Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, TX, 77843, USA.
Department of Nuclear Engineering, Texas A&M University, College Station, TX, 77843, USA.
Adv Sci (Weinh). 2024 Nov;11(41):e2404688. doi: 10.1002/advs.202404688. Epub 2024 Sep 13.
Isothermal PCR can be performed by imposing a static temperature gradient that continuously circulates reagents through denaturing, annealing, and extension conditions inside a PCR tube. But despite early promise, these systems have yet to demonstrate performance and repeatability sufficient for adoption in validated laboratory tests because the rate-limiting extension step is inherently short and cannot be increased independently of the other stages in a temperature cycle. Here, a discovery that enables isothermal PCR to be achieved with statistically robust repeatability that meets or exceeds diagnostic assay requirements (false positive/negative rate <8% at 95% confidence) by manipulating the interplay between the DNA replication biochemistry (via the amplicon GC content) and the microscale circulatory flow inside a PCR tube is reported. Surprisingly, optimal performance depends on selecting primer sequences that replicate high GC content amplicons, contradicting established PCR primer design rules. This innovative thermocycling approach accelerates PCR to speeds rivaling ultra-fast instruments, enabling rapid, repeatable isothermal DNA analysis across a range of targets relevant to diagnostics and pathogen detection.
等温 PCR 可以通过施加静态温度梯度来实现，该梯度通过在 PCR 管内的变性、退火和延伸条件下连续循环试剂来实现。但是，尽管早期有希望，但这些系统尚未表现出足够的性能和可重复性，无法在经过验证的实验室测试中采用，因为限速延伸步骤本身很短，并且不能独立于温度循环的其他阶段来增加。在这里，通过操纵 DNA 复制生物化学（通过扩增子 GC 含量）和 PCR 管内微尺度循环流动之间的相互作用，实现了具有统计学上稳健的可重复性的等温 PCR 的发现，这满足或超过了诊断分析物的要求（置信度为 95%时假阳性/阴性率<8%）。令人惊讶的是，最佳性能取决于选择复制高 GC 含量扩增子的引物序列，这与已建立的 PCR 引物设计规则相矛盾。这种创新的热循环方法将 PCR 加速到与超快速仪器相当的速度，能够在与诊断和病原体检测相关的一系列目标上进行快速、可重复的等温 DNA 分析。
