{"title":"Active diagnostic ingredients (ADIs) for PCR: A mini-bioreactor producing dNTPs with silica immobilized R5-kinases","authors":"Anna R. Bird, Elizabeth A. H. Hall","doi":"10.1002/bit.28837","DOIUrl":null,"url":null,"abstract":"<p>Low availability of routine nucleic acid amplification testing (NAAT) during infection outbreaks, especially in less resourced environments, was highlighted by the Covid pandemic. One of the barriers lies with the supply chain and cost of the active diagnostic ingredients (ADIs) that are the reagents for NAATs. This work explores a novel synthesis method to produce a key NAAT reagent, namely the 2′-deoxynucleoside 5′-triphosphate (dNTPs), via a reusable enzyme bioreactor, that can be integrated into a NAAT workflow. A self-immobilizing R5-silaffin kinase fusion enzyme was designed for immobilization on silica, converting dNMPs to their respective dNTP ADIs for PCR in a R5-kinase mini-bioreactor, designed to be implemented in a reusable device, stable over 2 months, when stored at 4°C. The performance is demonstrated for PCR reactions of the lambda genome and showed successful amplification up to 7.5 kb. In comparison with commercial dNTPs, in <i>Plasmodium malariae</i> NAATs, a high linear correlation was shown between the <i>C<sub>t</sub></i> value and the log(Copy Number), with lower incidence of false positives than with the commercial dNTPs. Overall a pathway to generate deoxynucleotides from monophosphate precursors was demonstrated, and an immobilized enzyme mini-bioreactor investigated as a proof-of-principle for work-flow integration with NAAT in low-resource research and diagnostics labs.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"121 12","pages":"3834-3847"},"PeriodicalIF":3.5000,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28837","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biotechnology and Bioengineering","FirstCategoryId":"5","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/bit.28837","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Low availability of routine nucleic acid amplification testing (NAAT) during infection outbreaks, especially in less resourced environments, was highlighted by the Covid pandemic. One of the barriers lies with the supply chain and cost of the active diagnostic ingredients (ADIs) that are the reagents for NAATs. This work explores a novel synthesis method to produce a key NAAT reagent, namely the 2′-deoxynucleoside 5′-triphosphate (dNTPs), via a reusable enzyme bioreactor, that can be integrated into a NAAT workflow. A self-immobilizing R5-silaffin kinase fusion enzyme was designed for immobilization on silica, converting dNMPs to their respective dNTP ADIs for PCR in a R5-kinase mini-bioreactor, designed to be implemented in a reusable device, stable over 2 months, when stored at 4°C. The performance is demonstrated for PCR reactions of the lambda genome and showed successful amplification up to 7.5 kb. In comparison with commercial dNTPs, in Plasmodium malariae NAATs, a high linear correlation was shown between the Ct value and the log(Copy Number), with lower incidence of false positives than with the commercial dNTPs. Overall a pathway to generate deoxynucleotides from monophosphate precursors was demonstrated, and an immobilized enzyme mini-bioreactor investigated as a proof-of-principle for work-flow integration with NAAT in low-resource research and diagnostics labs.
期刊介绍:
Biotechnology & Bioengineering publishes Perspectives, Articles, Reviews, Mini-Reviews, and Communications to the Editor that embrace all aspects of biotechnology. These include:
-Enzyme systems and their applications, including enzyme reactors, purification, and applied aspects of protein engineering
-Animal-cell biotechnology, including media development
-Applied aspects of cellular physiology, metabolism, and energetics
-Biocatalysis and applied enzymology, including enzyme reactors, protein engineering, and nanobiotechnology
-Biothermodynamics
-Biofuels, including biomass and renewable resource engineering
-Biomaterials, including delivery systems and materials for tissue engineering
-Bioprocess engineering, including kinetics and modeling of biological systems, transport phenomena in bioreactors, bioreactor design, monitoring, and control
-Biosensors and instrumentation
-Computational and systems biology, including bioinformatics and genomic/proteomic studies
-Environmental biotechnology, including biofilms, algal systems, and bioremediation
-Metabolic and cellular engineering
-Plant-cell biotechnology
-Spectroscopic and other analytical techniques for biotechnological applications
-Synthetic biology
-Tissue engineering, stem-cell bioengineering, regenerative medicine, gene therapy and delivery systems
The editors will consider papers for publication based on novelty, their immediate or future impact on biotechnological processes, and their contribution to the advancement of biochemical engineering science. Submission of papers dealing with routine aspects of bioprocessing, description of established equipment, and routine applications of established methodologies (e.g., control strategies, modeling, experimental methods) is discouraged. Theoretical papers will be judged based on the novelty of the approach and their potential impact, or on their novel capability to predict and elucidate experimental observations.