Center for Genomic Medicine, Rigshospitalet, Copenhagen, Denmark.
Institute of Physico-Chemical Biology (IBPC), CNRS/University Paris Diderot, Paris, France.
APMIS. 2021 Jul;129(7):393-400. doi: 10.1111/apm.13123. Epub 2021 Mar 17.
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.
SARS-CoV-2 大流行迫切需要用于检测病毒 RNA 的诊断测试。商业 RNA 提取试剂盒通常昂贵、供应有限，并且并不总是能完全使病毒失活。因此，需要开发更安全的 SARS-CoV-2 提取方法，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 RNA 提取方法，该方法结合了高摩尔酸性异硫氰酸胍（GITC）溶液、苯酚和氯仿。首先，我们确定了与鼻咽（NP）或口咽（OP）拭子样本中 B2M mRNA 的高效两步 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 RT-qPCR。此外，我们还测试了 SARS-CoV-2 稀释系列以确定检测阈值。该方法通过 RT-qPCR 具有高灵敏度（~4 个病毒 RNA 拷贝/RT-qPCR）来实现 SARS-CoV-2 的下游检测。该方案简单、安全，并扩大了分析能力，因为灭活的样本可用于未分类进行病毒工作的实验室的 RT-qPCR 检测测试。该方法从拭子到可用于 PCR 的病毒 RNA 大约需要 30 分钟，并且避免了对商业 RNA 纯化试剂盒的需求。
