Development and validation of cost-effective one-step multiplex RT-PCR assay for detecting the SARS-CoV-2 infection using SYBR Green melting curve analysis

Sarkar, S. L.; Alam, A. S. M. R. U.; Das, P. K.; Pramanik, M. H. A.; Al-Emran, H. M.; Jahid, I. K.; Hossain, M. A.

TaqMan probe-based expensive commercial real-time (RT) PCR kits are being used in COVID-19 diagnosis. The unprecedented scale of SARS-CoV-2 infections has urgently needed to meet the challenge of testing more persons at a reasonable cost. This study developed a rapid, simple, and cost-effective alternative diagnostic method based on melting curve analysis of SYBR green multiplex assay with a host-specific internal control. A total of 90 randomly selected samples were used for comparing the assay with an available commercial kit to analyse the variation and validity of this in-house developed method. Our customized designed primers specifically detected the virus as similar to commercial kit manufactured by Sansure Biotech Inc. We optimized separately the N, E, S, and RdRp genes by SYBR Green RT-PCR method based on melting curve analysis. Afterwards, a multiplex COVID-19 diagnosis method targeting N and E genes of the virus along with the {beta}-actin gene of the host as an internal control has been established. The total run-time of our proposed method was less than 90 minutes. The cost of each sample processing was less than $2. Overall, this one-step and one-tube method can revolutionize the COVID-19 diagnosis in developing countries.