A型肉毒梭菌atx基因TaqMan探针荧光定量PCR检测  

Detection of atx gene in Clostridium botulinum using TaqMan probe FQ-PCR

作　　者：王春晖[1] 赵素慧[1] 韦耀[1] 周莹[1] 万成松[1] 

机构地区：[1]南方医科大学公共卫生与热带医学学院中心实验室,广东广州510515

出　　处：《中国公共卫生》2012年第6期863-865,共3页Chinese Journal of Public Health

基　　金：广东省社会发展领域科技计划项目(2010B031000005);广州市科技亚运专项(2010U1-E00591)

摘　　要：目的采用TaqMan探针荧光定量PCR方法对A型肉毒梭菌atx基因进行检测。方法以atx基因为靶基因,设计引物和TaqMan探针,优化反应条件,制作定量标准曲线,进行灵敏度、特异性和重复性验证,建立A型肉毒梭菌TaqMan荧光定量PCR检测方法。结果构建质粒pUC57-△atx,标准曲线在103～107拷贝数之间有较好的线性关系,相关系数为0.997,灵敏度达到22个拷贝数,比普通PCR提高约100倍;能选择性检测A型肉毒梭菌,与其他5种食源性病原菌无交叉反应,结果与普通PCR一致;重复性试验表明,同一浓度的15个平行样品的变异系数为1.0%。结论 A型肉毒梭菌TaqMan探针荧光定量PCR方法具有灵敏度高、特异性强、重复性好等特点,可以快速、准确、定量地检测A型肉毒梭菌。Objective To detect the atx gene of Clostridium botulinum（C.botulinum） type A neurotoxin（bont/A） using TaqMan probe fluorescence quantitative polymerase chain reaction（FQ-PCR）.Methods By targeting the gene atx of bont/A,designing primer and TaqMan probe,optimizing the reaction conditions,making quantitative standard curve,and verifying sensitivity,specificity and repeatability of the method,we established a TaqMan FQ-PCR method to detect bont/A.Results Plasmid pUC57-△atx was constructed successfully.The standard curve established had good linearity when gene quantity was between 103-107 copies,and the coefficient of correlation was 0.997.The limit of detection for FQ-PCR was 22 copies.The sensitivity was about 100 times higher than that of ordinary PCR.The FQ-PCR method could selectively detect C.botulinum and there were no cross reactions with other five food-borne pathogen samples,consisting with the results of ordinary PCR.Repeatability tests showed that the coefficient of variation of 15 parallel samples in same concentration was only 1.0%.Conclusion A TaqMan probe FQ-PCR method with high sensitivity and specificity,and good repeatability was established for rapid,accurate and quantitative detectoin of C.botulinum type A.

关 键 词：肉毒梭菌 TAQMAN探针 atx基因 荧光定量PCR 

分 类 号：R-331[医药卫生]