中文摘要：

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ΔCT值（突变CT值-总体CT值）,采用ROC曲线分析突变CT值和ΔCT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ΔCT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ΔCT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ΔCT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。

英文摘要：

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 ΔCT （mutation CT-total CT） values were obtained by PNA-PCR/K-ras method. After the cut-off values of the mutation CT and ΔCT 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 ΔCT. 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 ΔCT 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 ΔCT being 41.7 and 15.4, respectively. The diagnostic criteria （mutation CT≤41.7 or ΔCT≤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.