Ultrafast RNA extraction-free SARS-CoV-2 detection by direct RT-PCR using a rapid thermal cycling approach (preprint)

The surging COVID19 pandemic has underlined the need for quick, sensitive, and high-throughput SARS-CoV-2 detection assays. Although many different methods to detect SARS-CoV-2 particles in clinical material have been developed, none of these assays are successful in combining all three of the above characteristics into a single, easy-to-use method that is suitable for large-scale use. Here we report the development of a direct RT-PCR SARS-CoV-2 detection method that can reliably detect minute quantities of SARS-CoV-2 gRNA in nasopharyngeal swab samples as well as the presence of human genomic DNA. An extraction-less validation protocol was carried out to determine performance characteristics of the assay in both synthetic SARS-CoV-2 RNA as well as clinical specimens. Feasibility of the assay and analytical sensitivity was first determined by testing a dilution series of synthetic SARS-CoV-2 RNA in two different solvents (water and AMIES VTM), revealing a high degree of linearity and robustness in fluorescence readouts. Following analytical performance using synthetic RNA, the limit of detection was determined at equal to or less than 1 SARS-CoV-2 copy/ul of sample in a commercially available sample panel that contains surrogate clinical samples with varying SARS-CoV-2 viral load. Lastly, we benchmarked our method against a reference qPCR method by testing 87 nasopharyngeal swab samples. The direct endpoint ultra-fast RT-PCR method exhibited a positive percent agreement score of 98.5% and a negative percent agreement score of 100% as compared to the reference method, while RT-PCR cycling was completed in 27 minutes/sample as opposed to 60 minutes/sample in the reference qPCR method. In summary, we describe a rapid direct RT-PCR method to detect SARS-CoV-2 material in clinical specimens which can be completed in significantly less time as compared to conventional RT-PCR methods, making it an attractive option for large-scale SARS-CoV-2 screening applications.

Ultra-fast one-step RT-PCR protocol for the detection of SARS-CoV-2 (preprint)

The COVID-19 pandemic resulted in lockdowns all over the world thus affecting nearly all aspects of social life and also had a huge impact on global economies. Since vaccines and therapies are still not available for the population, prevention becomes desperately needed. One important aspect for prevention is the identification and subsequent isolation of contagious specimens. The currently used methods for diagnostics are time consuming and also hindered by the limited availability of reagents and reaction costs, thus presenting a bottle neck for prevention of COVID-19 spread. Here, we present a new ultra-fast test method which is ten times faster than conventional diagnostic tests using real time quantitative PCR (RT-qPCR). In addition, this ultra-fast method is easy to handle as well as cost effective. We translated published SARS-CoV-2 testing protocols from the Centers of Disease Control and Prevention (Atlanta, Georgia, USA) and the Charite Berlin (Germany) to the NEXTGENPCR (NGPCR) machine and combined it with a fluorescence-based endpoint measurement. Fluorescence was measured with a commercial blue light scanner. We confirmed the NEXTGENPCR results with commercially available positive controls. In addition, we isolated RNA from SARS-CoV-2 infected patients and achieved similar results to clinical RT-qPCR assays. Here, we could show correlation between the results obtained by NEXTGENPCR and conventional RT-qPCR.