Zhang Likun, Ma Xueping, Liu Danni, Shan Jingwen, Chu Yanan, Zhang Jieyu, Qi Xiemin, Huang Xiaohui, Zou Bingjie, Zhou Guohua
Department of Clinical Pharmacy, Affiliated Jinling Hospital, State Key Laboratory of Analytical Chemistry for Life Science & Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing, 210002, China.
Key Laboratory of Drug Quality Control and Pharmacovigilance of Ministry of Education, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China.
J Biomed Nanotechnol. 2022 Feb 1;18(2):394-404. doi: 10.1166/jbn.2022.3258.
A simple and rapid genotyping method with less-instrumentation is essential for realizing point-of-care detection of personalized medicine-related gene biomarkers. Herein, we developed a rapid and visualized genotyping method by coupling recombinase polymerase amplification (RPA) with allele-specific invader reaction assisted gold nanoparticle probes assembling. In the method, the DNA targets were firstly amplified by using RPA, which is a rapid isothermal amplification technology. Then an allele-specific invasion reaction was performed to recognize the single nucleotide polymorphisms (SNPs) site in the amplicons, to produce signal molecules that caused discoloration of gold nanoparticle probes. As a result, genotyping was achieved by observing the color change of the reaction by using naked eye without the requirement for any expensive instrument. In order to achieve rapid genotyping detection, the genomic DNA from oral swab lysate samples were used for the RPA templates amplification. In this way, a visualized genotyping from "samples to results" within 25 min was realized. Two clopidogrel related SNPs and of 56 clinical samples were correctly genotyped by using this rapid visualized genotyping assay. In addition, the feasibility for this pathogen genotyping method was also verified by detecting plasmid DNA containing three SARS-COV-2 gene mutation sites, indicating that this method has the potential for clinical sample detection.
一种简单快速且仪器设备需求少的基因分型方法对于实现个性化医疗相关基因生物标志物的即时检测至关重要。在此，我们通过将重组酶聚合酶扩增（RPA）与等位基因特异性入侵反应辅助金纳米颗粒探针组装相结合，开发了一种快速且可视化的基因分型方法。在该方法中，首先使用RPA扩增DNA靶标，RPA是一种快速等温扩增技术。然后进行等位基因特异性入侵反应以识别扩增子中的单核苷酸多态性（SNP）位点，产生导致金纳米颗粒探针变色的信号分子。结果，无需任何昂贵仪器，通过肉眼观察反应的颜色变化即可实现基因分型。为了实现快速基因分型检测，将口腔拭子裂解液样本中的基因组DNA用作RPA模板进行扩增。通过这种方式，在25分钟内实现了从“样本到结果”的可视化基因分型。使用这种快速可视化基因分型检测方法对56份临床样本中的两个氯吡格雷相关SNP进行了正确的基因分型。此外，通过检测含有三个SARS-CoV-2基因突变位点的质粒DNA，也验证了这种病原体基因分型方法的可行性，表明该方法具有临床样本检测的潜力。
