An in-well direct lysis method for rapid detection of SARS-CoV-2 by real time RT-PCR in eSwab specimens

Diagnostic real time reverse transcription PCR (rRT-PCR) is usually done using nucleic acid (NA) purified from the sample. In the SARS-CoV-2 pandemic reagents and utensils for NA purification has been in short supply. This has generated interest in methods that eliminate the need for NA purification.

To investigate if addition of detergent to rRT-PCR master mix (MM) enabled in-well direct lysis and detection of SARS-CoV-2 in clinical eSwab specimens.

IGEPAL-CA-630 (IGEPAL) was added to SARS-CoV-2 MM to 0.3 % final concentration and crude sample was added directly to the PCR well containing MM. Cycle of positivity (Cp) and categorical agreement was compared in samples tested in standard rRT-PCR after NA purification and in in-well lysis, direct rRT-PCR.

In-well lysis direct rRT-PCR detected SARS-CoV-2 in 27/30 previously SARS-CoV-2+ samples with an average bias of 3.26 cycles (95 %CI: 0.08-6.43 cycles). All 30 previously test negative samples remained negative when tested in in-well lysis, direct PCR.

Supplementation of detergent to MM was shown to be useful for the detection of SARS CoV-2 in eSwab specimens (COPAN) by direct rRT-PCR without prior NA purification.