Bluetongue virus detection by two real-time RT-qPCRs targeting two different genomic segments
Bluetongue virus detection by two real-time RT-qPCRs targeting two different genomic segments
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DOI：
10.1016/j.jviromet.2006.11.007
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发表时间：
2007-03-01
影响因子：
3.1
通讯作者：
De Clercq, K.
中科院分区：
文献类型：
--
作者：
Toussaint, J. F.;Sailleau, C.;De Clercq, K.
通过常规方法检测蓝舌病病毒（BTV）是特别困难和劳动密集型的。由于BTV的24种血清型之间和内部具有高度的遗传多样性，因此分子诊断也很复杂。在本研究中，两个实验室联手开发和验证了两种新的RT-qPCR检测和扩增BTV片段1和5。2种检测试剂可检测所有24种血清型的菌株。它们的检测限均为0.01 ECE 50，来自无BTV山羊、绵羊和牛的所有114个样本均为阴性。这两种检测方法导致类似的C值时，测试生物样品收集的绵羊实验感染的BTV从地中海盆地的现场应变。平均而言，应用于24种血清型的2种方法获得的C1值彼此没有显著差异，但在少数菌株中观察到一些中度至高度差异。因此，这两种方法是互补的，可以并行使用，以确认诊断可能的新引进的BTV。还开发并验证了扩增β-肌动蛋白mRNA片段的RT-qPCR作为蓝舌病特异性测定的内部对照。所描述的三种测定法允许可靠且快速地检测BTV。(c)2006 Elsevier B. V.保留所有权利。
The detection of the bluetongue Virus (BTV) by conventional methods is especially difficult and labour-intensive. Molecular diagnosis is also complex because of the high genetic diversity between and within the 24 serotypes of BTV. In the present study, two laboratories joined forces to develop and validate two new RT-qPCRs detecting and amplifying BTV segments 1 and 5. The 2 assays detect strains from all 24 serotypes. They both have a detection limit of 0.01 ECE50 and all 114 samples from BTV-free goats, sheep and cattle were negative. The two assays resulted in similar C, values when testing biological samples collected in sheep infected experimentally with a field strain of BTV from the Mediterranean basin. On average, the C, values obtained with the 2 methods applied to the 24 serotypes were not significantly different from each other, but some moderate to high differences were seen with a few strains. Therefore these two methods are complementary and Could be used in parallel to confirm the diagnosis of a possible new introduction of BTV. An RT-qPCR amplifying a fragment of the beta-actin mRNA was also developed and validated as internal control for the bluetongue specific assays. The three assays described allow a reliable and rapid detection of BTV. (c) 2006 Elsevier B.V. All rights reserved.