登革-2型病毒一步法TaqMan荧光定量RT-PCR检测方法建立

Development and evaluation of TaqMan-based one-step reverse transcriptionpolymerase chain reaction assay for detection of dengue virus type 2

目的建立一种针对登革2型病毒(DENV-2)的快速、灵敏、特异的检测方法。方法从Gen Bank下载105株DENV-2病毒毒株全基因序列,应用生物软件Bioedit进行比对分析筛选出保守序列,在保守区域设计特异引物和探针,建立检测DENV-2的TaqMan荧光定量逆转录聚合酶链反应(RT-PCR)方法。结果该方法检测灵敏度达到102copies/μL;特异性验证中除登革2型病毒有明显扩增外,与墨累谷脑炎病毒、西尼罗病毒、蜱传脑炎病毒、登革病毒1、3、4型、黄热病毒和科萨努尔森林病毒、乙型脑炎病毒均无交叉反应。重复性实验结果表明该方法的组间和组内的变异系数(CV)均<2%。对人工感染DENV-2的87只蚊虫标本进行比对实验,结果荧光定量RT-PCR检测出56份阳性,传统RT-PCR只检测出16份阳性,差异有统计学意义(χ~2=37.908,P=0.000)。结论 TaqMan荧光定量RT-PCR方法检测DENV-2,特异性强、敏感性高。

Objective To develop a TaqMan-based reverse transcription-polymerase chain reaction assay （RT-PCR） for the detection of dengue virus type 2 （DENV-2）.Methods The whole genome sequences of DENV-2 were downloaded from the GenBank and aligned with the Bioedit software.Specific primers and probes were designed on the conserved region of DENV-2 based on the alignment results to develop a TaqMan-based RT-PCR assay.Results The detection limit of the assay was 102 copies/μL.Besides the obvious amplification of DENV-2,there were no cross reactions with Murray Valley encephalitis virus,Japanese encephalitis virus,West Nile virus,tick-borne encephalitis virus,other types of dengue virus,yellow fever virus,and Kyasanur Forest disease virus.The results of the stability assay showed that the between-and within-group coefficient of variation were less than 2%.For the 87 mosquito specimens detected with two different methods,56 positive samples were confirmed with fluorescence quantitative RT-PCR,while only 16 positive samples were confirmed with the traditional RT-PCR method,with a significant difference （χ2=37.908,P=0.000）.Conclusion The developed TaqMan-based one-step reverse transcription-polymerase chain reaction assay is of strong specificity and high sensitivity for molecular diagnosis of DENV-2.