Wittwer Carl T, Zhou Luming, Ye Felix, Millington Adam, de Cola Adrian, Kusukawa Noriko
Crestwood Technology, Camden, Maine, USA.
Department of Pathology, University of Utah, Salt Lake City, Utah, USA.
Int J Lab Hematol. 2025 Apr 22. doi: 10.1111/ijlh.14487.
Over the past 40 years, molecular diagnostic methods have evolved from multi-step, time-consuming protocols towards either rapid targeted tests or expansive, massively parallel testing.
Here we consider the speed limits of targeted molecular diagnostics, considering the three sequential required steps: nucleic acid preparation, amplification, and analysis.
MATERIALS & METHODS: Instead of the bind/wash/elute steps commonly used for nucleic acid extraction, simple alkaline lysis of whole blood results in a suspension ready for PCR in seconds that can be added directly to an appropriately buffered PCR master mix. For amplification, the time requirements of PCR are typically limited by the temperature cycling instrumentation and not by biochemistry.
RESULTS & DISCUSSION: By lowering sample volumes, increasing the surface area to volume ratio, decreasing the thickness of the sample container, decreasing the amplicon size, and inducing rapid temperature changes by a myriad of innovative means, 30 cycles of PCR can easily be completed in less than 5 min. By increasing primer and polymerase concentrations in synchrony with even faster cycling (< 2 s cycles), "extreme PCR" has amplified a 60 bp human genomic target in < 15 s (35 cycles) with high yield and specificity. For analysis, cumbersome, contamination-prone gel analysis can be replaced by melting curve analysis. Although melting curve analysis usually takes up to an hour on commercial instrumentation, precise temperature control can enable single base genotyping in 1-4 s.
These advances demonstrate the feasibility of sample-to-answer molecular diagnostics in seconds.
在过去40年里，分子诊断方法已从多步骤、耗时的方案发展为快速靶向检测或广泛的大规模平行检测。
在此，我们考虑靶向分子诊断的速度限制，考量三个连续的必要步骤：核酸制备、扩增和分析。
全血简单碱性裂解可得到能直接加入适当缓冲的PCR预混液中的悬浮液，数秒内即可用于PCR，而非通常用于核酸提取的结合/洗涤/洗脱步骤。对于扩增，PCR的时间要求通常受温度循环仪器限制，而非受生物化学限制。
通过降低样品体积、增加表面积与体积比、减小样品容器厚度、减小扩增子大小以及通过多种创新手段实现快速温度变化，30个循环的PCR能轻松在不到5分钟内完成。通过同步增加引物和聚合酶浓度并采用更快的循环（<2秒循环），“极限PCR”已在<15秒（35个循环）内以高产率和特异性扩增了一个60bp的人类基因组靶点。对于分析，繁琐且易污染的凝胶分析可被熔解曲线分析取代。尽管在商业仪器上熔解曲线分析通常需要长达一小时，但精确的温度控制可在1 - 4秒内实现单碱基基因分型。
这些进展证明了数秒内实现样本到答案分子诊断的可行性。
