Duplex SYBR RT-qPCR for SARS-CoV-2 detection in Malaysia

Helyatul Rasmah Mahali and Zarina Amin and Nur Athirah Yusof (2025) Duplex SYBR RT-qPCR for SARS-CoV-2 detection in Malaysia. Journal of Tropical Life Science, 15 (2). pp. 397-408. ISSN 2087-5517

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Abstract

TaqMan probe-based commercial real-time PCR kits are the gold standard for COVID-19 diagnosis but are expensive. The large scale of SARS-CoV-2 infections necessitates affordable testing for humans, animals, and environmental samples. COVID-19 symptoms overlap with other respiratory infections like influenza, common cold, MERS, and SARS, requiring precise diagnostic tests such as real-time PCR. This study introduces a cost-effective method using melting curve analysis of a SYBR green multiplex assay with gel electrophoresis. We developed a duplex SYBR Green real-time PCR assay to detect the spike (S), envelope (E), RNA-dependent RNA polymerase (RdRp), and nucleocapsid (N) gene of SARS-CoV-2 using two sets of tubes. Specific primers were designed from conserved regions of the SARS-CoV-2 genome sequences isolated from Malaysia, retrieved from the GISAID database. Initial single plex SYBR Green RT-PCR reactions for the N, E, S, and RdRp genes were optimized. After several rounds of optimization, we targeted the S and N genes in one tube and the E and RdRp genes in another. The assay uses real-time PCR to detect four genes with two primer sets distinguished by melting temperatures: 81.5°C and 88°C for the S and N genes, and 79.5°C and 85.5°C for the E and RdRp genes. It demonstrated high specificity and sensitivity, with a detection limit of 0.1 pg/μL of DNA. We compared this method to the Lyte Star 2019-nCoV RT-PCR Kit (China), approved by Malaysia's Ministry of Health. Both kits accurately detected all positive SARS-CoV-2 samples without false positives. In conclusion, the newly developed duplex SYBR Green I-based real-time PCR assay demonstrates high sensitivity, specificity, and reliability, making it a valuable and effective alternative for the detection of SARS-CoV-2.

Item Type:	Article
Keyword:	COVID-19, Gel electrophoresis, Melting curve analysis, SARS-CoV-2, SYBR Green Multiplex Assay, TaqMan probe-based real-time PCR
Subjects:	Q Science > QC Physics > QC1-999 Physics > QC1-75 General Q Science > QH Natural history > QH301-705.5 Biology (General) > QH426-470 Genetics R Medicine > RA Public aspects of medicine > RA1-1270 Public aspects of medicine > RA421-790.95 Public health. Hygiene. Preventive medicine
Department:	INSTITUTE > Biotechnology Research Institute (BRI)
Depositing User:	JUNAINE JASNI -
Date Deposited:	10 Jan 2026 17:32
Last Modified:	10 Jan 2026 17:32
URI:	https://eprints.ums.edu.my/id/eprint/45835

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