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Updated: Jun 23, 2026
Rapid Molecular Detection and Differentiation of Influenza Viruses A and B
Published on: January 30, 2017
Real-time RT-PCR assays for type and subtype detection of influenza A and B viruses
Luke T Daum1, Linda C Canas, Bernard P Arulanandam
1Department of Biology, The University of Texas at San Antonio, San Antonio, TX 78249, USA.
Abstract:
Influenza viruses type A (H3N2 and H1N1 subtypes) and B are the most prevalently circulating human influenza viruses. However, an increase in several confirmed cases of high pathogenic H5N1 in humans has raised concerns of a potential pandemic underscoring the need for rapid, point of contact detection. In this report, we describe development and evaluation of 'type,' i.e., influenza virus A and B, and 'subtype,' i.e., H1, H3, and H5, specific, single-step/reaction vessel format, real-time RT-PCR assays using total RNA from archived reference strains, shell-vial cultured and uncultured primary (throat swab/nasal wash) clinical samples. The type A and B specific assays detected all 16 influenza type A viruses and both currently circulating influenza B lineages (Yamagata and Victoria), respectively. 'Type' and 'subtype' specific assays utilize one common set of thermocycling conditions, are specific and highly sensitive (detection threshold of approximately 100 target template molecules). All clinical specimens and samples were evaluated using both the unconventional portable Ruggedized Advanced Pathogen Identification Device (RAPID) and standard laboratory bench LightCycler instruments. These potentially field-deployable assays could offer significant utility for rapid, point of care screening needs arising from a pandemic influenza outbreak.
Insights
Rapid real-time RT-PCR assays were developed for detecting influenza A and B viruses, including H5N1. These assays offer sensitive, specific, and field-deployable screening for pandemic influenza preparedness.
Area of Science:
- Virology
- Molecular Biology
- Epidemiology
Background:
- Human influenza is primarily caused by influenza A (H3N2, H1N1) and B viruses.
- Emerging highly pathogenic avian influenza A (H5N1) in humans raises pandemic concerns, necessitating rapid detection methods.
Purpose of the Study:
- To develop and evaluate single-step, real-time RT-PCR assays for rapid detection of influenza A and B virus types and H1, H3, H5 subtypes.
- To assess the utility of these assays for point-of-care screening during potential influenza outbreaks.
Main Methods:
- Development of type-specific (A and B) and subtype-specific (H1, H3, H5) real-time RT-PCR assays.
- Utilized total RNA from reference strains and clinical samples (throat swabs, nasal washes).
- Assays were validated on both standard laboratory instruments and a portable device (RAPID).
Main Results:
- Developed assays accurately detected all tested influenza A viruses and both influenza B lineages (Yamagata, Victoria).
- Assays demonstrated high specificity and sensitivity, with a detection threshold of approximately 100 target molecules.
- Both laboratory and portable instruments yielded comparable results for clinical specimens.
Conclusions:
- The developed real-time RT-PCR assays provide a rapid, sensitive, and specific method for influenza virus detection.
- These assays are suitable for field deployment, offering significant utility for point-of-care screening during pandemic influenza events.
- The assays contribute to enhanced preparedness for emerging influenza threats, including H5N1.
