Abstract
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 Gly12 Asp 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.(Chin J Lab Med,2013,36:704-707) Key words: Microfluidic analytical techniques; Polymerase chain reaction; Ligase chain reaction; Genes; ras; Genotype; Carcinoma; non-small-cell lung
Published Version
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