Detection of swine influenza virus (SIV) in commercial swine herds is important for understanding the infection status of the herd and for controlling disease. Current molecular diagnostics require that specimens be submitted to a laboratory which provides results to the growers after some time which is generally too late to intercede in disease control. Moreover, current diagnostic assays are time-consuming, typically costly, and require skilled technical expertise. We have instituted a reverse transcription loop-mediated isothermal amplification (RT-LAMP) diagnostic assay based on conserved regions of the SIV matrix (M) gene and H1N1 hemagglutinin (HA) sequences. The RT-LAMP assay was optimized to use both colorimetric and fluorescent endpoints and was validated. The M and HA RT-LAMP assays have a limit-of-detection (LOD) sensitive to 11 and 8-log-fold dilutions of viral RNA, respectively, and are capable of discriminating between H1 and H3 strains of SIV. Additionally, the RT-LAMP assay was optimized for direct amplification of SIV from field samples without the need for viral RNA isolation. The direct RT-LAMP detected >86 % of qRT-PCR validated SIV samples, and >66 % of negative samples when spiked with viral RNA or SIV. The diagnostic RT-LAMP assay is a rapid, sensitive, specific, and cost-effective method for the detection of SIV in herds substantially aiding diagnosis and surveillance.

在商业猪群中检测猪流感病毒 (SIV) 对于了解猪群的感染状况和控制疾病非常重要。当前的分子诊断要求将样本提交给实验室，该实验室在一段时间后向种植者提供结果，这通常为时已晚，无法干预疾病控制。此外，目前的诊断分析非常耗时，通常成本高昂，并且需要熟练的技术专长。我们已经建立了基于 SIV 基质 (M) 基因和 H1N1 血凝素 (HA) 序列的保守区域的逆转录环介导的等温扩增 (RT-LAMP) 诊断测定。RT-LAMP 检测经过优化，可同时使用比色和荧光端点，并得到验证。M 和 HA RT-LAMP 检测具有分别对 11 倍和 8 倍稀释的病毒 RNA 敏感的检测限 (LOD)，并且能够区分 SIV 的 H1 和 H3 株。此外，RT-LAMP 检测针对从现场样本中直接扩增 SIV 进行了优化，无需分离病毒 RNA。直接 RT-LAMP 检测到超过 86% 的 qRT-PCR 验证 SIV 样本，以及在添加病毒 RNA 或 SIV 时超过 66% 的阴性样本。诊断性 RT-LAMP 检测是一种快速、灵敏、特异且具有成本效益的方法，用于检测猪群中的 SIV，大大有助于诊断和监测。RT-LAMP 检测针对从现场样本中直接扩增 SIV 进行了优化，无需分离病毒 RNA。直接 RT-LAMP 检测到超过 86% 的 qRT-PCR 验证 SIV 样本，以及在添加病毒 RNA 或 SIV 时超过 66% 的阴性样本。诊断性 RT-LAMP 检测是一种快速、灵敏、特异且具有成本效益的方法，用于检测猪群中的 SIV，大大有助于诊断和监测。RT-LAMP 检测针对从现场样本中直接扩增 SIV 进行了优化，无需分离病毒 RNA。直接 RT-LAMP 检测到超过 86% 的 qRT-PCR 验证 SIV 样本，以及在添加病毒 RNA 或 SIV 时超过 66% 的阴性样本。诊断性 RT-LAMP 检测是一种快速、灵敏、特异且具有成本效益的方法，用于检测猪群中的 SIV，大大有助于诊断和监测。