title: Rapid influenza A detection and quantitation in birds using a one-step real-time reverse transcriptase PCR and High Resolution Melting.
abstract: 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.
authors.affiliation: Department of Ecology and Evolutionary Biology and Center for Tropical Research, Institute of the Environment and Sustainability, University of California, Los Angeles, 619 Charles E. Young Drive East, Los Angeles, CA 90095-1496, USA. eecurd@ucla.edu
authors.forename: Emily
authors.initials: E
authors.lastname: Curd
authors.forename: John
authors.initials: J
authors.lastname: Pollinger
authors.forename: Erin
authors.initials: E
authors.lastname: Toffelmier
authors.forename: Thomas
authors.initials: T
authors.lastname: Smith
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D001717:Birds / Q000821:virology*; D002645:Chickens / Q000821:virology*; D003959:Diamines; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics; D053127:Influenza A Virus, H9N2 Subtype / Q000235:genetics / Q000302:isolation & purification; D009980:Influenza A virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000821:virology*; D009930:Organic Chemicals; D011804:Quinolines; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors; D044366:Transition Temperature
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I