逆转录聚合酶螺旋反应快速检测蜱传脑炎病毒方法的建立及初步评价  

Establishment and preliminary evaluation of rapid detection of tick-borne encephalitis virus by reverse transcription polymerase spiral reaction

作　　者：闫美田 郑雨桐 万楠 YAN Meitian;ZHENG Yutong;WAN Nan(Department of Clinical Laboratory,General Hospital of Northern Theater Command,Shenyang,Liaoning 110016,China)

机构地区：[1]北部战区总医院检验科,辽宁沈阳110016

出　　处：《国际检验医学杂志》2022年第16期1935-1939,1944,共6页International Journal of Laboratory Medicine

基　　金：辽宁省重点研发联合计划技术专项(2021JH2/10300085)。

摘　　要：目的建立一种等温条件下蜱传脑炎病毒(TBEV)的现场快速检测的方法。方法针对TBEV NS1基因设计一对特异性螺旋引物,在等温条件下建立逆转录聚合酶螺旋反应(RT-PSR)检测方法,优化反应条件,对特异性和灵敏性进行分析,应用全血模拟标本评价RT-PSR检测的符合率。结果成功建立并优化了TBEV的RT-PSR检测方法,RT-PSR法在61℃的温度下只需一种酶可一步实现逆转录和扩增,55 min可完成TBEV的检测,并可通过SYBR GreenⅠ直接肉眼判读结果,该方法特异性强,灵敏性高,最低检测限为107.341×10^(-6)ng/μL,是PCR的100倍。全血模拟标本符合率为100%,灵敏性比RNA标准品低10倍。结论RT-PSR方法具有良好的特异性和灵敏性,可用于基层对TBEV的现场快速检测。Objective To establish a rapid method for point-of-care detection of virulent tick-borne encephalitis virus(TBEV)under isothermal conditions.Methods A pair of specific spiral primers were designed for TBEV gene NS1,and the reverse transcription polymerase spiral reaction(RT-PSR)was established under isothermal conditions.The reaction conditions were optimized,the specificity and sensitivity were analyzed,and the coincidence rate of RT-PSR assay was evaluated by whole blood simulation samples.Results The RT-PSR method for TBEV detection was successfully established and optimized.The RT-PSR method could achieve reverse transcription and amplification in one step with only one enzyme at 61℃,and the TBEV detection could be completed in 55 min,the results could be read directly with the naked eye by addition of SYBR GreenⅠ.The RT-PSR method had high specificity and high sensitivity,and the minimum detection limit was 107.341×10^(-6)ng/μL,which was 100 times higher than that of PCR.The coincidence rate of whole blood simulated samples was 100%,and the sensitivity was 10 times lower than that of RNA standard samples.Conclusion RT-PSR method has good specificity and sensitivity,and could be used for rapid point-of-care detection of TBEV.

关 键 词：蜱传脑炎病毒 逆转录聚合酶螺旋反应 快速检测 

分 类 号：R446.5[医药卫生—诊断学]