应用液相芯片技术联合多重PCR快速检测四种常见食源性致病菌的研究  

Application of a suspension array technology and the multiplex PCR for rapid detection of four common food-borne pathogens

作　　者：金玉娟[1] 陈应坚[1] 甘莉萍[1] 刘渠[1] 杨慧[1] 李静媚[1] 胡春凌 

机构地区：[1]深圳市龙岗区疾病预防控制中心,广东深圳518172 [2]深圳博睿祥晖生物技术有限公司,广东深圳518000

出　　处：《热带医学杂志》2015年第6期735-740,共6页Journal of Tropical Medicine

基　　金：深圳市科技计划项目(医疗卫生类)(201203347)

摘　　要：目的建立一种基于多重PCR结合液相芯片技术快速、准确的同时检测沙门菌、副溶血性弧菌、单增李斯特氏菌及肠出血性大肠埃希氏菌的方法。方法建立四种常见食源性致病菌的多重PCR扩增方法,将PCR产物与偶联核酸探针的微球混合物进行杂交,利用液相芯片检测仪来检测杂交结果。并对检测方法的灵敏度和特异性进行评价。结果建立了一种特异、快速、准确的检测沙门菌、副溶血性弧菌、单增李斯特氏菌及肠出血性大肠埃希氏菌的液相芯片方法,该方法对于副溶血性弧菌tdh和沙门菌inv A检测的灵敏度为1×104 CFU/ml;单增李斯特氏菌hly A、肠出血性大肠埃希氏菌stx1和stx2检测的灵敏度为1×105 CFU/ml。结果也表明该方法检测的特异性为100%。结论本研究建立的液相芯片检测方法能快速、准确、特异的同时检测沙门菌、副溶血性弧菌、单增李斯特氏菌及肠出血性大肠埃希氏菌四种常见食源性致病菌。Objective To develop a multiplex PCR combined suspension assay for the rapid, accurate and simultaneous detection of Salmonella sp p., Vibrio parahaemolyticus, Listeria monocytogenes and enterohemorrhage E. coli（EHEC）.Methods After a multiplex PCR amplification of four common food-borne pathogens, the PCR products hybridized with microsphere mixtures that have been coupled to gene-specific probes. The suspension array system was used to detect the results of hybridization. The sensitivity and specificity of the method was valued. Results The suspension array was developed for a specific, rapid, accurate detection of Salmonella spp., V. parahaemolyticus, L. monocytogenes and EHEC.The sensitivity of detection of tdh gene of V. parahaemolyticus and detection of inv A gene of Salmonella spp. was 1×10^4CFU /ml. The sensitivity of detection of hly A gene of L.monocytogenes and detection of stx1 and stx2 genes of EHEC was 1×10^5CFU / ml. The method showed good specificity. Conclusion The suspension array could rapidly, effectively, accurately,simultaneously detect four foodborne pathogens Salmonella spp., V. parahaemolyticus, L. monocytogenes and EHEC.

关 键 词：液相芯片 食源性致病菌 多重PCR 

分 类 号：R155.5[医药卫生—营养与食品卫生学]