摘要
目的应用多交叉置换扩增(MCDA)技术建立单增李斯特菌的简单、快速、敏感且特异的诊断方法,并对该方法进行敏感度、特异性及实际应用评价。方法根据单增李斯特菌的种特异性基因lmo0733设计引物,采用荧光染料颜色变化、实时浊度检测及琼脂糖凝胶电泳三种方法确认MCDA产物,对MCDA引物进行最佳反应条件、敏感度、特异性评估,并对实际样本进行检测。结果单增李斯特菌MCDA检测方法的最佳反应温度为61℃。单增李斯特菌MCDA检测方法的敏感度为10fg/反应,分别是环介导等温扩增(LAMP)技术和交叉引物恒温扩增(CPA)技术的25倍和250倍。对153份鼠粪便标本2次增菌培养物的检测结果证实,本研究建立的单增李斯特菌MCDA检测方法的检测能力与分离培养方法(ISO 11290-1)相同,且优于LAMP、CPA和PCR方法。结论 MCDA方法作为一种快速、敏感和高效的检测方法可以应用于食品行业及临床标本中单增李斯特菌的检测。
Listeria monocytogenes is a significant food-borne pathogen that is capable of causing severe listeriosis in both humans and animals.Traditional techniques such as culture-based approaches have good specificity and sensitivity,while they are time-consuming and tend to be tedious.Hence,we presented a simple and rapid molecular technique using multiple cross displacement amplification(MCDA)targeting L.monocytogenes-specific gene lmo 0733 for sensitive and specific detection of the pathogen.L.monocytogenes-MCDA assay used a set of ten primers spanning ten distinct regions of target sequence and was carried out at a constant temperature 61℃to evaluate the specificity,sensitivity and feasibility.A total of three methods were used to confirm MCDA products,including color change of Loopa mp Fluorescent Detection Reagent,real-time measurement of turbidity and agarose gel electrophoresis.The limit of detection(LoD)of the MCDA was 10 fg DNA,which was 25-fold and 250-fold more sensitive than that of the LAMP and CPA assay for detecting L.monocytogenes DNA,respectively.The detection ability of MCDA method was identical to the culture-biotechnical method for 153 rat intestinal feces samples,and was better than LAMP,CPA and conventional PCR.The results in this study indicated that the MCDA assay could be used as a rapid,sensitive and effective tool for the detection of L.monocytogenes in the food industry,medical institutions and field.
作者
李慧
王毅
王艳
李华
陈峥宏
叶长芸
LI Hui;WANG Yi;WANG Yan;LI Hua;CHEN Zheng-hong;YE Chang-yun(Department of Microbiology,School of Basic Medical Science,Guizhou Medical University/ Key Laboratory of Medical Microbiology and Parasitology of Guizhou Province,Guiyang 550004,China;State Key Laboratory of Infectious Disease Prevention and Control,National Institute for Communicable Disease Control and Prevention,Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases,Chinese Center for Disease Control and Prevention,Beijing 102206,China)
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2018年第2期99-104,共6页
Chinese Journal of Zoonoses
基金
国家重大传染病防治科技重大专项(No.2013ZX10004-101)、传染病预防控制国家重点实验室(No.2015SKLID507)和中国疾病预防控制中心传染病预防控制所(No.2016ZZKTB09)联合资助
