A Novel Quadriplex Reverse Transcription PCR Assay for Robust SARS-CoV-2 Diagnosis and Variant Detection: Experimental Optimization for Enhanced Sensitivity and Specificity

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has been identified as the causative agent of an ongoing global pandemic of acute respiratory distress syndrome, coronavirus disease 2019. Ranked as one of the top priorities is developing an accurate, reliable, and affordable laboratory diagnostic test for pathogen detection to facilitate disease tracking. In this study, a multiplex-based assay to detect SARS-CoV-2 using four primers/probes to amplify regions of the spike (S), nucleocapsid (N), and envelope (E) protein-coding genes, along with amplifying region of the 3' untranslated region (UTR) was designed and developed. To provide accurate detection of different SARS-CoV-2 variants, an in silico design and validation pipeline was utilized. These included multi-sequence alignments with Clustal Omega, specificity verification by the Basic Local Alignment Search Tool (primer-BLAST), and computational performance verification using Biopython. The primers and hydrolysis probes designed were experimentally tested against more than two million SARS-CoV-2 genome sequences obtained from the National Center for Biotechnology Information Virus Database, with >97% specificity across 2200 lineages, including all of the significant variants of concern and variants of interest. The experimental validation phase included synthetic SARS-CoV-2 constructs and ribonucleic acid samples from nasopharyngeal swabs of 20 patients, including both symptomatic and asymptomatic cases. The assay displayed 100% sensitivity and specificity, with the limit of detection as low as 10 copies/mL, which surpassed the detection limits of some commercially available reverse transcription quantitative polymerase chain reaction (RT-qPCR) kits. Amplification efficiency was optimized for all targets, ranging from 94.92% for the N gene, 98.84% for the S gene, 81.75% for the E gene, to 82.14% for the 3′UTR genes. Assay selectivity was verified by cross-reactivity against common respiratory viruses—SARS-CoV-1, Middle East respiratory syndrome coronavirus, Influenza A/B, and human coronaviruses (HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1)—and no off-target signal was observed. The ability to detect multiple genomic regions renders the assay resilient to mutation-based primer mismatch, making it extremely suitable for clinical diagnostics, epidemiological surveillance, and variant monitoring.