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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-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SYBR Green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SYBR Green实时荧光定量PCR方法的线性检测范围为1×101~1×10-7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 quantitative polymerase chain reaction (PCR) method for detecting prostate cancer antigen 3 (PCA3) gene. Methods PCA3 was amplified by reverse transcription-PCR and identified by agarose gel electrophoresis and gene sequencing. The detection of PCA3 by SYBR Green real-time quantitative PCR and the establishment of the corresponding standard curve, the use of melting curve analysis of the specificity of the amplified product. Meanwhile, the efficacy of the established PCA3 assay was verified in clinical specimens. Results Both agarose gel electrophoresis and gene sequencing suggested amplification products were specific. The linear detection range of SYBR Green real-time PCR for detection of PCA3 was 1 × 101 ~ 1 × 10-7 copies, the amplification efficiency was 98% and the determination coefficient was 0.999. Only one single peak was observed in the melting curve, confirming that the amplification product is specific. The intra-and inter-day CVs for this method were 2.1% and 2.7% respectively. PCA3 was used to detect PCA3 expression in 86 prostate cancer patients and 45 non-prostate cancer patients. The results showed that the performance of PCA3 in early diagnosis of prostate cancer was significantly better than that of serum prostate specific antigen (PSA). Conclusion A real-time quantitative PCR method for rapid, sensitive, high specificity, wide range quantitative and reproducible detection of PCA3 was established.