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.

Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection

“…Trizol extraction followed by isopropanol precipitation provides a high yield of purified RNA [ 5 ], however, it requires extensive labor, is difficult to scale to high-throughput, and involves hazardous materials. Simpler isopropanol precipitation methods, in which patient swab samples are first mixed with commercial or homemade lysis solutions, have been reported to give Ct values comparable to those obtained using commercial RNA purification kits [ 6 – 8 ].…”

An optimized and robust PEG precipitation method for detection of SARS-CoV-2 in wastewater

“…Differences in organic material, total dissolved solids and pH can influence virus survival and subsequently the detection of the SARS-CoV-2 RNA in wastewater ( Geller et al, 2012 ; Ye et al, 2016 ). For example, at a pH greater than 6, RNA is more susceptible to alkaline hydrolysis ( Wozniak et al, 2020 ), thus the stability and subsequent detection of viral RNA in highly alkaline wastewater may be compromised. As such, it is imperative that a method is found which is sufficiently robust to overcome matrix variability.…”

Design Of A Rapid And Reversible Fluorescence Assay To Detect Covid-19 And Other Pathogens

“…In practice, biological samples would have to be mixed with detergent with added salt and possibly protease. Heat treatment has been shown to release viral RNA, and if pure viral RNA is desired when coupling this method to others, treatment with acid can be used [15]. Our data show that the response of the beacon for viral RNA is close to 1:4 and because the beacon can be used in concentrated form (see below) and the viral load of infected patients can range from 10 4 −10 10 copies/mL [9, 14], we propose that viral RNA can be detected be by eye using a simple black light.…”