Development and optimisation of a duplex real-time reverse transcription quantitative PCR assay targeting the VP7 and NS2 genes of African horse sickness virus
Development and optimisation of a duplex real-time reverse transcription quantitative PCR assay targeting the VP7 and NS2 genes of African horse sickness virus
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DOI：
10.1016/j.jviromet.2010.03.009
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发表时间：
2010-07-01
影响因子：
3.1
通讯作者：
Guthrie, A. J.
中科院分区：
文献类型：
--
作者：
Quan, M.;Lourens, C. W.;Guthrie, A. J.
使用 2004 年至 2005 年期间收集的 52 个非洲马瘟病毒 (AHSV) 南非分离株（包括所有 9 个 AHSV 血清型的病毒）的核苷酸序列来设计和开发针对 AHSV VP7 (S8) 和 NS2 (S9) 基因的双重实时逆转录定量 PCR (RT-PCR) 测定。该测定法针对自然感染马的新鲜和冷冻血液中 AHSV 的检测进行了优化。使用随机六聚体而不是 RI 的基因特异性引物，并且在随机六聚体存在的情况下双链 RNA 变性，可以增强检测性能。该测定是有效的，线性范围至少有五个数量级。该测定的分析灵敏度为 132 个拷贝的目标基因（每毫升血液 4125 个拷贝），并且该测定的灵敏度比 BHK-21 细胞上病毒分离的灵敏度至少高 10 倍。该检测还具有高度特异性，因为它没有检测到相关的环状病毒，例如蓝舌病病毒和马脑病病毒。 (C) 2010 Elsevier B.V. 保留所有权利。
Nucleotide sequences of 52 South African isolates of African horse sickness virus (AHSV) collected during 2004-2005 and including viruses of all nine AHSV serotypes, were used to design and develop a duplex real-time reverse transcription quantitative PCR (RT-PCR) assay targeting the VP7 (S8) and NS2 (S9) genes of AHSV. The assay was optimized for detection of AHSV in fresh and frozen blood of naturally infected horses. Assay performance was enhanced using random hexamers rather than gene-specific primers for RI, and with denaturation of double-stranded RNA in the presence of random hexamers. The assay was efficient with a linear range of at least five orders of magnitude. The analytical sensitivity of the assay was 132 copies of the target genes (4125 copies per ml of blood), and the assay was at least 10-fold more sensitive than virus isolation on BHK-21 cells. The assay was also highly specific because it did not detect related orbiviruses, such as bluetongue and equine encephalosis viruses. (C) 2010 Elsevier B.V. All rights reserved.