Comparison of three template preparation methods for routine detection of beak and feather disease virus and avian polyomavirus with single and nested polymerase chain reaction in clinical specimens

Abstract

Direct comparisons are important when assessing the value of DNA extraction methods for diagnostic virology as the inhibitors present and the efficiency of extraction vary with the target infectious agent as well as the species and the site from which the clinical sample was obtained. Three DNA extraction methods were compared for routine polymerase chain reaction (PCR) detection of beak and feather disease virus (BFDV) in whole blood and feather samples and of avian polyomavirus (APV) in feather samples. Boiling in Chelex® 100 Resin was found to be the most sensitive method for detection of BFDV or APV DNA in both feather and blood samples. In combination with nested PCR it enabled detection of BFDV DNA in 13/13 positive whole blood samples and in 22/23 positive feather samples. It also enabled detection of APV in 31/31 samples detected as positive in this study. NucleoSpin® kits enabled detection of BFDV DNA in only 9/13 blood samples and in 18/23 feather samples. The lower rate of BFDV DNA detection when using NucleoSpin® kits was not a result of inhibition of PCR in most cases. The NucleoSpin® Tissue Kit enabled detection of APV DNA in 29/31 feather samples. Inhibition of DNA amplification was observed when using the DNAzol® Direct kit. Therefore, of the methods evaluated here, Chelex® 100 Resin treatment of samples was the best option for routine testing for BFDV and APV DNA in clinical samples. © 2008 Houghton Trust Ltd.