Multiplex real-time PCR using double-strand primers and probes for the detection of nucleic acids.
Multiplex real-time PCR using double-strand primers and probes for the detection of nucleic acids.
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DOI：
10.1039/d0ay01661f
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发表时间：
2020-10
期刊：
影响因子：
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通讯作者：
Zhang Zhang-Zhang;Juan Yao;Xiaoling Huang;Li Zhang;Ting Wang;Zhi Weng;G. Xie
中科院分区：
文献类型：
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作者：
Zhang Zhang-Zhang;Juan Yao;Xiaoling Huang;Li Zhang;Ting Wang;Zhi Weng;G. Xie
由于所有引物对在相同的退火温度下工作，多重PCR在引物设计上存在困难。在本研究中，我们建立了一种双链引物介导的多链位移反应，用于检测SARS-COV-2的ORF、N和E基因（举例）。双引物由不影响聚合酶延伸的5‘修饰荧光团链和不影响延伸的3’修饰猝灭链组成。在退火温度下，荧光团链与模板结合，延伸并导致荧光信号释放。结果表明，双链引物相对表现出较宽的退火温度范围和三对引物与探针之间良好的相容性。这些优点使得核酸的简单和多重实时荧光定量成为可能。检测限为400拷贝/mL，检测时间约为2 h。该方法具有极强的特异性和简便性，在多重PCR和SNP检测等方面应用广泛。
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.