Since the rapid onset of the COVID-19 pandemic, its causative virus, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), continues to spread and increase the number of fatalities. To expedite studies on understanding potential surface transmission of the virus and to aid environmental epidemiological investigations, we developed a rapid viability reverse transcriptase PCR (RV-RT-PCR) method that detects viable (infectious) SARS-CoV-2 from swab samples in <1 day compared to several days required by current gold-standard cell-culture-based methods. The method integrates cell-culture-based viral enrichment in a 96-well plate format with gene-specific RT-PCR-based analysis before and after sample incubation to determine the cycle threshold (C_T) difference (ΔC_T). An algorithm based on ΔC_T ≥ 6 representing ∼ 2-log or more increase in SARS-CoV-2 RNA following enrichment determines the presence of infectious virus. The RV-RT-PCR method with 2-hr viral infection and 9-hr post-infection incubation periods includes ultrafiltration to concentrate virions, resulting in detection of <50 SARS-CoV-2 virions in swab samples in 17 hours (for a batch of 12 swabs), compared to days typically required by the cell-culture based method. The SARS-CoV-2 RV-RT-PCR method may also be useful in clinical sample analysis and antiviral drug testing, and could serve as a model for developing rapid methods for other viruses of concern.

自 COVID-19 大流行迅速爆发以来，其致病病毒严重急性呼吸综合征冠状病毒-2 (SARS-CoV-2) 持续传播并增加了死亡人数。为了加快了解病毒潜在表面传播的研究并帮助环境流行病学调查，我们开发了一种快速活力逆转录酶 PCR (RV-RT-PCR) 方法，可从拭子样本中检测出活的（传染性）SARS-CoV-2与当前基于细胞培养的金标准方法所需的几天相比，只需 3C1 天。该方法将 96 孔板中基于细胞培养的病毒富集与样品孵育前后基于基因特异性 RT-PCR 的分析相结合，以确定循环阈值 (C _T ) 差异 (ΔC _T )。基于 ΔC _T ≥ 6 的算法代表富集后 SARS-CoV-2 RNA 增加约 2 个对数级或更多，从而确定感染性病毒的存在。 RV-RT-PCR 方法采用 2 小时病毒感染和 9 小时感染后潜伏期，包括超滤以浓缩病毒粒子，从而在 17 小时内检测到拭子样本中的 <50 SARS-CoV-2 病毒粒子（对于一批12 个拭子），与基于细胞培养的方法通常需要的天数相比。 SARS-CoV-2 RV-RT-PCR 方法也可用于临床样本分析和抗病毒药物测试，并可作为开发其他关注病毒的快速方法的模型。