Xie Feifei, Huang Jie, Qu Shoufang, Wu Weili, Jiang Jun, Wang Huagui, Wang Shujuan, Liu Qi, Zhang Senlin, Xu Lizhi, Gao Shangxian, Zhang Yunqing, Zhao Jinyin, Chen Weijun
Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China.
Division of In Vitro Diagnostic Reagents, National Institutes for Food and Drug Control (NIFDC), Beijing 100050, China.
Clin Biochem. 2014 Nov;47(16-17):237-42. doi: 10.1016/j.clinbiochem.2014.08.015. Epub 2014 Sep 2.
The objective of this study is to develop a novel and sensitive method for KRAS codon 12 mutation testing.
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.
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.
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.
本研究的目的是开发一种用于KRAS基因第12密码子突变检测的新型灵敏方法。
我们开发了一种灵敏的一步实时消化阻断TaqMan探针PCR（RTDB-PCR）技术，该技术使用一种耐热核酸内切酶和一种小沟结合剂（MGB）阻断剂来检测KRAS基因第12密码子突变。使用稀释模拟DNA板评估该技术的灵敏度。进行了RTDB-PCR方法，并与其他三种方法进行比较：PCR测序、突变富集PCR测序和突变富集PCR-质谱分析。还使用这四种方法对总共100份福尔马林固定石蜡包埋（FFPE）转移性结直肠癌（mCRC）标本进行了检测。
RTDB-PCR对低至0.01%的突变DNA敏感，显著高于其他方法。在检测的100份FFPE mCRC标本中，根据RTDB-PCR，45份KRAS基因第12密码子突变检测呈阳性，根据突变富集PCR测序和突变富集PCR-质谱分析，44份检测呈阳性，而根据PCR测序，只有26份样本检测呈阳性。
与突变富集PCR测序和突变富集PCR-质谱分析相比，RTDB-PCR更具成本效益，节省时间且易于使用，使其适用于临床低水平KRAS突变的检测。
