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

Publication date: Dec 01, 2024

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 4^0C. 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 C 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.

Concepts Keywords
5kb Bioreactors
Biotechnol COVID-19
Malariae dNTPs
Pcr Enzymes, Immobilized
Triphosphate Enzymes, Immobilized
Humans
low resource areas
mini‐bioreactor
Polymerase Chain Reaction
SARS-CoV-2
Silicon Dioxide
Silicon Dioxide

Semantics

Type Source Name
drug DRUGBANK Silicon dioxide
disease IDO nucleic acid
disease MESH infection
disease IDO reagent
disease MESH immobilization
disease MESH COVID-19

Original Article

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