Rapid Single-use PCR-based Nucleic Acid Testing

The goal of this project is to establish the first rapid PCR-based test kits where the reagents are co-packaged with a disposable thermocycling instrument.
Categories
Equipment and Supplies
Active Immunization Countermeasures

Industry Need

  • Increase the ability to perform nucleic acid analysis in a variety of PON settings 

Approach

The project objectives include:  

  1. Instrument and detection system design which will leverage recent technical breakthroughs to design and build a portable thermocycling instrument.  
    1. This will be achieved by integration of a hardware platform that imposes a prescribed temperature gradient to the PCR reagents using: 
      1. Resistive heating under battery power  
      2. Multi-well PCR tubes that support convective thermocycling and detection 
      3. Fluorescence analysis components 
  2. Assay development and validation which will be achieved by quantifying performance characteristics of thermocycling instrument and identify optimal design for protocol development. 
    1. This will involve evaluating a test panel of single- and multiplex amplification reactions to establish performance benchmarks pertinent to cell-based manufacturing, vaccine manufacturing, and diagnostic scenarios.  
    2. This will also establish a framework for optimal primer design and introduce protocols
  3. The platform including instrument configuration, user interface, and standardized assay test kits design will be finalized. 

Impacts

This project will catalyze a radical shift in biomanufacturing and diagnostics by enabling gold standard nucleic acid analysis to be routinely performed in virtually any PON setting.

In addition, the completion of this project will lay the groundwork for efforts to organize manufacturing partners, initiate regulatory processes, and engage in customer discovery so that deployment can begin immediately upon project completion.

Furthermore, the objectives of this project will lay the groundwork for efforts to organize manufacturing partners, initiate regulatory processes, and engage in customer discovery so that deployment can begin immediately upon project completion.

Outputs/Deliverables

  • The team applied microscale thermal convection to enable isothermal accelerated PCR, overcoming a critical barrier to portable PCR-based testing​
  • The team discovered how DNA amplification can be chemically programmed by manipulating the interplay between the PCR bio-chemistry and the microscale convective flow field​
  • The team discovered that chemical programmability is counterintuitively enabled by elevated amplicon GC contents that are significantly higher than the established recommended levels​
  • The team discovered that chemical programmability enables accelerated isothermal DNA amplification by prolonging extension, the rate-limiting step in the replication process, independent of all other stages in a temperature cycle, making it possible to achieve 100% repeatability at speeds that rival ultra-fast PCR instruments while using lower reagent concentrations than conventional protocols​
  • The team achieved rapid, highly repeatable PCR and RT-PCR with high portability potential, paving the way to deploy lab-quality nucleic acid-based diagnostics in decentralized settings​
  • The team produced prototype PCR tubes incorporating the newly discovered optimal design parameters and performed preliminary studies incorporate real-time melting curve analysis


Publications

Kim, M., Ravisankar, V., Hassan, Y. A., & Ugaz, V. M. (2024). Biochemically Programmable Isothermal PCR. Advanced Science, 11(41). https://doi.org/10.1002/advs.202404688

Kim, M., Ravisankar, V., Hassan, Y., & Ugaz, V. (2024) Biochemically Programmable Isothermal PCR, Wiley Advanced, https://doi.org/10.1002/advs.202404688

Posters

Kim, M.G, Ravisankar, V., Hassan, Y.A., and Ugaz, V.M. Isothermal Microfluidic PCR Enables Accelerated Pathogen Detection and Diagnostics. Poster M216.j. The 28th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2024. Montreal, Canada, October 14, 2024

Marsool, W., Hazeri, A.H., Ramamirtham, S.A.M., Ravisankar, V., Contreras-Naranjo, J.C., and Ugaz, V.M. Towards Rapid Real-Time DNA Analysis Using Quantitative Fluorescence in Convective PCR. Poster 174ao. 2024 Annual Meeting of the American Institute of Chemical Engineers, San Diego, CA, October 28, 2024

Ugaz, V., Presenter, ARP-23 Rapid Single-use PCR-based Nucleic Acid Testing, NIIMBL National Meeting, Washington, D.C., July 28, 2022.

Ugaz, V.M. Chemically Programmable Isothermal PCR. Poster PS2-12. 2024 NIIMBL National Meeting, June 26, 2024

Presentations

Kim, M.G., Ravisankar, V., Kim, M.G., Hassan, Y.A., and Ugaz, V.M., Presenter, Accelerating Pathogen Detection: Towards Portable, Rapid Isothermal PCR Diagnostics. Paper 436b. 2024 Annual Meeting of the American Institute of Chemical Engineers, San Diego, CA, October 29, 2024

Ugaz, V.M. Invited Keynote, Chemically Programmable Isothermal PCR Enables Rapid Nucleic Acid-based Bioanalysis at the Point of Need. Materials & Tools for Developing POC & Rapid Dx 2023. Laguna Hills, CA, November 30, 2023

Ugaz, V.M. Invited Oral Presentation, Chemically Programmable Isothermal PCR. NIIMBL Portfolio Snapshot of Member-Led Projects (Part II). 2024 NIIMBL National Meeting, June 26, 2024.

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Project Lead

Texas A&M University System

Texas A&M University System