Division for Diagnostics & Scientific Advice, National Veterinary Institute, Technical University of Denmark, Kongens Lyngby, Denmark.
Institute of Diagnostic Virology, Federal Research Institute for Animal Health, Friedrich-Loeffler Institute, Riems, Germany.
Front Cell Infect Microbiol. 2018 May 22;8:165. doi: 10.3389/fcimb.2018.00165. eCollection 2018.
Influenza A viruses (IAVs) are important human and animal pathogens with high impact on human and animal health. In Denmark, a passive surveillance program for IAV in pigs has been performed since 2011, where screening tests and subsequent subtyping are performed by reverse transcription quantitative real-time PCR (RT-qPCR). A disadvantage of the current subtyping system is that several assays are needed to cover the wide range of circulating subtypes, which makes the system expensive and time-consuming. Therefore, the aim of the present study was to develop a high-throughput method, which could improve surveillance of swine influenza viruses (swIAVs) and lower the costs of virus subtyping. Twelve qPCR assays specific for various hemagglutinin and neuraminidase gene lineages relevant for swIAV and six assays specific for the internal genes of IAV were developed and optimized for the high-throughput qPCR platform BioMark (Fluidigm). The qPCR assays were validated and optimized to run under the same reaction conditions using a 48.48 dynamic array (48.48DA). The sensitivity and specificity was assessed by testing virus isolates and field samples with known subtypes. The results revealed a performance of the swIAV 48.48DA similar to conventional real-time analysis, and furthermore, the specificity of swIAV 48.48DA was very high and without cross reactions between the assays. This high-throughput system provides a cost-effective alternative for subtyping of swIAVs.
甲型流感病毒（IAV）是重要的人兽病原体，对人类和动物的健康有很大的影响。在丹麦，自 2011 年以来，一直对猪进行 IAV 的被动监测计划，通过逆转录定量实时 PCR（RT-qPCR）进行筛选测试和随后的亚型鉴定。当前的亚型鉴定系统的一个缺点是，需要进行多种检测才能覆盖循环的广泛亚型，这使得该系统昂贵且耗时。因此，本研究的目的是开发一种高通量方法，该方法可以改善猪流感病毒（swIAV）的监测，并降低病毒分型的成本。开发并优化了 12 种针对 swIAV 相关各种血凝素和神经氨酸酶基因谱系的 qPCR 检测和 6 种针对 IAV 内部基因的 qPCR 检测，用于高通量 qPCR 平台 BioMark（Fluidigm）。通过使用 48.48 动态阵列（48.48DA）对 qPCR 检测进行验证和优化，以在相同的反应条件下运行。通过测试具有已知亚型的病毒分离株和现场样本评估了灵敏度和特异性。结果表明，swIAV 48.48DA 的性能与常规实时分析相似，此外，swIAV 48.48DA 的特异性非常高，并且各检测之间没有交叉反应。该高通量系统为 swIAV 的分型提供了一种具有成本效益的替代方法。
