计相应的引物及探针,制备寡核苷酸芯片。对多重PCR扩增条件进行优化,将PCR扩增产物与带有7种特异探针的基因芯片杂交。检测标准菌株25株,模拟粪标本及临床腹泻病粪便标本64份。结果经优化多重PCR扩增出7种腹泻病致病菌特异的致病基因片段,与基因芯片特异的探针杂交后,全部产生特异性杂交信号。标准菌株及模拟粪标本检测的阳性和阴性对照符合率均为100%。单一菌株最低检测量为10～100cfu/ml。64份腹泻病粪便标本检测出45份致病菌,其中33份志贺菌、12份副溶血弧菌,检出率为70.3%(45/64)。粪培养检出16份志贺菌,6份副溶血弧菌,检出率为34.4%(22/64),基因芯片法明显优于粪培养方法(P<0.01)。结论该基因芯片直接检测腹泻病致病菌,检出率高,达到了高效的检测目的,具有较好的实用性。

Objective To study the application of microarray on the direct detection of common pathogens of diarrhea.Methods Seven specific genes of pathogens of common diarrhea were selected as target genes for detection.They were ipaH gene of Shigella,hilA of Salmonella,tdh of Vibrio Parahemolyticus,stxA2 of enterohemorrhagic Escherichia coli,FlaA of Campylobacter jejuni,st of enterotoxigenic Escherichia coli,and ctxA2 of Vibrio cholera.The specific probes and primers were designed and the microarray was developed.The seven-multiplex PCR system was optimized and the amplified products were hybridized with the GeneChip containing the seven specific probes mentioned above.Twenty-five standard bacteria strains and 64 simulated or stool samples of diarrheal diseases were tested directly by using the system.Results Seven specific gene segments were amplified by the optimized multiplex PCR.All segments were successfully connected with each specific probe in the microarray system.All the results in the standard bacteria strains and simulated stool samples were in accordance with the positive and negative controls（100%）.The lowest limit of positive detection for single bacterial strain was 10-100cfu/ml.From 64 stool samples of diarrheal disease,a total of 45 pathogenic bacterial strains were detected by microarray,including 33 Shigella strains and 12 Vibrio parahaemolyticus strains.The detection rate was 70.3%（45/64）,significantly higher than that by conventional stool culture,from which only 16 Shigella strains and 6 Vibrio parahaemolyticus strains were detected with detection rate of 34.4%（22/64）.Conclusions The microarray system established in the present study is highly sensitive,specific and efficient in detection of clinical samples of diarrheal diseases,and it is suitable for clinical application.