摘要
目的 利用Taqman技术 ,建立荧光定量逆转录聚合酶链反应 (FQ RT PCR)技术 ,对前列腺癌病人外周血中前列腺特异性抗原基因 (PSAmRNA)进行定量检测。方法 在PSA基因的外显子 2和 3之间设计一对引物和一条Taqman探针 ;优化反应体系的条件 ,以不同LNCaP细胞含量为标准品和其循环阈值 (Ct值 )制作标准曲线 ,检测外周血中PSAmRNA含量 ;对本方法进行方法学评价及初步临床应用评价。结果 成功建立了FQ RT PCR检测PSAmRNA技术 ,本方法最低检测限为每毫升全血 5个LNCap细胞 ,线性范围为 5～ 1 0 8细胞 /ml,批内和批间变异系数分别为 9 5 %～1 4 3%和 1 5 2 %～ 2 0 1 % ,扩增产物克隆和测序分析显示本扩增片段为PSAmRNA序列的特异性片段。初步临床研究显示 ,1 0例经ECT ,检查证实骨转移的前列腺癌患者的外周血均检到PSAmRNA ,其含量变化为 8～ 1 0 4细胞 /ml,健康男性、女性各 5名外周血中PSAmRNA含量均小于本法最低检测限。结论 荧光定量逆转录聚合酶链反应检测PSAmRNA具有灵敏、特异、简便、结果数据化等特点 ,该方法可为前列腺癌微转移诊断、预后判断、指导治疗等奠定良好基础 。
Objective To develop fluorescence quantitative reverse transcriptase polymerase chain reaction(FQ RT PCR) for the measurement of prostate specific antigen(PSA) mRNA in peripheral blood of prostate carcinoma using the Taqman detection system Methods A pair of primers were designed to amplify a fragment of 145 bp between exon2 and exon3, A TaqMan probe modified with 6 Fam at 5' end and Tamra at its 3' end was designed, then, the detection system was developed The PCR product was cloned and sequenced with Beckman CEQ8000; Blood samples were collected from 10 patients with prostate carcinoma and from 5 female and male health controls respectively, the circulating prostate derived cells were isolated by the density gradient separation and PSAmRNA was measured Results The quantitative assay was developed using an external standard reference curve generated from the human prostate cell line LNCap, The assay's minimum limit was 5 cells/ml, the linear range was from 5 to 10 8cells/ml, the CV value is 9 5%～14 3% and 15 2%～20 1% respectively in intra and inter assay, the PCR product was specific for PSAmRNA sequence, PSAmRNA was detected from 8～10 4 cells/ml in patients with prostate carcinoma and not detected in healthy controls Conclusions The fluorescence quantitative reverse transcriptase polymerase chain reaction for the measurement of PSAmRNA was sensitive, specific and reliable procedure Circulating PSAmRNA is a promising marker for the detection and follow up of these patients
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2004年第3期148-151,共4页
Chinese Journal of Laboratory Medicine
基金
浙江省自然科学基金资助项目 (SZJ0 2 0 0 3 )
