A Commercial Real-Time PCR Kit Provides Greater Sensitivity than Direct Sequencing to Detect KRAS Mutations A Morphology-Based Approach in Colorectal Carcinoma
A Commercial Real-Time PCR Kit Provides Greater Sensitivity than Direct Sequencing to Detect KRAS Mutations A Morphology-Based Approach in Colorectal Carcinoma
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DOI：
10.2353/jmoldx.2010.090139
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发表时间：
2010-05-01
影响因子：
4.1
通讯作者：
Lopez-Rios, Fernando
中科院分区：
文献类型：
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作者：
Angulo, Barbara;Garcia-Garcia, Elena;Lopez-Rios, Fernando
KRAS突变检测已成为癌症患者治疗的标准程序。KRAS检测最常用的方法是对PCR产物进行直接测序。商业实时定量聚合酶链式反应试剂盒的开发提供了一个有用的替代方案，因为从理论上讲，它们比直接测序更敏感，而且它们避免了聚合酶链式反应后处理。我们介绍了我们作为研究KRAS突变的参考中心的经验，比较了直接测序和商业实时定量聚合酶链式反应试剂盒的使用，以及在临床实践中确定这两种程序的敏感性。Therascreen K-RAS突变试剂盒在170个肿瘤中发现了75个突变(44%)。3例K-RAS基因突变试剂盒检测为阳性，直接测序为阴性。然后，我们比较了试剂盒和直接测序的敏感性，使用了74个突变的肿瘤。该试剂盒能够在13.5%的肿瘤总DNA稀释1%的情况下检测到突变的存在，在84%的肿瘤中，在5%的稀释时发现KRAS突变。当突变DNA占总DNA的10%(20/74)(27%)时，测序能够检测到KRAS突变。当突变DNA占总DNA的30%时，测序可检测到56/74(76%)的突变。(摩尔诊断杂志2010年，12：292-299；doi：10.2353/jmoldx.2010.090139)
KRAS mutation testing has become a standard procedure in the management of patients with carcinomas. The most frequently used method for KRAS testing is direct sequencing of PCR products. The development of commercial real-time quantitative PCR kits offers a useful alternative since they are in theory much more sensitive than direct sequencing and they avoid post-PCR handling. We present our experience as a reference center for the study of KRAS mutations, comparing direct sequencing and the use of a commercial real-time quantitative PCR kit, as well as determining the sensitivity of both procedures in clinical practice. The TheraScreen K-RAS Mutation Kit identified mutations in 75 (44%) of the 170 tumors. Three cases were tested positive using TheraScreen K-RAS Mutation Kit and negative by direct sequencing. We then compared the sensitivity of the kit and that of direct sequencing using 74 mutant tumors. The kit was able to detect the presence of a mutation in a 1% dilution of the total DNA in 13.5% of the tumors and, in 84%, KRAS mutation was identified at a dilution of 5%. Sequencing was able to detect KRAS mutations when the mutant DNA represented 10% of the total DNA in 20/74 (27%) of the tumors. When the mutant DNA represented 30% of the total DNA, sequencing could detect mutations in 56/74 (76%). (J Mol Diagn 2010, 12:292-299; DOI: 10.2353/jmoldx.2010.090139)