Development of a colorimetric loop-mediated isothermal amplification assay for rapid and specific detection of Aves polyomavirus 1 from psittacine birds

Abstract

A colorimetric loop-mediated isothermal amplification (LAMP) assay was developed for the rapid and specific detection of the T gene of Aves polyomavirus 1 (APyV), a causative agent of budgerigar fledgling disease (BFD) in psittacine birds. The amplification can be completed in 40 min at 60 °C, and the results can be visually detected by the naked eye using hydroxyl naphthol blue as a colorimetric indicator. The assay specifically amplified APyV DNA but not other viral and bacterial nucleic acids. The limit of detection of the assay was 5 × 102 DNA copies/reaction, which was comparable to those of previously reported conventional polymerase chain reaction assays. In the clinical evaluation, the LAMP results showed 100% concordance with those of the previously reported PCR assays with regard to specificity, sensitivity, and percentage of overall agreement, with a kappa value of 1.0. These results indicate that the developed LAMP assay will be a valuable tool for the rapid, sensitive and specific detection of APyV from BFD-suspected psittacine bird samples even in resource-limited laboratories.