A comprehensive review of methodological and technological advancement in PCR during the last 15 years

Polymerase chain reaction (PCR) has evolved from a foundational DNA amplification tool to a sophisticated analytical platform driving precision medicine. This review highlights PCR from fundamental principles to advanced techniques and diverse applications. It summarized the development of PCR technologies including nested PCR, quantitative PCR, multiplex PCR, and various kinds of microfluidic PCR (e.g., convective PCR and continuous flow PCR) that collectively enhanced sensitivity from micrograms to single molecules. Advancements in technology have propelled the application of PCR in two key directions: (1) integration with microfluidic chips for point-of-care testing (POCT), and (2) the shift from qualitative to absolute quantification, enabling the detection of single DNA molecules and thereby advancing the field of precision medicine. Furthermore, we discussed the application of machine vision and neural networks in digital PCR systems significantly enhanced the accuracy of positive microchamber identification in chip-based analyses.