蓝舌病病毒、流行性出血病病毒和帕利亚姆血清群病毒三重RT-qPCR检测方法的建立及应用

Establishment and application of triplex RT-qPCR for detection of bluetongue virus, epizootic hemorrhagic disease virus and Palyam serogroup virus

【目的】建立蓝舌病病毒(Bluetongue virus,BTV)、流行性出血病病毒(Epizootic hemorrhagic disease virus,EHDV)及帕利亚姆血清群病毒(Palyam serogroup virus,PALV)快速筛查和诊断的三重RT-qPCR检测技术。【方法】选择BTV的NS3、EHDV的NS1以及PALV的VP7基因作为靶基因,设计3组特异性引物和探针,建立3种病毒的三重RT-qPCR检测方法。对检测方法的灵敏度、特异性和重复性进行验证,同时使用我国分离的BTV、EHDV与PALV不同血清型毒株和对应的阳性血液样本,与24个BTV血清型及6个EHDV血清型参考毒株对该三重法的检测效果进行验证。【结果】三重RT-qPCR的扩增效率均可达90%以上,对3种病毒核酸拷贝数的检测下限均为10μL-1级别,与单重法的检测灵敏度相当;与阿卡斑病毒、小反刍兽疫病毒、口蹄疫病毒和牛流行热病毒之间无交叉反应;Ct值批内、批间变异系数均在2%以内;可检测出24种BTV血清型、6种EHDV血清型与3种PALV血清型,具有群特异性;可有效检测血液中的病毒核酸,检测结果与单重法一致。【结论】本研究建立的BTV、EHDV与PALV三重RT-qPCR具有良好的敏感性、特异性和重复性,可用于临床样本中对3种病毒的同时诊断与筛查。

【Objective】To establish triplex RT-qPCR detection technique for rapid screening and diagnosis of bluetongue virus(BTV), epizootic hemorrhagic disease virus(EHDV) and Palyam serogroup virus(PALV)infection.【Method】NS3 gene of BTV, NS1 gene of EHDV and VP7 gene of PALV were used as target genes to design three sets of primers and probes for the establishment of a triplex RT-qPCR assay. The sensitivity,specificity and repeatability of this assay were evaluated, meanwhile different BTV, EHDV, PALV serotype strains isolated in China and their corresponding positive blood samples, as well as reference strains of 24 BTV serotypes and six EHDV serotypes were used to verify detection effects of the assay.【Result】The amplification efficiencies of the triplex RT-qPCR assay were all more than 90%, and the copy number detection limits of in vitro transcribed ssRNAs were all low to 10 μL-1, which was equivalent to those of single plex RTqPCR assay for each virus. There was no cross reaction with akabane virus, peste des petits ruminants virus,foot-and-mouth disease virus and bovine ephemeral fever virus. The intra-assay and inter-assay variable coefficients of Ct value were all less than 2%. The established triplex RT-qPCR successfully detected viral strains belonging to 24 BTV serotypes, six EHDV serotypes and three PALV serotypes, indicating that this assay possessed remarkable group specificity. The established triplex RT-qPCR assay could effectively detect the positive blood samples collected from animals infected by BTV, EHDV and PALV, and the results were consistent with those of monoplex RT-qPCR assay.【Conclusion】The triplex RT-qPCR detection assay for BTV, EHDV and PALV has good sensitivity, specificity and repeatability, and can be used for the simultaneous diagnosis and screening of these three viruses in clinical samples.