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

Zhenqing Li; Xingyu Yang; Bo Yang; Jing Yang; Chunxian Tao; Dawei Zhang; Yoshinori Yamaguchi

doi:10.1016/j.biotechadv.2025.108719

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

Biotechnol Adv. 2025 Dec:85:108719. doi: 10.1016/j.biotechadv.2025.108719. Epub 2025 Sep 27.

Authors

Zhenqing Li¹, Xingyu Yang¹, Bo Yang¹, Jing Yang², Chunxian Tao¹, Dawei Zhang³, Yoshinori Yamaguchi⁴

Affiliations

¹ Engineering Research Center of Optical Instrument and System, Key Lab of Optical Instruments and Equipment for Medical Engineering, Ministry of Education, Shanghai Key Lab of Modern Optical System, University of Shanghai for Science and Technology, Shanghai 200093, China.
² Faculty of Engineering, Anhui Sanlian University, Hefei 230000, China.
³ Engineering Research Center of Optical Instrument and System, Key Lab of Optical Instruments and Equipment for Medical Engineering, Ministry of Education, Shanghai Key Lab of Modern Optical System, University of Shanghai for Science and Technology, Shanghai 200093, China. Electronic address: dwzhang@usst.edu.cn.
⁴ Comprehensive Research Organization, Waseda University, Tokyo 162-0041, Japan.

PMID: 41022185
DOI: 10.1016/j.biotechadv.2025.108719

Abstract

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.

Keywords: Continuous flow PCR; Digital PCR; Natural convective PCR; Nested PCR; Oscillating flow PCR; Polymerase chain reaction.

Publication types

Review

MeSH terms

Humans
Lab-On-A-Chip Devices
Polymerase Chain Reaction* / methods