摘要
目的建立基于高通量液相芯片技术、可同时检测p53、p16、视网膜母细胞瘤（Rb）和表皮生长因子受体（EGFR）基因热点突变的方法，并探讨其在非小细胞肺癌（NSCLC）分子诊断中的意义。方法针对p53、p16、Rb和EGFR基因热点突变位点，分别设计正常序列和突变序列探针，将探针固定于标记不同比例荧光物的微球上。分别提取65例NSCLC患者（Ⅰ期23例，Ⅱ期20例，Ⅲ期22例）癌组织标本和其中20例患者癌旁正常组织标本基因组DNA，PCR扩增p53、p16、Rb和EGFR基因。将PCR产物与含寡核苷酸探针的微球混合后用Luminex100多功能流式点阵仪进行流式荧光检测分析。结果单独检测p53、p16、Rb和EGFR基因突变时，NSCLC标本突变检出率分别为53．8％（35／65）、20．O％（13／65）、7．7％（5／65）和35．4％（23／65）；癌旁正常组织标本为5．0％（1／20）、5．0％（1／20）、0和0。而四基因联合检测，敏感性为81．5％（53／65），特异性为90．0％（18／20），准确性为83．5％（71／85）；Ⅰ、Ⅱ、Ⅲ期NSCLC标本突变检出率分别为78．3％（18／23）、80．0％（16／20）和86．4％（19／22）。结论成功建立了具有较高敏感性和特异性、可同时检测临床NSCLC标本多基因突变的液相芯片检测方法，该方法有助于提高NSCLC分子诊断的效率，并可能有助于NSCLC的早期诊断。
Objective To construct a high-throughput suspension microarray for detecting the hotspot gene mutations of p53, p16, retinobiastoma （Rb） and epidermal growth factor receptor （EGFR） and to investigate the significance of this multimarker panel in molecular diagnosis of non-small-cell lung cancer （NSCLC）. Methods The specific probes of normal or mutated sequences targeting the hotspot mutation sites of p53, p16, Rb and EGFR were designed and immobilized to earboxylated Luminex microspheres （micro-beads）. Genomic DNA was extracted from 65 specimens of cancer tissues and 20 adjacent normal lung tissues, p53, p16, Rb and EGFR genes were amplified by PCR, hybridized with the specific probes on the beads and measured using Luminex 100. Results The single gene mutations of p53, p16, Rb or EGFR inNSCLC specimens were 53.8% （35/65）, 20. 0% （13/65）, 7.7% （5/65） or 35.4% （23/65） respectively. The para-tumor normal tissue specimens were 5.0% （1/20） , 5.0% （1/20） , 0 and 0 respectively. For combined detections of four genes, the sensitivity, specificity and accuracy were 81.5% （53/65） , 90.0% （18/20） and 83.5% （71/85 ）respectively. The mutation rates of this panel in stage Ⅰ , stage Ⅱ and stage Ⅲ were 78. 3% （18/23）, 80. 0% （16/20） and 86.4% （19/22） respectively. Conclusions A high-throughput suspension microarray with a higher specificity and sensitivity has been built. It may be used to simultaneously detect the gene mutations of p53, p16, Rb or EGFR in NSCLC specimens. This suspension microarray is helpful to improve the sensitivity of molecular diagnosis of NSCLC and guide the molecular targeting therapy of NSCLC.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2009年第48期3393-3396,共4页
National Medical Journal of China
基金
国家“十五”重大科技攻关项目（2001BA703811）
江苏省社会发展项目（Bs2006005）
关键词
癌
非小细胞肺
芯片分析技术
突变
分子诊断技术
Carcinoma, non-small-cell lung
Microarray analytical procedure
Mutation
Molecular diagnostic technique
