用于检测狂犬病及狂犬病相关病毒六种基因型的半巢式聚合酶链反应检测法
Heminested PCR assay for detection of six genotypes of rabies and rabies-related viruses.
作者信息
Heaton P R, Johnstone P, McElhinney L M, Cowley R, O'Sullivan E, Whitby J E
机构信息
Department of Virology, Veterinary Laboratories Agency, Addlestone, Surrey, United Kingdom.
出版信息
J Clin Microbiol. 1997 Nov;35(11):2762-6. doi: 10.1128/jcm.35.11.2762-2766.1997.
Abstract
A heminested reverse transcriptase PCR (hnRT-PCR) protocol which is rapid and sensitive for the detection of rabies virus and rabies-related viruses is described. Sixty isolates from six of the seven genotypes of rabies and rabies-related viruses were screened successfully by hnRT-PCR and Southern blot hybridization. Of the 60 isolates, 93% (56 of 60) were positive by external PCR, while all isolates were detected by heminested PCR and Southern blot hybridization. We also report on a comparison of the sensitivity of the standard fluorescent-antibody test (FAT) for rabies antigen and that of hnRT-PCR for rabies viral RNA with degraded tissue infected with a genotype 1 virus. Results indicated that FAT failed to detect viral antigen in brain tissue that was incubated at 37 degrees C for greater than 72 h, while hnRT-PCR detected viral RNA in brain tissue that was incubated at 37 degrees C for 360 h.
摘要
本文描述了一种半巢式逆转录聚合酶链反应（hnRT-PCR）方法，该方法快速且灵敏，可用于检测狂犬病病毒及狂犬病相关病毒。通过hnRT-PCR和Southern印迹杂交成功筛选出了来自狂犬病和狂犬病相关病毒七种基因型中六种基因型的60株病毒分离株。在这60株分离株中，93%（60株中的56株）通过外部PCR呈阳性，而所有分离株均通过半巢式PCR和Southern印迹杂交检测到。我们还报告了对狂犬病抗原标准荧光抗体试验（FAT）和hnRT-PCR对感染1型病毒的降解组织中狂犬病病毒RNA检测灵敏度的比较结果。结果表明，FAT无法检测到在37℃孵育超过72小时的脑组织中的病毒抗原，而hnRT-PCR可检测到在37℃孵育360小时的脑组织中的病毒RNA。
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