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

BACKGROUND: 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.

OBJECTIVES: 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.

STUDY DESIGN: 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.

RESULTS: 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.

CONCLUSIONS: 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.