Optimized digital droplet PCR for BCR-ABL.
Authors of this article are:
Maier J, Lange T, Cross M, Wildenberger K, Niederwieser D, Franke GN.
A summary of the article is shown below:
Quantitative real-time-PCR methods are commonly used to monitor BCR-ABL transcript levels in patients with chronic myelogenous leukemia. However, standard techniques involve separate measurements of target and reference DNAs, require standard curves, and are susceptible to PCR inhibition. An optimized duplex droplet digital (dd)PCR should provide absolute quantification without the need for standard curves. The combination of high sensitivity and low background is particularly important for reliable monitoring of minimal residual disease. 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.
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