实时荧光定量PCR检测尿液CK20方法的建立  

Development of fluorescent quantitation PCR method for detection of urinary CK20

作　　者：郭斌[1] 罗春丽[1] 荀春华[1] 谢建红[1] 吴小候[2] 蒲军[2] 

机构地区：[1]重庆医科大学医学检验系临床检验诊断学教育部重点实验室 [2]重庆医科大学附属第一医院泌尿外科,重庆400016

出　　处：《重庆医科大学学报》2009年第6期752-755,共4页Journal of Chongqing Medical University

摘　　要：目的:建立一种简便、快速、灵敏度和特异性较好的定量检测尿液CK20方法,用于膀胱移行细胞癌(Transitional cell car-cinoma of the bladder,TCCB)早期诊断、预后及复发的评估。方法:培养膀胱癌T24细胞,针对CK20基因保守序列设计合成两对引物和Taqman探针,将PCR扩增产物片段克隆,作为定量检测的标准品,优化反应条件,建立快速检测CK20mRNA的实时荧光定量PCR方法,并进行方法学评价。结果:荧光定量PCR检测尿液CK20方法能较好的定量检出尿液CK20mRNA,检测范围为102～109copies/μl,灵敏度为102copies/μl,特异性好,批内、天间重复性(Coefficientofvariation,CV)分别为0.89%和2.45%。结论:成功建立了快速检测尿液CK20的荧光定量PCR方法,为进一步开发出用于TCCB早期诊断、预后及复发评估的定量检测试剂盒奠定了基础。Objective： To develop a novel real-time fluorescence PCR method using Taqman probe for convenient, fast, sensitive and specific detection of urinary CK20 and use in early diagnosis and follow-up of transitional cell carcinoma of the bladder （TCCB）. Methods： CK20 gene from cultured T24 cells was amplified by conventional RT-PCR, the standard quantitative plasmid was constructed by T-Aclone method. Taqman probe and primers was designed according to the sequence of CK20 cloned gene,the real-time PCR method for determination of CK20 mRNA was established and evaluated. Results： The developed real-time PCR method showed high sensitivity （10^2copies/μ1） and good specificity;the linear range was 10^2-10^9copies/μ1, the coefficient variation （CV） was 0.89 % in intra-assay and 2.45% in day to day. Conclusion： A novel real-time fluorescence quantitative PCR method for detection of CK20 was established. It provided a basis for developing new diagnostic kits using the Taqman probe for clinical application.

关 键 词：膀胱移行细胞癌 CK20 实时荧光定量PCR 

分 类 号：R737.14[医药卫生—肿瘤]