Feifei Xie1, Jie Huang2, Shoufang Qu2, Weili Wu1, Jun Jiang1, Huagui Wang1, Shujuan Wang1, Qi Liu3, Senlin Zhang3, Lizhi Xu3, Shangxian Gao2, Yunqing Zhang4, Jinyin Zhao5, Weijun Chen6. 1. Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China. 2. Division of In Vitro Diagnostic Reagents, National Institutes for Food and Drug Control (NIFDC), Beijing 100050, China. 3. Beijing Macro & Micro Test Biotech Company, Beijing 101312, China. 4. Department of Dermatology, The Third Affiliated Hospital of Sun Yat-sen University, Guang Zhou, Guang Dong Province 510630, China. 5. Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: zhaojy526@gmail.com. 6. Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: chenwj@genomics.org.cn.
Abstract
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.
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.