Figure 4.

The qPCR assay can specifically detect the HPV type L1 in a complex mixture containing all 4 L1 amplicons. (a) Individual plasmids ranging from 2 × 10¹ to 2 × 10⁶ copies were run in multiplex reaction mix to generate standard curves. (b) Multiplex reactions were performed on mixtures containing all 4 plasmids carrying type-specific amplicons. Mixtures contained equal amounts of each plasmid ranging in concentrations from 2 × 10¹ to 2 × 10⁶ copies of each plasmid/reaction. The top panels show the standard curve amplification plots. The bottom panel shows the amplification plots for each plasmid mixture concentration range. CaSki and HeLa cell DNA were used as positive controls for HPV16 and 18, respectively. (c) Excess genomic DNA does not affect the performance of the multiplex HPV L1 qPCR assay. Reactions were performed using a mixture containing all 4 HPV L1 plasmids that ranged from 2 × 10⁶ to 2 × 10¹ copies. Additional reactions were performed that contained a mixture of 5 × 10⁵ copies of HPV6 L1, 5 × 10⁴ copies of HPV11 L1, 5 × 10³ copies of HPV16 L1 and 5 × 10² copies of HPV18 L1. The assay was performed in the presence or absence of 100 ng of DG-75 DNA to determine if excess human genomic DNA would affect the amplification and detection of type-specific HPV L1 DNA.