Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Bünteweg 17p, 30559 Hannover, Germany.
Genes (Basel). 2022 Nov 2;13(11):2007. doi: 10.3390/genes13112007.
The rapid rate of virus transmission and pathogen mutation and evolution highlight the necessity for innovative approaches to the diagnosis and prevention of infectious diseases. Traditional technologies for pathogen detection, mostly PCR-based, involve costly/advanced equipment and skilled personnel and are therefore not feasible in resource-limited areas. Over the years, many promising methods based on clustered regularly interspaced short palindromic repeats and the associated protein systems (CRISPR/Cas), i.e., orthologues of Cas9, Cas12, Cas13 and Cas14, have been reported for nucleic acid detection. CRISPR/Cas effectors can provide one-tube reaction systems, amplification-free strategies, simultaneous multiplex pathogen detection, visual colorimetric detection, and quantitative identification as alternatives to quantitative PCR (qPCR). This review summarizes the current development of CRISPR/Cas-mediated molecular diagnostics, as well as their design software and readout methods, highlighting technical improvements for integrating CRISPR/Cas technologies into on-site applications. It further highlights recent applications of CRISPR/Cas-based nucleic acid detection in livestock industry, including emerging infectious diseases, authenticity and composition of meat/milk products, as well as sex determination of early embryos.
病毒传播和病原体突变与进化的快速率突出了创新方法在传染病诊断和预防中的必要性。传统的病原体检测技术主要基于 PCR，涉及昂贵/先进的设备和熟练的人员，因此在资源有限的地区不可行。多年来，已经报道了许多基于成簇规律间隔短回文重复序列和相关蛋白系统 (CRISPR/Cas) 的有前途的方法，即 Cas9、Cas12、Cas13 和 Cas14 的同源物，用于核酸检测。CRISPR/Cas 效应物可以提供单管反应系统、无扩增策略、同时多重病原体检测、可视化比色检测和定量鉴定，作为定量聚合酶链反应 (qPCR) 的替代品。本综述总结了 CRISPR/Cas 介导的分子诊断的最新发展，以及它们的设计软件和读出方法，突出了将 CRISPR/Cas 技术集成到现场应用中的技术改进。它进一步强调了基于 CRISPR/Cas 的核酸检测在畜牧业中的最新应用，包括新发传染病、肉/奶制品的真实性和成分以及早期胚胎的性别鉴定。
