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Article

3′ Tth Endonuclease Cleavage Polymerase Chain Reaction (3TEC-PCR) Technology for Single-Base-Specific Multiplex Pathogen Detection using a Two-Oligonucleotide System

by 1,2,* and 1,2
1
Molecular Diagnostics Research Group, School of Natural Sciences, National University of Ireland, Galway, Ireland
2
Centre for One Health, Ryan Institute, National University of Ireland, Galway, Ireland
*
Author to whom correspondence should be addressed.
Academic Editor: Vasso Apostolopoulos
Int. J. Mol. Sci. 2021, 22(11), 6061; https://doi.org/10.3390/ijms22116061
Received: 16 May 2021 / Revised: 28 May 2021 / Accepted: 1 June 2021 / Published: 4 June 2021
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′ Tth endonuclease cleavage PCR (3TEC-PCR), a two-oligonucleotide PCR system incorporating a modified primer/probe and a thermostable cleavage enzyme, Tth 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 Tth endonuclease IV. View Full-Text
Keywords: nucleic acid diagnostics; polymerase chain reaction; multiplex detection; single-nucleotide polymorphism; bacterial meningitis nucleic acid diagnostics; polymerase chain reaction; multiplex detection; single-nucleotide polymorphism; bacterial meningitis
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MDPI and ACS Style

Higgins, O.; Smith, T.J. 3′ Tth Endonuclease Cleavage Polymerase Chain Reaction (3TEC-PCR) Technology for Single-Base-Specific Multiplex Pathogen Detection using a Two-Oligonucleotide System. Int. J. Mol. Sci. 2021, 22, 6061. https://doi.org/10.3390/ijms22116061

AMA Style

Higgins O, Smith TJ. 3′ Tth Endonuclease Cleavage Polymerase Chain Reaction (3TEC-PCR) Technology for Single-Base-Specific Multiplex Pathogen Detection using a Two-Oligonucleotide System. International Journal of Molecular Sciences. 2021; 22(11):6061. https://doi.org/10.3390/ijms22116061

Chicago/Turabian Style

Higgins, Owen, and Terry J. Smith. 2021. "3′ Tth Endonuclease Cleavage Polymerase Chain Reaction (3TEC-PCR) Technology for Single-Base-Specific Multiplex Pathogen Detection using a Two-Oligonucleotide System" International Journal of Molecular Sciences 22, no. 11: 6061. https://doi.org/10.3390/ijms22116061

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