多重PCR检测蜡样芽胞杆菌的研究  

Development of multiplex PCR assay for the detection of enterotoxic B. cereus group strains

作　　者：刘秀峰[1] 黄铭珊[2] 潘珍瑜[1] 林萍[1] 强华[3] 

机构地区：[1]福州市疾病预防控制中心福建医科大学教学基地,福建福州350004 [2]福建卫生职业技术学院,福建福州350101 [3]福建医科大学,福建福州350001

出　　处：《中国热带医学》2016年第3期221-225,共5页China Tropical Medicine

基　　金：福州市科技计划项目(No.2014-s-145)

摘　　要：目的建立多重PCR方法检测蜡样芽胞杆菌。方法根据蜡样芽胞杆菌(B.cereus)的hbl A、hbl C、nhe A、nhe B、cyt K、cer、ent FM、bce T基因和苏云金芽胞杆菌(B.thuringiensis)的cry基因设计引物,优化反应条件。应用单一引物PCR和国标法对多重PCR反应结果进行确认。结果蜡样芽胞杆菌溶血性基因hbl A和hbl C检出率为57.5%和25.0%,非溶血性基因nhe A、nhe B检出率分别为75.0%和80.0%,细胞毒素cyt K基因检出率为37.5%。肠毒素ent FM和bce T检出率分别为28.7%和18.7%。蜡样芽胞杆菌和苏云金芽胞杆菌cer基因检出率分别为97.5%和100.0%,cry基因检出率为分别为0.0%和100.0%。cer基因和cry基因同时检测,条带区分清晰,可作为蜡样芽胞杆菌和苏云金芽胞杆菌快速鉴别的首选方法。结论三重PCR结果非常好,三对引物所扩增出的DNA条带明显区分开,电泳条带清晰,敏感度高,特异性强。单重PCR和三重PCR检测结果没有显著性差异。Objective To establish a multiplex PCR assay for the detection of enterotoxic B.cereus group strains.Methods Primers were designed to amplify genes of B. cereus including hbl A, hbl C, nhe A, nhe B, cyt K, cer, ent FM and bce T,and cry gene of B. thuringiensis. The reaction conditions of multiplex PCRs were optimized. Multiplex PCR assay was validatedby comparing with single- PCR assays, and correlated to the classical and biochemical identification of the organisms.Results The detection rates of hemolytic hbl A and hbl C and nonhemolytic enterotoxin nhe A and nhe B were 57.5%, 25%,75.0% and 80.0% respectively. The detection rates for cyt K gene and enterotoxin ent FM and bce T were 37.5%, 28.7% and18.7% respectively. Cer gene had the highest detection rate which was 97.5% for B. cereus and 100% for B. thuringiensis. Crygene was detected in all B. thuringiensis but was undetectable in B. cereus stains. Duplex PCR for Cer and cry genes showeddistinct bands, and thus can be applied to distinguish B. cereus from B. thuringiensis. Conclusion Triplex PCR assay wasfast, sensitive and specific with no significant difference from single-PCRs in gene amplification.

关 键 词：蜡样芽胞杆菌 苏云金芽胞杆菌 多重PCR 毒力基因 腹泻 

分 类 号：R155.3[医药卫生—营养与食品卫生学]