title: Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses.
abstract: Multiplex PCR methods are attractive to clinical laboratories wanting to broaden their detection of respiratory viral pathogens in clinical specimens. However, multiplexed assays must be well optimized to retain or improve upon the analytic sensitivity of their singleplex counterparts. In this experiment, the lower limit of detection (LOD) of singleplex real-time PCR assays targeting respiratory viruses is compared to an equivalent panel on a multiplex PCR platform, the GenMark eSensor RVP. LODs were measured for each singleplex real-time PCR assay and expressed as the lowest copy number detected 95-100% of the time, depending on the assay. The GenMark eSensor RVP LODs were obtained by converting the TCID50/mL concentrations reported in the package insert to copies/μL using qPCR. Analytical sensitivity between the two methods varied from 1.2-1280.8 copies/μL (0.08-3.11 log differences) for all 12 assays compared. Assays targeting influenza A/H3N2, influenza A/H1N1pdm09, influenza B, and human parainfluenza 1 and 2 were most comparable (1.2-8.4 copies/μL, <1 log difference). Largest differences in LOD were demonstrated for assays targeting adenovirus group E, respiratory syncytial virus subtype A, and a generic assay for all influenza A viruses regardless of subtype (319.4-1280.8 copies/μL, 2.50-3.11 log difference). The multiplex PCR platform, the GenMark eSensor RVP, demonstrated improved analytical sensitivity for detecting influenza A/H3 viruses, influenza B virus, human parainfluenza virus 2, and human rhinovirus (1.6-94.8 copies/μL, 0.20-1.98 logs). Broader detection of influenza A/H3 viruses was demonstrated by the GenMark eSensor RVP. The relationship between TCID50/mL concentrations and the corresponding copy number related to various ATCC cultures is also reported.
authors.affiliation: Department of Health and Social Services, Division of Public Health, Alaska State Public Health Virology Laboratory, Fairbanks, Alaska, United States of America.|Department of Biology and Wildlife, Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, Alaska, United States of America.
authors.forename: Jayme
authors.initials: J
authors.lastname: Parker
authors.affiliation: Department of Health and Social Services, Division of Public Health, Alaska State Public Health Virology Laboratory, Fairbanks, Alaska, United States of America.
authors.forename: Nisha
authors.initials: N
authors.lastname: Fowler
authors.affiliation: Department of Health and Social Services, Division of Public Health, Alaska State Public Health Virology Laboratory, Fairbanks, Alaska, United States of America.
authors.forename: Mary Louise
authors.initials: ML
authors.lastname: Walmsley
authors.affiliation: Department of Health and Social Services, Division of Public Health, Alaska State Public Health Virology Laboratory, Fairbanks, Alaska, United States of America.
authors.forename: Terri
authors.initials: T
authors.lastname: Schmidt
authors.affiliation: Department of Health and Social Services, Division of Public Health, Alaska State Public Health Virology Laboratory, Fairbanks, Alaska, United States of America.
authors.forename: Jason
authors.initials: J
authors.lastname: Scharrer
authors.affiliation: Department of Health and Social Services, Division of Public Health, Alaska State Public Health Virology Laboratory, Fairbanks, Alaska, United States of America.
authors.forename: James
authors.initials: J
authors.lastname: Kowaleski
authors.affiliation: Department of Health and Social Services, Division of Public Health, Alaska State Public Health Virology Laboratory, Fairbanks, Alaska, United States of America.
authors.forename: Teresa
authors.initials: T
authors.lastname: Grimes
authors.affiliation: Department of Biology and Wildlife, Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, Alaska, United States of America.
authors.forename: Shanann
authors.initials: S
authors.lastname: Hoyos
authors.affiliation: Department of Health and Social Services, Division of Public Health, Alaska State Public Health Virology Laboratory, Fairbanks, Alaska, United States of America.|Department of Biology and Wildlife, Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, Alaska, United States of America.
authors.forename: Jack
authors.initials: J
authors.lastname: Chen
mesh_terms: D018628:Gene Dosage; D006801:Humans; D057230:Limit of Detection; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012141:Respiratory Tract Infections / Q000821:virology*; D012680:Sensitivity and Specificity; D014780:Viruses / Q000235:genetics / Q000302:isolation & purification*