Abstract：
Digital PCR (dPCR) has become indispensable in nucleic acid (NA) detection across various fields, including viral diagnostics and mutant detection. However, misclassification of partitions in dPCR can significantly impact accuracy. Despite existing methods to minimize misclassification bias, accurate classification remains elusive, especially for nonamplified target partitions. To address these challenges, this study introduces an innovative microdroplet-based competitive PCR platform for nucleic acid quantification in microfluidic devices independent of Poisson statistics. In this approach, the target concentration (T) is determined from the concentration of competitor DNA (C) at the equivalence point (E.P.), where C/T is 1. Competitive PCR ensures that the ratio of target to competitor DNA remains constant during amplification, reflected in the resultant fluorescence intensity, allowing the quantification of target DNA concentration at the equivalence point. The unique amplification technique eliminates Poisson distribution, addressing misclassification challenges. Additionally, our approach reduces the need for post-PCR procedures and shortens analytical time. We envision this platform as versatile, reproducible, and easily adaptable for driving significant progress in molecular biology and diagnostics.
摘要：
数字PCR(dPCR)已成为各种领域核酸(NA)检测中不可或缺的,包括病毒诊断和突变检测。然而,dPCR中分区的错误分类会显著影响准确性。尽管现有方法可以最大限度地减少误分类偏差，准确的分类仍然难以捉摸，特别是对于非扩增的目标分区。为了应对这些挑战，本研究引入了一种创新的基于微滴的竞争性PCR平台，用于微流体设备中的核酸定量，而与泊松统计无关。在这种方法中，目标浓度(T)由竞争物DNA(C)在等效点(E.P.)的浓度确定,C/T为1。竞争性PCR确保靶与竞争DNA的比例在扩增过程中保持恒定，反映在所得的荧光强度中，允许在等效点定量靶DNA浓度。独特的放大技术消除了泊松分布，解决错误分类的挑战。此外,我们的方法减少了对PCR后程序的需要,并缩短了分析时间.我们设想这个平台是多功能的，可重复,容易适应推动分子生物学和诊断的重大进展。
