【摘要】 本研究旨在建立一种快速、敏感和高特异性检测鸭坦布苏病毒(DTMUV)、鸭肠炎病毒(DEV)和番鸭细小病毒(MDPV)的TaqMan三重实时荧光定量PCR(q-PCR)的诊断方法并应用于临床疑似样品检测。根据DTMUV的E基因、DEV的UL2基因和MDPV的VP3基因保守区域,分别设计合成了3对特异性引物和探针,在建立单重q-PCR方法的基础上建立了三重q-PCR方法。运用三重q-PCR方法对198份来自江苏和安徽的鸭组织疑似病料进行检测,结果表明,建立的TaqMan三重q-PCR可同时检测这3种病毒,检测灵敏度至少达100个拷贝,相关系数(R~2)均在0.99以上,扩增效率为90%～110%。同时,该方法对H9亚型禽流感病毒(H9N2 AIV)、鸭甲肝病毒Ⅰ型(DHAV-1)、番鸭呼肠孤病毒(MDRV)、鸭呼肠孤病毒(DRV)、新城疫病毒(NDV)、鹅细小病毒(GPV)的检测均为阴性,表明该方法具备特异性强、灵敏度高、重复性好和快速等优点。与常规PCR检测方法相比,三重q-PCR方法灵敏度大约高100倍。临床疑似样品检测结果表明DTMUV的检出率最高,3种病毒混合感染亦常见。建立的TaqMan三重q-PCR检测方法为DTMUV、DEV和MDPV的临床样品检测提供了快速、有效、特异和灵敏的工具,也为临床分子流行病学调查及定量分析奠定了基础。更多还原

【Abstract】 This study aimed to establish a triple TaqMan real-time fluorescence quantitative PCR(q-PCR) method for the rapid, sensitive and specific detection of duck Tembusu virus(DTMUV), duck enteritis virus(DEV) and Muscovy duck parvovirus(MDPV). Based on the conserved regions of DTMUV E, DEV UL2 and MDPV VP3 genes, three pairs of specific primers and probes were designed and synthesized respectively. Furthermore, a triple q-PCR method was established based on the establishment of a single q-PCR method. The triple q-PCR method was used to detect 198 suspected samples from Jiangsu and Anhui. The results showed that the established TaqMan triple q-PCR method could detect these three viruses simultaneously. The detection sensitivity was at least 100 copies, the correlation coefficient(R~2) was above 0.99, and the amplification efficiency ragned from 90% to 110%. At the same time, the detection results of H9 N2 avian influenza virus(H9 N2 AIV), duck hepatitis A virus type I(DHAV-1), Muscovy duck reovirus(MDRV), duck reovirus(DRV), Newcastle disease virus(NDV) and goose parvovirus(GPV) were negative, it showed that the method had the advantages of high specificity, high sensitivity fastness good repeatability. The sensitivity of triple q-PCR method was approximately 100 times greater than that of conventional PCR assay. The results of clinical suspected samples indicated that the detection rate DTMUV was highest, and mixed infections of three viruses were also common. This triple TaqMan q-PCR assay provides a fast, efficient, specific and sensitive tool for the detection of DTMUV, DEV, MDPV and also lays the foundation for clinical molecular epidemiological investigation and quantitative analysis.更多还原