Fig. 2
Schematics of the semi-nested RT-PCR assay for CP mRNA quantification. The novel quantitative RT-PCR assay for CP mRNA involves two rounds of amplification to enhance sensitivity and specificity. The first round uses specific primers for CP and an internal control (IC) gene, incorporating non-complementary sequences at the 5’ ends (Fig. 2, panel i). These primers also function in reverse transcription to synthesize cDNA (Fig. 2, panel ii). The second round employs a common reverse primer (U-Ri) for both genes (Fig. 2, panel iv) and specific forward primers (CP-Fi and IC-Fi) (Fig. 2, panel iii). The amplified products are analyzed via DNA melting curve to distinguish CP mRNA and IC peaks (Fig. 2, panel v). The relative concentration of CP mRNA is quantified by calculating the ratio of the CP peak height to the IC peak height.
