Weldon School of Biomedical Engineering , Purdue University , West Lafayette , Indiana 47907 , United States.
Institute for Cellular and Molecular Biology, Center for Systems and Synthetic Biology, and Department of Chemistry , The University of Texas at Austin , Austin , Texas 78712 , United States.
Anal Chem. 2018 Jun 5;90(11):6580-6586. doi: 10.1021/acs.analchem.8b00269. Epub 2018 May 8.
Sensitive and specific detection of pathogens via nucleic acid amplification is currently constrained to laboratory settings and portable equipment with costly fluorescent detectors. Nucleic acid-detecting lateral flow immunoassay strips (LFIAs) offer a low-cost visual transduction strategy at points of need. Unfortunately, these LFIAs frequently detect amplification byproducts that can yield spurious results which can only be deciphered through statistical analysis. We integrated customizable strand displacement probes into standard loop mediated isothermal amplification (LAMP) assays to prevent byproduct capture on commercial LFIAs. We find that combining strand displacement with LAMP (SD-LAMP) yields LFIA test band intensities that can be unequivocally interpreted by human subjects without additional instrumentation, thereby alleviating the need for a portable reader's analysis. Using SD-LAMP, we capture target amplicons on commercially available LFIAs from as few as 3.5 Vibrio cholerae and 2 750 Escherichia coli bacteria without false positive or false negative interpretation. Moreover, we demonstrate that LFIA capture of SD-LAMP products remain specific even in the presence of complex sample matrixes, providing a significant step toward reliable instrument-free pathogen detection outside of laboratories.
通过核酸扩增对病原体进行敏感和特异的检测目前受到限于实验室环境和带有昂贵荧光探测器的便携式设备。核酸检测横向流动免疫分析条（LFIAs）在需要的地方提供了低成本的视觉转换策略。不幸的是，这些 LFIAs 经常检测到扩增副产物，这可能会产生虚假结果，只能通过统计分析来解读。我们将定制的链置换探针整合到标准的环介导等温扩增（LAMP）检测中，以防止商业 LFIAs 上捕获副产物。我们发现，将链置换与 LAMP（SD-LAMP）相结合，可以产生 LFIA 测试带强度，无需额外的仪器，人类可以明确地解释，从而无需便携式阅读器进行分析。使用 SD-LAMP，我们可以从仅 3.5 个霍乱弧菌和 2750 个大肠杆菌细菌中捕获商业上可用的 LFIAs 上的靶扩增子，而不会出现假阳性或假阴性解释。此外，我们证明即使在存在复杂的样品基质的情况下，SD-LAMP 产物的 LFIA 捕获仍然是特异性的，这为在实验室外实现可靠的无仪器病原体检测迈出了重要的一步。
