Background: Multiplex qPCR is often used for the simultaneous detection of respiratory viruses in clinical specimens. Economic and seasonally-driven pressures for faster, accurate sample testing at reduced costs have resulted in an increased need for highly sensitive and specific assays with high throughput for respiratory illnesses. The PlexPCR™ RV11 (beta) assay (SpeeDx, Australia) detects 11 virus-specific targets in a 2-well format which are highly snesitive and enable efficient qPCR multiplexing(Mokany et al 2013). Here we evaluated this platform in comparison with an in-house 10-virus targeted multiplex PCR assay which detects a broad range of respiratory viruses(Ratnamohan et al 2014).
Material/methods: PlexPCR™ Respi-Virus 11 (beta) assay was evaluated against in-house CIDMLS assay on prospective 204 nasopharyngeal swabs. The targets of the PlexPCR™ Respi-Virus 11 (beta) assay were: Well 1: Influenza A, Influenza B, RSV (A/B) Rhinovirus and internal control; Well 2: Human Metapneumovirus, Adenovirus and Human Parainfluenza virus 1-4. Targets contained in the in-house CIDMLS assay were Well 1: Influenza A, Influenza B, RSV (A/ B); Well 2: Parainfluenza virus 1-3; Well 3: Rhinovirus, Enterovirus, Human Metapneumovirus; and Well 4: Adenovirus and internal control. The discrepant results resolved using a commercial assay (GeneXpert FA/FB/RSV, Cepheid) and/or DNA sequencing.
Results: Compared against the CIDMLS in-house assay, the PlexPCR™ Respi-Virus 11 (beta) assay had a final sensitivity/specificity of: Influenza A 100%/100% (95%CI: 89-100%/98-100%), Influenza B 100%/100% (95%CI: 40-100%/98-100%), RSV 100%/100% (95%CI: 69-100%/98-100%), Rhinovirus 100%/98.9% (95%CI: 82-100%/97-100%) and a preliminary sensitivity/specificity for hMPV 100%/97.5% (95%CI: 29-100%/96-100%), HPIV1-4 100%/99.5% (95%CI: 29-100%/97-100%) & Adenovirus 100%/98.5% (95%CI: 29-100%/96-100%). Processing of the 204 samples required 4.25/8.5 plates equivalent to 6.4/12.8 hours for PlexPCR/in-house assay respectively.
Conclusions: The PlexPCR™ RV11 (beta) assay demonstrated high sensitivity and specificity even in complex multiplex assays with decreased reaction setup from 4 to 2 wells. We found the PlexPCR assay provided a good approach to qPCR with the advantages of a robust performance in multiplex. There are also advantages seen in higher throughput which would have additional cost savings.