Evaluation of Two Commercial Loop-Mediated Isothermal Amplification Assays for Detection of Avian Influenza H5 and H7 Hemagglutinin Genes

• Published in: Journal of Veterinary Diagnostic Investigation
• Main Authors: Postel, Alexander, Letzel, Tobias, Frischmann, Sieghard, Grund, Christian, Beer, Martin, Harder, Timm
• Format: Article in Journal/Newspaper
• Language: English
• Published: SAGE Publications 2010
• Subjects: Avian flu
• Online Access: http://dx.doi.org/10.1177/104063871002200110; http://journals.sagepub.com/doi/pdf/10.1177/104063871002200110; http://journals.sagepub.com/doi/full-xml/10.1177/104063871002200110

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 realtime 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.