基于核酸侵入反应偶联PCR的粪便样本基因甲基化定量分析方法及用于结直肠癌无创筛查

Quantitative Detection of Methylated Gene in Stool Samples Based on Invader Assay Coupled with Real-time Polymerase Chain Reaction and Its Application in Non-invasive Screening of Colorectal Cancer

从粪便中检测BMP3基因甲基化水平可实现对结直肠癌的无创筛查,但对检测方法的灵敏度与特异性要求很高。本研究将核酸侵入反应与实时荧光聚合酶链式反应(PCR)偶联,建立了BMP3基因甲基化检测方法,以ACTB基因作为参比基因,通过优化反应体系中探针浓度、Flap核酸内切酶与Taq酶用量,提高BMP3目标基因与ACTB参比基因的扩增效率至接近100%,实现了利用ΔCT法进行BMP3基因甲基化水平的相对定量,可对低至10 copies的甲基化BMP3基因进行检测,并可从非甲基化模板中准确定量0.01%的甲基化模板,非甲基化模板不产生非特异性信号,表明本方法具有极高的灵敏度与良好的特异性。将本方法用于16例结直肠癌患者、7例结直肠腺瘤患者及19例健康人粪便样本中BMP3基因甲基化检测,结果表明,有5例结直肠癌患者的粪便样本检出BMP3基因甲基化,2例结直肠腺瘤患者的粪便样本检出BMP3甲基化,健康人的粪便样本均没有检出甲基化,表明本方法可用于检测粪便样本中基因的甲基化水平,为临床开展基于粪便中基因甲基化检测的无创结直肠癌筛查提供了新方法。

Bone morphogenetic protein 3(BMP3)gene methylation in stool DNA is an effective biomarker for non-invasive screening of colorectal cancer.However,the detection of BMP3 gene methylation in stool DNA requires a highly sensitive and specific method.Here,we developed a BMP3 gene methylation quantification method by combining real-time polymerase chain reaction(PCR)with invasive reaction using Beta-actin(ACTB)gene as a reference.After optimizing the concentration of detection probes,FEN1 enzyme and Taq polymerase,both amplification efficiencies of BMP3 and ACTB genes were close to 100%,and the accurate relative quantification of methylation of BMP3 gene was achieved withΔCT algorithms.The method could detect as low as 10 copies of BMP3 fragment,and 0.01%methylated templates could be picked up from unmethylated templates,indicating that the method was highly sensitive and specific for the detection of BMP3 gene methylation.The method was successfully applied to detect BMP3 methylation in stool DNA samples from 16 colorectal cancer patients,7 adenoma patients and 19 healthy volunteers.The result showed that methylation of BMP3 occurred in 5 cases of 16 cancer patients and 2 cases of 7 adenoma patients,but was not observed in 19 cases of healthy volunteers.Therefore,this method could be used to quantify the gene methylation in stool samples,providing an effective technique for non-invasive screening of colorectal cancer.