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

A duplex polymerase chain reaction (PCR) was developed to measure Marek's d isease virus (MDV) load in two subpopulations of Marek's disease (MD) lymph oma cells from chickens. PCR primers were designed using the sequence of th e MDV-ICP4 gene and the chicken IFN gamma gene. Each set of primers was pre sent in the same reaction tube so that the 327 bp ICP4 product and the 420 bp IFN gamma product were co-amplified. Two different fluorescent dyes were used to 5'-end label one PCR primer of each pair to distinguish the IFN ga mma and ICP4 products by colour. The IFN gamma PCR product was used as an i nternal standard enabling comparisons of MDV-ICP4 products between differen t samples. Neither duplex PCR product was preferentially amplified and both reactions were in their exponential phases when stopped. The products coul d 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 gre ater MDV-loads than AV37(-) lymphoma cells. The principles used here should be applicable to any cell phenotype and/or cell-associated DNA virus. (C) 1999 Elsevier Science B.V. All rights reserved.