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

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Biochemistry

Background:

• The Covid pandemic highlighted low availability of nucleic acid amplification testing (NAAT) in resource-limited settings.
• Supply chain and cost of active diagnostic ingredients (ADIs), such as deoxynucleoside triphosphates (dNTPs), are key barriers to NAAT accessibility.

Purpose of the Study:

• To develop a novel synthesis method for producing dNTPs using a reusable enzyme bioreactor.
• To demonstrate the integration of this bioreactor into a NAAT workflow for improved diagnostics.

Main Methods:

• Designed a self-immobilizing R5-silaffin kinase fusion enzyme for silica immobilization.
• Developed an R5-kinase mini-bioreactor to convert deoxynucleoside monophosphates (dNMPs) to dNTP ADIs.
• Tested the bioreactor's stability and performance in PCR amplification of lambda genome and Plasmodium malariae NAATs.

Main Results:

• The reusable enzyme bioreactor demonstrated stability for over 2 months at 4°C.
• Successful PCR amplification up to 7.5 kb of the lambda genome was achieved.
• Plasmodium malariae NAATs showed a high linear correlation between Ct value and log(Copy Number) with fewer false positives compared to commercial dNTPs.

Conclusions:

• Demonstrated a viable pathway for generating deoxynucleotides from monophosphate precursors.
• Investigated an immobilized enzyme mini-bioreactor as a proof-of-principle for workflow integration.
• This approach offers a potential solution for low-resource research and diagnostics labs to enhance NAAT capabilities.