Development of real-time fluorescence quantitative RT-PCR assay for β-actin gene of Panax ginseng / 中草药

ABSTRACT

Objective: To construct a real-time fluorescence quantitative RT-PCR method for the β-actin gene of Panax ginseng. Methods: According to the β-actin gene of other higher plants available in Genbank, A pair of primers weredesigned and the amplified fragment of β-actin gene was linked with pMD20-T vector to construct recombinant plasmids. Then the positive plasmids were diluted and the standard curve was established. The sensitivity, specificity, and repeatability were detected. Results: The results showed that the lowest copy number for detection of β-actin gene with this method was 43.0 copies/μL, and there was a good linear relationship in a wide range from 43 to 4.3 × 107 copies/μL (R2 = 0.995 3). The melting curve showed a single peak with the temperature of (84.51 ± 0.01) ℃. The coefficient of variation (CV) of five different concentration of positive plasmids was 0.58% to 2.79% and 2.61% to 4.41% in intra-assay and inter-assay, respectively. Conclusion: The method established in this paper has the advantage of rapidity, sensitivity, specificity, high throughput, and good repeatability, which provides a methodological basis for the quantitative analysis on the functional genes of P. ginseng when β-actin gene is taken as a reference gene.