液态芯片与PCR—LDR技术对K—ras基因分型初探

The preliminary establishment and application of a new method for evaluating K-ras mutations based fluid chip

目的采用基于液态芯片的K—ras基因分型检测方法分析非小细胞肺癌（NSCLC）组织样本中的K—ras基因突变水平，指导非小细胞肺癌患者个体化靶向治疗和预后评估。方法实验诊断技术研究方法。对液态芯片的K—ras基因分型检测方法进行灵敏度和重复性的评估。选取2011年11月至2012年2月上海市胸科医院的100例非小细胞肺癌患者新鲜组织样本，提取基因组DNA，通过采用PCR-连接酶检测反应（LDR）结合液态芯片技术，对待测样品K—ras基因2号外显子上12、13密码子上8个突变位点进行分析，并通过测序验证结果。结果液相芯片技术检测灵敏度为10％～20％，高于传统测序法的1％。平均CV值〈1％，表现出很好的重复性。从100例NSCLC患者组织样本中共检出5例突变，其中3例为Gly12Val突变，2例为Gly12Asp突变。结论基于液态芯片与PCR—LDR技术的K—ras基因分型检测方法检测通量高、灵敏度高、重复性好，是NSCLC患者个体化治疗及预后判断的K—ras基因突变分析的合理方法。

Objective A new method for detecting K-ras mutations based liquid chip was used to evaluate K-ras mutations associated with non-small cell lung cancer （NSCLC） patients, to direct the personalized treatment and prognosis evaluation. Methods Take the diagnosis technology research methods, the sensitivity and repeatability of the liquid chip K-ras gene mutation detection method were assessed. A total of 100 NSCLC patients from Nov 2011 to Feb 2012 in Shanghai Chest hospital were included in this study, the fresh tumor tissues were collected for DNA extraction. The 2nd exon 12 and 13 codons, containing 8 K-ras mutations occuring in high frequency were amplified by polymerase chain reaction （PCR） , followed by ligation of the PCR products to a series of special probes using ligase detection reaction （ LDR）, then the PCR-LDR products were analyzed by liquid chip platform. Direct sequencing was applied to compare with the detection results. Results The sensitivity of liquid chip technology detection was 10% - 20%, higher than the traditional sequencing method by 1%. Average CV value was 4% -15% and showed good repeatability. 5 K-ras mutations in 100 patients （ 5% ） were detected using multiplex PCR-LDR combined fluid chip methods, including 3 Gly12Val and 2 Glyl2Asp mutations in exon 2. The 5 K-ras mutations were verified accurately by direct sequencing. Conclusions The novel detection method of K-ras mutations based PCR- LDR and fluid chip shows high throughput, high sensitivity, good repeatability and the results are reliable and accurate. This method can be used to accurately identified K-ras mutations for NSCLC patients prior to their targeted therapy with TKIs.