摘要
目的建立基于环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术的志贺菌快速检测方法。方法针对志贺菌特异保守ipaH基因设计引物,通过引物筛选和反应条件优化,建立检测志贺菌的实时荧光及肉眼目视观测结果的LAMP方法,对方法的检出限、有无交叉反应、符合率和抗干扰能力进行评价。结果60~65℃等温条件下30min内可完成LAMP反应。荧光和肉眼目视观察结果检出限分别为211和21.1 fg/μL,与22种非志贺菌无交叉反应。检测35份志贺菌污染的样品和30份未污染标品,荧光和肉眼目视检测阳性符合率分别为97.1%和94.3%,阴性符合率均为100%,总符合率分别为98.5%和96.9%。方法具备良好的抗干扰能力。结论在基层医院、灾区救治、感染事件现场,环境、设备和人员受限等条件下,建立的实时荧光和肉眼目视观察结果的LAMP方法可为志贺菌快速检测提供有力手段。
Objective To establish a rapid detection method for Shigella based on loop-mediated isothermal amplification(RT-LAMP)technique.Methods Primers were designed for Shigella’s specific conservative ipaH gene.Through a primer screening and reaction conditions optimization process,a LAMP method for detecting Shigella’s real-time fluorescence and visual observation results was established.We then evaluated the detection limit of the method,as well as whether there was a cross-reaction,coincidence rate and anti-interference ability.Results LAMP reaction was completed within 30 minutes at an isothermal temperature of 60-65℃.The detection limits of fluorescence and visual observation were 211 and 21.1 fg/μL,respectively,and there was no cross-reaction with 22 non-Shigella Strains.35 samples contaminated with Shigella and 30 Shigella-free samples were tested.The positive coincidence rates of fluorescence and visual inspection were 97.1%and 94.3%,respectively,the negative coincidence rates were both 100%,and the total coincidence rates were 98.5%and 96.9%,respectively.The detection method has a good anti-interference ability.Conclusion In primary hospitals,treatment of disaster-stricken areas,infection scenes,and restricted environments,also in the conditions of insufficient professional equipment and personnel,the established LAMP method of real-time fluorescence and visual observation of results can provide powerful means for the rapid detection of Shigella.
作者
欧红玲
王岩
白静
张巧云
贾连玲
李梦文
冯鑫鑫
王欣茹
OU Hongling;WANG Yan;BAI Jing;ZHANG Qiaoyun;JIA Lianling;LI Mengwen;FENG Xinxin;WANG Xinru(Department of Clinical Laboratory, PLA Rocket Characteristic Medical Center, Beijing 100088, China)
出处
《标记免疫分析与临床》
CAS
2021年第5期847-852,共6页
Labeled Immunoassays and Clinical Medicine
基金
全军医学科技青年培育计划课题(编号:16QNP030)。
关键词
环介导等温扩增
志贺菌
快速检测
Loop-mediated isothermal amplification
Shigella
Rapid detection
