King Donald P, Reid Scott M, Hutchings Geoffrey H, Grierson Sylvia S, Wilkinson Philip J, Dixon Linda K, Bastos Armanda D S, Drew Trevor W
Virology Department, Veterinary Laboratories Agency, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK.
J Virol Methods. 2003 Jan;107(1):53-61. doi: 10.1016/s0166-0934(02)00189-1.
A closed-tube polymerase chain reaction (PCR) was developed to allow the rapid detection of African swine fever virus (ASFV) DNA. This assay targets the VP72 gene of ASFV and uses the 5'-nuclease assay (TaqMan) system to detect PCR amplicons, avoiding tube opening and potential cross-contamination of post-PCR products. An artificial mimic was engineered with the TaqMan probe site replaced by a larger irrelevant DNA fragment allowing discrimination from ASFV by using two-colour TaqMan probe reporters. When added to the samples, successful amplification of this mimic demonstrated the absence of substances inhibitory to PCR, thereby validating negative results. Assay sensitivity was confirmed by obtaining positive signals with a representative selection of ASFV isolates. Many of the clinical and post-mortem features of ASF resemble those of classical swine fever (CSF) and porcine dermatitis and nephropathy syndrome (PDNS). Therefore, fast and reliable detection of ASFV is essential not only for the implementation of control measures to prevent the spread of ASF, but also in the differential diagnosis from CSF and PDNS. This assay should prove to be a valuable tool in the laboratory diagnosis of ASF and will complement existing molecular methods to provide rapid differential diagnosis in cases of suspected swine fever.
开发了一种闭管聚合酶链反应（PCR），用于快速检测非洲猪瘟病毒（ASFV）DNA。该检测方法针对ASFV的VP72基因，并使用5'-核酸酶检测（TaqMan）系统来检测PCR扩增产物，避免了打开管子以及PCR后产物的潜在交叉污染。构建了一种人工模拟物，将TaqMan探针位点替换为更大的无关DNA片段，通过使用双色TaqMan探针报告基因来区分ASFV。当添加到样品中时，该模拟物的成功扩增表明不存在抑制PCR的物质，从而验证了阴性结果。通过对一系列有代表性的ASFV分离株获得阳性信号，确认了检测方法的灵敏度。非洲猪瘟的许多临床和死后特征与经典猪瘟（CSF）和猪皮炎肾病综合征（PDNS）相似。因此，快速可靠地检测ASFV不仅对于实施预防非洲猪瘟传播的控制措施至关重要，而且对于与CSF和PDNS的鉴别诊断也很重要。该检测方法应被证明是非洲猪瘟实验室诊断中的一种有价值的工具，并将补充现有的分子方法，以便在疑似猪瘟病例中提供快速鉴别诊断。
