Nakayama T, Mori T, Yamaguchi S, Sonoda S, Asamura S, Yamashita R, Takeuchi Y, Urano T
Kitasato Institute, Department of Virology, Tokyo, Japan.
Virus Res. 1995 Jan;35(1):1-16. doi: 10.1016/0168-1702(94)00074-m.
A simple and sensitive method for the detection of measles virus genome was developed, amplifying the regions encoding the nucleocapsid (N) protein and hemagglutinin (H) protein of measles virus by reverse transcriptase-polymerase chain reaction (RT-PCR). We examined a variety of measles patients: 28 patients with natural infection, 4 with measles encephalitis and 1 with subacute sclerosing panencephalitis (SSPE). In 28 patients with natural measles infection a single step PCR amplifying the N region resulted in a high detection rate for all plasma samples (28/28) within 3 days of the onset of rash and 80% (20/25) even on day 7 of the onset of rash and later. Within 3 days of the onset of rash, 24/25 (96.0%) of nasopharyngeal secretions (NPS) and 27/28 (96.4%) of peripheral blood mononuclear cells (PBMC) were positive for the N region PCR and the positivity rate of PCR decreased in NPS and PBMC after 7 days of the rash. In acute measles infection, measles genome was detected in all cell fractions, CD4, CD8, B cells, and monocytes/macrophages by the H gene nested PCR. Measles genome was also detected from cerebrospinal fluids (CSF) in patients with measles encephalitis, SSPE, and acute measles by the H gene nested PCR. PCR products of the N and H regions were sequenced and we confirmed the presence of measles genome. Based on the sequence data, chronological sequence differences were observed over the past 10 years. The sequences obtained from the SSPE patient were closely related to those of the wild viruses that were circulating at the time when the patient initially acquired measles. RT-PCR for NPS, PBMC, CSF, and plasma provides a useful method for the diagnosis of measles and molecular epidemiological study in addition to virus isolation.
开发了一种简单且灵敏的检测麻疹病毒基因组的方法，通过逆转录聚合酶链反应（RT-PCR）扩增麻疹病毒编码核衣壳（N）蛋白和血凝素（H）蛋白的区域。我们检测了各类麻疹患者：28例自然感染患者、4例麻疹脑炎患者和1例亚急性硬化性全脑炎（SSPE）患者。在28例自然感染麻疹的患者中，单步PCR扩增N区域在出疹后3天内对所有血浆样本的检测率很高（28/28），甚至在出疹后第7天及以后仍有80%（20/25）的检测率。出疹后3天内，24/25（96.0%）的鼻咽分泌物（NPS）和27/28（96.4%）的外周血单个核细胞（PBMC）的N区域PCR呈阳性，出疹7天后NPS和PBMC中PCR的阳性率下降。在急性麻疹感染中，通过H基因巢式PCR在所有细胞组分（CD4、CD8、B细胞和单核细胞/巨噬细胞）中检测到麻疹基因组。通过H基因巢式PCR还在麻疹脑炎、SSPE和急性麻疹患者的脑脊液（CSF）中检测到麻疹基因组。对N和H区域的PCR产物进行测序，我们确认了麻疹基因组的存在。基于序列数据，观察到过去10年的时间序列差异。从SSPE患者获得的序列与该患者最初感染麻疹时流行的野生病毒序列密切相关。除病毒分离外，对NPS、PBMC、CSF和血浆进行RT-PCR为麻疹诊断和分子流行病学研究提供了一种有用的方法。
