Differential amplification and quantification of Marek’s Disease viruses using real-time polymerase chain reaction

Islam, Aminul, Harrison, Bruce, Cheetham, Brian F., Mahony, Timothy J., Young, Peter L. and Walkden-Brown, Stephen W. (2004) Differential amplification and quantification of Marek’s Disease viruses using real-time polymerase chain reaction. Journal of Virological Methods, 119 2: 103-113.


Author Islam, Aminul
Harrison, Bruce
Cheetham, Brian F.
Mahony, Timothy J.
Young, Peter L.
Walkden-Brown, Stephen W.
Title Differential amplification and quantification of Marek’s Disease viruses using real-time polymerase chain reaction
Journal name Journal of Virological Methods   Check publisher's open access policy
ISSN 1879-0984
Publication date 2004-05-06
Sub-type Article (original research)
DOI 10.1016/j.jviromet.2004.03.006
Volume 119
Issue 2
Start page 103
End page 113
Total pages 11
Place of publication Amsterdam, Netherlands
Publisher Elsevier
Formatted abstract Quantitative real-time PCR (qPCR) assays for the three serotypes of Marek's disease virus (MDV) have been developed. An internal control qPCR assay that detects chicken α2 (VI) collagen gene was also developed to allow quantitation of MDV. To reduce costs and time, the assays for MDV1 and the internal control were combined into a duplex assay. The sensitivity, specificity, precision, and reproducibility of each assay are reported. The MDV qPCR assays were specific to their target gene when compared using Australian field and vaccine strains of MDV and 10-100-fold more sensitive than standard PCR. Using DNA from infected spleen tissue, the lower limit of detection of total DNA (viral and host combined) was 0.025 ng for the MDV1 and collagen assays, and 0.25 ng for the HVT and MDV2 assays. All assays were found to be highly reproducible for Ct values, but less so for calculated concentrations. MDV1 and HVT were quantitated in spleen tissue of twenty experimentally infected chickens 7-35 days after infection. The relative abundance of MDV1 exhibited a clear peak at day 14 post-infection, whereas HVT displayed an increasing trend over the 35 days post-infection. The duplex assay was optimized such that it was able to accurately quantitate MDV1 in samples of very high, medium, and very low relative abundance of MDV1. These qPCR assays will be useful for reliable differentiation and quantitation of MDV for a range of research and industry applications.
Keyword Real-time PCR
Quantitation
Marek's disease virus serotypes
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: Queensland Alliance for Agriculture and Food Innovation
 
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Created: Mon, 10 Oct 2011, 12:52:56 EST by Dr Timothy Mahony on behalf of Qld Alliance for Agriculture and Food Innovation