Developing SYBR Green Real-Time PCR assay for wide range detection of human GAPDH gene / 国际检验医学杂志

ABSTRACT

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 .