CAS Key Laboratory of Quantitative Engineering Biology, Guangdong Provincial Key Laboratory of Synthetic Genomics and Shenzhen Key Laboratory of Synthetic Genomics, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
Anal Chem. 2023 May 23;95(20):8063-8069. doi: 10.1021/acs.analchem.3c01033. Epub 2023 May 11.
It is well-established that different detection modes are necessary for corresponding applications, which can effectively reduce matrix interference and improve the detection accuracy. Here, we reported a magnetic separation method based on recombinase polymerase amplification (RPA)-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a for dual-mode analysis of African swine fever virus (ASFV) genes, including colorimetry and fluorescence. The ASFV gene was selected as the initial RPA template to generate the amplicon. The RPA amplicon was then recognized by CRISPR-associated RNA (crRNA), activating the -cleavage activity of Cas12a and leading to the nonspecific cleavage of ssDNA as well as a significant release of alkaline phosphatase (ALP) in the ALP-ssDNA modified magnetic bead. The released ALP can catalyze -nitrophenyl phosphate to generate -nitrophenol, resulting in substantial changes in absorbance and fluorescence, both of which can be used for detection with the naked eye. This strategy allows the sensitive detection of ASFV DNA, with a 20 copies/mL detection limit; no cross-reactivity with other viruses was observed. A good linear relationship was obtained in serum. In addition, this sensor displayed 100% specificity and sensitivity for clinical sample analysis. This method integrates the high sensitivity of fluorescence with easy readout of colorimetry and enables a simple, low-cost, and highly sensitive dual-mode detection of viral nucleic acid, thereby providing a broad prospect for the practical application in the diagnosis of virus infection.
已经证实，不同的检测模式对于相应的应用是必要的，这可以有效地减少基质干扰并提高检测准确性。在这里，我们报道了一种基于重组酶聚合酶扩增（RPA）辅助的簇状规则间隔短回文重复序列（CRISPR）/Cas12a 的磁性分离方法，用于非洲猪瘟病毒（ASFV）基因的双模式分析，包括比色法和荧光法。ASFV 基因被选为初始 RPA 模板，以生成扩增子。然后，RPA 扩增子被 CRISPR 相关 RNA（crRNA）识别，激活 Cas12a 的切割活性，导致 ssDNA 的非特异性切割以及 ALP-ssDNA 修饰磁珠中碱性磷酸酶（ALP）的显著释放。释放的 ALP 可以催化 -硝基苯磷酸生成 -硝基苯酚，导致吸光度和荧光发生显著变化，两者均可肉眼检测。该策略允许对 ASFV DNA 进行灵敏检测，检测限为 20 个拷贝/mL；与其他病毒没有交叉反应。在血清中获得了良好的线性关系。此外，该传感器对临床样本分析显示出 100%的特异性和灵敏度。该方法将荧光的高灵敏度与比色法的易读性相结合，实现了病毒核酸的简单、低成本、高灵敏度的双模式检测，为病毒感染诊断的实际应用提供了广阔的前景。
