摘要
目的建立检测前列腺癌抗原3（PCA3）基因的实时定量聚合酶链反应（PCR）方法。方法应用反转录一PCR扩增PCA3后，通过琼脂糖凝胶电泳和基因测序进行鉴定。应用SYBRGreen实时荧光定量PCR检测PCA3和建立相应的标准曲线，使用熔解曲线分析扩增产物的特异性。同时在临床标本中验证所建立的PCA3检测方法的效能。结果琼脂糖凝胶电泳和基因测序均提示扩增产物是特异性的。建立的检测PCA3的SYBRGreen实时荧光定量PCR方法的线性检测范围为1×10^1～1×10^-7拷贝，扩增效率为98％，决定系数为0．999。熔解曲线仅见单一的峰，证实扩增产物是特异性的。该方法的日内和日间变异系数分别为2．1％和2．7％。利用所建立的PCA3检测方法分析86例前列腺癌患者和45例非前列腺癌患者尿液中的PCA3表达水平，结果表明PCA3早期诊断前列腺癌的性能显著优于血清前列腺特异性抗原（PSA）。结论建立了一种快速、灵敏、高特异性、宽定量范围和高重复性检测PCA3的实时定量PCR方法。
Objective To establish a real-time fluorescent polymerase chain reaction （PCR） method for determining mRNA expression of prostate cancer antigen 3 （PCA3）. Methods The PCR product of classical reverse transcription-PeR was verified by agarose gel electrophoresis and gene sequencing. SYBR Green real time fluorescent PCR was used to examine a serial dilution of purified PCA3 and to establish a standard curve, which was used to analyze the specificity of fluorescence signal. Then the efficacy of the established method for quantifying PCA3 was validated using clinical samples. Results The product of RT-PCR was consistent to the PCA3 sequence in gene bank as verified by agarose gel electrophoresis and gene sequencing. The linear range of the SYBR Green real time fluorescent PCR was from 1×10^1 to 1×10^-7copies/reaction, with the efficiency being 0.98 and R2 being 0. 999. The melting curve analysis showed a single peak, suggesting that the PCR product was specific for PCA3. The intra- and inter-test coefficients of variation of the established method were 2. 1% and 2.7%, respectively. In a cohort including 86 prostate cancer patients and 45 non-prostate cancer patients, we found that the urinary PCA3 significantly outperformed serum prostate specific antigen （PSA） in early diagnosis of prostate cancer. Conclusions A rapid, sensitive SYBR Green real-time PCR approach with high specificity, wide detection range and high reproducibility has been established for detecting PCA3 mRNA.
出处
《上海医学》
CAS
CSCD
北大核心
2011年第7期521-524,I0002,共5页
Shanghai Medical Journal
