Active diagnostic ingredients (ADIs) for PCR: A mini-bioreactor producing dNTPs with silica immobilized R5-kinases

IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Anna R. Bird, Elizabeth A. H. Hall
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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.

Abstract Image

用于 PCR 的活性诊断成分 (ADI):用硅胶固定 R5 激酶生产 dNTPs 的微型生物反应器
Covid 大流行凸显了在感染爆发期间,特别是在资源较少的环境中,常规核酸扩增检测(NAAT)的可用性较低。障碍之一在于作为 NAAT 试剂的活性诊断成分(ADI)的供应链和成本。这项研究探索了一种新的合成方法,通过可重复使用的酶生物反应器生产一种关键的 NAAT 试剂,即 2′-脱氧核苷酸 5′-三磷酸酯(dNTPs),该方法可集成到 NAAT 工作流程中。设计了一种固定在二氧化硅上的自固定 R5-丝氨酸激酶融合酶,在 R5 激酶微型生物反应器中将 dNMPs 转化为 PCR 所需的相应 dNTP ADIs。在对λ基因组进行 PCR 反应时证明了其性能,并成功扩增了 7.5 kb。与商用 dNTP 相比,在疟疾疟原虫 NAATs 中,Ct 值与 log(拷贝数)之间呈高度线性相关,假阳性发生率低于商用 dNTP。总之,从单磷酸前体生成脱氧核苷酸的途径已得到证实,并对固定化酶微型生物反应器进行了研究,作为低资源研究和诊断实验室与 NAAT 工作流程整合的原理验证。
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来源期刊
Biotechnology and Bioengineering
Biotechnology and Bioengineering 工程技术-生物工程与应用微生物
CiteScore
7.90
自引率
5.30%
发文量
280
审稿时长
2.1 months
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.
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