尿液中GSTP1基因甲基化数字PCR方法的建立和优化  

The Optimization of ddPCR Method for Detection of GSTP1 Gene Methylation

作　　者：黄薇 蒋涛[2] 逄瑷博 张朋军 田亚平[2] HUANG Wei;JIANG Tao;PANG Aibo;ZHANG Pengjun;TIAN Yaping(Graduate School,Chinese PLA General Hospital,Beijing 100853,China;Birth Defects Prevention and Control Technology Research Center,Medical Research and Innovation Department,Chinese PLA General Hospital,Beijing 100853,China;Key Laboratory of Pathogenesis and Transformation of Malignant Tumors,Department of Interventional Therapy,Peking University Cancer Hospital and Beijing Institute of Cancer Prevention and Control,Ministry of Education,Beijing 100142,China)

机构地区：[1]解放军医学院,北京100853 [2]中国人民解放军总医院医学创新研究部出生缺陷防控技术研究中心,北京100853 [3]北京大学肿瘤医院暨北京市肿瘤防治研究所介入治疗科恶性肿瘤发病机制及转化研究教育部重点实验室,北京100142

出　　处：《标记免疫分析与临床》2023年第10期1764-1769,共6页Labeled Immunoassays and Clinical Medicine

基　　金：国家自然科学基金(编号:8197081525)。

摘　　要：目的 基于微滴式数字PCR(droplet digital PCR,ddPCR)建立一种检测前列腺癌(prostatic cancer, PCa)尿液谷胱甘肽硫转移酶P1(glutathione S-transferase pi-1,GSTP1)基因甲基化的方法,并对其进行优化。方法 本研究收集20例前列腺癌尿液样本,设计并筛选引物和探针,通过调整亚硫酸氢盐转化时间及ddPCR反应体系中引物浓度、探针浓度、退火温度、循环次数、变性时间和退火延伸时间对ddPCR检测方法进行优化。结果 尿液DNA亚硫酸氢盐转化的最佳时间是2h 10min,最佳引物浓度是700nmol/L,最佳扩增温度为58.3℃,最佳循环次数是50次,最佳变性时间是30s,最佳退火延伸时间是60s,本研究范围内探针浓度对ddPCR影响不大,可根据实验自行调整探针浓度。结论 本研究建立并优化了ddPCR检测前列腺癌尿液样本GSTP1基因甲基化方法,提高了ddPCR检测尿液中痕量DNA的性能。Objective Based on Droplet digital PCR(ddPCR),a method for detecting prostatic cancer was established.The method of methylation of Glutathione S-transferase pi-1(GSTP1)gene in urine was then optimized.Methods In this study,urine samples from 20 cases of prostate cancer were collected.Primers and probes were designed and screened,and ddPCR detection method was optimized by adjusting the bisulfite conversion time,primer concentration,probe concentration,amplification annealing temperature,cycle times,denaturation time and annealing extension time in ddPCR reaction system.Results The best time point for urine DNA methylation bisulfite conversion was 2h 10min,while the best concentration of primers was 700nM,and the best amplification temperature was 58.3℃.The best number of cycles was 50 times,the best denaturation time was 30s,and the best annealing extension time was 60s.The probe concentration had little effect on ddPCR within the scope of this study.The probe concentration can be adjusted according to the experiment results.Conclusion In this study,a ddPCR method for detecting GSTP1 gene methylation in prostate cancer urine samples is established and optimized,and the performance of ddPCR for detecting trace DNA is improved.

关 键 词：尿液 DNA甲基化 数字PCR 谷胱甘肽硫转移酶P1 前列腺癌 

分 类 号：R-331[医药卫生]