Abstract

An intercalating dye-based qualitative and quantitative real time PCR assay was developed to detect and estimate the AML1/ETO fusion transcript in patients suffering from Acute myeloid leukaemia. Quantitation standard was prepared by molecular cloning of the fusion transcript in a multicopy cloning vector followed by its propagation in Escherichia coli. Nucleotide sequencing confirmed the AML1/ETO fusion point within the cloned insert and further, its serial dilution resulted in a predicted increase in cycle threshold value which was used to generate a standard curve. Secondary calibration of the standard with an CE-IVD approved, quantified control DNA allowed quantitative detection of AML1/ETO and ABL copy numbers of an AML1/ETO testing panel of samples and the percentage of the AML1/ETO was successfully ascertained. Qualitative detection data of the fusion transcripts from the panel indicated 100% concordance of data when compared with a published protocol that used hydrolysis probes (TaqMan) for detecting the AML1/ETO fusion transcript percentage in clinical samples. The study indicated the potential of using Evagreen as an intercalating dye for qualitative and quantitative detection of AML1/ETO and ABL transcripts for estimating Minimal Residual Disease (MRD) in AML patients.

Keywords:Evagreen; AML1/ETO; RT-PCR; Quantitation; Cloning

Keywords:E-RT-PCR: Evagreen Real Time PCR; CT: Cycle Threshold