Development and validation of a duplex quantitative real-time RT-PCR assay for simultaneous detection and quantitation of foot-and-mouth disease viral positive-stranded RNAs and negative-stranded RNAs
Development and validation of a duplex quantitative real-time RT-PCR assay for simultaneous detection and quantitation of foot-and-mouth disease viral positive-stranded RNAs and negative-stranded RNAs
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DOI：
10.1016/j.jviromet.2009.06.008
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发表时间：
2009-10-01
影响因子：
3.1
通讯作者：
Zheng, Congyi
中科院分区：
文献类型：
--
作者：
Li, Yong;Huang, Xuan;Zheng, Congyi
一种简化的、成本效益高的、两步双工实时定量RT-PCR方法可以同时检测和定量口蹄疫病毒的正链rna和负链rna，以改进对病毒感染和复制状态的研究。引物和Taqman探针分别取自2B基因和3D基因的编码区，这两个基因在血清型中差异最小。急性感染细胞、组织样本和单细胞样本用于评估该测定。在体外感染病毒的早期阶段，病毒复制水平在9 h.p.i.时达到峰值，直到3 h.p.i.时才可检测到阴性链。体内肝脏、肾脏和脾脏中阳性链与阴性链（+RNA/-RNA）的比率动力学相似，这表明三个器官的复制动力学相似。187份单细胞样本中有55份qPCR阳性和阴性，阳性/阴性比值（+RNA/-RNA）在15.6 ~ 1463.4之间，单细胞样本间差异较大，说明病毒在单细胞内的活性复制差异较大。双定量实时RT-PCR验证是有效和可靠的。(C) 2009 Elsevier B.V.版权所有
A simplified, cost-effective, two-step duplex quantitative real-time RT-PCR assay was shown to detect and quantify foot-and-mouth disease virus positive-stranded RNAs and negative-stranded RNAs simultaneously for improved investigation of the state of virus infection and replication. The primers and Taqman probes were selected from the coding regions of 2B gene and 3D gene respectively, which have the least variations among serotypes. Cells infected acutely, tissue samples and single cell samples were used for evaluation of the assay. At the early stages of virus infection in vitro, the replication level reached a peak at 9 h.p.i. and the negative strands were detectable until 3 h.p.i. The kinetics of ratios of positive strands to negative strands (+RNA/-RNA) in vivo in the liver, kidney and spleen were similar, which demonstrated that the replication dynamics were similar in the three organs. 55 single cell samples out of 187 were positive by both positive strands qPCR and negative strands qPCR, the ratios (+RNA/-RNA) ranged from 15.6 to 1463.4 which showed considerable difference among single cell samples, indicating that active viral replication differs greatly in single cells. A duplex quantitative real-time RT-PCR was validated as effective and reliable. (C) 2009 Elsevier B.V. All rights reserved.