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| 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 |
| 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* |
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(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
△共同第一作者
*通信作者) |
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| 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。 |
| 关键词: K-ras基因 突变 肽核酸类 聚合酶链反应 |
| DOI： |
| 投稿时间：2013-02-21修订日期：2013-07-22 |
| 基金项目:国家自然科学基金(30910103911, 81272663),上海市重点科技攻关项目(11441901800),国家科技支撑计划(2006BAI02A12). |
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| Detection of K-ras mutation by PNA-PCR/K-ras method in diagnosis of colorectal cancer tissues |
| LI Quan-jiang1△,HU Jia-jia1△,JIN Jing1,WU Hong-yu1,MAN Xiao-hua1,ZHU Ling2,GAO Jun1*,LI Zhao-shen1* |
(1. Department of Gastroenterology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
2. The China Table Tennis College, Shanghai University of Sport, Shanghai 200433, China
△Co-first authors.
*Corresponding author.) |
| 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 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. |
| Key words: K-ras gene gene mutation peptide nucleic acids polymerase chain reaction |
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