A quantitative duplex PCR technique for measuring amounts of cell-associated Marek's disease virus: differences in two populations of lymphoma cells

Abstract

A duplex polymerase chain reaction (PCR) was developed to measure Marek's disease virus (MDV) load in two subpopulations of Marek's disease (MD) lymphoma cells from chickens. PCR primers were designed using the sequence of the MDV-ICP4 gene and the chicken IFNγ gene. Each set of primers was present in the same reaction tube so that the 327 bp ICP4 product and the 420 bp IFNγ product were co-amplified. Two different fluorescent dyes were used to 5′-end label one PCR primer of each pair to distinguish the IFNγ and ICP4 products by colour. The IFNγ PCR product was used as an internal standard enabling comparisons of MDV-ICP4 products between different samples. Neither duplex PCR product was preferentially amplified and both reactions were in their exponential phases when stopped. The products could be distinguished by both size and colour. MD lymphoma cells were taken ex vivo and separated on the basis of expressing a novel host surface antigen recognised by the monoclonal antibody AV37. AV37 + lymphoma cells had greater MDV-loads than AV37 − lymphoma cells. The principles used here should be applicable to any cell phenotype and/or cell-associated DNA virus.