The fluorescence amplification strategy based on 3D DNA walker and CRISPR/Cas12a for the rapid detection of BRAF V600E

作者全名："Zhang, Wenxiu; Zhao, Shuhui; Xie, Zuowei; Chen, Siyi; Huang, Yuqi; Zhao, Zixin; Yi, Gang"

作者地址："[Zhang, Wenxiu; Zhao, Shuhui; Xie, Zuowei; Chen, Siyi; Huang, Yuqi; Zhao, Zixin; Yi, Gang] Chongqing Med Univ, Coll Lab Med, Key Lab Med Diagnost, Minist Educ, Chongqing 400016, Peoples R China"

通信作者："Yi, G (通讯作者)，Chongqing Med Univ, Coll Lab Med, Key Lab Med Diagnost, Minist Educ, Chongqing 400016, Peoples R China."

来源：ANALYTICAL SCIENCES

ESI学科分类：CHEMISTRY

WOS号：WOS:000807954700001

JCR分区：Q4

影响因子：1.6

年份：2022

卷号：　

期号：　

开始页：　

结束页：　

文献类型：Article; Early Access

关键词：Circulating tumor DNA; 3D DNA walker; CRISPR/Cas12a; Fluorescence

摘要："Circulating tumor DNA (ctDNA) is a fragment of single- or double-stranded DNA originating from tumor or circulating tumor cells and provides accurate information regarding the molecular characteristics of tumors. Therefore, sensitive detection of ctDNA is of great significance to mutation analysis and clinical diagnosis. Among various ctDNAs, the BRAF V600E is related to aggressive behavior, disease recurrence, and disease-specific mortality in papillary thyroid carcinoma. Herein, we selected the BRAF V600E gene sequence as an in vitro biomarker, and established a fluorescence detection strategy combined 3D DNA walker with CRISPR/Cas12a. In the presence of the target ctDNA, 3D DNA walker could identify and bind it, and thus released a large amount of output DNAs through cyclic cleavage with the assistance of specific endonuclease (Nb.BbvCI). The output DNAs were specifically bound to crRNA and activated the non-specific trans-cleavage activity of Cas12a. Finally, the fluorescence signal was significantly enhanced. Notably, this method can detect the BRAF V600E in a range of 1 fM similar to 20 nM with a detection limit of 0.37 fM without DNA polymerase. Due to the powerful amplification capability of 3D DNA walker and high specificity and programmability of CRISPR/Cas12a, the entire process took only a maximum of 70 min. Furthermore, it can be potentially used for the detection of ctDNA in human serum. In summary, this method not only provides a platform for the rapid detection of ctDNA, but also shows good potential for early clinical diagnosis and biomedical research."

基金机构：Key Project of Science and Technology Research Program of Chongqing Education Commission [KJZD-K202000404]

基金资助正文：This work was supported by the Key Project of Science and Technology Research Program of Chongqing Education Commission (Grant No. KJZD-K202000404).