Validation of a Novel Molecular Assay to the Diagnostic of COVID-19 Based on Real Time PCR with High Resolution Melting

Ferreira, B. I. d. S.; Gomes, N. L. d. S.; Coelho, W. L. d. C. N. P.; Costa, V. D.; Carneiro, V. C. d. S.; Kader, R. L.; Amaro, M. P.; Villar, L. M.; Miyajima, F.; Alves-Leon, S. V.; de Paula, V. S.; Leon, L. A. A.; Moreira, O. C.

With the emergence of the Covid-19 pandemic, the world faced an unprecedented need for RT-qPCR-based molecular diagnostic tests, leading to a lack of kits and inputs, especially in developing countries. Hence, the costs for commercial kits and inputs were overrated, stimulating the development of alternative methods to detect SARS-CoV-2 in clinical specimens. The availability of the complete SARS-CoV-2 genome at the beginning of the pandemic facilitated the development of specific primers and standardized laboratory protocols for Covid-19 molecular diagnostic. High-sensitive and cost-effective molecular biology technique based on the Melting Temperature differences between purine and pyrimidine bases can be used to the detection and genotyping of pathogens in clinical specimens. Here, a RT-qPCR assays with High Resolution Melting (HRM-RTqPCR) was developed for different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states in Brazil, in comparison to a commercial TaqMan RT-qPCR assay, as gold standard. The sensitivity of the HRM-RTqPCR assays targeting N, RdRp and E were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 targets, and diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, the HRM-RTqPCR emerges as an alternative and low-cost methodology to increase the molecular diagnostic of patients suspicious for Covid-19, especially in restricted-budget laboratories.