双重实时荧光逆转录聚合酶链反应检测尿液DD3／PSAmRNA比值及其应用

科研成果详情

题名	双重实时荧光逆转录聚合酶链反应检测尿液DD3／PSAmRNA比值及其应用
其他题名	Detection of urine DD3/PSA mRNA ratio by duplex TaqMan RT-PCR assay and evolution of its primary application
作者	陈凤萍 1; 陈俐丽 1; 沈默 1; 陈伟 2; 陶志华1; 吴秀玲3; 胡元平4; 李澄棣 2; 陈占国 1; 陈晓东1
发表日期	2008
发表期刊	中华医学杂志影响因子和分区
语种	中文
原始文献类型	期刊论文
关键词	前列腺肿瘤基因聚合酶链反应尿液
摘要	目的建立双重实时荧光逆转录聚合酶链反应（RT—PCR）检测尿液DD3／PSAmRNA比值，评价其初步应用。方法分别在前列腺特异性抗原（PSA）基因外显子1和2、差异显示编码3（DD3）基因外显子1和3之间设计一对引物和一条探针，PSA和DD3基因的TaqMan—MGB探针5’分别标记HEX和FAM荧光素，建立双重实时荧光RT—PCR检测尿液DD3／PSAmRNA比值的方法，并对方法学进行评价。对34例前列腺癌（PCa）、44例良性前列腺增生（BPH）患者前列腺按摩后尿液中的DD3／PSAmRNA比值进行检测，评价其临床应用价值。结果扩增产物经测序证明为PSA和DD3特异性片段。以LNCaP细胞cDNA作模板，PSAmRNA和DD3mRNA最低检测限分别为0，6细胞／反应和60细胞／反应。DD3／PSAmRNA比值批内、批间变异系数分别为3．8％-4，7％和4．1％-4，9％。PCa组尿液DD3／PSAmRNA比值明显高于BPH组（P〈0．01）。尿液DD3／PSAmRNA比值诊断PCa的ROC曲线下面积（AUC）为0．746（95％CI：0．630—0．862），当截断值为0．254时其敏感度和特异度分别为64．7％和77.3％，其阳性率与临床和病理分级均无关。结论成功建立了双重实时荧光RT—PCR检测尿液DD3／PSAmRNA比值的分子生物学诊断方法。该方法对PCa诊断具有较高特异度和敏感度，且节省操作时间、降低实验成本，有望成为PCa早期诊断的有效方法。
其他摘要	Objective To establish a duplex TaqMan RT-PCR assay for urine DD3/PSA mRNA ratio, and to evaluate its effect in diagnosis of prostate cancer （PCa）. Methods Urine samples were obtained from 34 patients with PCa and 44 patients with benign prostate hypertrophy （ BPH ） by prostate massage. A duplex TaqMan RT-PCR assay was developed： PCR primers were designed to amplify the fragments between the exonl and exon2 in the PSA mRNA and between the exonl and exon3 in the DD3 mRNA, and the PCR TaqMan-MGB probes were labeled with HEX and FAM respectively in 5＇ for PSA mRNA. LNCaP cells were used as template. DD3/PSA mRNA ratio was measured. Receiver operating characteristic curve （ROC） was drawn so as to evaluate its diagnostic efficacy. Results Sequencing showed that the PCR products were specific for PSA mRNA and DD3 mRNA. The minimum detection level was approximately 0.6 cells/reaction for PSA mRNA and was 60 cells/reaction for DD3 mRNA in the LNCaP cell eDNA. The intra- and inter-assay coefficients of variation of DD3/PSA mRNA were 3.8% - 4. 7% and 4.1% -4.9% respectively. Urine DD3/PSA mRNA ratio in PCa group was significantly higher than the BPH group（P 〈0.01 ）, When the cutoff value was defined as 0.254, the area under curve （AUC） of DD3/ PSA mRNA ratio was 0. 746 （95% CI：0. 630 -0.862） , and the sensitivity and specificity were 64.7% and 77.3% respectively. The urine DD3/PSA mRNA ratio positive rate was not correlated with clinical and pathological parameters. Condusion A duplex TaqMan RT-PCR assay for urine DD3/PSA mRNA ratio has been established with an excellent clinical performance and specificity for PCa, saving time and reducing costs. It may be a promising method in the early diagnosis of PCa.
ISSN	0376-2491
卷号	88 期号:4 页码:261-264
收录类别	CSCD
学科领域	医药、卫生 ; 临床医学
CSCD记录号	CSCD:3216160
引用统计
文献类型	期刊论文
条目标识符	https://kms.wmu.edu.cn/handle/3ETUA0LF/99021
专题	附属第一医院附属第一医院_超声影像科附属第一医院_泌尿外科
作者单位	1.温州医学院附属第一医院实验诊断中心, 325000 ; 2.温州医学院附属第一医院泌尿外科, 325000 ; 3.温州医学院附属第一医院病理科, 325000 ; 4.温州医学院附属第一医院超声影像科, 325000
第一作者单位	附属第一医院
第一作者的第一单位	附属第一医院
推荐引用方式 GB/T 7714	陈凤萍,陈俐丽,沈默,等. 双重实时荧光逆转录聚合酶链反应检测尿液DD3／PSAmRNA比值及其应用[J]. 中华医学杂志,2008,88(4):261-264.
APA	陈凤萍., 陈俐丽., 沈默., 陈伟., 陶志华., ... & 陈晓东. (2008). 双重实时荧光逆转录聚合酶链反应检测尿液DD3／PSAmRNA比值及其应用. 中华医学杂志, 88(4), 261-264.
MLA	陈凤萍,et al."双重实时荧光逆转录聚合酶链反应检测尿液DD3／PSAmRNA比值及其应用".中华医学杂志 88.4(2008):261-264.