An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip  

作　　者：Jiaojiao Gong Lijuan Kan Xiuming Zhang Ying He Jiaqiang Pan Liping Zhao Qianyun Li Menghao Liu Jie Tian Sili Lin Zhouyu Lu Liang Xue Chaojun Wang Guanghui Tang 

机构地区：[1]Yaneng Biotech,Co.,Ltd,Fosun Pharma,Shenzhen 518100,China [2]Department of Urology,The First Affiliated Hospital,Zhejiang University School of Medicine,Hangzhou 310003,China [3]Department of Laboratory Medicine,Luohu District People’s Hospital,Shenzhen 518001,China [4]Department of Laboratory Medicine,The Eighth Affiliated Hospital,Sun Yat-sen University,Shenzhen 518033,China [5]Department of Laboratory Medicine,Nanning First People’s Hospital,Nanning 530022,China [6]Department of Neurology,Hwa Mei Hospital,University of Chinese Academy of Sciences,Ningbo 315010,China [7]Nanobiological Medicine Center,Key Lab of Fuel Cell Technology of Guangdong Province,School of Chemistry and Chemical Engineering,South China University of Technology,Guangzhou 510641,China

出　　处：《Bioactive Materials》2021年第12期4580-4590,共11页生物活性材料（英文）

基　　金：This work was supported by the Discipline Construction Ability Promotion Project of Shenzhen Health and Population Family Planning Commission(NO.SZXJ2018031(Kan L.J.));Sanming Project of Medicine in Shenzhen(NO.SZSM201601062(Zhang X.M.));Shenzhen Key Medical Discipline Construction Fund(NO.SZXK054(Zhang X.M.)).

摘　　要：CRISPR-Cas12a system has been shown promising for nucleic acid diagnostics due to its rapid,portable and accurate features.However,cleavage of the amplicons and primers by the cis-and trans-activity of Cas12a hinders the attempts to integrate the amplification and detection into a single reaction.Through phosphorothioate modification of primers,we realized onepot detection with high sensitivity using plasmids of SARS-CoV-2,HPV16 and HPV18.We also identified the activated Cas12a has a much higher affinity to C nucleotide-rich reporter than others.By applying such reporters,the reaction time required for a lateral-flow readout was significantly reduced.Furthermore,to improve the specificity of the strip-based assay,we created a novel reporter and,when combined with a customized gold-nanopaticle strip,the readout was greatly enhanced owing to the elimination of the nonspecific signal.This established system,termed Targeting DNA by Cas12a-based Eye Sight Testing in an Onepot Reaction(TESTOR),was validated using clinical cervical scrape samples for human papillomaviruses(HPVs)detection.Our system represents a general approach to integrating the nucleic acid amplification and detection into a single reaction in CRISPR-Cas systems,highlighting its potential as a rapid,portable and accurate detection platform of nucleic acids.

关 键 词：CRISPR-Cas12a Onepot Visual detection 

分 类 号：R318[医药卫生—生物医学工程]