环介导等温扩增
清脆的
反式激活crRNA
核酸
DNA
计算生物学
病菌
生物
病毒学
核糖核酸
分子诊断学
微生物学
遗传学
基因组编辑
基因
作者
Linxian Li,Shiyuan Li,Dayong Gu,Yong Xu,Jin Wang
出处
期刊:Methods in molecular biology
日期:2023-01-01
卷期号:: 267-278
标识
DOI:10.1007/978-1-0716-2950-5_15
摘要
Rapid detection of infectious and zoonotic diseases is very important for pathogen identification and infection control. Molecular diagnostic assays are well-known for high accuracy and sensitivity; however, conventional methods such as real-time PCR may require professional instruments and operations, preventing their wide applications in scenarios including animal quarantine. The recently developed CRISPR diagnostic (CRISPR-Dx) methods, employing the trans-cleavage activities of either Cas12 (e.g., HOLMES) or Cas13 (e.g., SHERLOCK), have shown great potential in rapid and convenient nucleic acid detection. Guided by specially designed CRISPR RNA (crRNA), Cas12 binds target DNA sequences and trans-cleaves ssDNA reporters, generating detectable signals, while Cas13 recognizes target ssRNA and trans-cleaves ssRNA reporters. To achieve high detection sensitivity, both HOLMES and SHERLOCK systems can be combined with pre-amplification procedures including both PCR and isothermal amplifications. Here, we present the employment of the HOLMESv2 method for convenient detection of the infectious and zoonotic diseases. Specifically, target nucleic acid is first amplified by LAMP or RT-LAMP, and the products are then detected by the thermophilic Cas12b. In addition, Cas12b reaction can be combined with LAMP amplification to achieve one-pot reaction systems. In this chapter, we provide a step-by-step description of the HOLMESv2-mediated rapid and sensitive detection of Japanese encephalitis virus (JEV), an RNA pathogen as an example.
科研通智能强力驱动
Strongly Powered by AbleSci AI