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Updated: Jun 19, 2026
High-throughput Detection Method for Influenza Virus
Published on: February 4, 2012
Design and clinical application of a molecular method for detection and typing of the influenza A/H1N1pdm virus
Eleonora Lalle1, Licia Bordi, Concetta Castilletti
1National Institute for Infectious Diseases L. Spallanzani, Rome, Italy.
Abstract:
In March/April 2009, Mexico experienced an outbreak of respiratory illness, due to a new influenza of swine origin virus, which spread rapidly via human-to-human transmission, and became pandemic (A/H1N1pdm). Because of its unique genome composition, which includes gene segments of swine, avian and human origin, and to the considerable differences to the human influenza A viruses that have circulated so far, the currently used molecular methods proved inadequate. Based on published sequences, a primer set targeting the nucleoprotein gene was designed, which provided enhanced sensitivity for the new strain and proved suitable for sequence-based strain identification. The novel nucleoprotein reverse-transcription-PCR showed higher sensitivity for A/H1N1pdm than a commercial test for influenza A, and was comparable to the real-time-based method developed by the Centers for Disease Control and Prevention. It was used to screen 177 clinical samples referred to the laboratory for suspected A/H1N1pdm infection, detecting 17 (9.6%) infections that were confirmed by sequence analysis (100% sensitivity as compared to the real-time kit). The novel method is suitable for the diagnosis of A/H1N1pdm, and is also suitable, at least in the screening phase, for laboratories not equipped with the real-time PCR technology.
Insights
A new molecular diagnostic method was developed for the pandemic influenza A/H1N1 virus. This nucleoprotein reverse-transcription-PCR offers enhanced sensitivity for accurate A/H1N1pdm diagnosis.
Area of Science:
- * Virology and Molecular Diagnostics
- * Infectious Disease Epidemiology
Background:
- * The 2009 swine-origin influenza A (H1N1) pandemic (A/H1N1pdm) presented diagnostic challenges due to its novel genetic makeup.
- * Existing molecular methods were insufficient for detecting the new influenza strain effectively.
Purpose of the Study:
- * To develop and validate a sensitive molecular assay for A/H1N1pdm detection.
- * To enable sequence-based identification of the pandemic influenza strain.
Main Methods:
- * Design of a primer set targeting the nucleoprotein gene for reverse-transcription PCR (RT-PCR).
- * Evaluation of the novel RT-PCR assay's sensitivity and specificity against commercial and CDC-developed real-time PCR methods.
- * Screening of 177 clinical samples suspected of A/H1N1pdm infection.
Main Results:
- * The novel nucleoprotein RT-PCR demonstrated higher sensitivity for A/H1N1pdm compared to a commercial influenza A test.
- * The assay showed comparable performance to the CDC's real-time PCR method.
- * 17 out of 177 (9.6%) suspected cases were accurately identified, with 100% sensitivity confirmed by sequence analysis.
Conclusions:
- * A novel nucleoprotein RT-PCR assay is effective for diagnosing A/H1N1pdm infections.
- * This method is suitable for laboratories lacking real-time PCR technology, particularly for screening purposes.
