Molecular detection of SARS-CoV-2 using a reagent-free approach

Abstract

Shortage of reagents and consumables required for the extraction and molecular detection of SARS-CoV-2 RNA in respiratory samples has led many laboratories to investigate alternative approaches for sample preparation. Fomsgaard et al 2020 5 recently presented results using heat-processing of respiratory samples prior to RT-qPCR as an economical method enabling an extremely fast streamlining of the processes at virtually no cost.Here, we present our results using this method and highlight some major pitfalls that diagnostics laboratories should be aware of before proceeding with this technique. We first investigated various treatments using different temperatures, incubation times and sample volumes based on the above study to optimise the heattreatment conditions. Although the initial data confirmed the published results, further investigations revealed unexpected inhibitory properties of some commonly used virus transport media (VTMs) on some commercially available RT-qPCR mixes, emphasising the critical importance of a thorough validation process to determine the most adapted reagents to be used depending on the sample types to be tested.In conclusion, although the method works, with very consistent Ct values and an excellent sensitivity when compared to a conventional RNA extraction method, it is critical to include an internal control to check each sample for potential inhibition.

Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples

“…We caution that the study was done with a limited number of samples and amplifications should be closely monitored to avoid increasing the false negatives above that of the standard diagnosis based on RNA extractions ( Xie et al 2020 ). Despite that, diverse empirical assessments of our protocol and that proposed by Fomsgaard & Rosenstierne (2020) revealed that the quantitative results are highly comparable ( Calvez et al 2020 ). Remarkably, SARS-CoV-2 and SARS-CoV-1 show comparable environmental stability ( van Doremalen et al 2020 ), and several evidences suggest that SARS-CoV-1 ( Geller et al 2012 ) and SARS-CoV-2 ( Pastorino et al 2020 ) lose infectivity above 56 °C within short periods of time, and without any significant effect on the number of viral gene-copies detected by RT-qPCR below 92 °C, even after 30-60 min of pre-treatment ( Pastorino et al 2020 ).…”

Comparing lateral flow testing with a rapid RT-PCR method for SARS-CoV-2 detection in the UK

“…Samples were prepared for RT-PCR using a rapid heat-treatment method 6 . This involves heating samples to 95°C for 5 minutes and cooling to 4°C.…”

“…We developed a highly sensitive, rapid, UKAS accredited in-house RT-PCR assay utilising the N1 assay published by the CDC 6 . RNA is prepared using a rapid heat treatment method, removing the need for time-consuming and expensive nucleic acid extraction kits 6 .…”

“…Many of these published assays have been developed as commercial kits and are used globally for the detection of SARS-CoV-2 RNA. We developed a highly sensitive, rapid, UKAS accredited in-house RT-PCR assay utilising the N1 assay published by the CDC 6 . RNA is prepared using a rapid heat treatment method, removing the need for time-consuming and expensive nucleic acid extraction kits 6 .…”

RNA-extraction-free diagnostic method to detect SARS-CoV-2: an assessment from two States, India

“…The combination of two gene targets is recommended to enhance accuracy of diagnosis in the context of possible viral genetic variability; one from the conserved region of the virus and another from SARS CoV-2 specific region of the genome [8]. Several alternative protocols described ways to simplify RT-PCR test by excluding the RNA extraction step [11][12][13][14]. These modifications attempted to reduce the turn-around time for quickly obtaining test results and also to address the issue of shortage of RNA extraction kits when the demand runs high.…”