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

作     者：Xinyi Luo Ke Wang Yingying Xue Xiaobao Cao Jianhua Zhou Jiasi Wang 

作者机构：Guangdong Provincial Key Laboratory of Sensor Technology and Biomedical Instrument School of Biomedical Engineering Shenzhen Campus of Sun Yat-sen University School of Biomedical Engineering Sun Yat-sen University Guangzhou National LaboratoryGuangzhou International Bio Island 

出 版 物：《Chinese Chemical Letters》 (中国化学快报(英文版))

年 卷 期：2025年第36卷第2期

页      面：118-126页

核心收录：

学科分类：100208[医学-临床检验诊断学] 1002[医学-临床医学] 10[医学] 

基　　金：supported by the National Key Research and Development Program of China (Nos.2023YFC2307305,2021YFF0703300) the Shenzhen Medical Research Fund(No. B2303003) Shenzhen Research Funding Program (Nos.JCYJ20220818102014028, RCBS20210609104339043) National Natural Science Foundation of China (No. 22174167) Guangdong Basic and Applied Basic Research (No. 2024A1515011281) Fundamental Research Funds for the Central Universities (No.24qnpy087) from Sun Yat-sen University 

主　　题：Absolute quantification Digital bioassay Digital CRISPR/Cas Isothermal amplification Nucleic acid detection 

摘      要：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 singlemolecule level, include the digital isothermal amplification techniques (d IATs) and the digital clustered regularly interspaced short palindromic repeats (CRISPR) technologies. They combine the advantages of d PCR 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 sequencebased amplification (d NASBA) and the digital multiple displacement amplification (d MDA), 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.