Capillary Electrophoresis of PCR fragments with 5’-labelled primers for testing the SARS-Cov-2

Abstract

Abstract Background Due to the huge demand for SARS-Cov-2 determination, alternatives to the standard qtPCR tests are potentially useful for increasing the number of samples screened. Our aim was to develop a direct fluorescent PCR capillary-electrophoresis detection of the viral genome. We validated this approach on several SARS-Cov-2 positive and negative samples. Study design We isolated the naso-pharingeal RNA from 20 positive and 10 negative samples. The cDNA was synthesised and two fragments of the SARS-Cov-2 were amplified. One of the primers for each pair was 5’-end fluorochrome labelled. The amplifications were subjected to capillary electrophoresis in ABI3130 sequencers to visualize the fluorescent peaks. Results The two SARS-Cov-2 fragments were successfully amplified in the positive samples, while the negative samples did not render fluorescent peaks. Conclusion We describe and alternative method to identify the SARS-Cov-2 genome that could be scaled to the analysis of approximately 100 samples in less than 5 hours. By combining a standard PCR with capillary electrophoresis our approach would overcome the limits imposed to many labs by the qtPCR (lack of reactive and real-time PCR equipment) and increase the testing capacity.