含内参的登革病毒与基孔肯雅病毒3重荧光定量PCR检测方法的建立与评估  

Development of a multiplex real-time RT-PCR assay with an internal control for detection of dengue and Chikungunya viruses

作　　者：徐静[1,2] 谢翊[3] 管大伟[2] 苏健平 廖华乐[3] 柯昌文[1,2] 

机构地区：[1]南方医科大学公共卫生与热带医学学院,广东广州510515 [2]广东省疾病预防控制中心,广东广州511430 [3]佛山市疾病预防控制中心,广东佛山528000 [4]上海之江生物科技股份有限公司,上海200000

出　　处：《中国病原生物学杂志》2016年第4期301-304,311,共5页Journal of Pathogen Biology

基　　金：实验室感染性材料溯源和生物风险溯源关键技术和产品研究项目(No.2014AA021404)

摘　　要：目的建立一种含内参的,针对登革病毒和基孔肯雅病毒核酸检测的3重荧光定量PCR方法。方法针对登革病毒3'端非结构蛋白编码区和基孔肯雅病毒5'端非结构蛋白编码区设计引物与探针,建立一套含有内参的并且能同时检测登革病毒和基孔肯雅病毒的3重实时荧光定量RT-PCR方法。对其最低检测限和特异度进行验证,通过检测血清标本对该方法进行临床应用评估。结果建立的3重荧光定量PCR登革病毒与基孔肯雅病毒绝对定量标准曲线显示,病毒拷贝数的对数值与CT值之间具有良好的线性关系,梯度稀释后的标准品核酸检测均呈现典型的S型曲线。检测日本脑炎病毒、西尼罗病毒、黄热病毒、辛德毕斯病毒各1株,结果均为阴性,特异度为100%。用该方法检测294份登革热患者血清,灵敏度为100%,特异度为89.3%,阳性预测值为92.1%,阴性预测值为100%;测定33份基孔肯雅热病患者血清的灵敏度、特异度及阴、阳性预测值均为100%。结论建立的含有内参的能同时检测登革病毒与基孔肯雅病毒的3重荧光定量PCR方法具有较高的灵敏度和特异度,可用于登革病毒与基孔肯雅病毒感染检测,为早期筛查和鉴别登革病毒和基孔肯雅病毒感染提供依据。Objective To establish a multiplex real-time RT-PCR assay for detection of dengue and Chikungunya viruses in one tube. Methods Primers and a probe were designed based on the sequence of the 3＇-UTR of the dengue virus and the sequence of the 5＇-UTR of Chikungunya virus.A multiplex real-time RT-PCR assay was established to detect the dengue and Chikungunya viruses,the minimum detection limits and specificity were determined,and clinical specimens were tested to assess the clinical usefulness of this approach. Results An absolute quantitative standard curve for the dengue virus and the Chikungunya virus showed that the log of the number of viral copies and the CT value had a good linear relationship.After gradient dilution,standard nucleic acid detection produced a typical S curve.The assay was used to detect the Japanese encephalitis virus,west Nile virus,yellow fever virus,and Sindbis virus strains and the results were all negative.The assay had a specificity of 100%.The assay was used to clinically evaluate 294 cases of suspected dengue fever.The assay had a sensitivity of 100%,a specificity of 89.3%,apositive predictive value of 92.1%,and a negative predictive value of 100%.The assay was used to test specimens from 33 patients infected with the Chikungunya virus.The assay had a sensitivity of 100%,a specificity of 100%,apositive predictive value of 100%,and a negative predictive value of 100%. Conclusion This study established a multiplex real-time RT-PCR assay with an internal control for detection of dengue and Chikungunya viruses.This assay had a high level of sensitivity and specificity and it can be used to molecularly detect the dengue virus and Chikungunya virus.This assay provides a basis for early screening and identification of infection with the dengue virus and Chikungunya virus.

关 键 词：3重荧光定量RT-PCR 登革病毒 基孔肯雅病毒 

分 类 号：R373.33[医药卫生—病原生物学]