Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR

Abstract

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The most widely used method of COVID-19 diagnostics is a reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay, detecting the presence of SARS-CoV-2 RNA in patient samples, typically from nasopharyngeal swabs. The RNA extraction is a major bottleneck in current COVID-19 testing, in terms of turn-around, logistics, component availability and cost, which delays or completely precludes COVID-19 diagnostics in many settings. Efforts to simplify the current methods are important, as increased diagnostic availability and efficiency is expected to benefit patient care and infection control. Here, we describe methods to circumvent RNA extraction in COVID-19 testing by performing RT-qPCR directly on heat-inactivated subject samples as well as samples lysed with readily available detergents. Our data, including cross-comparisons with clinically diagnosed patient samples, suggest that direct RT-qPCR is a viable option to extraction-based COVID-19 diagnostics. We argue that significant savings in terms of time and cost can be achieved by embracing RNA-extraction-free protocols, that feeds directly into the established PCR-based testing pipeline. This could aid the expansion of COVID-19 testing.

Improved and Simplified Diagnosis of Covid-19 using TE Extraction from Dry Swabs

“…This approach can be employed as rapid and economical method for diagnosis which does not require any RNA extraction step. Our results are in line with the earlier reports of RNA extraction-free RT-PCR (Alcoba-Florez et al, 2020;Bruce et al, 2020;Smyrlaki et al, 2020), but here we have done a very extensive (n=40) and thorough standardization of this procedure which is now consistent and compelling.…”

Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP

“…Several groups are optimizing workarounds to avoid the RNA extraction step for PCR based SARS-CoV-2 testing while maintaining sensitivity 13,15,17,29 . Here, we have demonstrated a variety of saliva pre-treatment protocols that enable sensitive detection of SARS-CoV-2 by both RT-LAMP and qRT-PCR.…”

“…We found that a 30-minute incubation provided a more reliable readout. To neutralize or otherwise reduce inhibitors in human saliva, we tested several approaches that have been demonstrated to improve viral RNA detection in crude samples including saliva [13][14][15][16][17] . We tested simple dilution of particle-containing saliva into water, and various heat and chemical treatments.…”

An Inexpensive RT-PCR Endpoint Diagnostic Assay for SARS-CoV-2 Using Nested PCR: Direct Assessment of Detection Efficiency of RT-qPCR Tests and Suitability for Surveillance

“…Hence the RT-nPCR test was able to detect presence of all three samples in a pool of 1:5. Direct testing of samples by RT-qPCR without RNA isolation has been described in recent reports (Bruce et al, 2020;Merindol et al, 2020;Smyrlaki et al, 2020). We performed direct testing of heat inactivated positive samples by the RT-nPCR test.…”