Sandvik T, Paton D J, Lowings P J
Department of Virology and Serodiagnostics, Central Veterinary Laboratory, Oslo, Norway.
J Virol Methods. 1997 Feb;64(1):43-56. doi: 10.1016/s0166-0934(96)02136-2.
Based on published gene sequences of bovine viral diarrhoea virus (BVDV) type I and classical swine fever virus (CSFV), genus- and species-specific primers were designed to detect and identify pestivirus cDNA sequences in a nested polymerase chain reaction (PCR). The PCR primers were validated using cDNA synthesized from 146 pestivirus isolates, comprising representatives of all four so far described genotypes (BVDV type I, BVDV type II, CSFV and border disease virus), as well as others of uncertain classification. PCR products of the predicted size were amplified from all viruses with the genus-specific primers. All 53 cattle isolates, including 5 typed antigenically as BVDV type II were amplified by the internal BVDV-specific primers, but not the CSFV-specific primers. The same result was found for other BVDV type I and II viruses isolated from sheep and pigs. Seventy-seven CSF viruses were amplified by their respective internal primers. Available information strongly indicate that 4 CSF viruses also amplified by the BVDV-specific primers had been contaminated with BVDV in cell cultures. Border disease viruses were mostly not detected by the BVDV-specific primers, but were detected weakly by the CSFV-specific primer pair. Using carrier RNA for extraction of viral RNA, the sensitivity of detection of the single and nested PCR was, respectively, 5 and 50 times higher than obtained with a cell culture assay. The RT-PCR also detected BVDV in all of 15 commercial batches of fetal calf serum examined, and verified three earlier diagnoses of CSFV by detecting specific gene sequences in 30 year old frozen archival organ samples.
根据已发表的牛病毒性腹泻病毒1型（BVDV）和经典猪瘟病毒（CSFV）的基因序列，设计了属特异性和种特异性引物，用于在巢式聚合酶链反应（PCR）中检测和鉴定瘟病毒cDNA序列。使用从146种瘟病毒分离株合成的cDNA对PCR引物进行了验证，这些分离株包括迄今描述的所有四种基因型（BVDV 1型、BVDV 2型、CSFV和边境病病毒）的代表，以及其他分类不确定的病毒。用属特异性引物从所有病毒中扩增出预测大小的PCR产物。所有53株牛分离株，包括5株抗原性鉴定为BVDV 2型的分离株，均被BVDV内部特异性引物扩增，但未被CSFV特异性引物扩增。从绵羊和猪分离的其他BVDV 1型和2型病毒也得到了相同的结果。77株CSF病毒被其各自的内部引物扩增。现有信息强烈表明，4株也被BVDV特异性引物扩增的CSF病毒在细胞培养中已被BVDV污染。边境病病毒大多未被BVDV特异性引物检测到，但被CSFV特异性引物对微弱检测到。使用载体RNA提取病毒RNA，单重PCR和巢式PCR的检测灵敏度分别比细胞培养检测高5倍和50倍。RT-PCR还在检测的15个商业批次胎牛血清中全部检测到了BVDV，并通过检测30年冻存的存档器官样本中的特异性基因序列，证实了3例早期CSFV诊断。
