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

摘要

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.