Hofmann Martin A
Institute of Virology and Immunoprophylaxis, Sensemattstrasse 293, CH-3147 Mittelhaeusern, Switzerland.
J Virol Methods. 2003 Dec;114(1):77-90. doi: 10.1016/j.jviromet.2003.09.004.
RT-PCR is used widely as a diagnostic method to detect and differentiate pestiviruses. The construction of two chimeric classical swine fever virus (CSFV) recombinants based on a marker virus constructed previously [J. Virol. 72 (1998) 5318-5322] is described. These viruses, termed vA187CAT_5UTRBVD and vA187CAT_IRESBVD, contain the entire 5' untranslated region (5'UTR) or the internal ribosome entry site (IRES) of bovine viral diarrhea virus (BVDV), respectively. Both chimeric viruses proved to be infectious in cell culture. Hence, the 5'UTR as well as the IRES element only of BVDV can substitute for the corresponding genome region of CSFV. Next, two sets of primers and corresponding dual-labeled TaqMan probes were designed; one detecting specifically a conserved but CSFV-specific area within the 5'UTR of wild-type CSFV, the other one targeting the CAT gene inserted in vA187CAT_5UTRBVD. The two primer/probe sets were combined in a closed-tube multiplex one-step RT-PCR. To monitor the entire extraction and detection process limited amounts of vA187CAT_5UTRBVD were added directly to clinical samples before RNA extraction. The multiplex RT-PCR proved to be as sensitive as the single primer/probe set method, but allowed the validation of each sample tested individually, based on the detection of the CAT marker gene. vA187CAT_5UTRBVD was also used successfully for foot-and-mouth disease virus (FMDV) TaqMan RT-PCR. Therefore, it is considered a universal internal positive control for RT-PCR assays to exclude loss of RNA during extraction, or failure of amplification due to inhibitory substances present in the sample.
逆转录聚合酶链反应（RT-PCR）作为一种诊断方法被广泛用于检测和区分瘟病毒。本文描述了基于先前构建的一种标记病毒[《病毒学杂志》72 (1998) 5318 - 5322]构建两种嵌合经典猪瘟病毒（CSFV）重组体的过程。这些病毒，分别称为vA187CAT_5UTRBVD和vA187CAT_IRESBVD，分别包含牛病毒性腹泻病毒（BVDV）的整个5'非翻译区（5'UTR）或内部核糖体进入位点（IRES）。两种嵌合病毒在细胞培养中均被证明具有感染性。因此，仅BVDV的5'UTR以及IRES元件可以替代CSFV的相应基因组区域。接下来，设计了两组引物和相应的双标记TaqMan探针；一组特异性检测野生型CSFV 5'UTR内保守但CSFV特异的区域，另一组靶向插入vA187CAT_5UTRBVD中的CAT基因。这两组引物/探针组合在一个封闭管多重一步RT-PCR中。为了监测整个提取和检测过程，在RNA提取前将有限量的vA187CAT_5UTRBVD直接添加到临床样本中。多重RT-PCR被证明与单引物/探针组方法一样灵敏，但基于对CAT标记基因的检测，可以对每个单独检测的样本进行验证。vA187CAT_5UTRBVD也成功用于口蹄疫病毒（FMDV）的TaqMan RT-PCR。因此，它被认为是RT-PCR检测的通用内部阳性对照，以排除提取过程中RNA的损失或样本中存在的抑制物质导致的扩增失败。
