Song Najie, Zhong Xueting, Li Qingge
Engineering Research Centre of Molecular Diagnostics, Ministry of Education, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
PLoS One. 2014 Apr 25;9(4):e96420. doi: 10.1371/journal.pone.0096420. eCollection 2014.
Detection of somatic mutations for targeted therapy is increasingly used in clinical settings. However, due to the difficulties of detecting rare mutations in excess of wild-type DNA, current methods often lack high sensitivity, require multiple procedural steps, or fail to be quantitative. We developed real-time bidirectional pyrophosphorolysis-activated polymerization (real-time Bi-PAP) that allows quantitative detection of somatic mutations. We applied the method to quantify seven mutations at codons 12 and 13 in KRAS, and 2 mutations (L858R, and T790M) in EGFR in clinical samples. The real-time Bi-PAP could detect 0.01% mutation in the presence of 100 ng template DNA. Of the 34 samples from the colon cancer patients, real-time Bi-PAP detected 14 KRAS mutant samples whereas the traditional real-time allele-specific PCR missed two samples with mutation abundance <1% and DNA sequencing missed nine samples with mutation abundance <10%. The detection results of the two EGFR mutations in 45 non-small cell lung cancer samples further supported the applicability of the real-time Bi-PAP. The real-time Bi-PAP also proved to be more efficient than the real-time allele-specific PCR in the detection of templates prepared from formalin-fixed paraffin-embedded samples. Thus, real-time Bi-PAP can be used for rapid and accurate quantification of somatic mutations. This flexible approach could be widely used for somatic mutation detection in clinical settings.
用于靶向治疗的体细胞突变检测在临床环境中越来越常用。然而，由于在野生型DNA过量的情况下检测罕见突变存在困难，目前的方法往往缺乏高灵敏度，需要多个程序步骤，或者无法进行定量。我们开发了实时双向焦磷酸解激活聚合反应（实时Bi-PAP），可对体细胞突变进行定量检测。我们将该方法应用于定量临床样本中KRAS基因第12和13密码子处的7种突变以及EGFR基因中的2种突变（L858R和T790M）。在存在100 ng模板DNA的情况下，实时Bi-PAP能够检测到0.01%的突变。在34例结肠癌患者的样本中，实时Bi-PAP检测到14例KRAS突变样本，而传统的实时等位基因特异性PCR漏检了2例突变丰度<1%的样本，DNA测序漏检了9例突变丰度<10%的样本。45例非小细胞肺癌样本中两种EGFR突变的检测结果进一步支持了实时Bi-PAP的适用性。在检测从福尔马林固定石蜡包埋样本制备的模板时，实时Bi-PAP也被证明比实时等位基因特异性PCR更有效。因此，实时Bi-PAP可用于快速、准确地定量体细胞突变。这种灵活的方法可广泛应用于临床环境中的体细胞突变检测。
