Nath Babu K, Das Shubhagata, Das Tridip, Forwood Jade K, Raidal Shane R
School of Agricultural, Environmental and Veterinary Sciences, Faculty of Science and Health, Charles Sturt University, Wagga, New South Wales 2678, Australia.
School of Dentistry and Medical Sciences, Faculty of Science and Health, Charles Sturt University, Wagga, New South Wales 2678, Australia.
J Virol Methods. 2022 Oct;308:114588. doi: 10.1016/j.jviromet.2022.114588. Epub 2022 Jul 21.
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.
基于TaqMan探针的定量聚合酶反应（TaqMan qPCR）是一种用于检测和定量目标DNA拷贝的强大且可靠的技术。对基因多样的单链DNA（ssDNA）病毒，如鸽圆环病毒（PiCV）进行定量分子诊断可能具有挑战性，因为临床样本中引物结合困难或模板DNA拷贝丰度低。已经描述了几种检测PiCV的方法，其中qPCR是最简单和可靠的。据了解，迄今为止描述的两种qPCR系统基于SYBR green。本研究报告了一种针对Rep的高灵敏度TaqMan qPCR的开发和验证，该方法用于检测鸽样本中高度多样的PiCV，具有出色的重现性、特异性和灵敏度。检测限低至2个质粒拷贝。基于对60个样本（已知PiCV阳性，n = 30；已知PiCV阴性，n = 20；喙羽病病毒（BFDV）阳性样本，n = 5；犬圆环病毒阳性样本，n = 5）的qPCR结果，获得了100%特异性和100%灵敏度的估计值。在同一次检测中，Ct值的变异系数（CV）在0.27%至0.78%之间，在不同检测中为1.84 - 2.87%。
