发热伴血小板减少综合征病毒实时荧光定量RT-PCR检测方法的建立及应用  

Establishment and application of real-time fluorescent quantitative RT-PCR method for detection of severe fever with thrombocytopenia syndrome virus

作　　者：李鹏飞 李全 李梦兰 吴玲 李志锋[2] LI Pengfei;LI Quan;LI Menglan;WU Ling;LI Zhifeng(Department of Clinical Laboratory,Jiangsu Province Hospital of Chinese Medicine,Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing,Jiangsu 210029,China;Department of acute infectious diseases control and prevention,Jiangsu Provincial Center for Disease Prevention and Control,Nanjing,Jiangsu 210009,China)

机构地区：[1]南京中医药大学附属医院江苏省中医院检验科,江苏南京210029 [2]江苏省疾病预防控制中心急性传染病预防控制所,江苏南京210009

出　　处：《中华卫生杀虫药械》2025年第2期149-153,209,共6页Chinese Journal of Hygienic Insecticides and Equipments

基　　金：国家自然科学基金青年项目(81403136);国家中医临床研究基地(江苏省中医院)开放课题(JD2022SZ11);南京中医药大学自然科学基金重点项目(XZR2024010)。

摘　　要：目的建立一种基于Taqman探针的高灵敏度一步法实时荧光定量RT-PCR方法,用于检测发热伴血小板减少综合征病毒(SFTSV)。方法靶向发热伴血小板减少综合征病毒基因组S片段保守区域设计引物和Taqman探针建立实时荧光定量RT-PCR方法。评价方法的灵敏度、特异性和重复性,并应用临床样本进行应用评价。结果该方法最低检测限为100拷贝/ml,其灵敏度为普通RT-PCR的1000倍。在线性范围内,循环阈值(Ct值)与病毒RNA标准品之间表现出较强的线性相关性(R 2>0.999)。批内和批间重复性的变异系数均小于1.0%。该方法还具有高度特异性,不与其他病毒核酸交叉反应。应用发热伴血小板减少综合征确诊患者和健康志愿者血清样本对该方法进行了评估,结果显示,该方法检测灵敏度和特异度均为100.0%。此外,用该方法对288例疑似SFTSV感染患者的样本进行检测,有14.9%的患者感染了SFTSV。该方法速度较快,包括核酸提取步骤在内仅需2 h。结论本研究建立的实时荧光定量RT-PCR检测方法可作为SFTSV感染早期诊断的可靠方法。Objective To establish a highly sensitive one-step real-time fluorescent quantitative reverse transcription PCR(RT-PCR)method based on Taqman probe for detection of severe fever with thrombocytopenia syndrome virus(SFTSV).Methods Primers and Taqman probes were designed targeting the conserved region of the S segment of the severe fever with thrombocytopenia syndrome virus genome to establish a real-time fluorescent quantitative RT-PCR method.The sensitivity,specificity and repeatability of the method were evaluated.And the clinical samples were used to verify the method.Results The limit of detection of this method was 100 copies/ml,and its sensitivity was 1000 times than that of conventional RT-PCR.Within the linear range,there was a strong correlation(R 2>0.999)between the cyclic threshold(Ct value)and the viral RNA standard.The coefficient of variation for intra-and inter batch repeatability was all less than 1.0%.This method also exhibited high specificity and did not yield cross-reacting signals from other viral nucleic acids.And this method was evaluated using serum samples from severe fever with thrombocytopenia syndrom confirmed patients and healthy volunteers.The results showed that the detection sensitivity and specificity of this method were 100.0%.Furthermore,this method was used to detect the samples from 288 suspected SFTSV infected patients.It demonstrated that 14.9% of the patients were infected with SFTSV.This method was fast and only took 2 hours,including the nucleic acid extraction step.Conclusion The real-time fluorescent quantitative RT-PCR assay established in our study is a reliable method for the early diagnosis of SFTSV infection.

关 键 词：发热伴血小板减少综合征病毒 实时荧光定量RT-PCR 特异性 检测限 

分 类 号：R183.5[医药卫生—流行病学]