Towards Routine Molecular Diagnostics: Multiplex SYBR Green Real-time PCR for Rapid Molecular Screening of Recurrent Translocations in Acute Myeloid Leukemia

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AuthorFarzaneh Tavakolien
AuthorHamide Rahmani Serajien
AuthorElham Roshandelen
AuthorFatemeh Karimpouren
AuthorHoda Hasheminasaben
AuthorNader Vazifeh Shiranen
AuthorMozhdeh Mohammadianen
AuthorMohammad Hossein Mohammadien
OrcidFarzaneh Tavakoli [0000-0002-5898-591X]en
OrcidHamide Rahmani Seraji [0000-0001-6871-1747]en
OrcidElham Roshandel [0000-0002-3698-4342]en
OrcidFatemeh Karimpour [0000-0001-8594-2950]en
OrcidHoda Hasheminasab [0009-0002-0149-3516]en
OrcidNader Vazifeh Shiran [0000-0003-0438-4438]en
OrcidMozhdeh Mohammadian [0000-0003-4957-8797]en
OrcidMohammad Hossein Mohammadi [0000-0002-8697-1371]en
Issued Date2025-12-31en
AbstractBackground: Accurate detection of recurrent chromosomal translocations is essential for risk stratification and therapeutic decision-making in acute myeloid leukemia (AML). Although innovative techniques are becoming increasingly accessible, further improvements in conventional assays remain valuable. Although numerous translocations have been identified, the most frequent ones with prognostic value are limited. Objectives: In the present study, we developed a screening assay applicable in multiplex formats, using the same reagents, instruments, and conditions for screening the three most frequent translocations: RUNX1-RUNX1T1, PML-RARA, and CBFB-MYH11. Methods: A SYBR-Green real-time multiplex PCR was developed to simultaneously screen four frequent translocation variants in AML, including RUNX1-RUNX1T1, PML-RARα, bcr1, PML-RARα, bcr3, and CBFB-MYH11 type A. The ABL gene served as a reference and was amplified in separate reactions. Fifty newly diagnosed AML patients were chosen based on the availability of sufficient RNA quantity and quality, along with confirmed cytogenetic or molecular diagnosis according to the World Health Organization (WHO) 2022 classification criteria. The assay was validated by comparing the results with those obtained from singleplex real-time PCR in AML patients. Results: All four targets could successfully amplify in the master mix, containing seven primers, with no interference in their respective melting curves. Specificity was 100% for all targets. The intra-assay and inter-assay coefficient of variation (CV) for cycle threshold (Ct) values ranged from 0.68% to 1.12% and 0.22% to 1.02%, respectively. The validation assessments reported excellent consistency between singleplex and multiplex real-time PCR. However, the assay does not detect all CBFB-MYH11 variants or the bcr2 isoform of PML-RARα because of overlapping melting temperature (Tm). Despite these limitations, this multiplex assay could be considered a valuable first-line screening tool. Conclusions: This method provides a simple, accurate, cost-effective, and informative tool for screening these translocations in AML. Since the primers were previously validated in the singleplex method, this assay is simple and does not require particular materials or instruments. This novel assay may improve patient stratification, support more precise therapeutic planning, and represent an important advancement in molecular diagnostics.en
DOIhttps://doi.org/10.5812/ijcm-166704en
KeywordAcute Myeloid Leukemiaen
KeywordMultiplex Real-time Polymerase Chain Reactionen
KeywordMolecular Diagnostic Techniquesen
KeywordGenetic Testingen
PublisherBrieflandsen
TitleTowards Routine Molecular Diagnostics: Multiplex SYBR Green Real-time PCR for Rapid Molecular Screening of Recurrent Translocations in Acute Myeloid Leukemiaen
TypeResearch Articleen

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