The SARS-CoV-2 pandemic has created an urgent need for diagnostic tests to detect viral RNA. Commercial RNA extraction kits are often expensive, in limited supply, and do not always fully inactivate the virus. Together, this calls for the development of safer methods for SARS-CoV-2 extraction that utilize readily available reagents and equipment present in most standard laboratories. We optimized and simplified a RNA extraction method combining a high molar acidic guanidinium isothiocyanate (GITC) solution, phenol and chloroform. First, we determined the GITC/RNA dilution thresholds compatible with an efficient two-step RT-qPCR for B2M mRNA in nasopharyngeal (NP) or oropharyngeal (OP) swab samples. Second, we optimized a one-step RT-qPCR against SARS-CoV-2 using NP and OP samples. We furthermore tested a SARS-CoV-2 dilution series to determine the detection threshold. The method enables downstream detection of SARS-CoV-2 by RT-qPCR with high sensitivity (~4 viral RNA copies per RT-qPCR). The protocol is simple, safe, and expands analysis capacity as the inactivated samples can be used in RT-qPCR detection tests at laboratories not otherwise classified for viral work. The method takes about 30 min from swab to PCR-ready viral RNA and circumvents the need for commercial RNA purification kits.

中文翻译：

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一种简单、安全、灵敏的 SARS-CoV-2 灭活和 RNA 提取方法用于 RT-qPCR

SARS-CoV-2 大流行已经迫切需要检测病毒 RNA 的诊断测试。商业 RNA 提取试剂盒通常价格昂贵，供应有限，而且并不总是能完全灭活病毒。总之，这需要开发更安全的 SARS-CoV-2 提取方法，该方法利用大多数标准实验室中现成的试剂和设备。我们优化并简化了一种结合高摩尔酸性异硫氰酸胍 (GITC) 溶液、苯酚和氯仿的 RNA 提取方法。首先，我们确定了与鼻咽 (NP) 或口咽 (OP) 拭子样本中 B2M mRNA 的有效两步 RT-qPCR 兼容的 GITC/RNA 稀释阈值。其次，我们使用 NP 和 OP 样本优化了针对 SARS-CoV-2 的一步式 RT-qPCR。我们还测试了 SARS-CoV-2 稀释系列以确定检测阈值。该方法能够通过 RT-qPCR 以高灵敏度（每个 RT-qPCR 约 4 个病毒 RNA 拷贝）对 SARS-CoV-2 进行下游检测。该协议简单、安全并扩展了分析能力，因为灭活的样本可用于实验室的 RT-qPCR 检测测试，而不是其他分类为病毒工作的实验室。该方法从拭子到可用于 PCR 的病毒 RNA 大约需要 30 分钟，并且无需商业 RNA 纯化试剂盒。