Review of rapid molecular diagnostic tools for avian influenza

Authors: Suarez, David; Das, Amaresh; Ellis, Elizabeth

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/6/2006
Publication Date: 4/6/2007
Citation: Suarez, D.L., Das, A., Ellis, E.H. 2007. Review of rapid molecular diagnostic tools for avian influenza. Avian Diseases. 51:201-208.

Interpretive Summary: Avian influenza is a virus naturally found in wild birds where it seldom causes disease. On rare occasions the virus can be transmitted to poultry, including chickens and turkeys, where it can cause disease. The virus in poultry can cause a severe form of disease called highly pathogenic avian influenza. The virus is considered highly infectious and early diagnosis of infection is one of the keys for rapid control of an outbreak in poultry. Recent molecular diagnostics tests, including PCR tests, have provided for a rapid and sensitive diagnosis of the disease. The use of these molecular tests is increasing and more research to improve these tests is on going. The paper describes some of the recent advances and common problems observed with molecular testing technology.

Technical Abstract: Molecular diagnostics tests are commonly used to diagnose avian influenza virus (AIV) because they are sensitive, can be performed rapidly, with high throughput, and at a moderate cost. Molecular diagnostic tests have recently proven themselves to be invaluable in controlling disease outbreaks around the world. Several different methods, including traditional reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR (RRT-PCR), and nucleic acid sequence based amplification (NASBA) among others, have been described for the diagnosis of avian influenza in poultry with many different variations of primer, probes, enzymes etc. Few of these tests have been validated, with the understanding that validation should be described as a level of comparison testing to show “fitness for purpose”. None of the molecular diagnostic tests are validated for all species or specimen types that might be presented to a diagnostic laboratory. The sensitivity and specificity for all the molecular tests are governed by three critical control points, including RNA extraction, enzymes used for amplification, and the sequence of primers and probe. The RNA extraction step is of particular concern, since high quality RNA is needed for any of the molecular tests. Some samples types, Includeing cloacal (fecal) swabs and tissue samples, are difficult to process, and issues of poor RNA extraction or PCR inhibitors are common with these types of samples. The development of internal controls, robotics, and bead reagents are providing improved performance of existing tests and new technologies will likely provide better tests for the future. With any molecular test, assay assurance must be performed on an ongoing basis, which includes the use of proficiency panels to measure test performance.