Development and applications of a TaqMan based quantitative real-time PCR for the rapid detection of Pigeon circovirus (PiCV)

Abstract

TaqMan probe based quantitative polymerase reaction (TaqMan qPCR) is a robust and reliable technique for detecting and quantifying target DNA copies. Quantitative molecular diagnosis of genetically diverse single stranded DNA (ssDNA) virus such as Pigeon circovirus (PiCV) can be challenging owing to difficulties in primer binding or low abundance of template DNA copies in clinical specimens. Several methods have been described for the detection of PiCV, being qPCR the most simple and reliable. As far as is known, two qPCR systems described until now are based on SYBR green. This study reports development and validation of a highly sensitive TaqMan qPCR targeted to Rep for the detection of highly diverse PiCV in pigeon samples with excellent reproducibility, specificity, and sensitivity. The limit of detection was determined as low as 2 (two) plasmid copies. Estimations of 100 % specificity and 100 % sensitivity were obtained based on the qPCR results with panel of 60 samples (known PiCV positive, n = 30; known PiCV negative, n = 20; samples positive to Beak and feather disease virus (BFDV), n = 5 and samples positive to canine circovirus, n = 5). Co-efficient of variation (CV) for Ct values ranged between 0.27 % and 0.78 % in the same assay and 1.84–2.87 % in different assays.