五种致病菌流式液相芯片检测方法的建立

Development of A Suspension Array Method for Detection of Five Pathogen

用DNAStar软件对GenBank中沙门氏菌的侵袭蛋白invA基因、单增李斯特菌的Hly基因、金黄色葡萄球菌的sa442基因、霍乱弧菌的hlyA基因、副溶血弧菌toxR基因进行序列分析,设计针对这些基因的特异性探针和引物,并标记生物素、探针,分别与不同编号的荧光编码微球偶联后再与相应的PCR产物杂交反应,用液相芯片检测仪（Liquichip 200）检测荧光信号,建立沙门氏菌、单增李斯特菌、金黄色葡萄球菌、霍乱弧菌、副溶血弧菌的快速液相芯片检测方法。检测结果显示,该方法具有较好的特异性,偶联特异性探针的微球只与相应的病菌基因的PCR产物反应,检测灵敏度达到50~100copies/mL。该方法的建立为其它致病细菌和病毒的快速高通量检测提供了借鉴和经验。

InvA gene of Salmonella.spp,Hly gene of Listeria monocytogenes,sa442 gene of Staphylococcus aureus,hlyA gene of Vibrio cholerae and toxR gene of Vibrio parahaemolyticus in the GenBank were analyzed by using the software DNAStar specific gene probes of all the five bacteria labled with biotin were prepared and coupled with fluorescencecoded microspheres.The probes were used for hybridization reaction to PCR products of the five bacteria,and then the liquichip detection technique for detection of all these pathogenic bacteria was established by using liquichip 200 to detect fluorescence signals in the reaction system.The results showed that this method displayed high specificity to PCR products of correspondent bacteria when being used for detection.The sensitivity test indicated that the detecting limitation for the viruses could reach to 50~100 copies/mL.The rapidly high throughput liquichip detection will provide foundation and exploration for further research to detect other pathogenic bacteria and viruses with the same technique.