A SYBR Green-Based Multiplex RT-qPCR Assay for the Simultaneous Screening of Prognostically Significant Translocations in Acute Lymphoblastic Leukemiafig

Abstract

Background: Acute lymphoblastic leukemia (ALL) is driven by recurrent chromosomal translocations that serve as key diagnostic and prognostic markers. Conventional detection methods are accurate but costly and labor-intensive. SYBR Green-based real-time PCR offers a simpler, probe-free alternative. Objectives: This study aimed to develop and validate a SYBR Green-based multiplex real-time PCR assay for simultaneous detection of four major ALL translocations — ETV6-RUNX1, TCF3-PBX1, KMT2A-AF4, and BCR-ABL1 — to provide an affordable diagnostic tool suitable for resource-limited settings. Methods: To concurrently identify prevalent chromosomal translocations of ALL, a SYBR Green-based real-time multiplex PCR assay was developed. The HPRT gene, characterized by a distinct melting temperature, was amplified separately and served as the internal control. Upon optimization of the multiplex protocol, its performance was validated across a cohort of 30 newly diagnosed ALL patients, with pre-established translocation-positive and translocation-negative status, and 10 healthy donors. Results: The optimized multiplex assay showed robust performance, with all five targets successfully co-amplified in a single master mix containing nine primers. Amplification efficiencies for all targets remained within the acceptable 90–110% range, with specificity verified at 100%. The assay exhibited strong reproducibility, supported by low intra- and inter-assay variation. Conclusions: Our findings confirm that the designated SYBR Green-based multiplex PCR assay is a precise and reliable alternative for the simultaneous detection of major ALL-associated translocations. This method shows promise as a cost-effective, high-performance diagnostic tool. However, to firmly establish its clinical utility and reproducibility, future validation with a larger, multi-center patient cohort is warranted.