The use of innovative tools to facilitate the amplification and quantification of Minor, m-BCR:BCR-ABL1 (p190) rearrangement has brought great advancements in the field of molecular biology. This is particularly significant in the detection of minimal residual disease (MRD) and the optimization of RNA extraction protocols.
Using a specifically designed kit, lab scientists can accurately calculate the number of copies of the rearrangement and the endogenous ABL gene, contributing to the overall understanding of diseases like chronic myeloid leukemia (CML).
The Role and Function of the Detection Kit
The detection kit, designated as IMG-122, employs a blend of oligonucleotides and hydrolysis probes to amplify and quantify the Minor, m-BCR:BCR-ABL1 (p190) rearrangement via real-time PCR. This tool enables the user to calculate both the number of copies of the rearrangement and the number of copies of the endogenous ABL gene. Importantly, this analysis aids in the detection of minimal residual disease (MRD), with sensitivity levels contingent upon the maximum number of copies of the ABL1 reference gene that can be detected.
Technical Specifications and Validations
The kit has been validated using cDNA samples synthesized from the retrotranscription of total RNA, extracted from peripheral blood samples of healthy patients and those diagnosed with chronic myeloid leukemia (CML). This kit specifically detects fusion products and the reference gene (ABL1).
Kit validation was performed using reagents not included in the kit itself, such as M-MLV RT (Moloney murine leukemia virus reverse transcriptase) and TaqMan Environmental Master Mix 2.0 (ThermoFisher Scientific). The detection limit (LOD) of this analysis has been set at 5 total copies of ABL1 and m-BCR-ABL1 (p190), employing a synthetic DNA standard, pIMEGEN plasmid, calibrated with certified reference material from IRMM (ERM®-AD623).
The quantification limit (LOQ) is the minimum quantifiable value, established at 50 total copies. Hence, the most diluted point included in the standard curve corresponds with the LOQ for both the ABL1 reference gene and the m-BCR rearrangement. This kit is suitable for detecting a molecular response (MR) corresponding to a 4 log reduction from the International Randomized Study, defined as:
Detectable disease ≤ 0.01% BCR-ABL1
Undetectable disease in cDNA with ≥ 10,000 copies of the ABL1 reference gene
The kit contains the following reagents required to perform 48 real-time PCR reactions for each of the two targets analyzed in this assay: