Aim:
PlexPlus® technology significantly enhances standard qPCR readout capabilities, making it valuable for diagnostic applications. This advanced technology employs universal nucleic acid probes that fluoresce at either low or high temperatures, with fluorescence data acquired at two distinct temperatures across multiple wavelengths. The data collected within each channel at each temperature is discrete, ensuring no cross talk between temperatures. Utilizing the RespiV PlexPlus® prototype assay as a model, the objective was to evaluate the capability to simultaneously detect 14 respiratory viral targets in a single well, thereby improving the efficiency of viral diagnosis in clinical settings.
Methods:
Fluorescence data were acquired at 52°C and 76°C in six channels during qPCR cycling on the QuantStudioTM Real-Time PCR system. The assay was characterized by retrospective analysis of 680 nasopharyngeal swab samples using SpeeDx’s PlexPCR® RespiVirus and PlexPCR® Flu/RSV/SARS-CoV-2 tests as comparators, with a subset of non-concordant results resolved by sequencing.
Results:
The assay had the capacity to detect down to 5-20 copies of target across the entire panel in preliminary analytical studies. Analysis of the nasopharyngeal swabs showed overall concordance of 96% or greater for all 14 targets. Further, the assay successfully detected and discriminated Human Rhinovirus and Enterovirus in the nasopharyngeal specimens and in a QCMD 2023 Rhinovirus RNA EQA Programme (RVRNA23S) panel.
Conclusions:
The single-well, highly multiplexed assay delivered comparable results on 680 samples to two current IVD-cleared assays, demonstrating its reliability for clinical diagnosis. It enables high-throughput analysis, determining up to 1,302 results for 14 respiratory viral targets in 93 patient samples simultaneously in one 96-well plate on a standard PCR instrument. This technology enhances the efficiency of viral diagnosis making it a valuable tool for laboratory testing.