Abstract
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.
Highlights
Ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), are hereditary macromolecule biopolymers composed of nucleotide subunits
DNA consists of two antiparallel nucleotide strands in a double helix structure joined together by hydrogen bonds that link complementary nitrogenous bases: guanine with cytosine and adenine with thymine [1,2]
The H. influenzae 3TEC-polymerase chain reaction (PCR) wild-type assay successfully detected each duplicate of the 10-fold serially diluted type strain H. influenzae DNA concentrations tested at 104, 103, 102, and 101 genome copies per reaction, and detected one of the duplicates tested at 100 genome copies (Figure 2)
Summary
Ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), are hereditary macromolecule biopolymers composed of nucleotide subunits. DNA consists of two antiparallel nucleotide strands in a double helix structure joined together by hydrogen bonds that link complementary nitrogenous bases: guanine with cytosine and adenine with thymine [1,2]. This complementary DNA base pairing is highly specific and utilised in molecular diagnostics for infectious disease pathogen detection [3]. Nucleic acid amplification diagnostics, such as polymerase chain reaction (PCR), are used to target pathogen specific genomic nucleotide sequences and amplify these biomarkers to detectable levels [4]. PCR was first conceived in the 1980s [5,6,7,8], it remains the most commonly used nucleic acid amplification technology and has been the primary diagnostic tool of choice during the global COVID-19 pandemic [9,10,11]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
