TY - JOUR
T1 - Development and applications of a TaqMan based quantitative real-time PCR for the rapid detection of Pigeon circovirus (PiCV)
AU - Nath, Babu K.
AU - Das, Shubhagata
AU - Das, Tridip
AU - Forwood, Jade K.
AU - Raidal, Shane R.
N1 - Funding Information:
The authors are grateful to the referring veterinary clinicians who recognised the history and clinical condition as a unique opportunity to thoroughly investigate and for collecting and sending samples for analysis.
Publisher Copyright:
© 2022 Elsevier B.V.
Copyright © 2022 Elsevier B.V. All rights reserved.
PY - 2022/10
Y1 - 2022/10
N2 - 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.
AB - 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.
KW - pigeon circovirus
KW - Real-time PCR
KW - TaqMan
KW - Validation
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U2 - 10.1016/j.jviromet.2022.114588
DO - 10.1016/j.jviromet.2022.114588
M3 - Article
C2 - 35870671
AN - SCOPUS:85135125292
SN - 0166-0934
VL - 308
SP - 114588
JO - Journal of Virological Methods
JF - Journal of Virological Methods
M1 - 114588
ER -