核酸
清脆的
核酸检测
检出限
核糖核酸
计算生物学
生物
化学
色谱法
生物化学
基因
作者
Long Thanh Nguyễn,Jeevan Gurijala,Santosh R. Rananaware,Brianna L.M. Pizzano,Brandon Stone,Piyush Jain
出处
期刊:Methods
[Elsevier]
日期:2022-07-01
卷期号:203: 116-124
被引量:9
标识
DOI:10.1016/j.ymeth.2021.02.001
摘要
Rapid detection of nucleic acids is essential for clinical diagnosis of a wide range of infectious and non-infectious diseases. CRISPR-based diagnostic platforms are well-established for rapid and specific detection of nucleic acids but suffer from a low detection sensitivity without a target pre-amplification step. Our recently developed detection system, called CRISPR-ENHANCE, employs engineered crRNAs and optimized conditions to achieve a significantly higher sensitivity and enable femtomolar levels of nucleic acid detection even without target pre-amplification. Using the CRISPR-ENHANCE platform and following the methodology detailed in this paper, nucleic acid detection for low copy numbers can be achieved in less than an hour through either a fluorescence-based detection or a lateral flow assay. The step-by-step instructions provided, in addition to describing how to perform both assays, incorporate details on a LAMP/RT-LAMP-based target amplification step to enable detection of RNA, ssDNA and dsDNA. Furthermore, a protocol for in-house expression and purification of LbCas12a using CL7/lm7-based affinity chromatography, which has been used to achieve a high yield and purity of the enzyme in a single-step, is provided.
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