人FHIT基因实时荧光定量RT-PCR检测方法的建立

ESTABLISHMENT OF A REAL-TIME FLUORESCENT QUANTITATIVE RT-PCR ASSAY FOR DETECTION OF HUMAN FHIT GENE

目的:建立检测人脆性组氨酸三联体(fragile histidine triad,FHIT)基因的荧光定量RT-PCR方法。方法:根据GenBank中人FHIT基因序列,在保守区设计合成一对特异引物,将PCR扩增得到的FHIT基因克隆至PMD18-T载体上,构建的重组质粒标准品倍比稀释后运用SYBR Green I染料法绘制标准曲线,并进行融解曲线分析。结果:FHIT基因的标准品稀释度在1×107～1×103copies/μl范围内具有良好的线性关系,Ct值线性范围为14.28～28.36,相关系数为0.998,融解曲线分析表明,产物为特异的单峰,Tm为92.3±0.1℃。结论:建立了人FHIT基因实时荧光定量RT-PCR方法,为在mRNA水平对人FHIT的定量分析奠定了基础。

Objective： To develop a real-time quantitative polymerase chain reaction assay for detecting fragile histidine triad（FHIT） gene of human.Methods： According to the human FHIT gene sequence available in GenBank,a pair of primers was designed and the amplified fragment of FHIT were linked with PMD18-T vector to construct recombined plasmid.Then the positive plasmid was diluted and the standard curve was established using SYBR Green I.The melting curve was also analyzed.Results： The standard curve had a good linear relationship（r2=0.998） with the standard samples from 1×107 to 1×103 copies/ μl and the linear range of standard curve Ct was from 14.28 to 28.36.The melting curve showed a single peak with the temperature of 92.3± 0.1.Conclusion： The method for detecting human FHIT gene with fluorescence quantitative polymerase chain reaction was developed successfully and could provide the basis for quantitative analysis of FHIT gene mRNA expression.