环介导等温扩增技术检测炭疽芽孢杆菌的研究  

Detection of Bacillus anthracis by LAMP technique

作　　者：褚红娜[1] 王伟[1] 韩会强[1] 魏峰[1] 马樱子[1] 商静[1] 王海连[1] CHU Hongna;WANG Wei;HAN Huiqiang;WEI Feng;MA Yingzi;SHANG Jing;WANG Hailian(Chengde Centre for Disease Control and Prevention,Chengde,Hebei 067000,China)

机构地区：[1]承德市疾病预防控制中心,河北承德067000

出　　处：《医学动物防制》2023年第11期1085-1088,1092,共5页Journal of Medical Pest Control

基　　金：河北省2023年度医学科学研究课题项目(20231189);2021年承德市科学技术研究与发展计划项目(202109A013)。

摘　　要：目的建立环介导等温扩增技术(loop mediated isothermal amplification,LAMP)检测炭疽芽孢杆菌。方法应用Primer Explorer V5软件设计针对炭疽杆菌的保守区引物,建立LAMP检测方法,检测其特异度和灵敏度,并与荧光定量PCR法进行比较。采用荧光定量PCR法、LAMP和细菌培养鉴定,对45份炭疽疫情标本进行检测。结果LAMP、荧光定量PCR法和细菌培养鉴定的检测结果差异无统计学意义(χ^(2)=3.202,P=0.229)。LAMP扩增产物通过紫外凝胶成像系统显示,炭疽芽孢杆菌强毒株和弱毒株均呈现特异性梯状条带,而其他菌株如痢疾志贺菌、肠炎沙门杆菌和金黄色葡萄球菌等菌株电泳均无条带,扩增结果为阴性。LAMP与荧光定量PCR法检测炭疽芽孢杆菌的检测限均为4.0×10^(2)CFU/ml。结论LAMP使用恒温水浴箱进行等温扩增,反应结束后可在可见光或紫外灯下肉眼观察结果。LAMP灵敏特异,简单快速,无需特殊仪器,可广泛用于基层实验室甚至疫情现场。Objective To establish and apply LAMP for the detection of Bacillus anthracis.Methods Primer Explorer V5 software was used to design the primers for the conserved regions against Bacillus anthracis,LAMP was established,and the specificity and sensitivity of LAMP were tested and compared with fluorescent quantitative PCR.A total of 45 anthrax specimens were detected by fluorescence quantitative PCR,LAMP and bacterial culture identification.Results There was no significant difference in the detection results of LAMP,fluorescence quantitative PCR method and bacterial culture identification(χ^(2)=3.202,P=0.229).The LAMP amplified products showed by the UV Gel imaging system that the strong and weak strains of Bacillus anthracis showed specific ladder like bands,while other strains such as Shigella dysentery,Salmonella enteritis and Staphylococcus aureus and other strains showed no bands on electrophoresis,and the amplification results were negative.The detection limits of LAMP and fluorescence quantitative PCR were all 4.0×10^(2)CFU/ml.Conclusion The LAMP uses a thermostatic water bath for isothermal amplification,and the results can be observed by naked eye under visible light or ultraviolet light after the reaction.The LAMP is sensitive,specific,simple and rapid,and does not require special instruments.Therefore,it can be widely used in primary laboratories and even epidemic sites.

关 键 词：炭疽芽孢杆菌 环介导等温核酸扩增技术 荧光定量PCR法 检测 调查 

分 类 号：R378.72[医药卫生—病原生物学]