(160aa) Reliability and Reproducibility of Amplification in Rapid PCR-Based Nucleic Acid Testing

The polymerase chain reaction (PCR) is a gold-standard technique that plays a critical role in pathogen diagnostics and biomanufacturing. And PCR has been implemented in numerous systems with high portability potential (e.g., microfluidic approaches). But very few of these systems have been adopted for routine use because of a lack of quantitative data to support their validation as accepted laboratory methods. We have recently discovered a thermocycling approach that overcomes this barrier by exploiting the ability to isothermally perform the PCR via convective flow in a format that dramatically reduces complexity and electrical power consumption, and can be readily interfaced with most detection methods. Here, we present the results of new studies that establish a link between the PCR tube design (which governs the physics of convective flow and thermocycling) and the primer design (which regulates the PCR biochemistry). These studies’ results make it possible to identify operating conditions where ~100% reproducibility is achievable, providing critical quantitative data to support the adoption of convective thermocycling as part of validated laboratory methods. These insights also point to convective thermocycling’s potential to deliver superior results in applications (e.g., GC-rich amplicons) where PCR replication is challenging.