Optimal preparation of SARS-CoV-2 viral transport medium for culture.
Optimal preparation of SARS-CoV-2 viral transport medium for culture.
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DOI：
10.1186/s12985-021-01525-z
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发表时间：
2021-03-10
期刊：
影响因子：
4.8
通讯作者：
Strugnell R
中科院分区：
文献类型：
--
作者：
McAuley J;Fraser C;Paraskeva E;Trajcevska E;Sait M;Wang N;Bert E;Purcell D;Strugnell R
The sudden arrival of the COVID-19 pandemic placed significant stresses on supply chains including viral transport medium (VTM).迫切需要 VTM 来支持病毒复制以及其他常规诊断方法。我们描述了用于快速扩展诊断测试的 VTM 的准备和验证测试，其中保持了 VTM 保持病毒完整性、用于培养、分离和全序列分析的能力。 VTM was prepared using different methods of sterilization then ‘spiked’ with virus.使用 Vero 细胞中的病毒培养物来研究 VTM，并通过定量 PCR 进行核酸检测。通过过滤和高压灭菌器灭菌可获得最佳结果。事实证明，VTM 对于基于培养的分析来说是稳健的，前提是接种的 VTM 储存在 4°C 下，并在 48 小时内进行测试。当模拟接种的 VTM 在室温下保存时，过滤的 VTM 还支持基于 PCR 的诊断至少 5 天。 VTM 生产的手动处理（包括灌装和灭菌）得到了优化。将 SARS-CoV-2 添加到 VTM 中，以评估不同的灭菌方法并测量储存时间和温度对 VTM 性能的影响。虽然大多数诊断方案不需要具有复制能力的病毒，但高质量 VTM 的使用将允许在 COVID-19 大流行中进行下一阶段的实验室分析，包括重新分离的 SARS-CoV-2 的药物和抗体敏感性分析，以及疫苗逃逸突变体的测试。
The sudden arrival of the COVID-19 pandemic placed significant stresses on supply chains including viral transport medium (VTM). The VTM that was urgently required needed to support viral replication, as well as other routine diagnostic approaches. We describe the preparation and validation testing of VTM for rapidly expanding diagnostic testing, where the capacity of the VTM to preserve viral integrity, for culture, isolation and full sequence analysis, was maintained. VTM was prepared using different methods of sterilization then ‘spiked’ with virus. The VTM was investigated using viral culture in Vero cells, and for nucleic acid detection by quantitative PCR. The best results were obtained by filter and autoclave-based sterilization. The VTM proved robust for culture-based analyses provided the inoculated VTM was stored at 4 °C, and tested within 48 h. The filtered VTM also supported PCR-based diagnosis for at least 5 days when the mock inoculated VTM was held at room temperature. The manual handling of VTM production, including filling and sterilization, was optimized. SARS-CoV-2 was spiked into VTM to assess different sterilization methods and measure the effects of storage time and temperature upon VTM performance. While most diagnostic protocols will not require replication competent virus, the use of high quality VTM will allow for the next phase of laboratory analysis in the COVID-19 pandemic, including drug and antibody susceptibility analysis of re-isolated SARS-CoV-2, and for the testing of vaccine escape mutants.
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