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

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.