Department of Chemistry, Colorado State University, Fort Collins, CO, 80523, USA.
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia.
Anal Bioanal Chem. 2020 May;412(13):3051-3061. doi: 10.1007/s00216-020-02569-w. Epub 2020 Mar 19.
Pathogen detection is crucial for human, animal, and environmental health; crop protection; and biosafety. Current culture-based methods have long turnaround times and lack sensitivity. Nucleic acid amplification tests offer high specificity and sensitivity. However, their cost and complexity remain a significant hurdle to their applications in resource-limited settings. Thus, point-of-need molecular diagnostic platforms that can be used by minimally trained personnel are needed. The nuclease protection assay (NPA) is a nucleic acid hybridization-based technique that does not rely on amplification, can be paired with other methods to improve specificity, and has the potential to be developed into a point-of-need device. In traditional NPAs, hybridization of an anti-sense probe to the target sequence is followed by single-strand nuclease digestion. The double-stranded target-probe hybrids are protected from nuclease digestion, precipitated, and visualized using autoradiography or other methods. We have developed a paper-based nuclease protection assay (PB-NPA) that can be implemented in field settings as the detection approach requires limited equipment and technical expertise. The PB-NPA uses a lateral flow format to capture the labeled target-probe hybrids onto a nitrocellulose membrane modified with an anti-label antibody. A colorimetric enzyme-substrate pair is used for signal visualization, producing a test line. The nuclease digestion of non-target and mismatched DNA provides high specificity while signal amplification with the reporter enzyme-substrate provides high sensitivity. We have also developed an on-chip sample pretreatment step utilizing chitosan-modified paper to eliminate possible interferents from the reaction and preconcentrate nucleic acids, thereby significantly reducing the need for auxiliary equipment. Graphical abstract.
病原体检测对于人类、动物和环境健康、作物保护和生物安全至关重要。目前基于培养的方法具有较长的周转时间且缺乏敏感性。核酸扩增测试具有高特异性和敏感性。然而，它们的成本和复杂性仍然是在资源有限的环境中应用的重大障碍。因此，需要能够由训练有素的人员在现场使用的即时分子诊断平台。核酸酶保护分析（NPA）是一种基于核酸杂交的技术，不依赖于扩增，可以与其他方法结合使用以提高特异性，并有可能开发成即时检测设备。在传统的 NPA 中，反义探针与靶序列的杂交随后是单链核酸酶消化。双链靶标-探针杂交体免受核酸酶消化，通过放射性自显影或其他方法沉淀并可视化。我们开发了一种基于纸张的核酸酶保护分析（PB-NPA），可以在现场环境中实施，因为检测方法需要有限的设备和技术专业知识。PB-NPA 使用侧流格式将标记的靶标-探针杂交体捕获到带有抗标记抗体的硝酸纤维素膜上。使用比色酶底物对用于信号可视化，产生测试线。非靶标和错配 DNA 的核酸酶消化提供了高特异性，而报告酶底物的信号放大提供了高灵敏度。我们还开发了一种基于芯片的样品预处理步骤，利用壳聚糖修饰的纸张消除反应中的可能干扰物并预浓缩核酸，从而大大减少对辅助设备的需求。图表摘要。
