单核苷酸多态性(SNP)检测是诊断疾病的关键,快速准确的诊断工具的开发对于治疗和预防至关重要。等位基因特异性聚合酶链反应(AS-PCR)被广泛用于检测具有多重功能的SNP,而基于CRISPR的技术在通过特异性指导RNA(gRNA)靶向突变位点方面提供了高灵敏度和特异性。在这项研究中,我们将CRISPR技术的高灵敏度和特异性与AS-PCR的多重功能相结合,实现了十个单碱基突变的同时检测。至于多重AS-PCR,我们的研究发现,靶向相同基因座的引物的竞争性抑制,加上这些引物不同的扩增效率,可能导致扩增效率降低。因此,我们调整并优化了引物组合和比例,以提高Multi-AS-PCR的扩增效率.最后,我们成功开发了一种新的巢式多AS-PCR-Cas12a方法用于多重SNP检测。为了评估这种方法在现实世界中的临床实用性,我们将其用于诊断利福平耐药的结核病(TB)。巢式多AS-PCR-Cas12a的检测限(LoD)为102aM,实现灵敏度,特异性,正预测值,阴性预测值为100%,93.33%,90.00%,100%,分别,与测序相比。总之,通过采用创新设计,将通用反向引物与十种不同的正向等位基因特异性引物结合在一起,巢式多AS-PCR-Cas12a技术有助于10个rpoB基因SNP的平行检测。这种方法还具有广泛的潜力,用于检测传染病和肿瘤中的耐药基因突变,以及特定遗传疾病的筛查。
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.