Abstract
The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-based molecular diagnostic testing, placing a strain on the supply chain and the availability of commercially available PCR testing kits and reagents. The effect of limited molecular diagnostics-related supplies has been felt across the globe, disproportionally impacting molecular diagnostic testing in developing countries where acquisition of supplies is limited due to availability. The increasing global demand for commercial molecular diagnostic testing kits and reagents has made standard PCR assays cost prohibitive, resulting in the development of alternative approaches to detect SARS-CoV-2 in clinical specimens, circumventing the need for commercial diagnostic testing kits while mitigating the high-demand for molecular diagnostics testing. The timely availability of the complete SARS-CoV-2 genome in the beginning of the COVID-19 pandemic facilitated the rapid development and deployment of specific primers and standardized laboratory protocols for the molecular diagnosis of COVID-19. An alternative method offering a highly specific manner of detecting and genotyping pathogens within clinical specimens is based on the melting temperature differences of PCR products. This method is based on the melting temperature differences between purine and pyrimidine bases. Here, RT-qPCR assays coupled with a High Resolution Melting analysis (HRM-RTqPCR) were developed to target 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 within Brazil; a larger validation group than that used in the development to the commercially available TaqMan RT-qPCR assay which is considered the gold standard for COVID-19 testing. The sensitivity of the HRM-RTqPCR assays targeting the viral N, RdRp and E genes were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 SARS-CoV-2 genome targets, and a diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, HRM-RTqPCR emerges as an attractive alternative and low-cost methodology for the molecular diagnosis of COVID-19 in restricted-budget laboratories.
Highlights
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense singlestranded RNA virus [1] causative of coronavirus disease 2019 (COVID-19), the airbornetransmitted respiratory illness responsible for the COVID-19 pandemic [2]
High Resolution Melting (HRM) curve was generated for each target, with Tm 80.8, 83.5, 77.6 and 86 ̊C for N, RNA-dependent RNA polymerase (RdRp), E and RNAse P, respectively (Table 1 and Fig 1A)
It was possible to observe the same approximate limit of detection (LOD)–the lowest concentration detected in the linearity assay, around 10 copies/ μL, between the three gene targets of SARS-CoV-2
Summary
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense singlestranded RNA virus [1] causative of coronavirus disease 2019 (COVID-19), the airbornetransmitted respiratory illness responsible for the COVID-19 pandemic [2]. The availability of the complete SARS-CoV-2 genome during the initial phase of the pandemic facilitated the development of specific primers and standardized laboratory protocols for COVID-19 molecular diagnostic [5, 6]. All the recommended protocols are TaqMan-based methods that use probes designed for the different regions of the viral genome Of these assays, only one uses an internal human control (RNAse P gene, CDC-USA protocol) that serves as a control for the nucleic acid extraction, which aids in successfully validating a truly negative result. Only one uses an internal human control (RNAse P gene, CDC-USA protocol) that serves as a control for the nucleic acid extraction, which aids in successfully validating a truly negative result Using these various approaches, a variety of molecular diagnostic kits were produced and validated in record time. The development of low-cost alternative diagnostic technologies would be beneficial for current and future medical needs
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