| 摘要: |
| 目的:应用肽核酸钳制PCR技术检测胰腺癌Kras基因突变,探讨其诊断价值。方法:结合特异性肽核酸(针对野生型序列)与实时定量PCR技术建立一步Kras基因检测法,与传统限制性片段长度多态性PCR法检测结果进行比较,评价新方法的诊断价值。结果:肽核酸钳制PCR法可同时对胰腺癌标本中Kras基因的第12、13密码子突变情况进行检测,操作简便；可在105倍的野生型Kras基因背景下检测出突变,灵敏度为0.001%,可检测突变基因最低限为102拷贝；该方法可同时进行较大规模的样品检测,单次检测时间仅需1 h。而传统方法可检测的突变基因最低限为103拷贝,检测灵敏度为0.01%；需花费约2 d完成同样数量的样本检测。结论:肽核酸钳制实时定量PCR法较限制性片段长度多态性PCR法有明显优势,适用于胰腺癌大规模临床样本的Kras基因突变检测,可作为胰腺癌筛查的有效手段。 |
| 关键词: 胰腺肿瘤 肽核酸 K ras基因 实时定量PCR |
| DOI：10.3724/SP.J.1008.2009.0762 |
| 投稿时间：2009-02-10修订日期：2009-04-24 |
| 基金项目: |
|
| Peptide nucleic acid mediated one step PCR assay in detection of K ras mutation |
| LIN Han,LI Zhaoshen*,GAO Jun,GONG Yanfang,JIN Jing |
| (Department of Gastroenterology,Changhai Hospital,Second Military Medical University,Shanghai 200433,China) |
| Abstract: |
| Objective:To establish a peptide nucleic acidmediated onestep PCR assay for detecting Kras mutation,and to evaluate its diagnostic value.Methods: We developed a onestep polymerase chain reaction (PCR) approach with melting curve analysis using wildtype specific peptide nucleic acid (PNA) and fluorescent dye SYBR Green Ⅰ to determine the genotypes in codon 12 and 13 of Kras oncogene. The result of our method was compared with that of restriction fragment length polymorphism (RFLP) analysis.Results: Our method simultaneously examined condon 12 and 13 of Kras oncogene,and was easy to perform.The sensitivity of our method was 0.001% when in a 105fold excess of wildtype Kras DNA.The detection limit was 102 copies.Our method could be used for large sample test,with the time of a single test being 1 hour.The limit of traditional method was 103copies and the sensitivity was 0.01%.The testing time was about 2 days for samples which needed only 1 hour using our method.Conclusion: Our method has obvious advantage over RFLP analysis; it is suitable for detecting Kras mutation in large samples and can be used as an effective method for early diagnosis of pancreatic cancer. |
| Key words: pancreatic neoplasms peptide nucleic acid K ras gene real time quantitative PCR |
