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. Many groups recently presented results using heat processing method 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 methodology. We first investigated various treatments using different temperatures, incubation times and sample volumes to optimise the heat treatment conditions. Although the initial data confirmed results published elsewhere, further investigations revealed unexpected inhibitory properties of some commonly used universal transport media (UTMs) on some commercially available RT-qPCR mixes, leading to a risk of reporting false-negative results. This emphasises the critical importance of a thorough validation process to determine the most suitable reagents to use depending on the sample types to be tested. In conclusion, a heat processing method is effective with very consistent Ct values and a sensitivity of 96.2% when compared to a conventional RNA extraction method. It is also critical to include an internal control to check each sample for potential inhibition.

Fig 1. Correlation between NA extraction-based and heat treatment-based detection of SARS-CoV-2.

A) Correlation of Ct values from NA/RT-qPCR (NA/ABI) and heat treatment/RT-qPCR (HT/Meridian Fast 1-Step). The Ct values obtained for the samples positive for SARS-CoV-2 using both methods (S6 Table) were plotted against each other. B) Comparison of Ct values in paired samples that were positive in both NA/ABI and Heat-treatment/RT-qPCR. The paired samples are individually plotted as red and blue lines if Heat-treatment/RT-qPCR Ct was earlier or later than NA/RT-qPCR, respectively. Paired t-test, t = -4.0787, df = 100, p = 0.00009.