New design of probe and central-homo primer pairs to improve TaqMan™ PCR accuracy for HBV detection.

Quantitative PCR (qPCR) assay using TaqMan™ probe was widely used in the detection of different nucleic acids. However, this technology has several drawbacks, including false negative results caused by primer-dimer (PD) and false positive issues due to primer-probe aggregations. Here, we designed a modified TaqMan™-Molecular Beacon probe by adding an antisense base and a new type of primer pair named central-homo primer pairs bearing 5-10 bases homologous sequence on the 3' end. Using the HBV qPCR assay as a proof of concept, the new design significantly improved the accuracy of the TaqMan™ qPCR assay for HBV detection. Application of the central-homo primer pair led to significantly delayed Ct values by 5-10 cycles compared with conventional primer design. The modified probe containing an antisense base did not produce any detectable signal in repeating primer-probe aggregation experiments. Furthermore, the use of the central-homo primer pair and the non-competitive internal control could solve the false negative problem caused by PD formation. We validated this customized duplex qPCR system using 208 clinical samples collected from patients in clinic showing accuracy was higher than that of the conventional qPCR method.