{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T02:45:55Z","timestamp":1779245155091,"version":"3.51.4"},"reference-count":50,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2021,6,4]],"date-time":"2021-06-04T00:00:00Z","timestamp":1622764800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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\u2032 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.<\/jats:p>","DOI":"10.3390\/ijms22116061","type":"journal-article","created":{"date-parts":[[2021,6,4]],"date-time":"2021-06-04T11:12:13Z","timestamp":1622805133000},"page":"6061","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["3\u2032 Tth Endonuclease Cleavage Polymerase Chain Reaction (3TEC-PCR) Technology for Single-Base-Specific Multiplex Pathogen Detection using a Two-Oligonucleotide System"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9420-8368","authenticated-orcid":false,"given":"Owen","family":"Higgins","sequence":"first","affiliation":[{"name":"Molecular Diagnostics Research Group, School of Natural Sciences, National University of Ireland, Galway, Ireland"},{"name":"Centre for One Health, Ryan Institute, National University of Ireland, Galway, Ireland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4956-3299","authenticated-orcid":false,"given":"Terry","family":"Smith","sequence":"additional","affiliation":[{"name":"Molecular Diagnostics Research Group, School of Natural Sciences, National University of Ireland, Galway, Ireland"},{"name":"Centre for One Health, Ryan Institute, National University of Ireland, Galway, Ireland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,6,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1039\/a904630e","article-title":"Nucleic acid conformation diversity: From structure to function and regulation","volume":"30","author":"Belmont","year":"2001","journal-title":"Chem. 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