Alternative RNA extraction-free techniques for the real-time RT-PCR detection of SARS-CoV-2 in nasopharyngeal swab and sputum samples

Villota, Stephany D.; Nipaz, Victoria E.; Carrazco-Montalvo, Andrés; Hernandez, Sarah; Waggoner, Jesse J.; Ponce, Patricio; Coloma, Josefina; Orlando, Alberto; Cevallos, Varsovia

Standard diagnoses of SARS-CoV-2 infections are done by RNA extraction and real-time RT-PCR (rRT-PCR). However, the need for RNA extraction complicates testing due to increased processing time, high cost, and limited availability of commercial kits. Therefore, alternative methods for rRT-PCR detection of SARS-CoV-2 without RNA extraction were investigated. Nasopharyngeal and sputum samples were used to compare the sensitivity of three techniques: Trizol RNA extraction, thermal shock, and the direct use of samples with an RNase inhibitor. Direct, extraction-free use of primary samples plus the RNase inhibitor produced diagnostic values of 100 % sensitivity and specificity compared to standard protocols, and these findings were validated in a second, independent laboratory.