Real-time RT-PCR detection of Bovine Viral Diarrhoea virus in whole blood using an external RNA reference.
Real-time RT-PCR detection of Bovine Viral Diarrhoea virus in whole blood using an external RNA reference.
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DOI：
10.1016/j.jviromet.2006.08.008
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发表时间：
2006-12
影响因子：
3.1
通讯作者：
Brownlie J
中科院分区：
文献类型：
--
作者：
Young NJ;Thomas CJ;Collins ME;Brownlie J
开发了一种使用SYBR®绿色I的新型两步实时RT-PCR检测方法，用于检测牛全血中的急性牛病毒性腹泻病毒（BVDV）感染。在感染期间，动物经历特征性的短暂白细胞减少症，并且每体积血液中的细胞数量随时间变化;因此通过参考细胞管家基因来定量病毒载量并不理想，因为这可能隐藏显著的动物间变异。因此，为了便于比较不同样本，使用外部RNA参考品进行标准化，其中在RNA提取前，每份样本均加标RNA病毒犬肠道冠状病毒（CECov），以进行比较。使用设计用于扩增BVDV 5′-UTR的156 bp区域或CECov核衣壳蛋白基因的280 bp区域的两组引物进行实时RT-PCR。确定了试验的线性和有效性，并使用BVDV攻毒小牛样品评估了该方法。在攻毒后第6天和第14天通过实时RT-PCR定量病毒RNA。通过传统细胞培养的感染性病毒分离在第7天后为阴性。本研究证明了快速、灵敏和可靠检测急性BVDV感染的令人鼓舞的结果，并提供了用于全血样本或无法获得合适管家基因的样本的替代实时RT-PCR方法。
A novel two-step real-time RT-PCR assay using SYBR® Green I was developed for the detection of acute Bovine Viral Diarrhoea virus (BVDV) infection in whole blood from cattle. During infection animals experience a characteristic transient leucopenia and the number of cells per volume of blood changes over time; so quantitation of viral load by reference to a cellular housekeeping gene is not ideal as this may hide significant animal to animal variation. Therefore, to facilitate comparison of different samples, an external RNA reference was used for normalisation whereby each sample was spiked with the RNA virus, Canine Enteric Coronavirus (CECov), prior to RNA extraction, for comparative purposes. Real-time RT-PCR was carried out with two primer sets designed to amplify either a 156 bp region of the BVDV 5′-UTR or a 280 bp region of the CECov nucleocapsid protein gene. Linearity and efficiency of the assay was established and the method assessed using samples from BVDV-challenged calves. Viral RNA was quantified on days 6 and 14 post-challenge by real-time RT-PCR. Infectious virus isolation by traditional cell culture was negative after day 7. This study demonstrates encouraging results for rapid, sensitive and reliable detection of acute BVDV infection and provides an alternative real-time RT-PCR method for use on whole blood samples or samples where suitable housekeeping genes are not available.
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影响因子：
3.1
作者：
Bae, HG;Nitsche, A;Niedrig, M
通讯作者：
Niedrig, M
影响因子：
4.1
作者：
Thellin, O;Zorzi, W;Heinen, E
通讯作者：
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影响因子：
3.1
作者：
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通讯作者：
Kerkhofs, P
影响因子：
4.8
作者：
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通讯作者：
Niesters, HGM
影响因子：
2.7
作者：
Cleland, A;Nettleton, P;Simmonds, P
通讯作者：
Simmonds, P