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Quantitative denaturing high performance liquid chromatography (Q-dHPLC) detection of APC long DNA in faeces from patients with colorectal cancer
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Murugan Kalimutho
Published/Copyright:
May 27, 2010
Abstract
Background: Colorectal cancer (CRC) is the second leading cause of cancer-related deaths. However, prevention is possible by early detection. In the present work, we have demonstrated and validated a novel quantitative method based on a DNA integrity assay and mutation in faeces of CRC patients using denaturing high performance liquid chromatography (dHPLC).
Methods: Faecal DNA (fDNA) was isolated from 28 CRC, 96 healthy and 61 patients with adenomas. Adenomatosis polyposis coli (APC)-Long-DNA and its mutations were analysed using dHPLC and the Sanger sequencing method. The diagnostic performance was assessed using receiver operating characteristic curve analysis.
Results: We detected APC-Long-DNA in 21/28 CRC subjects with a sensitivity of 75% and specificity of 91.7%. A cut-off ratio of 0.2317 was used for APC/β-actin. The Q-dHPLC detection limit was 0.02 ng/injection. The average initial fDNA presence based on a single gene of β-actin was 26.12±13.39 ng/mL for healthy, and 49.61±46.28 ng/mL for CRC subjects, with a sensitivity of 71.4% and a specificity of 84.4% at a cut-off value >29 ng/mL. We also detected a novel mutation at codon 1576 Lys/Glu using dHPLC.
Conclusions: This study highlights a novel application of Q-dHPLC in the DNA integrity assay, which demonstrates high performance, good reproducibility, and low cost for the CRC detection using faeces. Further studies in a larger population are needed to confirm these results.
Clin Chem Lab Med 2010;48:1303–11.
Keywords: adenomatosis polyposis coli (APC); colorectal cancer; denaturing HPLC; faecal DNA; faeces; quantitative
Received: 2009-12-10
Accepted: 2010;-3-15
Published Online: 2010-05-27
Published in Print: 2010-09-01
©2010 by Walter de Gruyter Berlin New York
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Keywords for this article
adenomatosis polyposis coli (APC);
colorectal cancer;
denaturing HPLC;
faecal DNA;
faeces;
quantitative
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- Genetics and Molecular Diagnostics
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- Transfer of myxovirus-protein-A mRNA assay for interferon-β bioactivity measurement in multiple sclerosis patients to routine laboratory practice. A 4-year experience
- General Clinical Chemistry and Laboratory Medicine
- Transferrin receptor expression on reticulocytes as a marker of iron status in dialyzed patients
- Quartz crystal microbalance-based biosensor for the detection of α-thalassemia 1 (SEA deletion)
- Retrospective analysis of 88,429 serum and urine glucose EQA results obtained from professional laboratories and medical offices participating in surveys organized by three European EQA centers between 1996 and 2007
- Liquid phase immunoassays utilizing magnetic markers and SQUID magnetometer
- L-Phenylalanine concentration in blood of phenylketonuria patients: a modified enzyme colorimetric assay compared with amino acid analysis, tandem mass spectrometry, and HPLC methods
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