Development and validation of novel RT-PCR assay for molecular diagnostic of viral variants using SARS-CoV-2 as a case study

Abstract

Emerging viruses have long posed significant challenges to global public health, frequently leading to widespread morbidity and mortality. The ongoing evolution of viruses driven by genetic mutations is critical in the emergence of these novel pathogens. Among the numerous viruses that have demonstrated this capability the SARS-CoV-2 responsible for the COVID-19 pandemic is the prime example of how viral mutations can profoundly impact disease dynamics, transmission, and control measures. In this study, we present the development of a multiplex RT-PCR assay, using allele-specific primer-probe tailored for molecular diagnostic of viral variants using SARS−CoV-2 as a case study. We conducted a comprehensive evaluation to validate the assay performance using a diverse panel of leftover clinical samples, including a few coded reference samples from external providers. This multiplex PCR typing method detects seven unique mutations of Omicron and two unique mutations of Delta strain with allele-specific primers and probe sets against the spike protein’s receptor-binding domain (RBD). The assay exhibits high analytical sensitivity, detecting about 1 x 10² copies/mL of SARS-CoV-2 RNA for each genetic variant tested, and possesses 100 % analytical specificity. Comparative analysis with existing commercial RT-PCR kits demonstrated better performance, particularly in detecting omicron and delta variants. This research highlights the translational potential of our approach in advancing diagnostic capabilities for emerging viral infections, enhancing public health responses to future outbreaks.