B-234 Detection of multiple targets in a single fluorescent channel: Respiratory virus multiplex RT-qPCR assay

Abstract Background Here we present a novel single-channel multiplex qPCR assay with real-time detection in 3 separate readouts in a single optical channel. Traditional multiplex qPCR technologies are limited to detection of one target per optical channel, limiting throughput and utility of existing instruments. This assay utilizes multiple probes that each fluoresce at a specific temperature in a single optical channel such as FAM, but only in the presence of their respective targets. The fluorescence signals from each probe are contained within their discrete temperature range, ensuring no cross talk between temperatures. Probes are universal and cleaved by target-specific PlexZymes®, making this probe system adaptable for any targets of interest. A reverse transcriptase qPCR assay (3T Respi) was developed for detection of 5 viral targets in 3 readouts in the FAM channel; Respiratory Syncytial Viruses A and B (RSV A and B) were detected at the first temperature (45°C); Influenza A (Flu A) and Influenza B (Flu B) were detected at the second temperature (61°C); and SARS-CoV-2 (ORF1ab and RdRp gene targets) was detected at the third temperature (78°C). Methods The assay performance was assessed in a retrospective evaluation of 657 nasopharyngeal swab (NPS) extracts provided by symptomatic individuals, on the CFX96™ Touch Real-Time PCR Detection System (Bio-Rad) against the CE-IVDR approved comparator PKamp™ Respiratory SARS-CoV-2 RT-PCR panel 1 (Revvity). The samples had been previously extracted with the Chemagic™ Viral DNA/RNA 300 Kit (Revvity). Any discordant extracts were tested with the CE-IVDR approved PlexPCR® Flu/RSV/SARS-CoV-2 (SpeeDx) on the Applied Biosystems™ QuantStudio5 Real-Time PCR instrument. Sensitivity of detection was established using commercial genomic RNA. Results The 3T Respi showed a positive percent agreement (PPA) of 83.5%, 92.9% and 96.0% for RSV A/B, Flu A/B and SARS-CoV-2 respectively with NPS extracts. Sample degradation over prolonged storage in -80°C was suspected as the reason for the PPA below 90% with RSV A/B. The negative percent agreement (NPA) was 99.8%, 95.8% and 99.1% for RSV A/B, Flu A/B and SARS-CoV-2 respectively. Further, the 3T Respi detected down to 9 copies of target per reaction (cps/rxn) of RSV A, FluA (H1N1), FluB and SARS-CoV-2, and 3 cps/rxn of RSV B and FluA (H3N2). Conclusion The 3T Respi assay showed detection of 5 viral targets in 3 independent readouts in a single optical channel, with detection down to 9 copies of target per reaction. In a retrospective clinical evaluation study against a comparator qPCR test, this assay demonstrated a PPA >90% for Flu A/B and SARS-CoV-2 targets, and a PPA of 83.5% for RSV A/B; lower PPA for RSV A/B was likely due to sample degradation over prolonged storage. NPA was over 95% for all targets. This assay can be run on standard qPCR instruments as demonstrated, or on point-of-care qPCR instruments typically with less optical channels. Overall, this data exemplifies the suitability of the utilised single-channel multiplex qPCR probe system for clinical applications, adaptable to any targets of interest.