SYBR Green实时荧光定量聚合酶链反应宽范围检测人GAPDH基因体系的建立

Developing SYBR Green Real-Time PCR assay for wide range detection of human GAPDH gene

目的建立一种快速、灵敏的人3-磷酸甘油醛脱氢酶（GAPDH）基因实时荧光定量-聚合酶链反应（RT-PCR）方法。方法根据GeneBank数据库中提供的人GAPDH基因（NC000012）mRNA序列,在其保守区域设计一对用于RT-PCR引物,通过优化反应体系和反应条件,成功建立了检测人GAPDH基因的SYBR Green RT-PCR方法。结果实验结果表明,该检测方法检测人GAPDH基因的最低检测拷贝数可达到15copies/μL,在一个较宽的浓度范围内（1.5×1011.5×107）具有良好的线性关系（r=0.992）。溶解曲线呈现了清晰的单一峰型,Tm值为（84.5±0.2）℃。结论该研究为人GAPDH基因作为内参基因进行人功能基因与病原基因表达的定量分析提供了一种更为快速、灵敏的方法。

Objective To develop a quick and sensitive real-time fluorescent quantitative polymerase chain reaction(RT-PCR) method for detecting human GAPDH gene .Methods According to the published GAPDH gene(NC_000012) mRNA sequence in GeneBank ,a pair of primers was designed in the conserved region .After optimization of reaction system and condition ,the method for detection of human GAPDH gene by SYBR Green RT-PCR was established .Results The measuring range lower limit of GAP-DH gene could reach 15 copies per microlitre and there was a nice linear relationship in statistics between the Ct value and the con-centration gradient of standard plasmid DNA specimen was from 1 .5 × 101 to 1 .5 × 107 per microlitre(r=0 .992) .The melting curve present a single and clear peck and the Tm value was (84 .5 ± 0 .2)℃ .Conclusion The method established in this research is rapid and sensitive ,which provides a methodological basis for quantitative analysis of human functional and etiological gene using GAP-DH as reference gene .