四重逆转录重组酶聚合酶等温扩增技术快速联检四种难鉴别蚊媒病毒的效果观察  

Establishment of a quadruple RT-RPA technique for rapid and simultaneous detection of 4 mosquito-borne viruses

作　　者：韩明月 林源吉 吴新新[1] 魏晨星 董学妍 王晓明 万海同[5] 余道军 HAN Mingyue;LIN Yuanji;WU Xinxin;WEI Chenxing;DONG Xueyan;WANG Xiaoming;WAN Haitong;YU Daojun(The Fourth Clinical Medical College of Zhejiang Chinese Medical University,Hangzhou 310006,China;不详)

机构地区：[1]浙江中医药大学第四临床学院,杭州310053 [2]杭州铭创生命科技有限公司 [3]浙江大学医学院 [4]浙江大学医学院附属杭州市第一人民医院检验科 [5]浙江中医药大学生物工程学院

出　　处：《浙江医学》2022年第10期1042-1049,共8页Zhejiang Medical Journal

基　　金：国家自然科学基金重点项目(81930111);浙江省医药卫生科技计划项目(2022PY016);杭州市医药卫生科技项目(Z2021005)。

摘　　要：目的建立针对登革病毒(DENV)、黄热病毒(YFV)、基孔肯雅病毒(CHIKV)、寨卡病毒(ZIKV)的四重逆转录重组酶聚合酶扩增(RT-RPA)技术,用于同时快速检测上述4种病毒。方法通过正交设计建立检测4种蚊媒病毒(DENV、YFV、CHIKV、ZIKV)的四重RT-RPA反应体系。通过质粒标准品、体外转录RNA和临床血清验证单重RT-RPA和四重RT-RPA的检测灵敏度、特异度、重复性和临床应用符合率。结果单重RT-RPA对DENV、YFV、CHIKV、ZIKV质粒DNA的检出限分别为1×10^(2)、1×10^(2)、1×10^(2)、<1×10^(2) Copies/ml,对DENV、YFV、CHIKV和ZIKV体外合成RNA的检出限分别为1×10^(2)、1×10^(2)、1×10^(2)、1×10^(2) Copies/ml,四重RT-RPA对DENV、YFV、CHIKV和ZIKV质粒DNA的检出限分别为1×10^(2)、1×10^(2)、1×10^(2)、1×10^(2) Copies/ml,对体外合成RNA的检出限分别为5×10^(2)、5×10^(2)、5×10^(2)、5×10^(2) Copies/ml。本研究建立的单重及四重RT-RPA反应体系与其他常见病毒不存在交叉反应,RT-RPA法测定的阈值时间值(TT)的变异系数为1.76%~5.96%。四重RT-RPA法对不同病毒体外转录RNA检出率不同,DENV、YFV和CHIKV为100.00%,ZIKV为83.33%。单重RT-RPA、四重RT-RPA、RT-PCR对DENV临床样本的检出率无统计学差异(P>0.05)。结论本研究建立的针对DENV、YFV、CHIKV和ZIKV的单重RT-RPA和四重RT-RPA检测方法特异度好、灵敏度高、重复性好,对蚊媒传染病的早期诊断及鉴别诊断具有重要作用,同时也为其他新发再发传染病的快速诊断方法建立提供研究方向。Objective To establish a quadruple reverse transcriptase recombinase polymerase isothermal amplification(RT-RPA)method for simultaneous and rapid detection of four mosquito-borne viruses.Methods Through orthogonal design,a quadruple RT-RPA system was established by optimizing parameters for detection of 4 mosquito-borne viruses:dengue virus(DENV),yellow fever virus(YFV),chikungunya virus(CHIKV)and Zika virus(ZIKV).The detection sensitivity,specificity,reproduci-bility and clinical coincidence rate of single RT-RPA and quadruple RT-RPA were verified by plasmid standards in transcribed RNA and serum samples.Results The detection limits of single RT-RPA for DENV,ZIKV,YFV and CHIKV plasmid DNA were 1×10^(2),1×10^(2),1×10^(2) and<1×10^(2) Copies/ml,for DENV,YFV,CHIKV and ZIKV in vitro transcribed RNA were 1×10^(2),1×10^(2),1×10^(2) and 1×10^(2) Copies/ml.The detection limits of quadruple RT-RPA for DENV,YFV,CHIKV and ZIKV,plasmid DNA were 1×10^(2),1×10^(2),1×10^(2) and 1×10^(2) Copies/ml,for DENV,YFV,CHIKV and ZIKV in vitro transcribed RNA were 5×10^(2),5×10^(2),5×10^(2) and 5×103 Copies/ml,respectively.There was no cross-reaction with other common viruses,and the coefficient of variation of threshold time value determined by RT-RPA method was 1.76%-5.96%.The detection rates of in vitro transcript RNA by quadruple RT-RPA for DENV,YFV and CHIKV were 100.00%,for ZIKV was 83.33%.There was no significant difference in the detection rate of DENV clinical samples among single RT-RPA,quadruple RT-RPA and RT-PCR(P>0.05).Conclusion The quadruple RT-RPA method for DENV,CHIKV,YFV and ZIKV established in this study have good specificity,high sensitivity and good repeatability.It can be used in the early diagnosis and differential diagnosis of mosquito-borne infectious diseases,and it also provides reference for the establishment of rapid diagnosis methods of other emerging and recurrent infectious diseases.

关 键 词：四重逆转录重组酶聚合酶扩增 蚊媒传染病 登革病毒 黄热病毒 基孔肯雅病毒 寨卡病毒 

分 类 号：R373.3[医药卫生—病原生物学]