title: Evaluation of two commercial loop-mediated isothermal amplification assays for detection of avian influenza H5 and H7 hemagglutinin genes.
abstract: Real-time reverse transcription loop-mediated isothermal amplification (real-time RT-LAMP) holds substantial potential as a highly sensitive, specific, and easy-to-perform molecular technique for pathogen detection in clinical samples. In the current study, the analytical and diagnostic performance of 2 commercial real-time RT-LAMP kits, Avian Flu H5 and Avian Flu H7, in detecting Avian influenza virus (AIV) infections were evaluated and compared with validated real-time reverse transcription polymerase chain reaction (RT-PCR) assays using RNA from reference virus isolates of subtypes H5 (n = 24) and H7 (n = 25) and of phylogenetically related subtypes (n = 20). When real-time RT-LAMP was carried out according to the recommendations of the manufacturer, 3 out of 24 H5 isolates and 8 out of 25 H7 reference strains were not detected. Prolonging the amplification phase resulted in detection of all H5 isolates but also in false positive detection of 2 non-H5 isolates. Real-time RT-LAMP specific to H7 failed to detect 2 H7 isolates after prolonged amplification. According to the examination of RNA log dilutions, the sensitivity of the real-time RT-LAMP assays, for a number of historic but also recent strains, was considerably lower compared with subtype-specific real-time RT-PCR assays. Application of the real-time RT-LAMP assays for analysis of diagnostic samples from wild birds confirmed their lower sensitivity. Commercial real-time RT-LAMP as tested in this study with a broad range of AIV H5 and H7 strains of phylogenetically diverse yet recent origin, holds some promise for routine veterinary diagnostic purposes, although real-time RT-LAMP was markedly more vulnerable to a reduction of detection limits because of strain-specific sequence variation than subtype-specific real-time RT-PCR.
authors.affiliation: O.I.E. and National Reference Laboratory for Avian Influenza Virus, Institute for Diagnostic Virology, Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Südufer 10, D-17493 Greifswald, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Postel
authors.forename: Tobias
authors.initials: T
authors.lastname: Letzel
authors.forename: Sieghard
authors.initials: S
authors.lastname: Frischmann
authors.forename: Christian
authors.initials: C
authors.lastname: Grund
authors.forename: Martin
authors.initials: M
authors.lastname: Beer
authors.forename: Timm
authors.initials: T
authors.lastname: Harder
mesh_terms: D000818:Animals; D001717:Birds; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics* / Q000302:isolation & purification; D009980:Influenza A virus / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D005585:Influenza in Birds / Q000821:virology*; D021141:Nucleic Acid Amplification Techniques / Q000662:veterinary*; D012680:Sensitivity and Specificity
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; C109963:hemagglutinin, avian influenza A virus