(436b) Accelerating Pathogen Detection: Towards Portable, Rapid Isothermal PCR Diagnostics

The polymerase chain reaction (PCR) is a gold standard for nucleic acid-based diagnostics, but portability and accessibility are constrained by the electrical power associated with cycling through different temperatures to denature, anneal, and extend the DNA strands. This constraint significantly limits the portability and accessibility of PCR, especially critical in pandemic response and public health monitoring. Consequently, the ability to perform PCR isothermally has long been sought-after to overcome these barriers. However, the PCR-like isothermal DNA replication methods developed thus far (e.g., loop-mediated isothermal amplification (LAMP)), while promising in specific applications, have yet to consistently match the performance and reliability of PCR. Here, we report a discovery that overcomes these limitations by enabling rapid isothermal PCR to be achieved with 100% repeatability. This breakthrough is accomplished by manipulating the interplay between the PCR biochemistry (via the amplicon GC content) and the lava-lamp-like microscale flow established inside a PCR tube whose upper and lower surfaces are isothermally maintained at annealing and denaturing temperatures. Surprisingly, we find that optimal performance critically depends on selecting primers that replicate amplicons with high GC content, contradicting established PCR primer design rules. Our approach accelerates PCR to speeds that rival ultra-fast instruments, enabling rapid, repeatable isothermal PCR across various targets relevant to diagnostics and pathogen detection. By allowing rapid repeatable isothermal PCR, our findings pave the way to make nucleic acid-based analysis systems accessible, reliable, and efficient so that lab-quality diagnostics can be broadly and equitably deployed.