Molecular Diagnostics Research Group, School of Natural Sciences, National University of Ireland, Galway, Ireland.
Centre for One Health, Ryan Institute, National University of Ireland, Galway, Ireland.
Int J Mol Sci. 2021 Jun 4;22(11):6061. doi: 10.3390/ijms22116061.
Polymerase chain reaction (PCR) is the standard in nucleic acid amplification technology for infectious disease pathogen detection and has been the primary diagnostic tool employed during the global COVID-19 pandemic. Various PCR technology adaptations, typically using two-oligonucleotide dye-binding methods or three-oligonucleotide hydrolysis probe systems, enable real-time multiplex target detection or single-base specificity for the identification of single-nucleotide polymorphisms (SNPs). A small number of two-oligonucleotide PCR systems facilitating both multiplex detection and SNP identification have been reported; however, these methods often have limitations in terms of target specificity, production of variable or false-positive results, and the requirement for extensive optimisation or post-amplification analysis. This study introduces 3' endonuclease cleavage PCR (3TEC-PCR), a two-oligonucleotide PCR system incorporating a modified primer/probe and a thermostable cleavage enzyme, endonuclease IV, for real-time multiplex detection and SNP identification. Complete analytical specificity, low limits of detection, single-base specificity, and simultaneous multiple target detection have been demonstrated in this study using 3TEC-PCR to identify bacterial meningitis associated pathogens. This is the first report of a two-oligonucleotide, real-time multiplex PCR technology with single-base specificity using endonuclease IV.
聚合酶链反应（PCR）是用于传染病病原体检测的核酸扩增技术的标准方法，也是全球 COVID-19 大流行期间主要采用的诊断工具。各种 PCR 技术的改进，通常使用双寡核苷酸染料结合方法或三寡核苷酸水解探针系统，实现了实时多重靶标检测或单碱基特异性，用于识别单核苷酸多态性（SNP）。已经报道了少数几种能够同时进行多重检测和 SNP 鉴定的双寡核苷酸 PCR 系统；然而，这些方法在靶标特异性、产生可变或假阳性结果以及需要广泛优化或扩增后分析方面存在局限性。本研究介绍了 3' 内切酶切割 PCR（3TEC-PCR），这是一种双寡核苷酸 PCR 系统，包含了经过修饰的引物/探针和一种热稳定的内切酶 4 型内切核酸酶，用于实时多重检测和 SNP 鉴定。本研究使用 3TEC-PCR 来鉴定细菌性脑膜炎相关病原体，证明了其具有完全的分析特异性、低检测限、单碱基特异性和同时多重靶标检测能力。这是首次报道使用 4 型内切核酸酶实现单碱基特异性的双寡核苷酸实时多重 PCR 技术。
