Establishment of a real-time fluorescent recombinase polymerase amplification (RT-RPA) assay for BCR-ABL fusion gene detection

This study aims to establish a rapid and highly sensitive real-time fluorescence recombinase polymerase amplification (RT-RPA) method for the qualitative and quantitative analysis of BCR-ABL fusion genes. The method is based on the design of primers and probes for the BCR and ABL (ABL1/ABL2) gene breakpoints, and takes advantage of the high sequence homology of the ABL1/ABL2 binding region to achieve universal reverse primer and probe design for the two fusion genes. Through optimization of primer design, reaction system, and amplification conditions, the study successfully realized specific amplification of BCR-ABL fusion genes, with a detection limit as low as 10 copies/μL and a total reaction time of 20 minutes. In comparison to conventional quantitative real-time (qRT-PCR) methodology, RT-RPA circumventes the need for complex thermal cycling equipment, making it suitable for resource-limited settings. These findings demonstrate that this novel method provides efficient technical support for the early diagnosis and therapeutic monitoring of chronic myeloid leukemia (CML).