肽核酸钳制-PCR检测K-ras突变法的阳性判断阈值及检测下限的研究

Identification of cut off value and lower limit of detection for K-ras mutation by PNA-PCR clamping method

目的确定肽核酸钳制-PCR（PNA-PCR）检测K-ras突变的阳性判断阈值（Cutoff）和检测下限。方法将含K-ras基因12或13密码子突变的胰腺癌细胞株PANCl、SWl990基因组DNA以不同的浓度分别与K-ras野生型的胎盘DNA混合配制成含不同突变率（0、0．1％、0．2％、0．4％、0．8％、1．6％、3．1％、6．25％、12．5％、25％、50％）的样本，并制备1％突变率PANCl细胞及30％突变率SWl990细胞DNA总量分别为50、20、5、1ng和50、10、5、1ng的样本。应用PNA-PCR方法检测样本中的K-ras基因12、13密码子突变，收集它们的突变ct值、总体ct值，并计算出ACt值（突变ct值-总体ct值）。实验重复10次。采用受试者工作特征（ROC）曲线分析ACt值，确定K-ras基因突变的最适Cutoff值，计算诊断阳性率及确定诊断下限。结果所有不同突变率的PANCl细胞12密码子突变以及SWl990细胞13密码子突变的突变ct值及ACt值与阴性对照样本的差异均有统计学意义（P值均〈0．05），而总体ct值与阴性对照样本的差异无统计学意义。诊断K-ras基因12密码子突变的ROC曲线下面积（AUC）为0．926，最适Cutoff值为11，相应的敏感度和特异度为84％和100％，检出下限为0．4ng；诊断K-ras基因13密码子突变的AUC为0．906，最适Cutoff值为9．5，相应的敏感度和特异度为71％和100％，检测下限为1．5ng。固定突变率的检测结果进一步确定上述的检测下限。结论成功确立了PNA-PCR法检测K-ras基因12、13密码子突变的Cutoff值和检测下限，达到临床应用的要求。

Objective To determine the lower limit of detection （LLOD） and cut off values of K-ras mutation detection by peptide nucleic acid （PNA） elamping-PCR. Methods The genomic DNA of pancreatic cancer cell lines （PANC1 and SW1990） with codonl2, 13 mutation and the genomic DNA of placenta with K- ras wild type were mixed and diluted serially into samples with different mutation rate （0, 0.1% , 0.2% , 0.4%, 0.8%, 1.6%, 3.1%, 6.25%, 12.5%, 25%, 50%）, PANC1 cells with 1% mutation rate and SW1990 ceils with 30% mutation rate and 4 samples with the quantity of DNA was 50, 20, 5, 1 ng and 50, 10, 5, 1 ng was prepared. Codon 12, 13 mutation of K-ras was determined by PNA-PCR, and the mutation Ct values, overall Ct values were collected, and the ACt values （mutation Ct values-overall Ct values ） were calculated, and the tests were repeated for 10 times. ROC curve was used to analyze the ACt values and. determine the best cut off values for K-ras mutation, and the positive diagnostic rate, LLOD was evaluated. Results The mutation Ct, ACt values of codon 12 mutation of PANC1 and eodon 13 mutation of SW1990 of all the different mutation rates were statistically significantly different （P 〈 0.05 ） when compared with negative control group, but the overall Ct values were not statistically significantly different from that of negative control group. For detection of K-ras codon 12 mutation by ROC curve, the relevant area of ROC curve （AUC） was 0.926, the optimum cut off value of ACT was 11, the sensitivity and specificity were 84% and 100%, respectively, and the LLOD was 0.4 ng. For detection of K-ras codon 13 mutation by ROC curve, the relevant AUC was 0.906, the optimum cut off value of ACT was 9.5, the sensitivity and specificity were 71% and 100%, respectively, and the LLOD was 1.5 ng. The mutation detection results of fixed rate further confirmed the LLOD. Conclusions This study successfully defines LLOD and cut off value of PNA clamping-PCR/K-ras method in detection of K-ras 12 and 13 codon mutations. This method meets the requirement of clinical application.