多重PCR检测蜡样芽孢杆菌毒力基因方法学建立与初步应用  

Establishment of multiplex PCR for detection of virulence genes in Bacillus cereus and its preliminary application

作　　者：陈雨珊 向琳 晏嘉 王娟 徐淼玲 刘加伟 李齐光 徐令清 李林海 CHEN Yu-shan;XIANG Lin;YAN Jia;WANG Juan;XU Miao-ling;LIU Jia-wei;LI Qi-guang;XU Ling-qing;LI Lin-hai(Guangzhou University of Chinese Medicine,Guangzhou,Guangdong 510006,China;不详)

机构地区：[1]广州中医药大学,广东广州510006 [2]清远市人民医院检验科,广东清远500518

出　　处：《中华医院感染学杂志》2024年第5期646-651,共6页Chinese Journal of Nosocomiology

基　　金：广东省中医药局基金资助项目(20201407);广东省医学科学技术研究基金资助项目(A2021490);清远市科技计划基金资助项目(200808114560452,2022808114560471);清远市人民医院医学科研基金资助项目(20190209,202301-201,318)。

摘　　要：目的 建立且优化一种能同时检测蜡样芽孢杆菌三种毒力基因的多重聚合酶链式反应(PCR)体系,初步应用于医院内环境中蜡样芽孢杆菌毒力基因检测。方法 以蜡样芽孢杆菌的细胞毒素K(cytK)、肠毒素FM(entFM)、非溶血性肠毒素(nhe)三种毒力基因为靶基因,进行多重PCR,优化体系的引物浓度、退火温度、电泳时间和电压,验证该多重PCR体系灵敏度、特异性以及稳定性,并检测医院环境中蜡样芽孢杆菌毒力基因携带情况。结果 成功建立多重PCR体系,nhe、cytK和entFM最佳引物浓度分别为8、20和6μm,退火温度为56℃,电泳条件为80V、30min。该体系灵敏度可达10^(-1)ng/μl,重复性好,特异性良好,可在-20℃稳定保存1个月。医院环境中蜡样芽孢杆菌毒力基因携带情况为:cytK基因携带率为75.00%,entFM基因携带率为66.67%,nhe基因携带率为16.67%。结论 多重PCR可同时检测蜡样芽孢杆菌三种毒力基因,操作简便,重复性好,具有灵敏、特异、稳定的优点;医院环境中蜡样芽孢杆菌的毒力基因携带率高,提示应加强医院环境的清洁与消毒。此文为蜡样芽孢杆菌毒力基因的快速检测和芽孢感染的预防提供依据。OBJECTIVE To establish and optimize a multiplex polymerase chain reaction(PCR)system that can de-tect three types of virulence genes in Bacillus cereus and preliminarily apply it in the detection of virulence genes in B.cereus strains from the hospital environment.METHODS The three types of virulence genes cytotoxin(cytK),enterotoxin FM(entFM)and non-hemolytic enterotoxin(nhe)were set as the target genes,the multiplex PCR was carried out,the concentration of primers,annealing temperature,electrophoresis time and voltage of the sys-tem were optimized.The sensitivity,specificity and stability of the multiplex PCR system were verified,and the virulence genes in the B.cereus strains isolated from the hospital environment were detected.RESULTS The multi-plex PCR system was successfully established;the optimal primer concentrations of nhe,cytKand entFM were 8,20 and 6μm,respectively.The annealing temperature was 56℃,and the electrophoresis condition was 80 V,30 min.The sensitivity of the system was as high as up to 10^(-1) ng/μl,with the repeatability favorable,the speci-ficity high,and it could be stored for 1 month at-20℃.The carrying rate of cytK gene of the B.cereus strains isolated from the hospital environment was 75.00%,the carrying rate of entFM gene was 66.67%,and the carry-ing rate of nhe gene was 16.67%.CONCLUSION Three types of virulence genes can be detected in the B.cereus strains by multiplex PCR.The system is simple,repeatable,sensitive,specific and stable.The carrying rates of virulence genes of the B.cereus strains isolated from the hospital environment are high,indicating that the cleaning and disinfection of the hospital environment should be strengthened.The paper may provide bases for rapid detec-tion of the virulence genes and prevention of B.cereus infection.

关 键 词：蜡样芽孢杆菌 细胞毒素K 肠毒素FM 非溶血性肠毒素 多重聚合酶链反应 快速检测 

分 类 号：R446.5[医药卫生—诊断学]