Shi Qingyuan, Li Jie, Liu Chunchen, Zhai Xuanpei, Chen Long, Zhang Ye, Feng Dezhi, Zhang Rong, Li Jian, Ling Shengjie, Zheng Lei, Luo Yuan, Liu Yifan
State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai, 200050, China; School of Physical Science and Technology, ShanghaiTech University, Shanghai, 201210, China.
School of Physical Science and Technology, ShanghaiTech University, Shanghai, 201210, China.
Biosens Bioelectron. 2023 Dec 1;241:115702. doi: 10.1016/j.bios.2023.115702. Epub 2023 Sep 22.
Digital PCR (dPCR) is considered the next generation of nucleic acid detection for its ability of absolute quantification and high sensitivity. However, when compared to the current gold standard, quantitative PCR (qPCR), dPCR is falling behind by several orders of magnitude in dynamic range, which limits its clinical applicability. Here we present fluorescence-coded logarithmic-dilution digital droplet PCR (Flodd-PCR) that features a dynamic range across 7 orders of magnitude, over 2 orders higher than conventional dPCR (4-5 log range) and approaching that of qPCR (7-8 log range). Flodd-PCR realizes such a wide dynamic range by dividing ∼20,000 droplets into 4 groups, each featuring a unique dilution factor of the loaded DNA template and thus a shifted dynamic range. This is achieved by a microfluidic chip that performs multi-step serial dilution (20-925 folds) and droplet generation. The post-PCR droplets can be clustered in silico based on their dilution indicator fluorescence and analyzed independently. Experimentally, Flodd-PCR can detect 4-20,000,000 copies/μL (cp./μL) of the synthetic human papillomavirus (HPV) DNA and outperforms standard dPCR when analyzing clinical HPV samples. Furthermore, Flodd-PCR can be implemented with existing dPCR system set-up with minimal adjustment, and therefore will also have wide practicality in different applications which conventional dPCR has already demonstrated.
数字PCR（dPCR）因其绝对定量能力和高灵敏度而被视为下一代核酸检测技术。然而，与当前的金标准定量PCR（qPCR）相比，dPCR在动态范围上落后了几个数量级，这限制了其临床适用性。在此，我们展示了荧光编码对数稀释数字液滴PCR（Flodd-PCR），其动态范围跨越7个数量级，比传统dPCR（4-5个对数范围）高出2个多数量级，接近qPCR（7-8个对数范围）。Flodd-PCR通过将约20,000个液滴分为4组来实现如此宽的动态范围，每组具有加载的DNA模板的独特稀释因子，从而具有偏移的动态范围。这是通过一个微流控芯片实现的，该芯片执行多步连续稀释（20-925倍）并生成液滴。PCR后的液滴可以基于其稀释指示荧光在计算机上进行聚类并独立分析。实验表明，Flodd-PCR可以检测合成人乳头瘤病毒（HPV）DNA的4-20,000,000拷贝/微升（cp./μL），并且在分析临床HPV样本时优于标准dPCR。此外，Flodd-PCR可以在现有dPCR系统设置下进行最小调整来实现，因此在传统dPCR已证明的不同应用中也将具有广泛的实用性。
