College of Biophotonics & School of Life Sciences , South China Normal University , Guangzhou 510631 , China.
Guangdong Laboratory for Lingnan Modern Agriculture , Guangzhou 510642 , China.
ACS Nano. 2020 Feb 25;14(2):2497-2508. doi: 10.1021/acsnano.0c00022. Epub 2020 Feb 14.
The lateral flow assay is one of the most convenient analytical techniques for analyzing the immune response, but its applicability to precise genetic analyses is limited by the false-positive signal and tedious and inefficient hybridization steps. Here, we introduce the CRISPR (clustered regularly interspaced short palindromic repeats) /Cas system into the lateral flow assay, termed CRISPR/Cas9-mediated lateral flow nucleic acid assay (CASLFA), to address such issues. In this study, CASLFA is utilized to identify , genetically modified organisms (GMOs), and African swine fever virus (ASFV) at a detection limit of hundreds of copies of genome samples with high specificity within 1 h. We further evaluated the performance of CASLFA in a nonlaboratory environment and successfully confirmed 27 ASFV-infected samples from 110 suspected swine serum samples, with an accuracy of 100% when compared to real-time PCR (RT-PCR) assay. CASLFA satisfies some of the characteristics of a next-generation molecular diagnostics tool due to its rapidity and accuracy, allowing for point-of-care use without the need for technical expertise and complex ancillary equipment. This method has great potential for gene analysis in resource-poor or nonlaboratory environments.
侧向流动分析是分析免疫反应最方便的分析技术之一，但由于假阳性信号和繁琐低效的杂交步骤，其在精确的遗传分析中的适用性受到限制。在这里，我们将 CRISPR（成簇规律间隔短回文重复）/Cas 系统引入侧向流动分析中，称为 CRISPR/Cas9 介导的侧向流动核酸分析（CASLFA），以解决这些问题。在这项研究中，CASLFA 用于在 1 小时内以高特异性识别转基因生物（GMO）和非洲猪瘟病毒（ASFV），检测限低至数百个基因组样本拷贝。我们进一步评估了 CASLFA 在非实验室环境中的性能，并成功地从 110 份疑似猪血清样本中确认了 27 份 ASFV 感染样本，与实时 PCR（RT-PCR）检测相比，准确率为 100%。CASLFA 由于其快速性和准确性，满足了下一代分子诊断工具的一些特征，允许在没有技术专业知识和复杂辅助设备的情况下进行现场使用。该方法在资源匮乏或非实验室环境中的基因分析方面具有巨大潜力。
