文献类型：期刊文献

英文题名：A multi-AS-PCR-coupled CRISPR/Cas12a assay for the detection of ten single-base mutations

作者：Wu, Yaozhou[1,2];Chang, Yanbin[2];Sun, Yingying[3];Wang, Yulin[4];Li, Keke[2];Lu, Zhangping[4];Liu, Qianqian[3];Wang, Fang[2];Wei, Lianhua[1,2]

第一作者：Wu, Yaozhou

通信作者：Wei, LH[1];Wang, F[2];Wei, LH[2]

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

第一机构：Lanzhou Univ, Sch Clin Med 1, Lanzhou 730000, Peoples R China

通信机构：[1]corresponding author), Lanzhou Univ, Sch Clin Med 1, Lanzhou 730000, Peoples R China;[2]corresponding author), Gansu Prov Hosp, Dept Clin Lab, 204 Donggang West Rd, Lanzhou 730000, Peoples R China.

年份：2024

卷号：1320

外文期刊名：ANALYTICA CHIMICA ACTA

收录：;EI(收录号：20243116793280);Scopus(收录号：2-s2.0-85199868963);WOS:【SCI-EXPANDED(收录号:WOS:001285716900001)】；

基金：This work was supported by the Hospital Fund of Gansu Provincial Clinical Research Center for Laboratory Medicine (No. 21GSSYC-41) and Gansu Provincial Clinical Research Center for Laboratory Medicine (No. 21JR7RA676) . The funders had no role in study design, data collection, and analysis, the decision to publish, or the preparation of the manuscript.

语种：英文

外文关键词：Mycobacterium tuberculosis; CRISPR-Cas12a; Multiple allele-specific polymerase chain reaction; rpoB; SNP

摘要：Single-nucleotide polymorphism (SNP) detection is critical for diagnosing diseases, and the development of rapid and accurate diagnostic tools is essential for treatment and prevention. Allele-specific polymerase chain reaction (AS-PCR) is widely used for detecting SNPs with multiplexing capabilities, while CRISPR-based technologies provide high sensitivity and specificity in targeting mutation sites through specific guide RNAs (gRNAs). In this study, we have integrated the high sensitivity and specificity of CRISPR technology with the multiplexing capabilities of AS-PCR, achieving the simultaneous detection of ten single-base mutations. As for MultiAS-PCR, our research identified that competitive inhibition of primers targeting the same loci, coupled with divergent amplification efficiencies of these primers, could result in diminished amplification efficiency. Consequently, we adjusted and optimized primer combinations and ratios to enhance the amplification efficacy of Multi-AS-PCR. Finally, we successfully developed a novel nested Multi-AS-PCR-Cas12a method for multiplex SNPs detection. To evaluate the clinical utility of this method in a real-world setting, we applied it to diagnose rifampicin-resistant tuberculosis (TB). The limit of detection (LoD) for the nested Multi-AS-PCR-Cas12a was 102 2 aM, achieving sensitivity, specificity, positive predictive value, and negative predictive value of 100 %, 93.33 %, 90.00 %, and 100 %, respectively, compared to sequencing. In summary, by employing an innovative design that incorporates a universal reverse primer alongside ten distinct forward allele-specific primers, the nested Multi-AS-PCR-Cas12a technique facilitates the parallel detection of ten rpoB gene SNPs. This method also holds broad potential for the detection of drug-resistant gene mutations in infectious diseases and tumors, as well as for the screening of specific genetic disorders.