Rapid and sensitive detection of Avibacterium paragallinarum in the presence of other bacteria using a 5' Taq nuclease assay: a new tool for diagnosing infectious coryza.

A 5' Taq nuclease assay specific for Avibacterium paragallinarum was designed and optimized for use in diagnosing infectious coryza. The region chosen for assay design was one of known specificity for Av. paragallinarum. The assay detected Av. paragallinarum reference strains representing the three Page and the eight Kume serovars, and field isolates from diverse geographical locations. No cross-reactions were observed with other Avibacterium species, with other bacteria taxonomically related to Av. paragallinarum nor with bacteria and viruses likely to be present in swabs collected from suspected infectious coryza cases. The detection limit for the assay was 6 to 60 colony-forming units per reaction. Twenty-two out of 53 swabs collected from sick birds reacted in the 5' Taq nuclease assay, whereas Av. paragallinarum was not isolated from any of the swabs. All of the 22 swabs yielded other bacteria in culture. The presence of Av. paragallinarum in the swabs was also demonstrated by sequencing, thereby confirming the ability of the assay to detect Av. paragallinarum in the presence of other bacteria. The ability to quantify bacterial load in the swabs using the 5' Taq nuclease assay was demonstrated.

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.