1. A multi-AS-PCR-coupled CRISPR/Cas12a assay for the detection of ten single-base mutations.
- Author
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Wu, Yaozhou, Chang, Yanbin, Sun, Yingying, Wang, Yulin, Li, Keke, Lu, Zhangping, Liu, Qianqian, Wang, Fang, and Wei, Lianhua
- Subjects
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GENETIC testing , *GENETIC mutation , *POLYMERASE chain reaction , *MYCOBACTERIUM tuberculosis , *CRISPRS - Abstract
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 Multi-AS-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 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. [Display omitted] • The key strength of this study was the combination of CRISPR/Cas12a and Multi-AS-PCR tech to detect 10 gene mutations. • The Multi-AS-PCR-Cas12a method was established for multiple rpoB gene mutations detection and visualized with a UV lamp. • We first used Multi-AS-PCR-Cas12a to detect 10 gene mutations by designing primers, minimizing primer dimer formation. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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