title: Validated H5 Eurasian real-time reverse transcriptase-polymerase chain reaction and its application in H5N1 outbreaks in 2005-2006.
abstract: Real time reverse transcriptase (RRT)-polymerase chain reaction (PCR) for the detection of Eurasian H5 avian influenza virus (AIV) isolates was adapted from an existing protocol, optimized, and validated using a number of genetically diverse H5 isolates (n = 51). These included 34 "Asian lineage" H5N1 highly pathogenic avian influenza (HPAI) viruses (2004-2006), plus 12 other H5 isolates from poultry outbreaks and wild birds in the Eastern Hemisphere (1996-2005). All 51 were positive by H5 Eurasian RRT-PCR. Specificity was assessed by testing representative isolates from all other AL virus subtypes (n = 52), non-AI avian pathogens (n = 8), plus a negative population of clinical specimens derived from AI-uninfected wild birds and poultry (n = 604); all were negative by H5 Eurasian RRT-PCR. RNA was directly extracted from suspect HPAI H5N1 clinical specimens (Africa, Asia, and Europe; 2005-2006; n = 58) from dead poultry and wild birds, and 55 recorded as positive by H5 Eurasian RRT-PCR: Fifty-one of these 55 were in agreement with positive AIV isolation in embryonated chickens' eggs. H5 Eurasian RRT-PCR was invaluable in H5 outbreak diagnosis and management by virtue of its rapidity and high degree of sensitivity and specificity. This method provides a platform for automation that can be applied for large-scale intensive investigations, including surveillance.
authors.affiliation: Virology Department, Veterinary Laboratories Agency, Woodham Lane, Addlestone, Surrey KT15 3NB, United Kingdom.
authors.forename: M J
authors.initials: MJ
authors.lastname: Slomka
authors.forename: T
authors.initials: T
authors.lastname: Pavlidis
authors.forename: J
authors.initials: J
authors.lastname: Banks
authors.forename: W
authors.initials: W
authors.lastname: Shell
authors.forename: A
authors.initials: A
authors.lastname: McNally
authors.forename: S
authors.initials: S
authors.lastname: Essen
authors.forename: I H
authors.initials: IH
authors.lastname: Brown
mesh_terms: D000818:Animals; D001717:Birds / Q000821:virology; D004196:Disease Outbreaks / Q000662:veterinary*; D053124:Influenza A Virus, H5N1 Subtype / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000453:epidemiology* / Q000821:virology; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013997:Time Factors