Chen Tai-Long, Chang John Wen-Cheng, Wang Chih-Liang, Yang Cheng-Ta, Wang Mei-Chia, Chiou Chiuan-Chian
Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan.
Division of Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan; School of Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
Anal Biochem. 2016 Nov 15;513:61-67. doi: 10.1016/j.ab.2016.09.002. Epub 2016 Sep 4.
EGFR exon 19 deletion is an important indicator for tyrosine kinase inhibitor treatment in non-small cell lung cancer. However, detection of exon 19 deletions faces a challenge: there are more than 30 types of mutations reported at the hotspot. Moreover, considering the application in body fluid samples, assays with high sensitivity and specificity are necessary for the detection of rare mutant alleles. Here, we describe a single tube reaction which could detect at least 29 types of exon 19 deletions with only an unlabeled peptide nucleic acid (PNA) clamp and a pair of DNA probes. The PNA clamp was used to inhibit amplification of wild-type templates; and the DNA probes were used to generate melting peaks for multiple types of mutations. Under optimal condition, the assay was able to detect as low as 0.01% mutant DNA in wild-type background, and had a limit of detection of 10 pg genomic DNA. Feasibility of the assay was tested in body fluid samples from lung cancer patients. The assay detected 100% and 60% of deletions in pleural effusions and plasma, respectively. We believe the present assay can be used in the clinical laboratories and has potential to be adapted for a microfluidic device.
表皮生长因子受体（EGFR）第19外显子缺失是非小细胞肺癌中酪氨酸激酶抑制剂治疗的重要指标。然而，检测第19外显子缺失面临一项挑战：在该热点区域已报道有30多种类型的突变。此外，考虑到在体液样本中的应用，对于罕见突变等位基因的检测，需要高灵敏度和特异性的检测方法。在此，我们描述了一种单管反应，该反应仅使用一个未标记的肽核酸（PNA）夹和一对DNA探针就能检测至少29种类型的第19外显子缺失。PNA夹用于抑制野生型模板的扩增；DNA探针用于产生多种类型突变的熔解峰。在最佳条件下，该检测方法能够在野生型背景中检测低至0.01%的突变DNA，检测限为10 pg基因组DNA。在肺癌患者的体液样本中测试了该检测方法的可行性。该检测方法分别在胸腔积液和血浆中检测到100%和60%的缺失。我们相信目前的检测方法可用于临床实验室，并有可能适用于微流控设备。
