猪瘟病毒荧光RT-PCR快速检测方法的建立

Development of fluorescent real-time RT-PCR for rapid detection of classical swine fever virus

以猪瘟病毒5′端非编码区为靶核酸序列设计引物和探针,建立了一步法荧光RT-PCR检测猪瘟病毒。荧光RT-PCR仅检测出猪瘟C株、T株,未能检测出牛病毒性腹泻病毒(BVDV)、猪呼吸系统冠状病毒、猪传染性胃肠炎病毒、猪细小病毒、伪狂犬病病毒、猪生殖与呼吸综合征病毒、PK-15细胞和牛睾丸原代细胞;对猪瘟病毒T株的扩增反应产物进行了测序分析,与预期序列相符。荧光RT-PCR的检测极限可达到1 TCID50/mL,整个试验流程只需2 h。采用荧光RT-PCR和抗原捕获ELISA同时检测临床病料、猪副产品共207份样本,两种方法的检出率分别为17.4%和13.5%,两者符合率为95.7%(198/207);荧光RT-PCR的检出率高于ELISA,两者差异显著。结果表明,建立的荧光RT-PCR可用于猪产品、临床病料中猪瘟病毒的快速检测。

A fluorescent real time RT-PCR for quick detection of classical swine fever virus （CSFV） was developed. Primers and FAM-labeled Taqman probes specific for CSFV were selected. Optimal con-centrations of transcriptase, Taq polymerase, Mg^2＋ and dNTPs; annealing temperature; and PCR cycles of the real-time RT-PCR were identified using the Thiveral strain （T strain） of CSFV. Specificity evaluation of the fluorescent RT-PCR showed that the assay detected T and the lapinized Chinese （C） strains of CSFV; no amplification signal was observed in any of the non-CSFV pestiviruses which included the following： bovine viral diarrhoea virus, porcine respiratory and reproductive syndrome virus（PRRSV）, porcine parvovirus, swine transmissible gastroenteritis virus （TGEV）, porcine respiratory coronavirus （PRCV）, pseudorabies virus （PRV）, and cell lines such as PK-15 cells and primary bovine testicular cells. Sequence of the PCR product of T strain was expected. The detection limit of the assay was 1 TCID50/mL. Of 75 visceral organ samples from suspected-CSFV infected pigs （e. g. , liver, spleen, heart and kidney） and 132 other swine products tested, the RT-PCR and the commercial antigen-capture ELISA assay showed a concordance of 95.7% （198/207）. The RT-PCR assay could be performed within 2 h or less. The above results demonstrated that the RT-PCR could be used as a method for quick diagnosis and detection of CSFV.