Citation: Xinyi Luo, Ke Wang, Yingying Xue, Xiaobao Cao, Jianhua Zhou, Jiasi Wang. Digital PCR-free technologies for absolute quantitation of nucleic acids at single-molecule level[J]. Chinese Chemical Letters, ;2025, 36(2): 109924. doi: 10.1016/j.cclet.2024.109924 shu

Digital PCR-free technologies for absolute quantitation of nucleic acids at single-molecule level

Xinyi Luo ^{a, b, c} ,
Ke Wang ^{a, b} ,
Yingying Xue ^c ,
Xiaobao Cao ^{c, *,} ,
Jianhua Zhou ^{a, b, *,} ,
Jiasi Wang ^{a, b, *,}

a.
Guangdong Provincial Key Laboratory of Sensor Technology and Biomedical Instrument, School of Biomedical Engineering, Shenzhen Campus of Sun Yat-sen University, Shenzhen 518107, China
b.
School of Biomedical Engineering, Sun Yat-sen University, Guangzhou 510275, China
c.
Guangzhou National Laboratory, Guangzhou International Bio Island, Guangzhou 510005, China

cao_xiaobao@gzlab.ac.cn

zhoujh33@mail.sysu.edu.cn

wangjs8@mail.sysu.edu.cn

Received Date: 13 September 2023
Revised Date: 17 April 2024
Accepted Date: 24 April 2024
Available Online: 3 June 2024

Figures(3)

Ultrasensitive detection of nucleic acids is of great significance for precision medicine. Digital polymerase chain reaction (dPCR) is the most sensitive method but requires sophisticated and expensive instruments and a long reaction time. Digital PCR-free technologies, which mean the digital assay not relying on thermal cycling to amplify the signal for quantitative detection of nucleic acids at the single-molecule level, include the digital isothermal amplification techniques (dIATs) and the digital clustered regularly interspaced short palindromic repeats (CRISPR) technologies. They combine the advantages of dPCR and IATs, which could be fast and simple, enabling absolute quantification of nucleic acids at a single-molecule level with minimum instrument, representing the next-generation molecular diagnostic technology. Herein, we systematically summarized the strategies and applications of various dIATs, including the digital loop-mediated isothermal amplification (dLAMP), the digital recombinase polymerase amplification (dRPA), the digital rolling circle amplification (dRCA), the digital nucleic acid sequence-based amplification (dNASBA) and the digital multiple displacement amplification (dMDA), and evaluated the pros and cons of each method. The emerging digital CRISPR technologies, including the detection mechanism of CRISPR and the various strategies for signal amplification, are also introduced comprehensively in this review. The current challenges as well as the future perspectives of the digital PCR-free technology were discussed.
- Digital bioassay,
- Isothermal amplification,
- Nucleic acid detection,
- Digital CRISPR/Cas,
- Absolute quantification
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沈阳化工大学材料科学与工程学院沈阳 110142