Optimized digital droplet PCR for BCR-ABL

We report here primer probe set testing ad step by step optimization of a duplex ddPCR for BCR-ABL/ABL. The optimization of ddPCR parameters increased ABL and BCR-ABL fluorescence signals by 2 and 5 fold, respectively, and enhanced the resolution between positive and negative drops. The optimized procedure generates a background false positive rate of 5% of samples and reliably detects BCR-ABL/ABL down to 1/100000 (CV<10%), with a single BCR-ABL copy being detected in 54% of reactions performed in duplicate from an MR5 sample (limit of detection = 1). Assay of duplicates resulted in a detection rate of 100% and 92% for MR4 and MR4.5, respectively. Detection of MR4.5 was increased to 100% by analyzing quadruplicates. Selection of an optimal primer/probe combination and stepwise optimization of the ddPCR conditions has yielded a robust low background duplex ddPCR procedure for BCR-ABL/ABL.
Source: The Journal of Molecular Diagnostics - Category: Pathology Source Type: research