Melting curve-based assay as an alternative technique for the accurate detection of SARS-CoV-2

Document Type : Original Article


1 Department of Medical Parasitology and Mycology, School of Medicine, & Research Core Facilities Laboratory, Isfahan University of Medical Sciences, Isfahan, Iran

2 Infectious Diseases and Tropical Medicine Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

3 Department of Medical Microbiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

4 Department of Environmental Health Engineering, Faculty of Health, Kashan University of Medical Sciences, Isfahan, Iran

5 Department of Pharmaceutical Biotechnology, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran

6 Department of Environmental Health Engineering, School of Health, Isfahan University of Medical Sciences, Isfahan, Iran


Background: Early and cost-effective diagnosis and monitoring of the infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are critically important to anticipate and control the disease. We aimed to set up a SYBR Green-based one-step real-time polymerase chain reaction (PCR) as a lower-cost alternative method to detect the virus. Materials and Methods: An in-house SYBR Green-based PCR assay targeting the envelope (E) and RNA-dependent RNA polymerase (RdRp) genes, was set up to diagnose the infection, and was compared with the reference probe-based PCR method. Results: When the commercial probe-based assay was considered as the reference method, SYBR Green-based PCR had a slightly lower sensitivity (81.98% and 86.25% for E and RdRp targets, respectively) and a good specificity (100% and 94.44% for E and RdRp targets, respectively). For both gene targets, three different melting temperature (Tm) patterns were found in the PCRs of the nasopharyngeal/oropharyngeal swab samples, but no size polymorphism was seen in agarose gel electrophoresis. Conclusion: Further studies to improvement of the assay are needed to make it an inexpensive and reliable tool for the diagnosis of COVID-19.


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