Background: In duplex real time quantitative PCR (qPCR), there are several factors affecting the sensitivity of duplex qPCR, such as sharing primer, quantity of the internal control (IC) gene, and the primer dimer (PD). The aim of the study is to find out the relationship of interference among templates with different primer pairs, the internal control gene, and the PD in duplex PCR.
Methods: We designed and synthesized plasmids with partial same sequence and different types of primers include central-homo primer pair, ordinary primer pair, and complementary primer pair. Then we compared the amplification efficiencies of different kinds of primer pairs. Besides, we adjusted the amount of IC plasmid and IC primer to find out the key factor that influences the sensitivity of the target template.
Results: The concentration ratios of two plasmids showed interference in sharing the universal primer pair, sharing one forward primer, and sharing no primer were 50:1, 200:1 and 500:1, respectively. The amplification efficiency of the ordinary primer pair was higher than that of the universal primer pair for the plasmid. Sensitivity of the duplex qPCR remained unchanged when the amount of PDs increased, but it declined rapidly when the quantity of the IC increased.
Conclusions: IC is the major factor influencing the sensitivity of the duplex qPCR and it would be better to use one universal primer for IC and target template to achieve minimum interference.