Evaluation of a single-tube fluorogenic RT-PCR assay for detection of bovine respiratory syncytial virus in clinical samples
Evaluation of a single-tube fluorogenic RT-PCR assay for detection of bovine respiratory syncytial virus in clinical samples
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DOI：
10.1016/j.jviromet.2004.09.016
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发表时间：
2005-02-01
影响因子：
3.1
通讯作者：
Belák, S
中科院分区：
文献类型：
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作者：
Hakhverdyan, M;Hägglund, S;Belák, S
牛呼吸道合胞体病毒（BRSV）在所有年龄段的未免疫牛中引起严重疾病，并且是犊牛呼吸道疾病综合征中的常见病原体。简化的快速BRSV诊断方法将鼓励在疫情暴发期间进行采样，从而有助于加深对该病毒的了解。在本研究中，基于TaqMan原理开发了一种BRSV荧光定量逆转录聚合酶链反应（fRT - PCR）检测方法，并在大量临床样本上进行了评估，这些样本代表了自然和实验性BRSV感染的各种情况。通过使用单步闭管形式，周转时间大幅缩短，并且在交叉污染风险最小的情况下获得了结果。根据比较分析，fRT - PCR的检测限与巢式PCR处于同一水平，且灵敏度相对高于常规PCR、抗原酶联免疫吸附测定（Ag - ELISA）和病毒分离（VI）。穿插的阴性对照样本、来自健康动物的样本以及8种有症状或遗传相关的病毒均呈阴性，证实了该检测方法具有高度特异性。综上所述，数据表明fRT - PCR检测方法可应用于临床标本中的常规病毒检测，并为BRSV研究提供了一种快速且有价值的工具。（C）2004爱思唯尔公司。版权所有。
Bovine respiratory syncytial virus (BRSV) causes severe disease in naive cattle of all ages and is a common pathogen in the respiratory disease complex of calves. Simplified methods for rapid BRSV diagnosis would encourage sampling during outbreaks and would consequently lead to an extended understanding of the virus. In this study, a BRSV fluorogenic reverse transcription PCR (fRT-PCR) assay, based on TaqMan principle, was developed and evaluated on a large number of clinical samples, representing various cases of natural and experimental BRSV infections. By using a single-step closed-tube format, the turn-around time was shortened drastically and results were obtained with minimal risk for cross-contamination. According to comparative analyses, the detection limit of the fRT-PCR was on the same level as that of a nested PCR and the sensitivity relatively higher than that of a conventional PCR, antigen ELISA (Ag-ELISA) and virus isolation (VI). Interspersed negative control samples, samples from healthy animals and eight symptomatically or genetically related viruses were all negative, confirming a high specificity of the assay. Taken together, the data indicated that the fRT-PCR assay can be applied to routine virus detection in clinical specimens and provides a rapid and valuable too] in BRSV research. (C) 2004 Elsevier B.V. All rights reserved.