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

Ioanna Smyrlaki; Martin Ekman; Martin Vondracek; Natali Papanicoloau; Antonio Lentini; Johan Aarum; Shaman Muradrasoli; Jan Albert; Björn Högberg; Björn Reinius

Currently, the widely used method of SARS-CoV-2 detection in clinical diagnostics is an RT-qPCR assay, detecting the presence of viral RNA in patient samples. Although RT-qPCR is widely implemented for the detection of pathogens, including viruses [3] in clinical samples, the implementation of the specific assay for the detection of SARS-CoV-2 has only recently been established. The currently used protocol was developed and optimized for the detection of the novel coronavirus at the Charité University Hospital, in collaboration with several other laboratories in Germany, the Netherlands, China, France, UK and Belgium [4] . Additionally, the existing protocol was further optimized by the Center for Disease Control (CDC) in the United States through the comprehensive comparison and validation of alternative available kits for nucleic acid extraction and the use of alternative probe and primer sets for efficient SARS-CoV-2 detection in clinical samples [5] [6] . Routinely, the application of qPCR for the relative quantification of a transcript of interest in a sample, is preceded by a) the isolation of total RNA from the sample and b) the use of purified RNA in a reverse-transcription (RT) reaction resulting in complementary DNA (cDNA) from the template RNA, which is then utilized for the qPCR reaction. However, nucleic acid purification and RT of the resulting RNA into cDNA are not only laborious and time-consuming, but the additional steps requiring manual handling can result in experimental errors. In the case of clinical sampling and diagnostics, the use of a single-reaction kit combining the RT and qPCR reactions (such as the TaqPath 1-step RT-qPCR) is therefore customary. Although the single-step RT-qPCR removes the need for a separate RT reaction, RNA isolation from clinical samples constitutes a significant chokepoint in the diagnostic process, as it remains both manually laborious and expensive. Specifically, both the Charité University Hospital and the CDC protocols require the use of RNA purification kits, which not only results in a significant cost increase but has already led to a major supply shortage of such kits. It is therefore crucial that a new test is not only affordable, quick and efficient, but that it keeps the use of industrial kits to the minimum. Recent attempts have been made to circumvent RNA extraction in COVID-19 detection [7] [8] [9] [10] .