Clinical and Applied Virology Group, Department of Infection and Immunity, Luxembourg Institute of Health, L-4354 Esch-sur-Alzette, Luxembourg.
Laboratoire de Médecine Vétérinaire de l'Etat, Luxembourg Veterinary and Food Administration (ALVA), L-3555 Dudelange, Luxembourg.
Viruses. 2024 Sep 10;16(9):1442. doi: 10.3390/v16091442.
Since early 2024, a multistate outbreak of highly pathogenic avian influenza H5N1 has been affecting dairy cattle in the USA. The influenza viral RNA concentrations in milk make it an ideal matrix for surveillance purposes. However, viral RNA detection in multi-component fluids such as milk can be complex, and optimization of influenza detection methods is thus required. Raw bulk tank milk and mastitis milk samples were artificially contaminated with an avian influenza strain and subjected to five extraction methods. HCoV-229E and synthetic RNA were included as exogenous internal process controls. Given the high viral load usually observed in individual raw milk samples, four out of five tested methods would enable influenza detection in milk with normal texture, over a time window of at least 2 weeks post-onset of clinical signs. Nevertheless, sample dilution 1:3 in molecular transport medium prior to RNA extraction provided the best results for dilution of inhibitory substances and a good recovery rate of influenza RNA, that reached 12.5 ± 1.2% and 10.4 ± 3.8% in two independent experiments in bulk milk and 11.2 ± 3.6% and 10.0 ± 2.9% on two cohorts of mastitis milk samples. We have also shown compatibility of an influenza RT-qPCR system with synthetic RNA detection for simultaneous validation of the RNA extraction and RT-qPCR processes.
自 2024 年初以来，美国发生了多州高致病性禽流感 H5N1 疫情，影响了奶牛。牛奶中的流感病毒 RNA 浓度使其成为监测目的的理想基质。然而，牛奶等多组分液体中的病毒 RNA 检测可能很复杂，因此需要优化流感检测方法。将原始散装奶和乳腺炎奶样品人工污染禽流感株，并进行了五种提取方法的检测。将 HCoV-229E 和合成 RNA 作为外源性内部过程对照。鉴于通常在单个原始牛奶样品中观察到的高病毒载量，在临床症状出现后至少 2 周的时间窗口内，五种测试方法中的四种将能够检测到正常质地的牛奶中的流感。然而，在 RNA 提取之前，将分子运输培养基稀释 1:3 可有效稀释抑制物质，并且流感 RNA 的回收率良好，在两个独立的散装奶实验中达到 12.5 ± 1.2%和 10.4 ± 3.8%，在两个乳腺炎奶样本组中分别达到 11.2 ± 3.6%和 10.0 ± 2.9%。我们还表明，流感 RT-qPCR 系统与合成 RNA 检测兼容，可同时验证 RNA 提取和 RT-qPCR 过程。
