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Multiplex real-time PCR using double-strand primers and probes for the detection of nucleic acids

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Abstract

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.

Graphical abstract: Multiplex real-time PCR using double-strand primers and probes for the detection of nucleic acids

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Supplementary files

Article information


Submitted
02 Sep 2020
Accepted
10 Oct 2020
First published
13 Oct 2020

Anal. Methods, 2020, Advance Article
Article type
Paper

Multiplex real-time PCR using double-strand primers and probes for the detection of nucleic acids

Z. Zhang, J. Yao, X. Huang, L. Zhang, T. Wang, Z. Weng and G. Xie, Anal. Methods, 2020, Advance Article , DOI: 10.1039/D0AY01661F

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