摘要
目的建立实时荧光定量PCR(fluorescent quantitative PCR,FQ-PCR)联合Sanger测序法快速检测结直肠癌患者表皮生长因子受体(epidermal growth factor receptor,EGFR)、KRAS和BRAF基因突变的方法。方法根据结直肠癌中EGFR、KRAS和BRAF基因常见突变类型设计引物及其TaqMan探针序列,以提取的86份结直肠癌患者肿瘤组织DNA为模板,进行FQ-PCR扩增,PCR产物经SAP酶和EXOⅠ酶作用后,收集酶切产物,进行测序PCR扩增,扩增产物经乙醇/醋酸钠法纯化后进行测序,测序结果采用Bioedit软件进行分析;检测EGFR、KRAS及BRAF基因突变,并与FQ-PCR法检测KRAS12/13密码子突变进行比较。结果成功建立了FQ-PCR联合Sanger测序法检测结直肠癌患者样本中EGFR、KRAS和BRAF基因突变的方法,各基因PCR扩增曲线均为标准的"S"型荧光扩增曲线,测序峰图清晰、稳定。86份结直肠癌样本中,EGFR基因突变概率2.3%(2/86),均为第21号外显子点突变,突变类型为L858R和L861Q;KRAS基因突变概率33.7%(29/86),其中第12位密码子突变占79.3%(23/29),突变类型为G12D和G12V,第13位密码子突变占10.3%(3/29),突变类型为G13D,第12和13位密码子同时突变1例(3.4%),第61位密码子未检测到突变,第146位密码子仅检测到2例突变,占6.9%(2/29),突变类型均为A146V;BRAF基因未检测到突变。FQ-PCR法检测KRAS12/13密码子突变概率高于FQ-PCR联合Sanger测序法,但差异无统计学意义(P>0.05),两种检测方法的总体符合率为97.7%(84/86)。结论实时FQ-PCR联合Sanger测序法可快速检测结直肠癌患者中EGFR、KRAS和BRAF基因突变,检测方法稳定、可靠。
Objective To develop a real-time fluorescent quantitative PCR（FQ-PCR）combined with Sanger sequencing method for determination of epidermal growth factor receptor（EGFR）,KRAS and BRAF gene mutations in colorectal cancer. Methods Primers and TaqMan probes were designed based on common mutations of EGFR,KRAS and BRAF gene in colorectal cancer for FQ-PCR amplification using the tumor tissue DNAs of 86 patients with colorectal cancer as templates. The PCR products were digested with SAP and EXOⅠ and purified by ethanol / sodium acetate method,then sequenced. The sequencing result was analyzed by Bioedit software. The mutations of EGFR, KRAS and BRAF genes were tested, and the results were compared with that of test for KRAS12 / 13 codon mutation by FQ-PCR. Results A method for determination of EGFR,KRAS and BRAF gene mutations in colorectal cancer by FQ-PCR combined with Sanger sequencing was successfully developed. The PCR amplification curves of various genes were in standard＂S＂shape,and the sequencing peaks were clear and stable. The probability of EGFR gene mutation in 86 colorectal cancer specimens was 2. 3%（2 / 86）,both of which were found in exon 21,and expressed as L858 R and L861 Q respectively.However,the probability of KRAS gene mutation was 33. 7%（29 / 86）,of which 79. 3%（23 / 29）in codon 12（G12Dand G12V）,10. 3%（3 / 29）in codon 13（G13D）,and 3. 4% in both codons 12 and 13. No mutations in codon 61 were observed,while those in codon 146 were observed in 2 of 29 specimens（6. 9%）,both of which were expressed as A146 V. No mutations of BRAF gene were found. The probability of KRAS12 / 13 mutation determined by FQ-PCR was insignificantly higher than that by FQ-PCR combined with Sanger sequencing（P 〉 0. 05）,with a total coincidence rate of 97. 7%（84 / 86）. Conclusion Real-time FQ-PCR combined with Sanger sequencing may be used for rapid determination of EGFR,KRAS and BRAF gene mutations in colorectal cancer,which was stable and reliable.
出处
《中国生物制品学杂志》
CAS
CSCD
2014年第8期1086-1091,共6页
Chinese Journal of Biologicals
基金
"艾滋病与病毒性肝炎等重大传染病防治"科技重大专项(2009ZX10004-006)
关键词
荧光定量PCR
结直肠癌
基因突变
测序
Fluorescent quantitative PCR （FQ-PCR）
Colorectal cancer
Gene mutation
Sequencing
