Quantitative detection of t(14;18)-positive cells by real-time quantitative PCR using fluorogenic probes

To detect t(14;18)-positive cells present in human lymphoma tissue, bone ma rrow aspirates and peripheral blood mononuclear cells (PBMNC), we have esta blished an automated, real-time quantitative PCR using double-labeled fluor ogenic probes. In relation to t(14;18)-positive genomic DNA or a cloned t(1 4;18)-DNA fragment, highly reproducible results can be obtained with initia l copy numbers between 10 to 10(5). The detection of single copies has been verified by the stochastic multiple-tube approach. PBM-NC cells obtained d uring clinical follow-up of patients with follicular lymphoma were analyzed by the one-step, real-time quantitative PCR and a two-step, semi-nested PC R combined with a limiting dilution assay. The quantitative results obtaine d by both assays correlate very well. Real-time quantitative PCR has severa l advantages: (i) it involves less critical pipetting steps, (ii) is less t ime-consuming and (iii) UTP, in combination with uracil-N-glycosylase, can be used to control carryover contamination. The higher specificity is due t o optimized primer annealing conditions and MgCl2 concentration and the use of AmpliTaq Gold (TM). The sensitivity is at least as high as by the two-s tep PCR. Real-time quantitative PCR will be very helpful in large epidemiol ogical studies and in research for molecular staging and the detection of m inimal residual tumor cells, including the analysis of blood stem-cell prep arations to be used for transplantation after myelo-ablative therapy.