Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR

GENOUD, VALERIA; STORTZ, MARTIN; WAISMAN, ARIEL; BERARDINO, BRUNO G.; VERNERI, PAULA; DANSEY, VIRGINIA; SALVATORI, MELINA; REMES LENICOV, FEDERICO; LEVI, VALERIA

PLOS ONE

PUBLIC LIBRARY SCIENCE

Año: 2021 vol. 16

1932-6203

AbstractReal-time reverse transcription PCR (RT-qPCR) is the gold-standard technique for severeacute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngealswabs specimens. The analysis by RT-qPCR usually requires a previous extraction step toobtain the purified viral RNA. Unfortunately, RNA extraction constitutes a bottleneck forearly detection in many countries since it is expensive, time-consuming and depends on theavailability of commercial kits. Here, we describe an extraction-free protocol for SARS-CoV-2 detection by RT-qPCR from nasopharyngeal swab clinical samples in saline solution. Themethod includes a treatment with proteinase K followed by heat inactivation (PK+HIDmethod). We demonstrate that PK+HID improves the RT-qPCR performance in comparisonto the heat-inactivation procedure. Moreover, we show that this extraction-free protocol canbe combined with a variety of multiplexing RT-qPCR kits. The method combined with a multiplexingdetection kit targeting N and ORF1ab viral genes showed a sensitivity of 0.99 and aspecificity of 0.99 from the analysis of 106 positive and 106 negative clinical samples. Inconclusion, PK+HID is a robust, fast and inexpensive procedure for extraction-free RTqPCRdeterminations of SARS-CoV-2. The National Administration of Drugs, Foods andMedical Devices of Argentina has recently authorized the use of this method.