肽核酸钳制-PCR/K-ras突变检测方法在结直肠癌组织中的诊断应用
Detection of K-ras mutation by PNA-PCR/K-ras method in diagnosis of colorectal cancer tissues
李泉江 1胡佳佳 1金晶 1吴洪玉 1满晓华 1朱玲 2高军 1李兆申1
作者信息
- 1. 第二军医大学长海医院消化内科,上海200433
- 2. 上海体育学院中国乒乓球学院,上海200433
- 折叠
摘要
目的 确定本实验室建立的肽核酸钳制(PNA)-PCR/K-ras突变检测方法的阳性判断标准,并评价其对结直肠癌组织的诊断价值.方法 将含K-ras基因12密码子突变的质粒和野生质粒以不同比例(突变/总体:0,1/3 200,1/1 600,1/800,1/400,1/200,1/100)混合作为标准样品,独立配制6个批次并分别进行PNA-PCR/K-ras突变检测,收集K-ras突变CT值及K-ras总体CT值,计算△CT值(突变CT值-总体CT值),采用ROC曲线分析突变CT值和△CT值诊断K-ras突变的最适Cut-off值,联合两者最适Cut-off值,设定该方法的最终阳性判断标准.分别采用该方法和直接测序法对35例结直肠癌组织及对应癌旁组织进行K-ras突变检测并比较分析.结果 突变模板浓度为1/800及以上的标准样品突变CT值和△CT值与阴性标准品之间差异存在统计学意义(P＜0.05);突变CT值和△CT值的最适Cut-off值分别为41.7和15.4.最终阳性判断标准为突变CT值≤41.7或△CT值≤15.4,对应的ROC曲线下面积为0.955(P=0.001),以此判断标准,各标准样品(0,1/3 200,1/1 600,1/800,1/400,1/200,1/100)的阳性检测率分别为0％、66.7％、83.3％、100％、100％、100％、100％,检测下限为1/800.在结直肠癌及癌旁组织标本中,该方法阳性检测率为45.7％(32/70),与直接测序法(18.6％,13/70)比较差异具有统计学意义(P=0.000).结论 PNA-PCR/K-ras突变检测方法具有较高的检测灵敏度,在结直肠癌组织样本中具有较直接测序法更高的阳性检出率.
Abstract
Objective To determine the positive judgement standard of K-ras mutation detection method peptide nucleic acid (PNA)-PCR/K-ras (previously established in our laboratory) and to assess its diagnostic value for colorectal cancer tissues.Methods Plasmids with K-ras codon 12 mutation and plasmids with K-ras wild-type plasmids were mixed and serially diluted into standard samples (mutation/total:0,1/3 200,1/1 600,1/800,1/400,1/200,1/100) for six independent tests.The mutation CT,total CT and △CT (mutation CT-total CT) values were obtained by PNA-PCR/K-ras method.After the cut-off values of the mutation CT and △CT for K-ras diagnosis were identified by ROC analysis,the diagnostic criteria for K-ras mutation was defined by combining both the cut-off values of the mutation CT and △CT.A comparison was made between K-ras diagnostic rate by PNA-PCR/K-ras method and direct sequencing for 35 colorectal cancer tissues and their corresponding adjacent noncancerous tissues.Results The mutation CT and △CT values for 1/800 and the above standard samples were significantly different from those of the negative samples(P＜0.05),with the optimum cut-off values of mutation CT and △CT being 41.7 and 15.4,respectively.The diagnostic criteria (mutation CT≤41.7 or △CT≤15.4) for K-ras mutation was set up as AUC-ROC 0.955 (P=0.001).According this diagnostic criteria,the K-ras mutation diagnostic rates in each concentration gradient of the standard samples (0,1/3 200,1/1 600,1/800,1/400,1/200,and 1/100) were 0％,66.7％,83.3％,100％,100％,100％,and 100％,receptivity,with the upper diagnostic limit being 1/800.The diagnostic rates of K-ras mutation by our method and by direct sequence method for colorectal cancer tissues were 45.7％ (32/70) and 18.6％ (13/70),respectively,showing significant difference (P=0.000).Conclusion PNA PCR/K-ras method has higher sensitivity and positive detection rate than direct sequencing method for colorectal cancer tissues.
关键词
K-ras基因/突变/肽核酸类/聚合酶链反应Key words
K-ras gene/gene mutation/peptide nucleic acids/polymerase chain reaction引用本文复制引用
基金项目
国家自然科学基金(30910103911)
国家自然科学基金(81272663)
上海市重点科技攻关项目(11441901800)
国家科技支撑计划(2006BAI02A12)
出版年
2013
NETL
NSTL
维普
万方数据