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Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field
Published on: August 2, 2011
Rapid molecular haemagglutinin subtyping of avian influenza isolates by specific real-time RT-PCR tests
Maia Elizalde1, Montserrat Agüero2, Dolores Buitrago2
1Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos, 28130 Madrid, Spain.
Abstract:
Sixteen haemagglutinin (HA) subtypes of avian influenza viruses (AIV) have been described to date. Rapid subtype identification of any AIV is of major interest because of the possible serious consequences for the poultry industry and even public health. Molecular techniques currently allow immediate accurate subtype characterisation prior to virus isolation. In this study, a set of fourteen specific real-time RT-PCR methods were developed and evaluated for AIV HA subtyping (H1-H4, H6-H8, H10-H16), H5 and H9 being excluded on the basis of the current validity of the European Union (EU) recommended specific assays. Specific primers and probes sets for each HA-subtype were designed to hybridise the largest isolates range within each single subtype, considering the Eurasian lineage as a major target. The robustness and general application of the 14 HA-subtype methods were verified by the analysis of 110 AIV isolates belonging to all 16 HA-subtypes, performed in different laboratories. The developed real-time RT-PCR assays proved to be highly specific and revealed suitable sensitivity, allowing direct HA-subtyping of clinical material. In summary, this study provides for the first time a panel of molecular tests using specific hydrolysis probes for rapid and complete AIV HA-subtype identification.
Insights
This study developed 14 real-time RT-PCR assays for rapid avian influenza virus (AIV) HA subtyping. These molecular tools enable quick and accurate identification of AIV subtypes, crucial for public health and the poultry industry.
Area of Science:
- Veterinary Virology
- Molecular Diagnostics
- Public Health Microbiology
Background:
- Avian influenza viruses (AIV) pose significant threats to poultry and public health.
- Accurate and rapid HA subtype identification is critical for effective disease control.
- Existing molecular methods may not cover all relevant HA subtypes.
Purpose of the Study:
- To develop and evaluate a comprehensive panel of real-time RT-PCR assays for avian influenza virus (AIV) HA subtyping.
- To provide rapid, specific, and sensitive molecular tools for identifying AIV HA subtypes H1-H4 and H6-H16.
- To establish a robust method for direct HA subtyping of clinical samples.
Main Methods:
- Design and validation of 14 specific real-time RT-PCR assays targeting AIV HA subtypes (H1-H4, H6-H8, H10-H16).
- Utilized specific primers and probes for hybridization with a broad range of isolates, focusing on the Eurasian lineage.
- Tested the assays on 110 AIV isolates across all 16 HA subtypes in multiple laboratories.
Main Results:
- The developed real-time RT-PCR assays demonstrated high specificity and suitable sensitivity for AIV HA subtyping.
- The assays successfully identified 14 distinct HA subtypes (excluding H5 and H9).
- Validated methods allowed for direct HA-subtyping of clinical material, confirming robustness and broad applicability.
Conclusions:
- This study presents the first panel of molecular tests for complete and rapid AIV HA-subtype identification using specific hydrolysis probes.
- The developed real-time RT-PCR assays offer a valuable tool for immediate characterization of AIV, aiding in disease surveillance and control.
- These molecular diagnostic tools are essential for managing the risks associated with AIV in poultry and potential public health impacts.
