Clustered\nRegularly Interspaced Short Palindromic\nRepeats/Cas9-Mediated Lateral Flow Nucleic Acid Assay

Abstract

The\nlateral flow assay is one of the most convenient analytical\ntechniques for analyzing the immune response, but its applicability\nto precise genetic analyses is limited by the false-positive signal\nand tedious and inefficient hybridization steps. Here, we introduce\nthe CRISPR (clustered regularly interspaced short palindromic repeats)\n/Cas system into the lateral flow assay, termed CRISPR/Cas9-mediated\nlateral flow nucleic acid assay (CASLFA), to address such issues.\nIn this study, CASLFA is utilized to identify <i>Listeria monocytogenes</i>, genetically modified organisms (GMOs), and African swine fever\nvirus (ASFV) at a detection limit of hundreds of copies of genome\nsamples with high specificity within 1 h. We further evaluated the\nperformance of CASLFA in a nonlaboratory environment and successfully\nconfirmed 27 ASFV-infected samples from 110 suspected swine serum\nsamples, with an accuracy of 100% when compared to real-time PCR (RT-PCR)\nassay. CASLFA satisfies some of the characteristics of a next-generation\nmolecular diagnostics tool due to its rapidity and accuracy, allowing\nfor point-of-care use without the need for technical expertise and\ncomplex ancillary equipment. This method has great potential for gene\nanalysis in resource-poor or nonlaboratory environments.