Quantitative detection of PML-RARα fusion transcript by real-time PCR with a single primer pair

Quantitative detection of minimal residual disease has prognostic value for some leukemias. Acute promyelocytic leukemia (APL) is characterized by the specific PML-RARα fusion gene from t(15;17). Added to three PML-RARα isoforms, alternative spliced forms of PML exons give rise to multiple isoforms even within a single patient. To date, multiple primer pairs for the detection of the various PML-RARα transcripts have been designed, potentially generating some nonspecific amplification products. Here, we established a real-time quantitative PCR (RQ-PCR) strategy with a single primer pair using LightCycler (sp-RQ-PCR), which could simultaneously detect three isoforms with equal specificity and sensitivity as well as alternative spliced forms. Results obtained with sp-RQ-PCR for 39 samples from 15 APL patients and 31 non-APL samples were compared with those with TaqMan™ assay with three primer pairs. In two of the APL samples, PML-RARα was detected in the TM, but not in the sp-RQ-PCR or nested PCR. Furthermore, the sp-RQ-PCR showed no positive results for the 31 non-APL samples, whereas the TM identified 13% (4/31) as positive. Electrophoresis detected some artifacts in the TM, which do not correspond to PML-RARα. We conclude that our sp-RQ-PCR is specific enough to identify various forms of PML-RARα and yields no false-positive results. J. Clin. Lab. Anal. 23:223–230, 2009. © 2009 Wiley-Liss, Inc.