Design of a 2D Melting Curve-Based Multiplex PCR Assay for Detection of SARS-CoV-2/RSV/Influenza A-B.

Background: Recent advances in detecting respiratory pathogens have allowed for the simultaneous identification of multiple agents, enabling quick and accurate diagnosis to start timely treatment. This study aimed to design a novel two-dimensional (2D) multiplex reverse transcription quantitative PCR (RT-qPCR) assay. This assay allows for the concurrent detection of SARS-CoV-2, RSV, and Influenza A-B using molecular beacon technology in a single-well format. Methods: We used 550 nasopharyngeal swab samples from the Kocaeli University, Research and Educational Hospital, PCR laboratory, along with synthetic plasmids for SARS-CoV-2, RSV, Influenza A-B, and internal control (RNase P). DNA products generated after amplification interacted with intermediate probes containing specific enzymatic cleavage sites and fluorescent markers, producing characteristic melting temperature (Tm) values for melting curve analysis. Results: Distinct Tm values were identified for SARS-CoV-2 (72°C), RSV (66°C), Influenza A (56°C), Influenza B (68°C), and internal control (80.5°C). The accuracy was confirmed by testing laboratory-confirmed samples and synthetic plasmids, with no cross-reactivity or false positives observed. Conclusions: This melting curve-based assay can differentiate among various pathogens within a single well and fluorescence channel by utilizing the unique Tm of each target. Consequently, this novel assay may serve as a cost-effective, high-throughput PCR testing method compared to traditional diagnostics.