Journal
CLINICAL BIOCHEMISTRY
Volume 47, Issue 16-17, Pages 237-242Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.clinbiochem.2014.08.015
Keywords
KRAS mutation detection; Low-level mutation; Colorectal cancer
Categories
Funding
- Major State Basic Research Development Program of China (973 Program) [2010CB529101]
- Chinese 863 Program [2012AA02A201]
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Objectives: The objective of this study is to develop a novel and sensitive method for KRAS codon 12 mutation testing. Design and methods: We developed a sensitive one-step real-time digestion-and-block TaqMan probe PCR (RTDB-PCR) technique that uses a thermostable endonuclease and a minor groove binder (MGB) blocker to detect KRAS codon 12 mutations. Dilution mimic DNA panels were used to assess the sensitivity of this technique. The RTDB-PCR method was performed and compared with three other methods: PCR sequencing, mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray. A total of 100 formalin-fixed paraffin-embedded (FFPE) metastatic colorectal cancer (mCRC) specimens were also tested by all four methods. Results: The RTDB-PCR was sensitive to as little as 0.01% mutant DNA, significantly higher than other methods. Among the 100 FFPE mCRC specimens examined, 45 tested positive for KRAS codon 12 mutations according to RTDB-PCR, 44 tested positive according to mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, and only 26 samples tested positive according to PCR sequencing. Conclusions: Compared with mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, RTDB-PCR is more cost effective, saves time, and is easier to use, making it suitable for the detection of low-level KRAS mutations in the clinic. (C) 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
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