281. Validation of Real-time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) Assays for the Detection and Characterization of Influenza Strains
Session: Poster Abstract Session: Diagnostic Microbiology; Novel Molecular Methods
Thursday, October 3, 2013
Room: The Moscone Center: Poster Hall C
  • MSA_VALID Poster_IDWeek2013_v2.pdf (2.0 MB)
  • Background: Turnaround time and simplicity of real-time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) assays make them essential tools for influenza vaccine effectiveness (VE) studies. Specimens tested for presence of Influenza A or B strains, and further characterization of subtype or lineage allows VE measurement against vaccine matched or mismatched strains and calculation of strain-specific disease burden.

    Methods: Singleplex rRT-PCR assays were selected and their specificity verified by “in silico” analysis of the primers and probes using Basic Local Alignment Search Tool (BLAST) analysis. Cycling conditions were optimized from the original conditions for multiplexing. The primary screening assay targets the Matrix gene of Influenza A or B viruses, followed by a secondary assay targeting the HA gene to subtype the virus. Subtyping involves two reactions, either to identify influenza A subtypes pH1N1, H3N2, or to determine the B lineages (Victoria and Yamagata). Four virus strains, detectable in the secondary assays, were tested as follows: 24 replicas made from six 10-fold dilution points (24 X 6) per virus were tested across four experiment days.  Each experiment was equally split between two operators. The Limit of Detection (LoD) was defined as the Cp (Crossing point) value where 95% of replicas were detected and was calculated using a Probit analysis for each of the detectors. Intra and inter-assay reproducibility was calculated as percent coefficient of variation (CV) for Cp values that were above the LoD. Assay specificity was evaluated by testing a panel of nucleic acid extracted from other respiratory organisms and viruses.

    Results: No unexpected similarities were observed with the primer and probe sequences, nor were there any dimer formations detected using BLAST. The Cp cut off values for the LoD, fell between 34.5 and 38.9 for all six assays.  The % CV for intra- and inter-assay precision was <6%. No cross-reactivity was observed with other respiratory pathogens (analytical specificity of 100%).

    Conclusion: This validation enhances our ability to serve as the central laboratory for the PCIRN Serious Outcomes Surveillance Network, and provides essential infrastructure for ongoing assessment of strain-specific effectiveness of Canadian influenza immunization programs.

    May Elsherif, MD1, Shelly Mcneil, MD, FIDSA1, Serge Durviaux2, Jessica Mclellan, PhD1, Amanda Lang, PhD1, Todd Hatchette, MD1, Jason Leblanc, PhD1 and on behalf of the Public Health Agency of Canada/Canadian Institutes of Health Research (PCIRN ) Serious Outcomes Surveillance Network Investigators, (1)Canadian Center for Vaccinology, IWK Health Centre and Capital Health, Dalhousie University, Halifax, NS, Canada, (2)GlaxoSmithKline Biologicals, Wavre, Belgium


    M. Elsherif, GlaxoSmithKline Biologicals: Grant Investigator, Grant recipient

    S. Mcneil, GlaxoSmithKline : Grant Investigator, Grant recipient

    S. Durviaux, GlaxoSmithKline: Employee, Salary

    J. Mclellan, GlaxoSmithKline: Grant Investigator, Research grant

    A. Lang, GlaxoSmithKline: Grant Investigator, Research grant

    T. Hatchette, GlaxoSmithKline: Grant Investigator, Research grant

    J. Leblanc, GlaxoSmithKline: Grant Investigator, Research grant

    Findings in the abstracts are embargoed until 12:01 a.m. PST, Oct. 2nd with the exception of research findings presented at the IDWeek press conferences.