双重实时荧光逆转录聚合酶链反应检测尿液DD3/PSA mRNA比值及其应用

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。

Detection of urine DD3/PSA mRNA ratio by duplex TaqMan RT-PCR assay and evolution of its primary application

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 exon1 and exon2 in the PSA mRNA and between the exon1 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 cDNA. 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. CONCLUSION: 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.