Review of rapid molecular diagnostic tools for avian influenza virus

Molecular diagnostic tests are commonly used to diagnose avian influenza virus because they are sensitive and can be performed rapidly, with high throughput, and at a moderate cost. Molecular diagnostic tests recently have proven themselves to be invaluable in controlling disease outbreaks around the world. Several different methods, including traditional reverse transcription-polymerase chain reaction (PCR), real-time reverse transcription-polymerase chain reaction, and nucleic acid sequence-based amplification among others, have been described for the diagnosis of avian influenza in poultry with many different variations of primers, 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 probes. The RNA extraction step is of particular concern, since high-quality RNA is needed for any of the molecular tests. Some sample types, including cloacal (fecal) swabs and tissues, are difficult to process, with issues of poor RNA extraction or PCR inhibitors being common. 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.