Multiplex real-time PCR using double strand displacing nucleic acids as primers and probes for the detection of nucleic acids
Multiplex PCR encounters difficulties in primers design that all primers pairs working at the same annealing temperature. In this article, we have developed a double strand primers mediated multiple strand displacement reaction for the detection of SARS-COV-2 ORF, N, and E gene as example. The double primer is composed of 5’ modified fluorophore strand thus does not impact polymerase extension and 3’ modified quencher strand thus cannot elongation. In annealing temperature, fluorophore strand combined template and extended thus resulted fluorescence signal release. Results showed that the double strand primer shows relative wide annealing temperature range, good compatibility between three pairs primers and probes. These merits allows simple and multiplex real-time fluorescent quantification of nucleic acids. The detection limit was 400 copies/mL, and the detection time was approximately 2 hour. In addition to its extreme specificity and simplicity, it has wide range of applications such as multiple PCR and SNPs detection.