Efficient influenza A viral surveillance of wild and domestic birds requires rapid viral detection and quantitation of high and low quality samples. Current influenza A qPCR-based detection protocols specified by CDC, OIE and USDA utilize fluorogenic hydrolysis probe based real-time reverse transcription PCR (RRT-PCR) assays for detection and quantitation. The sequence diversity of this virus, even in the conserved matrix gene M1, makes primer and probe designs challenging. In this report it was determined that false RRT-PCR positives are possible with this method. This is particularly problematic when surveying non-cultured or inactivated avian tracheal and cloacal mucosal samples with low concentrations of virus and large proportions of background nucleic acids. This report presents a modification of a one-step RRT-PCR detection method for influenza A using SYBR green intercalating dye-based target amplification detection. High Resolution Melting (HRM), amplicon size quantitation and sequence verification is used to screen for non-target amplification (false positives). The resulting protocol has the sensitivity of hydrolysis probe methods, allows for flexible primer design and verification of target amplification, and provides high confidence in positive results. A multiplex subtype detection method using the RRT-PCR HRM assay is also demonstrated. Overall, this method is both time and cost effective while providing an extra measure of confidence in surveillance results through the implementation of target verification.
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