甲基化特异性PCR检测PRAME基因启动子低甲基化30例及其临床应用  

Methylation-specific PCR detection for hypomethylation in PRAME gene promoter of 30 cases and its clinical application

作　　者：朱照辉[1] 林江[2] 钱军[1] 姚冬明[3] 钱震[1] 王雅丽[1] 肖高飞[2] 

机构地区：[1]江苏大学附属人民医院血液科,江苏212002 [2]江苏大学附属人民医院中心实验室,江苏212002 [3]江苏大学附属人民医院检验科,江苏212002

出　　处：《重庆医学》2010年第23期3165-3167,共3页Chongqing medicine

基　　金：江苏省医学重点人才资助项目(RC2007035);镇江市社会发展项目(SH2006032);江苏省卫生厅‘科教兴卫工程’--苏州大学附属第一医院血液学学科开放课题基金项目(KF200944);江苏大学临床医学科技发展基金资助项目(JLY20080048)

摘　　要：目的建立实时定量甲基化特异性PCR（RQ-MSP）法检测急性髓系白血病（AML）患者黑色素瘤优先表达的抗原（PRAME）基因低甲基化水平,初步评价其临床意义。方法用亚硫酸氢盐修饰后的基因组DNA为模板,建立EvaGreen染料法RQ-MSP检测PRAME基因低甲基化水平方法 ,检测18例（对照组）和30例（AML组）患者的骨髓单个核细胞中PRAME基因启动子低甲基化水平。结果 RQ-MSP融解曲线呈单一峰,标准品5×10^7-5×10^2copy/μL重复性良好;AML患者PRAME基因低甲基化水平（0.96-4 508.96,中位772.42）明显高于对照组（0-100.00,中位25.29）,差异有统计学意义（P=0.002）。结论建立的PRAME基因低甲基化RQ-MSP法特异性、重复性和灵敏性良好,可用于AML初诊和病情监测。Objective To establish and evaluate the method of real-time quantitative methylation-specific PCR（RQ-MSP）for detecting hypomethylation of PRAME gene promoter in the patients with acute myeloid leukemia（AML）.Methods Genomic DNA modified by sodium bisulfite was used as a template.RQ-MSP with EvaGreen dye was established to detect the hypomethylation of PRAME promoter.The samples from 30 AML patients and 18 controls were detected.Results The melting cure showed a single peak.In repeated tests,maximal sensitivities of 5×10^7-5×10^2 copy/μL were obtained.Preliminary studies have showed that the hypomethylation level of PRAME promoter was significantly higher in AML（0.96-4 508.96,median 772.42）than that in controls（0-100.00,median 25.29）（P=0.002）.Conclusion The RQ-MSP for detecting hypomethylation of PRAME promoter was established with good specificity,reproducibility and sensitivity,which could be applied in the detection of samples at initial diagnosis and in monitoring the minimal residual disease of AML patients.

关 键 词：甲基化特异性PCR 基因 黑色素瘤优先表达抗原 低甲基化 

分 类 号：R440[医药卫生—诊断学]