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Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting
Jayme Parker1,2, Nisha Fowler1, Mary Louise Walmsley1
1Department of Health and Social Services, Division of Public Health, Alaska State Public Health Virology Laboratory, Fairbanks, Alaska, United States of America.
Plos One
|November 17, 2015
Summary
Multiplex PCR assays offer broader respiratory virus detection but require optimization. This study compared singleplex and multiplex PCR methods, finding comparable sensitivity for some viruses but significant differences for others, with the multiplex platform showing improved detection for specific strains.
Area of Science:
- Clinical Virology
- Molecular Diagnostics
- Respiratory Pathogen Detection
Background:
- Multiplex PCR assays are increasingly adopted by clinical laboratories for expanded respiratory viral pathogen detection.
- Optimizing multiplexed assays is crucial to maintain or enhance the analytical sensitivity compared to singleplex assays.
Purpose of the Study:
- To compare the analytical sensitivity, specifically the lower limit of detection (LOD), of singleplex real-time PCR assays against a multiplex PCR platform (GenMark eSensor RVP).
- To evaluate the performance of multiplexed assays for detecting various respiratory viruses in clinical specimens.
Main Methods:
- Determined the LOD for individual singleplex real-time PCR assays targeting respiratory viruses.
- Obtained LODs for the GenMark eSensor RVP by converting manufacturer-reported TCID50/mL concentrations to copies/μL using qPCR.
- Compared the analytical sensitivity (LODs) between singleplex assays and the multiplex platform across 12 different viral targets.
Main Results:
- Analytical sensitivity varied significantly between methods, ranging from 1.2 to 1280.8 copies/μL.
- Assays for influenza A (H3N2, H1N1pdm09), influenza B, and human parainfluenza viruses 1 and 2 showed comparable LODs (<1 log difference).
- The GenMark eSensor RVP demonstrated improved analytical sensitivity for influenza A/H3, influenza B, human parainfluenza virus 2, and human rhinovirus.
- Largest differences in LOD were observed for adenovirus group E, respiratory syncytial virus A, and a generic influenza A assay.
Conclusions:
- While multiplex PCR offers advantages in pathogen detection breadth, careful optimization is needed to match singleplex assay sensitivity.
- The GenMark eSensor RVP platform shows promise for improved detection of specific respiratory viruses, including influenza A/H3.
- Understanding the quantitative relationship between TCID50/mL and copy number is important for assay validation.
