摘要
目的建立改良的酶切富集PCR-焦磷酸测序技术,检测非小细胞肺癌(NSCLC)患者癌组织与血浆中K-ras基因的突变情况。方法配对提取43例NSCLC患者的癌组织和血浆DNA,采用酶切富集PCR-焦磷酸测序技术检测K-ras基因第2外显子12及13密码子点突变,利用混合突变/野生型K-ras基因的细胞系(A549/HCC827)测定该方法的灵敏度。结果 NSCLC患者癌组织中K-ras突变率为9.3%(4/43),血浆中K-ras突变率为7.0%(3/43),均为12密码子突变,突变一致性达75%。混合细胞系检测显示,酶切富集PCR-焦磷酸测序技术的检测灵敏度达0.1%。结论酶切富集PCR-焦磷酸测序技术具有快速、准确、灵敏度高等优点,可用于临床NSCLC患者K-ras突变的筛查。
Objective To establish a modified method, mutant-enriched PCR combined with pyrosequencing, for the detection of K-ras mutations in plasma and cancer tissue of patients with NSCLC. Methods the cancer tissue and matched blood samples were collected and DNA was extracted from 43 NSCLC patients. the mutations of codon 12 and 13 in expressed region 2 of K-ras gene were detected by the established mutant-enriched PCR-pyrosequencing method. Cell line possessing mutant type/wild type of K-ras gene （A549/HCC827） was used to evaluate the sensitivity of the newly developed method. Results Four K-ras mutations were detected from cancer tissue samples （4/43, 9.3%）, and 3 mutations were detected from plasma samples （3/43, 7.0%）, all the mutations were 12 codon mutation. The consistency of K-ras mutation observed in both plasma and its matched tissue was 75%, and the sensitivity of the newly developed method was 0.1%. Conclusion The newly developed mutant-enriched PCR-pyrosequencing method has such advantages as rapid, accurate and high sensitivity. So it may be used for clinical screening ofK-ras gene mutation in NSCLC patients.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2014年第3期202-205,共4页
Medical Journal of Chinese People's Liberation Army
基金
军队医院临床高新技术重大项目(2010gxjs070)~~
