The development of multiplex one-step real-time rt-pcr for simultaneous detection variants of severe acute respiratory syndrome-coronavirus-2

Abstract

This study aimed to develop a multiplex one-step real-time RT-PCR test for the identification of SARS-CoV-2 variants that is quick and less expensive compared to the efficiency of the standard approach, genome sequencing. Moreover, the developed test would be used to investigate the epidemiology of the variants of SARS-CoV-2 in Thailand. Multiplex one-step real-time RT-PCR for variant detection of SARS-CoV-2 was developed with the plasmid positive control, and validated the sensitivity and specificity by using 155 SARS-CoV-2 positive RNA samples. The cross-reactivity of the other viral respiratory pathogen and the limit of detection were evaluated. Moreover, this test was used to investigate the SARS-CoV-2 variants from April 2021 to April 2022 by using 1,200 positive SARS-CoV-2 samples that outbreak each month. The developed test demonstrated that each SARS-CoV-2 variant has a high sensitivity and specificity. There was no cross-reactivity to other respiratory viral pathogens. According to the epidemiology investigation, the primary SARS-CoV-2 outbreak in Thailand between 2021 and 2022 began with the Alpha variation followed by Delta and Omicron (BA.1) and Omicron (BA.2) variants, respectively. The developed multiplex one-step real-time RT-PCR assay described in this study made it possible to simultaneously detect SARS-CoV-2 variants with high sensitivity, and specificity, and help with SARS-CoV-2 surveillance and outbreak. IMPLICATION OF THE THESIS: Suggestions for further research: SARS-CoV-2 generates several novel varieties due to its high rate of mutation. Therefore, to find the new variations and use as a screening method for SARS-CoV-2, a one-step real-time RT-PCR with specific primers and probes could be developed.