Abstract
SummaryCurrent conventional detection of SARS-CoV-2 involves collection of a patient’s sample with a nasopharyngeal swab, storage of the swab during transport in a viral transport medium, extraction of RNA, and quantitative reverse transcription PCR (RT-qPCR). We developed a simplified preparation method using a chelating resin, Chelex, which obviates RNA extraction during viral testing. Direct detection RT-qPCR and digital droplet PCR were compared to the current conventional method with RNA extraction for simulated samples and patient specimens. The heat treatment in the presence of Chelex markedly improved detection sensitivity as compared to heat alone, and lack of RNA extraction shortens the overall diagnostic workflow. Furthermore, the initial sample heating step inactivates SARS-CoV-2 infectivity, thus improving workflow safety. This fast RNA preparation and detection method is versatile for a variety of samples, safe for testing personnel, and suitable for standard clinical collection and testing on high-throughput platforms.
Highlights
Diagnostic testing for SARS-CoV-2 is crucial to combat the current COVID-19 pandemic, even as vaccines are being distributed
Direct detection RT-qPCR and digital droplet PCR were compared to the current conventional method with RNA extraction for simulated samples and patient specimens
The Centers for Disease Control and Prevention (CDC) protocol for detecting SARSCoV-2 (COVID-19) involves sample collection, RNA extraction, and one-step real-time reverse transcription PCR (CDC-006-00,019, Revision 06 accessed on 6/21/2021: https://www.fda.gov/ media/134922/download)
Summary
Diagnostic testing for SARS-CoV-2 is crucial to combat the current COVID-19 pandemic, even as vaccines are being distributed. Samples collected in a viral transport medium (VTM) have been successfully used for direct RT-qPCR detection after heating (Alcoba-Florez et al, 2020; Beltran-Pavez et al, 2020; Bruce et al, 2020; Fomsgaard and Rosenstierne, 2020; Grant et al, 2020; Hasan et al, 2020; Lubke et al, 2020; Merindol et al, 2020; Smyrlaki et al, 2020) Despite these noteworthy results, the Ct values were 4 or more cycles higher than those of the standard protocol utilizing the RNA extraction step.
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