Validation of an influenza virus A 5'Taq nuclease assay for the detection of equine influenza virus A RNA in nasal swab samples.

OBJECTIVE

Describe the in-house validation of a previously reported influenza virus type A 5'Taq nuclease assay for detecting equine influenza virus A RNA in nasal swab material.

METHODS

The validation compares the 5'Taq nuclease assay with a gel-based reverse transcription nested polymerase chain reaction (PCR) previously reported by the Irish Equine Centre for detection of H3N8 and H7N7 equine influenza viruses. This test was chosen because it targets a different region of the viral genome to the real-time test, so it is not merely a repeat of the same test in a different format. Moreover, nested PCRs are commonly considered to have similar sensitivity to real-time PCRs and are therefore ideal for evaluation comparisons.

RESULTS

The sensitivity of the nested PCR was comparable to the 5'Taq nuclease test. Known positive samples and known negative samples reacted with both tests with 100% correlation. Parallel testing of 276 nasal swab samples showed 98% agreement.

CONCLUSION

The specificity of the nested amplicons was confirmed by nucleotide sequencing and showed >99.5% identity with the same region of previously published equine influenza virus A sequences. The results of this work are appropriate validation for the acceptance of the real-time PCR for equine influenza A virus in equine nasal swabs.