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?:abstract
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Multiplex PCR encounters difficulties in primer designing with all the primer pairs working at the same annealing temperature. In this study, we have developed a double-strand primer-mediated multiple strand displacement reaction for the detection of SARS-COV-2 ORF, N and E genes (as examples). The double primer is composed of a 5\'-modified fluorophore strand, which does not impact polymerase extension and a 3\'-modified quencher strand, which cannot impact elongation. At the annealing temperature, the fluorophore strand combined with the template, extended and resulted in fluorescence signal release. Results showed that the double-strand primer relatively exhibits a wide annealing temperature range and good compatibility between three pairs of primers and probes. These merits allow the simple and multiplex real-time fluorescence quantification of nucleic acids. The detection limit was 400 copies/mL, and the detection time was approximately 2 h. In addition to its extreme specificity and simplicity, this method has a wide range of applications such as multiple PCR and SNP detection.
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