文献类型：期刊文献

英文题名：Development of a CRISPR/Cas12a one-tube POCT method with RCA for multiplex detection of SNPs

作者：Wu, Yaozhou[1,2];Sun, Yingying[1,3];Wang, Shijun[4];Wang, Xin[1];Li, Keke[1];Wang, Yulin[5];Lu, Zhangping[1];Liu, Qianqian[1];Wang, Wenjie[1];Dao, Yongchun[6];Chang, Yanbin[1];Wei, Lianhua[1,2]

第一作者：Wu, Yaozhou

通信作者：Chang, YB[1];Wei, LH[1]

机构：[1]Gansu Prov Hosp, Dept Clin Lab, 204 Donggang West Rd, Lanzhou 730000, Peoples R China;[2]Lanzhou Univ, Sch Clin Med 1, Lanzhou 730000, Peoples R China;[3]Gansu Univ Chinese Med, Sch Publ Hlth, Lanzhou 730000, Peoples R China;[4]Lanzhou Pulm Hosp, Dept Clin Lab, Lanzhou 730000, Peoples R China;[5]Ningxia Med Univ, Sch Clin Med 1, Yinchuan 750000, Peoples R China;[6]Huan Cty Peoples Hosp, Dept Clin Lab, Qingyang 745700, Peoples R China

第一机构：Gansu Prov Hosp, Dept Clin Lab, 204 Donggang West Rd, Lanzhou 730000, Peoples R China

通信机构：[1]corresponding author), Gansu Prov Hosp, Dept Clin Lab, 204 Donggang West Rd, Lanzhou 730000, Peoples R China.

年份：2025

卷号：210

外文期刊名：MICROCHEMICAL JOURNAL

收录：;Scopus(收录号：2-s2.0-85217733776);WOS:【SCI-EXPANDED(收录号:WOS:001428391700001)】；

基金：This work was supported by Multidisciplinary Collaborative Project of the Intramural Fund of Gansu Provincial Hospital (No. 16GSSY2-6) , Gansu Province Health Sector Project (No.GZK-2012-17) , and Health Commission of Gansu Province (GSWSKY2021-019) . The funders had no role in study design, data collection, and analysis, the decision to pub-lish, or the preparation of the manuscript.

语种：英文

外文关键词：CRISPR/Cas12a; Multiple; SNP; POCT; RCA

摘要：Background: The resistance of Mycobacterium tuberculosis (MTB) is becoming increasingly problematic, primarily due to single base mutations. To address this challenge, we have combined Rolling Circle Amplification (RCA) technology with the CRISPR/Cas12a system. Results: By optimizing ligation time, temperature, as well as the concentration and ratio of Padlock Probe probes, a method (RCA-Cas12a-Multi-SNP) was successfully developed that can detect four single nucleotide polymorphisms (SNPs) simultaneously. This method achieved a detection sensitivity of up to 104 aM and exhibits excellent specificity, with no cross-reactivity with DNA from other common clinical bacteria. In addition, we designed a system that integrates all reaction steps into a "cassette," enabling a one-tube point-of-care testing (POCT) method for detecting rifampicin-resistant genes in MTB. Compared with sequencing results, this method showed a sensitivity, specificity, positive predictive value, and negative predictive value of 100 %, 83.3 %, 90.9 %, and 100 %, respectively. Significance: Overall, we have established a thermostatic, multiplex, simple, and specific one-tube POCT method for SNPs, which can be used for rapid screening of rifampicin resistance in MTB. Additionally, this method can be applied to the rapid screening of genetic diseases, mutation detection for personalized medicine, monitoring cancer-related mutations, genetic screening, and genetic polymorphism analysis in agricultural and biological research.