酶促重组等温扩增实时荧光法快速检测发热伴血小板减少综合征病毒方法的建立  

Development of real-time fluorescent enzymatic recombinaseamplification method for severe fever with thrombocytopeniasyndrome virus detection

作　　者：王春芳 罗正汉 汪雨荷 张锦海 叶福强 徐敏志 蒯月璋 韩一芳 汪春晖 WANG Chunfang;LUO Zhenghan;WANG Yuhe;ZHANG Jinhai;YE Fuqiang;XU Minzhi;KUAI Yuezhang;HAN Yifang;WANG Chunhui(School of Public Health,Nanjing Medical University,Nanjing,Jiangsu 211100,China;Center for Disease Control and Prevention of Eastern Theater Command,Nanjing,Jiangsu 210002,China)

机构地区：[1]南京医科大学公共卫生学院,江苏南京211100 [2]东部战区疾病预防控制中心,江苏南京210002

出　　处：《中华卫生杀虫药械》2024年第6期554-558,共5页Chinese Journal of Hygienic Insecticides and Equipments

基　　金：疾控中心逸启指令性项目(2024YQZL01)。

摘　　要：目的建立一种可用于检测发热伴血小板减少综合征病毒(severe fever with thrombocytopenia syndrome virus,SFTSV)的逆转录-酶促重组等温扩增(reverse transcription enzymatic recombinase amplification,RT-ERA)方法。方法以SFTSV非结构蛋白NSs基因的保守序列为靶标,根据RT-ERA反应原理设计、合成荧光探针及对应引物,以pUC18为载体构建靶标质粒并在体外转录后作为阳性对照品,将阳性对照品倍比稀释后筛选出扩增效率最高的探针引物组合。利用筛选的探针引物组合,对不同拷贝数的阳性对照品进行荧光RT-ERA法扩增,评价该方法的最低检出限;接着以新型布尼亚病毒、新型冠状病毒、森林脑炎病毒、登革病毒、基孔肯雅病毒、寨卡病毒的RNA为模板进行荧光RT-ERA法扩增,评价该方法的特异性;最后用26例临床样本,检测与RT-qPCR的一致性。结果在42℃恒温条件下,对于SFTSV RNA,40 min内最低检出限可达100 copies/μl。当SFTSV核酸扩增为阳性时,其他病毒检测结果为阴性;对26例怀疑为发热伴血小板减少综合征患者样本的检测显示,RT-ERA检测的敏感性和特异性分别约为88.9%和100.0%;kappa值为92.3%。结论建立了一种酶促重组等温扩增实时荧光技术的SFTSV核酸检测方法,具有简单快速、敏感特异等优点。该方法在SFTS病原监测上有一定的应用前景。Objective To establish a RT-ERA method for detection of severe fever with thrombocytopenia syndrome virus(SFTSV).Methods Taking the conserved sequence of NSs gene of SFTSV as the target,the fluorescent probe and the corresponding primer were designed and synthesized according to the RT-ERA principle.The standard plasmid was constructed with pUC18 as the vector and transcripted in vitro as a positive control.The optimal probe primer combinations was screened after the gradient dilution of positive control product.The positive control product with different copy numbers were used to evaluate the sensitivity of RT-ERA.Then,the RNA of novel bunyavirus,novel coronavirus,forest encephalitis virus,dengue virus,chikungunya virus and Zika virus were used as templates to evaluate the specificity of the method.Finally,26 clinical samples were used to test the consistency with RT-qPCR.Results At 42℃,the fluorescence RT-ERA method could detect the minimum sensitivity of SFTSV RNA was 100 copies/μl within 40 minutes.When the SFTSV nucleic acid amplification was positive,the test results of other viruses were negative.A test of 26 serum samples of suspected severe fever with thrombocytopenia syndrome patients revealed that the sensitivity and specificity of RT-ERA were about 88.9%and 100.0%,respectively.The kappa value was 92.3%.Conclusion Considering the rapid,specific and sensitive advantages of RT-ERA,this method has certain application prospects in the detection of SFTSV.

关 键 词：发热伴血小板减少综合征病毒 酶促重组等温扩增实时荧光法 快速检测 

分 类 号：R183.5[医药卫生—流行病学]