In-house Modification and Improvement of the CDC Realtime PCR Diagnostic Assay for SARS-CoV-2 Detection

Objective: 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.
Methods: 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. 
Results: 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 RTPCR 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.
Conclusion: We believe this modified protocol allows for more sensitive detection of the virus which in turn will be useful for pandemic management.