Advanced Biomedical Research

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

Authors

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

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

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

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

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

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

Abstract

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.

Keywords

1.
Wu C, Zheng S, Chen Y, Zheng M. Single-cell RNA expression profiling of ACE2, the putative receptor of Wuhan 2019-nCoV, BioRxiv. 2020 Jan 1  Back to cited text no. 1
    
2.
Gorbalenya AE, Baker SC, Baric RS, de Groot RJ, Drosten C, Gulyaeva AA, et al. The species severe acute respiratory syndrome-related coronavirus: Classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol 2020;5:536.  Back to cited text no. 2
    
3.
Fakhim H, Nasri E, Aboutalebian S, Gholipour S, Nikaeen M, Vaezi A, et al. Asymptomatic carriers of coronavirus disease 2019 among healthcare workers in Isfahan, Iran. Future Virol 2021;16:93-8.  Back to cited text no. 3
    
4.
Ren X, Liu Y, Chen H, Liu W, Guo Z, Zhang Y, et al. Application and optimization of RT-PCR in diagnosis of SARS-CoV-2 infection. SSRN Electron J 2020 Feb 25.  Back to cited text no. 4
    
5.
Parasher A. COVID-19: Current understanding of its pathophysiology, clinical presentation and treatment. Postgrad Med J 2021;97:312-20.  Back to cited text no. 5
    
6.
Toptan T, Hoehl S, Westhaus S, Bojkova D, Berger A, Rotter B, et al. Optimized qRT-PCR approach for the detection of intra-and extra-cellular SARS-CoV-2 RNAs. Int J Mol Sci 2020;21:4396.  Back to cited text no. 6
    
7.
Dharavath B, Yadav N, Desai S, Sunder R, Mishra R, Ketkar M, et al. A one-step, one-tube real-time RT-PCR based assay with an automated analysis for detection of SARS-CoV-2. Heliyon 2020;6:e04405.  Back to cited text no. 7
    
8.
Jiang M, Pan W, Arastehfar A, Fang W, Fang H, Daneshnia FF, et al. Development and validation of a rapid single-step reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) system potentially to be used for reliable and high-throughput screening of COVID-19. medRxiv 2020; Jun 16;10:331.  Back to cited text no. 8
    
9.
Chen Z, Wu Q, Chen J, Ni X, Dai J. A DNA aptamer based method for detection of SARS-CoV-2 nucleocapsid protein. Virol Sin 2020;35:351-4.  Back to cited text no. 9
    
10.
Zhang F, Abudayyeh OO, Gootenberg JS. A protocol for detection of COVID-19 using CRISPR diagnostics. https://www.broadinstitute .org/files/publications/special/COVID-19%20detection%20(updated).pdf  Back to cited text no. 10
    
11.
Chen Y, Liu Q, Guo D. Emerging coronaviruses: Genome structure, replication, and pathogenesis. J Med Virol 2020;92:418-23.  Back to cited text no. 11
    
12.
Shen M, Zhou Y, Ye J, Abdullah Al-Maskri AA, Kang Y, Zeng S, et al. Recent advances and perspectives of nucleic acid detection for coronavirus. J Pharm Anal 2020;10:97-101.  Back to cited text no. 12
    
13.
Pereira-Gómez M, Fajardo Á, Echeverría N, López-Tort F, Perbolianachis P, Costábile A, et al. Evaluation of SYBR green real time PCR for detecting SARS-CoV-2 from clinical samples. J Virol Methods 2021;289:114035.  Back to cited text no. 13
    
14.
Eschbaumer M, Li WM, Wernike K, Marshall F, Czub M. Probe-free real-time reverse transcription polymerase chain reaction assays for the detection and typing of porcine reproductive and respiratory syndrome virus in Canada. Can J Vet Res 2015;79:170-9.  Back to cited text no. 14
    
15.
Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: Implications for virus origins and receptor binding. Lancet 2020;395:565-74.  Back to cited text no. 15
    
16.
Tang YW, Schmitz JE, Persing DH, Stratton CW. Laboratory diagnosis of COVID-19: Current issues and challenges. J Clin Microbiol 2020;58:e00512-20.  Back to cited text no. 16
    
17.
Tahamtan A, Ardebili A. Real-time RT-PCR in COVID-19 detection: Issues affecting the results. Expert Rev Mol Diagn 2020;20:453-4.  Back to cited text no. 17
    
18.
Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill 2020;25:2000045.  Back to cited text no. 18
    
19.
Gholipour S, Nikaeen M, Mohammadi Manesh R, Aboutalebian S, Shamsizadeh Z, Nasri E, et al. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contamination of high-touch surfaces in field settings. Biomed Environ Sci 2020;33:925-9.  Back to cited text no. 19
    
20.
Qiu H, Wu J, Hong L, Luo Y, Song Q, Chen D. Clinical and epidemiological features of 36 children with coronavirus disease 2019 (COVID-19) in Zhejiang, China: An observational cohort study. Lancet Infect Dis 2020;20:689-96.  Back to cited text no. 20
    
21.
Guo YR, Cao QD, Hong ZS, Tan YY, Chen SD, Jin HJ, et al. The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak – An update on the status. Mil Med Res 2020;7:11.  Back to cited text no. 21
    
22.
Lai CC, Shih TP, Ko WC, Tang HJ, Hsueh PR. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019 (COVID-19): The epidemic and the challenges. Int J Antimicrob Agents 2020;55:105924.  Back to cited text no. 22
    
23.
Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 2020;579:270-3.  Back to cited text no. 23
    
24.
Zhen W, Manji R, Smith E, Berry GJ. Comparison of four molecular in vitro diagnostic assays for the detection of SARS-CoV-2 in nasopharyngeal specimens. J Clin Microbiol 2020;58:e00743-20.  Back to cited text no. 24
    
25.
Dorlass EG, Monteiro CO, Viana AO, Soares CP, Machado RR, Thomazelli LM, et al. Lower cost alternatives for molecular diagnosis of COVID-19: Conventional RT-PCR and SYBR green-based RT-qPCR. Braz J Microbiol 2020;51:1117-23.  Back to cited text no. 25
    
26.
Fajardo A, Pereira-Gomez M, Echeverria N, Lopez-Tort F, Perbolianachis P, Aldunate F, et al. Evaluation of SYBR green real time PCR for detecting SARS-CoV-2 from clinical samples. bioRxiv 2020; Jan 1.  Back to cited text no. 26
    
27.
Sarkar SL, Alam AR, Das PK, Pramanik MH, Al-Emran HM, Jahid IK, et al. 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. medRxiv 2021; Jan 1.  Back to cited text no. 27
    
28.
Marinowic DR, Zanirati G, Rodrigues FV, Grahl MV, Alcará AM, Machado DC, et al. A new SYBR green real-time PCR to detect SARS-CoV-2. Sci Rep 2021;11:2224.  Back to cited text no. 28
    
29.
Zou L, Ruan F, Huang M, Liang L, Huang H, Hong Z, et al. SARS-CoV-2 viral load in upper respiratory specimens of infected patients. N Engl J Med 2020;382:1177-9.  Back to cited text no. 29
    
30.
Wang W, Xu Y, Gao R, Lu R, Han K, Wu G, et al. Detection of SARS-CoV-2 in different types of clinical specimens. JAMA 2020;323:1843-4.  Back to cited text no. 30
    
31.
Wan Z, Zhang Y, He Z, Liu J, Lan K, Hu Y, et al. A melting curve-based multiplex RT-qPCR assay for simultaneous detection of four human coronaviruses. Int J Mol Sci 2016;17:1880.  Back to cited text no. 31
    
32.
Woo PC, Lau SK, Huang Y, Yuen KY. Coronavirus diversity, phylogeny and interspecies jumping. Exp Biol Med (Maywood) 2009;234:1117-27.  Back to cited text no. 32
    
33.
Wang ZG, Li LJ, Luo Y, Zhang JY, Wang MY, Cheng SY, et al. Molecular biological analysis of genotyping and phylogeny of severe acute respiratory syndrome associated coronavirus. Chin Med J (Engl) 2004;117:42-8.  Back to cited text no. 33
    
34.
Shang L, Qi Y, Bao QY, Tian W, Xu JC, Feng MG, et al. Polymorphism of SARS-CoV genomes. Yi Chuan Xue Bao 2006;33:354-64.  
Advanced Biomedical Research
Volume 2022, November
November 2022
Pages 1-5