Kamolvarin N, Tirawatnpong T, Rattanasiwamoke R, Tirawatnpong S, Panpanich T, Hemachudha T
WHO Collaborating Centre for Research on Rabies Pathogenesis and Prevention, Queen Saovabha Memorial Institute, Bangkok, Thailand.
J Infect Dis. 1993 Jan;167(1):207-10. doi: 10.1093/infdis/167.1.207.
A simple, sensitive, and specific polymerase chain reaction (PCR) protocol for detection of rabies virus is described. The process consists of sample preparation, reverse transcription, two-step DNA amplification, and detection of the amplified product. RNA was extracted from animal and human brain by phenol-chloroform using guanidinium thiocyanate. Viral RNA was then amplified in a two-step PCR that used two sets of nested primers designed to amplify rabies nucleocapsid (N) sequence. Rabies nucleocapsid sequence was amplified from all brain samples from 95 dogs and 3 humans with rabies confirmed by fluorescent antibody (FAT) and mouse inoculation tests (MIT). Rabies-negative brain samples (110 dogs, 2 humans) were PCR-negative. The process requires < 24 h. Detection of viral RNA was still possible in brain material that was left at room temperature for 72 h. As little as 8 pg of rabies virus RNA could be detected. This technique could have practical applications as a confirmatory test to FAT at busy rabies diagnostic centers.
本文描述了一种用于检测狂犬病病毒的简单、灵敏且特异的聚合酶链反应（PCR）方法。该过程包括样本制备、逆转录、两步DNA扩增以及扩增产物的检测。使用异硫氰酸胍通过苯酚 - 氯仿从动物和人类脑组织中提取RNA。然后在两步PCR中扩增病毒RNA，该PCR使用两组巢式引物来扩增狂犬病核衣壳（N）序列。从95只狗和3名经荧光抗体（FAT）和小鼠接种试验（MIT）确诊为狂犬病的人的所有脑组织样本中扩增出狂犬病核衣壳序列。狂犬病阴性的脑组织样本（110只狗，2人）PCR检测为阴性。该过程耗时不到24小时。在室温下放置72小时的脑材料中仍可检测到病毒RNA。最低可检测到8 pg的狂犬病病毒RNA。这项技术在繁忙的狂犬病诊断中心作为FAT的确认试验可能具有实际应用价值。
