ARS | Publication request: Evaluation of primer and probe mismatches in sensitivity of select RRT-PCR tests for avian influenza

Submitted to: American Association of Avian Pathologists
Publication Type: Abstract Only
Publication Acceptance Date: July 1, 2011
Publication Date: July 17, 2011
Citation: Suarez, D.L. 2011. Evaluation of primer and probe mismatches in sensitivity of select RRT-PCR tests for avian influenza [abstract]. American Association of Avian Pathologists Meeting, July 16-19, 2011, St. Louis, Missouri. CD-ROM.

Technical Abstract: The recent outbreak of pH1N1 in animals highlighted an imperfection of the matrix real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) that has become the primary screening test for avian and swine influenza viruses. Four mismatches in one primer resulted in an important loss of sensitivity in the test. Bioinformatics for the matrix RRT-PCR test shows the forward primer and probe are highly conserved for almost all influenza isolates. The reverse primer, however, has 6 sites with important sequence variation. Two mismatches in the reverse primer are not uncommon in avian influenza samples, and 3 mismatches are observed in some lineages of virus. As mentioned previously the pandemic H1N1 viruses have 4 mismatches. In experimental testing of different variant viruses, viruses with 3 and 4 mismatches in the reverse primer had decreases in test sensitivity of 3-4 logs, which is unacceptable. Modifications of the original test were developed to improve sensitivity for all viruses. Bioinformatics analysis of H5 and H7 viruses also show important sequence variations that can affect sensitivity. Because sensitivity and specificity of RRT-PCR tests are dependant on proper annealing of primers and probes, efforts need to continue to evaluate tests against circulating strains of virus to assure proper test performance.