PCR protocols for the detection of avian influenza virus (AIV)

Introduction

It is important to consider how to select and to use a given method in the context of AI diagnosis or surveillance as applied to either poultry or wild birds. Details of the relevant AI scientific context are outlined in the EU diagnostic manual. This is also consistent with the:-

All the prescribed methods have been validated, and incorporated in the EU diagnostic manual.

See the reference list (24kb pdf) for more information. They have shown to be effective for the detection of AIV through blind AI PCR trials (conducted within the EU-funded AVIFLU project) and formal PCR  proficiency testing programmes organised within the EU.

Protocols

A simple “yes / no” PCR result for the presence of AIV in clinical specimens is provided by the highly sensitive and specific M gene real-time PCR protocol, essentially as described by Spackman (see references). This test can detect viruses of all haemagglutinin (HA) subtypes (1-16) since it targets the matrix gene that is conserved amongst all influenza A viruses.

To provide specific information on the HA subtype of a virus in relation to notifiable avian influenza there are subtype-specific real-time RT-PCRs for detecting H5 and H7 AIVs of Eurasian origin. Conventional H5 and H7 PCR protocols which facilitate molecular pathotyping across the H5 and H7 cleavage site region of the HA gene are also included.

  • Real time RT-PCR for the detection of AI subtype H5, H7, H9 made available by IZSV laboratory, Padova, Italy.

Any technical enquiries about these protocols should be sent to: aiwrl@vla.defra.gsi.gov.uk