A simple RNA preparation method for SARS-CoV-2 detection by RT-qPCR

Abstract

The technique RT-qPCR for viral RNA detection is the current worldwide strategy used for early detection of the novel coronavirus SARS-CoV-2. RNA extraction is a key pre-analytical step in RT-qPCR, often achieved using commercial kits. However, the magnitude of the COVID-19 pandemic is causing disruptions to the global supply chains used by many diagnostic laboratories to procure the commercial kits required for RNA extraction. Shortage in these essential reagents is even more acute in developing countries with no means to produce kits locally. We sought to find an alternative procedure to replace commercial kits using common reagents found in molecular biology laboratories. Here we report a method for RNA extraction that takes about 40 min to complete ten samples, and is not more laborious than current commercial RNA extraction kits. We demonstrate that this method can be used to process nasopharyngeal swab samples and yields RT-qPCR results comparable to those obtained with commercial kits. Most importantly, this procedure can be easily implemented in any molecular diagnostic laboratory. Frequent testing is crucial for individual patient management as well as for public health decision making in this pandemic. Implementation of this method could maintain crucial testing going despite commercial kit shortages.

Figure 1

Quantitative assessment of performance for selected RNA extraction methods. Cq values obtained by RT-qPCR with 45 cycles using TaqMan probe and primers against RNase P gene in saliva samples for TRIzol (27.39 + /- 0.34), BSA-based (35.3 + /- 0.79), acid pH-based (27.68 + /- 0.90), high temperature-based (n.d.) and direct (n.d.) methods. n.d.; not determined (no Cq reported). Control corresponds to a negative control with water instead of template. Bars show mean plus standard deviation of the mean for two biological and three technical replicates each (6 measurements).

Figure 2

The acid-pH method provides comparable results to commercial kits in clinical samples. Bars represent the mean + /- standard deviation Cq values for each RT-qPCR target gene N1, N2, and RNase P, for samples with Cq N1 ≤ 36 (A) and with Cq N1 > 36 (B). Each dot represents one sample. Orange bars show results obtained with High Pure Viral RNA Kit (Roche). Blue bars show results obtained with the acid pH method. Pairwise comparisons of mean Cq values for each target gene were done using a two-tailed paired Student’s t-test, with a confidence level of 95%. ‘ns’ means no statistically significant differences.

Figure 3

Schematic diagram of the validated acid-pH method for RNA extraction compatible with SARS-CoV2 RT-PCR testing. Steps carried out in the acid pH RNA extraction protocol.