Development of multiplex real-time polymerase chain reaction assay for the detection of leukemia-associated translocation genes

A BSTRACT Objective: Leukemia diagnosis involves diverse methods, including cytomorphology, flow cytometry, cytogenetics, and molecular diagnostics. While cytogenetics plays an indispensable role, it is limited by a high false-negative rate. In contrast, molecular diagnostics, such as real-time polymerase chain reaction (PCR), have become practical and widely used in clinical settings. This study aims to establish a multiplex real-time PCR system to detect critical leukemia-related chromosomal translocations, encompassing seven genes and 12 mutation types. Materials and Methods: Bioinformatics tools were employed for nucleic acid sequence analysis and primer design using reference sequences from NCBI and COSMIC databases. Primers were designed to flank frequent breakpoints. Performance testing involved gradient temperature PCR to optimize conditions and establish detection limits. A multiplex system was configured into six reaction tubes using a Rotor-Gene Q analyzer. Specific sequence plasmids were synthesized as quality control standards.