Absolute quantitation of Marek's disease virus and Herpesvirus of turkeys in chicken lymphocyte, feather tip and dust samples using real-time PCR.
The further development of Taqman quantitative real-time PCR (qPCR) assays for the absolute quantitation of Marek's disease virus serotype 1 (MDV1) and Herpesvirus of turkeys (HVT) viruses is described and the sensitivity and reproducibility of each assay reported. Using plasmid DNA copies, the lower limit of detection was determined to be 5 copies for the MDV1 assay and 75 copies for the HVT assay. Both assays were found to be highly reproducible for Ct values and calculated copy numbers with mean intra- and inter-assay coefficients of variation being less than 5% for Ct and 20% for calculated copy number. The genome copy number of MDV1 and HVT viruses was quantified in PBL and feather tips from experimentally infected chickens, and field poultry dust samples. Parallelism was demonstrated between the plasmid-based standard curves, and standard curves derived from infected spleen material containing both viral and host DNA, allowing the latter to be used for absolute quantification. These methods should prove useful for the reliable differentiation and absolute quantitation of MDV1 and HVT viruses in a wide range of samples.
Islam
Aminul
A
Centre for Animal Health and Welfare, School of Rural Science and Agriculture, University of New England, Armidale, NSW 2351, Australia. aislam@une.edu.au
Cheetham
Brian F
BF
Mahony
Timothy J
TJ
Young
Peter L
PL
Walkden-Brown
Stephen W
SW
D000704:Analysis of Variance; D000818:Animals; D002645:Chickens; D004279:DNA, Viral / Q000032:analysis*; D004391:Dust; D004783:Environmental Microbiology; D005241:Feathers / Q000821:virology*; D005260:Female; D026002:Herpesvirus 1, Meleagrid / Q000235:genetics / Q000302:isolation & purification*; D008381:Herpesvirus 2, Gallid / Q000235:genetics / Q000302:isolation & purification*; D008214:Lymphocytes / Q000821:virology*; D008380:Marek Disease / Q000821:virology; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013154:Spleen / Q000821:virology
D004279:DNA, Viral; D004391:Dust