基于微滴式数字PCR检测肠癌病人游离环状DNA KRAS突变的新方法

Detection of KRAS mutation in colorectal cancer patients’ cfDNA with droplet digital PCR

摘要:

基于微滴式数字聚合酶链式反应 (Droplet digital polymerase chain reaction, ddPCR) 设计一种检测肠癌游离循环DNA (Circulating cell free DNA, cfDNA) 中 KRAS (V-Ki-ras2 Kirsten ratsarcoma viral oncogene homolog) 基因突变的新方法并评估其灵敏度和准确性。根据肠癌病人KRAS基因的突变类型设计并合成，采用ddPCR扩增并评估其灵敏度和准确性；根据AMRS-PCR引物设计原理设计KRAS基因的实时定量PCR扩增引物并评估其准确性，进而比较ddPCR和qPCR二者之间的优缺点；最后针对52例肠癌病人的cfDNA采用ddPCR进行检测，研究ddPCR在cfDNA KRAS基因突变检测的应用。成功使用ddPCR和qPCR两种方法对KRAS野生型及7种突变型建立检测方法，使用质粒标准品及实际样品验证该两种方法可行并对其假阳性率、线性范围及检测下限等性能进行了评价，最后成功对52例临床患者和20例正常人的血浆cfDNA样本进行检测，临床灵敏度为97.64%，临床特异性为81.43%。ddPCR的检测性能优于qPCR，LOD达到个位数DNA拷贝，最低可确认突变浓度达到0.01%–0.04%。样本提取效率在方法学建立中也十分重要，直接影响到灵敏度和Cut Off值的判定。临床患者检测结果显示其KRAS突变率接近报道水平。

Abstract:

This study aims to develop a new method for the detection of KRAS mutations related to colorectal cancer in cfDNA, and to evaluate the sensitivity and accuracy of the detection. We designed a method of cfDNA based KRAS detection by droplets digital PCR (ddPCR). The theoretical performance of the method is evaluated by reference standard and compared to the ARMS PCR method. Two methods, ddPCR and qPCR, were successfully established to detect KRAS wild type and 7 mutants. Both methods were validated using plasmid standards and actual samples. The results were evaluated by false positive rate, linearity, and limit of detection. Finally, 52 plasma cfDNA samples from patients and 20 samples from healthy people were tested, the clinical sensitivity is 97.64%, clinical specificity is 81.43%. ddPCR method shows higher performance than qPCR. The LOD of ddPCR method reached single digits of cfDNA copies, it can detect as low as 0.01% to 0.04% mutation abundance.