SARS-CoV-2 characteristics and detection, facts and/or misconceptions

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and caused COVID-19 pandemic. So, reliable and accurate testing for SARS-CoV-2 was the principal prerequisite for preventing the spread of virus. Real Time RT-PCR unquestionably represent the most reliable, rapid and sensitive method for detection of SARS-CoV-2 RNA. However, there are numerous different assays, protocols, instruments and analysis methods in use without certified standards, standardized RNA extraction and reporting procedures. In practice, the reliability of RT-qPCR results depends on a number of parameters that include sample collection and processing, method of RNA extraction, choice of assay, etc. Here we present comparative analyses of the efficiency and sensitivity of 10 different amplification assays, as well as the relevance of manual RNA extractions compared to automatic one. Our results revealed that manual viral RNA extraction and amplification assays targeting three viral genes should be a method of choice for high sensitivity. Interestingly, RT-qPCR was exclusively used as qualitative diagnostic test for SARSCoV-2. Why, it is a quantitative method? We think that the ideal testing regimen should involve reliable and meaningful quantitative reporting of SARS-CoV-2 viral load. Now, a key question arises, why the medical community has never agreed on a cut-off value (viral load) for a positive result that would have clinical significance? Can we identify cut-off value for SARS-CoV-2 viral load that generates massive microthrombosis throughout the body, seen by our pathologist?