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A multi-AS-PCR-coupled CRISPR/Cas12a assay for the detection of ten single-base mutations.
Wu, Yaozhou; Chang, Yanbin; Sun, Yingying; Wang, Yulin; Li, Keke; Lu, Zhangping; Liu, Qianqian; Wang, Fang; Wei, Lianhua.
Afiliação
  • Wu Y; First School of Clinical Medicine, Lanzhou University, Lanzhou, 730000, PR China; Department of Clinical Laboratory, Gansu Provincial Hospital, Lanzhou, 730000, PR China.
  • Chang Y; Department of Clinical Laboratory, Gansu Provincial Hospital, Lanzhou, 730000, PR China.
  • Sun Y; First School of Clinical Medicine, Ningxia Medical University, Yinchuan, 750000, PR China.
  • Wang Y; School of Public Health, Gansu University of Chinese Medicine, Lanzhou, 730000, PR China.
  • Li K; Department of Clinical Laboratory, Gansu Provincial Hospital, Lanzhou, 730000, PR China.
  • Lu Z; School of Public Health, Gansu University of Chinese Medicine, Lanzhou, 730000, PR China.
  • Liu Q; First School of Clinical Medicine, Ningxia Medical University, Yinchuan, 750000, PR China.
  • Wang F; Department of Clinical Laboratory, Gansu Provincial Hospital, Lanzhou, 730000, PR China. Electronic address: 524865826@qq.com.
  • Wei L; First School of Clinical Medicine, Lanzhou University, Lanzhou, 730000, PR China; Department of Clinical Laboratory, Gansu Provincial Hospital, Lanzhou, 730000, PR China. Electronic address: 3752131518@qq.com.
Anal Chim Acta ; 1320: 343027, 2024 Sep 01.
Article em En | MEDLINE | ID: mdl-39142774
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.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Polimorfismo de Nucleotídeo Único / Sistemas CRISPR-Cas Limite: Humans Idioma: En Revista: Anal Chim Acta Ano de publicação: 2024 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Polimorfismo de Nucleotídeo Único / Sistemas CRISPR-Cas Limite: Humans Idioma: En Revista: Anal Chim Acta Ano de publicação: 2024 Tipo de documento: Article