In-house modification and improvement of the CDC real-time PCR diagnostic assay for SARS-CoV-2 detection

The world is currently facing an unprecedented pandemic caused by the novel coronavirus SARS-CoV-2 (COVID-19) which was first reported in late 2019 by China to the World Health Organization (WHO). The containment strategy for COVID-19, which has non-specific flu-like symptoms and where upwards of 80% of the affected has either mild or no symptoms, is critically centered upon diagnostic testing, tracking and isolation. Thus, the development of specific and sensitive diagnostic tests for COVID-19 is key towards the first successful step of disease management. Public health organizations like the WHO and the US-based Centers for Disease Control and Prevention (CDC) have developed real-time PCR (RT-PCR) based diagnostic tests to aid in the detection of acute infection. In this study we sought to modify the CDC RT-PCR diagnostic assay protocol to increase its sensitivity and to make the assay directly portable to health care providers in a community-based hospital setting. A number of modifications to the original protocol were tested. Increasing the RT-PCR annealing temperature by 7°C to 62°C was associated with the most significant improvement in sensitivity, wherein the cycle-threshold (Ct) value for the N2 assay was reduced by ∼3 units, in effect both reducing the overall number of inconclusive results and yielding N1/N2 assays to have similar Ct values. The limit of detection of the modified assay was also improved (0.86 RNA copies/µl for both nCoV 2019_N1/N2 assays) compared to the CDC RT-PCR diagnostic assay (1 and 3.16 RNA copies/µl for nCoV 2019_N1 and N2 assay, respectively). Using this modification, there was no significant effect on SARS-CoV-2 detection rate when viral RNA extraction was performed either manually or through an automated extraction method. We believe this modified protocol allows for more sensitive detection of the virus which in turn will be useful for pandemic management.