数字PCR检测尿液循环DNA甲基化的方法学性能评价

An Evaluation of Performanc of Digital PCR for Detection ofCirculating DNA Methylation in Urine

目的基于尿液中的谷胱甘肽硫转移酶P1(glutathione S-transferase pi-1,GSTP1)基因,对微滴式数字PCR(droplet digital PCR,ddPCR)检测循环DNA甲基化的方法学进行性能评价。方法根据甲基化检测试剂盒相关IVD产品的注册指南,主要从引物探针特异性、灵敏度、批内精密度、批间精密度、准确度5个方面对数字PCR检测尿液循环DNA甲基化进行性能评价。结果GSTP1基因仅在前列腺癌样本中生成明显阳性微滴,在胃癌、肝癌、肾细胞癌、肺癌、膀胱癌、尿道癌、结直肠癌和胰腺癌中不生成阳性微滴,引物探针特异性良好;GSTP1基因的检测限为0.1ng/孔,当DNA输入量大于等于0.1ng/孔时,DNA甲基化情况都可以被检测到,灵敏度较好;批内精密度变异系数均小于6.25%,批间精密度变异系数均小于8.33%,符合CLSI参考文件的标准,表示精密度良好;通过比较数字PCR与荧光定量PCR(Methylight)检测前列腺癌尿液样本GSTP1甲基化情况,相关系数为0.9949,表明两种方法相关性较好,证明数字PCR准确度良好。结论数字PCR检测尿液循环GSTP1基因甲基化的方法在特异性、灵敏度、精密度、准确度均表现良好,符合临床实验室标准,是一种检测尿液循环DNA甲基化的可靠方法。

Objective To evaluate the performance of droplet digital PCR(ddPCR)for DNA methylation detection based on Glutathione S-transferase pi-1(GSTP1)gene in urine.Methods In this study,according to the registration guidelines of IVD products related to methylation detection,the performance of digital PCR for detecting circulating DNA methylation in urine was evaluated from five aspects:primer probe specificity,sensitivity,intra-batch precision,inter-batch precision,and accuracy.Results GSTP1 gene only generated positive droplet in prostate cancer samples,but did not generate positive droplet in gastric cancer,liver cancer,renal cell carcinoma,lung cancer,bladder cancer,urethral cancer,colorectal cancer and pancreatic cancer.The specificity of primer probe was decent.The detection limit of GSTP1 gene was 0.1ng/well.When the input amount of DNA was greater than or equal to 0.1ng/well,DNA methylation could be detected,and the sensitivity was good.The variation coefficients of intra-batch precision were less than 6.25%,and the variation coefficients of inter-batch precision were less than 8.33%,which met the standards of CLSI reference document,indicating a good precision.By comparing the methylation of GSTP1 in prostate cancer urine samples with digital PCR and fluorescence quantitative PCR(MethyLight),the correlation coefficient was 0.9949,indicating a good correlation between the experimental results,which demonstrated a good accuracy of digital PCR.Conclusion The specificity,sensitivity,precision and accuracy of digital PCR for detecting GSTP1 gene methylation are good,which meet the need of clinical laboratory standards,and can serve as a reliable method for detecting circulating DNA methylation in urine.