前列腺癌特异DD3基因实时荧光定量PCR检测方法建立及初步应用

Establishment and application of DD3 gene detection with real-time fluorescence quantified reverse transcription polymerase chain reaction in patients with prostate cancer

目的:建立实时荧光定量PCR检测特异DD3基因方法,探讨其在前列腺癌早期诊断中的应用价值.方法:根据Genebank中的DD3mRNA全序列,设计DD3基因的特异引物和探针,构建用于制备DD3基因检测的定量标准品的克隆载体;建立DD3基因的实时荧光定量方法,分别用于前列腺癌患者全血、尿液、前列腺液标本的检测.结果:经稳定性、特异性、重复性和敏感性实验评价,DD3基因实时荧光定量PCR方法的最低检测限为1.64×103拷贝/L,线性范围为1.64×103～1.64×1015拷贝/L.对临床收集的19份前列腺癌患者,20份非前列腺癌患者、30份健康者的全血、尿液及前列腺液标本进行检测.19份前列腺癌患者标本中阳性率分别为100%(全血)、80%(尿液)、100%(前列腺液);20份非前列腺癌患者标本中只有1份前列腺液出现低拷贝值(1×105拷贝/L);30份健康者标本均为阴性.在全血、尿液、前列腺液标本中,前列腺癌患者DD3mRNA含量明显高于非前列腺癌患者和健康者(P<0.05).结论:DD3基因是一种用于前列腺癌早期诊断的特异性指标,可用于生物体液中少量癌细胞的检测,在前列腺癌早期诊断、微转移诊断、预后判断、指导治疗等方面均具有潜在的应用价值.

AIM： To establish the method of detecting DD3 gene with real-time fluorescence quantified reverse transcription polymerase chain reaction and to employ it in the early diagnosis of prostate cancer. METHODS： Specific primer and marked probes for DD3 gene were designed according to full DD3 mRNA sequence in Genbank. The vector to prepare the quantitative standard for DD3 gene detection was constructed and quantitative real-time fluorescence methods were employed to detect blood, urine and prostatic fluid specimens. RESULTS： By laboratory evaluation of the stability, specificity, repeatability and sensitivity, the lowest limit for detection of DD3 gene by real-time fluorescence quantitative PCR was 1.64×10^3 copy/L. Its line scope ranged from 1.64×10^3 to 1.64×10^15 copy/L. The specimens of blood, urine and prostatic fluid from 19 patients with prostate cancers, 20 non-prostate cancer patients and 30 healthy volunteers were assessed. The positive rate was 100% （blood）, 80% （urine）and 100% （prostatic fluid ）. Among the prostate gland liquid specimens from the 20 non-prostate cancer patients, only one specimen showed low copy（ 1 ×10^5 copy/L）. Specimens from the 30 healthy volunteers were all negative. In the specimens of blood, urine and prostatic fluid, the DD3 mRNA content of the prostate cancer patient was significantly higher than that of non-prostate cancer patients and healthy volunteers. （P 〈0. 05 ）. CONCLUSION： DD3 gene can be used as a specific marker for early prostate cancer diagnosis and it can be used for the examination for blood, seminal fluid, urine and prostatic fluid. The examination can be employed for early prostate cancer diagnosis, micro translocation diagnosis, treatment and prognosis.