Simultaneous detection of 45 fusion genes in leukemia by dual-color fluorescence real-time PCR.

ABSTRACT

INTRODUCTION: Detection of recurrent genetic abnormalities is of great significance for a refined diagnosis and assessment of prognosis in leukemia. Conventional nested reverse transcription PCR is labor intensive and time-consuming. METHODS: We have developed a novel dual-color TaqMan probe-based real-time PCR method for the simultaneous screening of 45 fusion transcripts in 12 parallel reactions. The method was tested and validated with cell lines carrying known fusion transcripts and patient samples. RESULTS: A multiplex real-time PCR method was successfully developed for rapid detection of 45 fusion genes and validated for 15 of the more commonly detected fusion genes. Intra-assay reproducibility assessed for the most frequent rearrangements ranged from 0.41% to 0.74% for the coefficient of variation (CV) of cycle threshold (Ct) and the interassay reproducibility ranged from 1.62% to 2.83% in five separate experiments. The lowest detection limit for the translocations tested ranged between 1 16 000 and 1 32 000. Validation of the method with 213 patient samples showed 100% specificity and excellent consistence with conventional nested RT-PCR. CONCLUSION: Overall, we believe that this method is easily applicable, cost-effective, and clinically useful for a rapid screening of fusion genes in the initial diagnostic phase of leukemia. Its use can also be extended to the monitoring of minimal residual disease.