A DNA Real-Time Quantitative PCR Method Suitable for Routine Monitoring of Low Levels of Minimal Residual Disease in Chronic Myeloid Leukemia

Paul Bartley, Susan Latham, Brad Budgen, David Ross, Elizabeth Hughes, Susan Branford, Deborah White, Timothy Hughes, Alexander Morley

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    16 Citations (Scopus)

    Abstract

    The BCR-ABL1 sequence has advantages over the BCR-ABL1 transcript as a molecular marker in chronic myeloid leukemia and has been used in research studies. We developed a DNA real-time quantitative PCR (qPCR) method for quantification of BCR-ABL1 sequences, which is also potentially suitable for routine use. The BCR-ABL1 breakpoint was sequenced after isolation by nested short-range PCR of DNA from blood, marrow, and cells on slides, obtained either at diagnosis or during treatment, or from artificial mixtures. PCR primers were chosen from a library of presynthesized and pretested BCR (n = 19) and ABL1 (n = 568) primers. BCR-ABL1 sequences were quantified relative to BCR sequences in 521 assays on 266 samples from 92 patients. For minimal residual disease detectable by DNA qPCR and RT-qPCR, DNA qPCR gave similar minimal residual disease results as RT-qPCR but had better precision at low minimal residual disease levels. The limit of detection of DNA qPCR depended on the amount of DNA assayed, being 10-5.8 when 5 μg was assayed and 10-7.0 when 80 μg was assayed. DNA qPCR may be useful and practical for monitoring the increasing number of patients with minimal residual disease around or below the limit of detection of RT-qPCR as the assay itself is simple and the up-front costs will be amortized if sequential assays are performed.

    Original languageEnglish
    Pages (from-to)185-192
    Number of pages8
    JournalJournal of Molecular Diagnostics
    Volume17
    Issue number2
    DOIs
    Publication statusPublished - 1 Mar 2015

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