Development of quantitative gene-specific real-time RT-PCR assays for the detection of measles virus in clinical specimens
Development of quantitative gene-specific real-time RT-PCR assays for the detection of measles virus in clinical specimens
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DOI：
10.1016/j.jviromet.2005.10.006
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发表时间：
2006-03-01
影响因子：
3.1
通讯作者：
Rota, PA
中科院分区：
文献类型：
--
作者：
Hummel, KB;Lowe, L;Rota, PA
建立了麻疹病毒（MV）核蛋白（N）、融合蛋白（F）和血凝素（H）基因的实时荧光定量RT-PCR检测方法。评估了N、F和H基因的四种引物和探针组，并优化了反应条件。使用合成RNA的稀释系列，确定每个靶RNA/反应的检测限约为10个拷贝。Ci值和RNA浓度之间的关系在10-10（6）RNA拷贝/反应范围内呈线性，并且测定内和测定间变异性较低。N基因特异性实时检测在确诊麻疹病例的100%临床样本中检测到MV RNA，而标准RT-PCR为41%。MV H和F基因特异性实时检测分别在93%和82%的这些标本中检测到MV RNA。实时检测可以检测来自代表MV的每个活性基因型的菌株的RNA，并且也具有高度特异性，因为当检测已知含有其他呼吸道病毒的样品时，没有发现假阳性。实时RT-PCR检测将可用于支持常规麻疹实验室监测，促进需要敏感和定量检测MV RNA的发病机制研究项目，并有助于调查自然麻疹感染或接种含麻疹疫苗导致的严重疾病后遗症。(c)2005 Elsevier B. V.保留所有权利。
Real-time RT-PCR assays targeting sequences in the measles virus (MV) nucleoprotein (N), fusion (F), and hemagglutinin (H) genes were developed for the detection of MV RNA in clinical specimens. Four primer and probe sets each for the N, F, and H genes were evaluated and reaction conditions optimized. Using dilution series of synthetic RNAs, the limits of detection were determined to be approximately 10 copies for each target RNA/reaction. The relationship between C, values and RNA concentration was linear within a range of 10-10(6) RNA copies/reaction, and intra- and inter-assay variability was low. The N gene-specific real-time assay detected MV RNA in 100% of clinical samples from confirmed measles cases compared to 41% by standard RT-PCR. The MV H and F gene-specific real-time assays detected MV RNA in 93% and 82% of these specimens, respectively. Real-time assays could detect RNA from strains representing each active genotype of MV and were also highly specific, as no false positives were identified when samples known to contain other respiratory viruses were tested. Real-time RT-PCR assays will be available to support routine measles laboratory surveillance, to facilitate research projects on pathogenesis that require sensitive and quantitative detection of MV RNA, and to aid in the investigation of serious disease sequelae resulting from natural measles infection or vaccination with measles-containing vaccines. (c) 2005 Elsevier B.V. All rights reserved.