重组酶介导的肠出血性大肠杆菌O157:H7等温扩增方法的建立  

Establishment of recombinase-aided amplification method for EHEC O157:H7

作　　者：蔡欣 王源林 赵杰 匡衡 孙学伟 刘宗财 崔茜 杨展 汪茂荣 CAI Xin;WANG Yuanlin;ZHAO Jie;KUANG Heng;SUN Xuewei;LIU Zongcai;CUI Qian;YANG Zhan;WANG Maorong(Department of Hepatology,Qinhuai Medical District,General Hospital of Eastern Theater Command,Bayi Clinical College,Anhui Medical University,Hefei 230000;Center for Disease Control and Prevention of Eastern Theater Command,Nanjing 210002;Medical Clinic of PLA Air Force 93801 Military Hospital,Xianyang 712201;Department of Infectious Diseases,the First Clinical College of Nanjing Medical University,Nanjing 210029;School of Basic Medical Sciences,Binzhou Medical College,Yantai 264003;Department of Military Preventive Medicine,Air Force Military Medical University,Xi’an 710032;Department of Respiratory Medicine,Air Force Hospital of Eastern Theater Command,Nanjing 210001,China)

机构地区：[1]安徽医科大学八一临床学院东部战区总医院秦淮医疗区肝病科教研室,安徽合肥230000 [2]东部战区疾病预防控制中心,江苏南京210002 [3]中国人民解放军空军93801部队医院医疗所,陕西咸阳712201 [4]南京医科大学第一临床医学院感染病科,江苏南京210029 [5]滨州医学院基础医学院,山东烟台264003 [6]空军军医大学军事预防医学系,陕西西安710032 [7]东部战区空军医院呼吸内科,江苏南京210001

出　　处：《南京医科大学学报（自然科学版）》2022年第9期1235-1239,1252,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(81871242);江苏省社会发展项目(BE2016617)。

摘　　要：目的:建立新的快速检测常见食源性致病微生物肠出血性大肠杆菌(enterohemorrhagic E.coli,EHEC)O157:H7的方法。方法:根据EHECO157:H7保守区序列,结合重组酶介导的恒温扩增(recombinase-aided amplification,RAA)和荧光定量或胶体金侧向流免疫层析试纸条(lateral flow dipstick,LFD),建立两种EHEC O157:H7检测方法(RAA荧光法和RAA-LFD法)并进行优化,评估这两种方法的灵敏性及特异性。结果:两种检测方法均可在39℃恒温条件下16 min内完成检测,两种方法的检测限均可达1×10~4拷贝数,具有较高的灵敏性。两种方法与其他常见的肠道致病菌(金黄色葡萄球菌、沙门氏杆菌、弯曲菌、耶尔森菌、志贺杆菌)质粒模板无交叉反应,具有良好的特异性。结论:本研究建立的两种检测EHEC O157:H7的方法,检测时间短并具有较好的特异性、灵敏性和重复性,可用于EHEC O157:H7的快速检测。Objective:This study aims to to develop rapid detection methods and establish a new method for a common foodborne pathogenic microorganism enterohemorrhagic E.coli(EHEC)O157:H7 detection.Methods:Two EHEC O157:H7 detection methods were established and optimized based on the conserved region sequence of EHEC O157:H7 combined with recombinase-aided amplification(RAA)and fluorescence quantitative method or lateral flow dipstick(LFD),then the sensitivity and specificity of the two methods(RAA fluorescence method and RAA-LFD method)were evaluated.Results:Both detection methods can be completed within 16 min at 39℃,and can detect 1×10~4 copies,showing high sensitivity.The two methods did not cross react with plasmid templates of other common intestinal pathogens(Staphylococcus aureus,Salmonella,Campylobacter,Yersinia,Shigella)and showed good specificity.Conclusion:The two EHEC O157:H7 detection methods established in this study have short detection time,good specificity,sensitivity and repeatability,and can be used for rapid detection of EHEC O157:H7.

关 键 词：EHEC O157:H7 RAA 快速检测 

分 类 号：R516.1[医药卫生—内科学]