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