人类SUZ12基因实时荧光定量PCR检测方法的建立

Quantification of human SUZ12 with SYBR Green I real-time fluorescent polymerase chain reaction

目的：建立SYBR GreenI实时荧光PCR定量检测人类SUZ12基因的方法．方法：将SUZ12RT-PCR扩增片段克隆入载体pGEM—T后，经测序鉴定正确后，进行纯化和定量及系列稀释，应用SYBRGreenI实时荧光定量PCR检测Suz12，建立标准曲线，熔解曲线分析产物的特异性．结果：该法检测的最低拷贝数为14．2，线性范围为1．42× 10^-1．42× 10^8拷贝，相关系数7为-1．00，1．42× 10^7拷贝／L标本的批内变异系数（CV）和日间CV分别为1．8％和2．8％．熔解曲线分析显示单一的峰，熔解温度（meltingtemperature，Tm）为（81．37±0．16）℃．结论：实时荧光定量PCR方法检测SUZ12基因，快速有效、灵敏度高、特异性好．

AIM： To establish a real-time fluorescent polymerase chain reaction （PCR） for quantifying human SUZ12. METHODS： The SUZ12 fragment in pure form from classical RT-PCR was cloned to pGEM-T vector, and recombinant plasmid pGEM-SUZ12 was purified and quantified by spectrophotometry. A standard curve was established using a serial dilution of quantified plasmids to measure SUZ12, using SYBR Green I real-time fluorescent PCR, and the characteristics of the specific SUZ12 amplicon were analyzed by the melting curve. RESULTS： The method could detect as few as 14.2 copies. Good linearity was found from 14.2 to 1.42 × 10^8 copies/reaction, and the correlation coeffecient was -1.00. The intra- and interassay variation of 1.42 × 10^4 copies/reaction was 1.8% and 2.8%, respectively. Melting curve analysis showed a single peak, and melting temperature （Tm） was （81.37 ± 0.16）℃. CONCLUSION： SYBR Green I real-time fluorescent quantitative PCR is rapid, specific, sensitive and accurate, and can be used for research on human SUZ12.