A CRISPR-Cas12a-Assisted Fluorescence Platform for Rapid and Accurate Detection of Nocardia cyriacigeorgica.

Xueping Liu,Yutong Kang,Xiaotong Qiu,Shuai Xu,Lichao Han,Fang Li,Yuan Yue,Zhenjun Li,Yanlin Che,Shenglin Chen

doi:10.3389/fcimb.2022.835213

Abstract

Nocardia cyriacigeorgica has gradually become a common pathogen in clinical microbial infections. Identification of Nocardia at the species level is essential to assess the susceptibility and pathogenicity of antimicrobials. However, there is no suitable method for rapid and accurate laboratory detection of N. cyriacigeorgica. In this study, we combined PCR amplification with the CRISPR-Cas12a system to establish a novel detection platform, named CRISPR-PCR, and applied it to the detection of N. cyriacigeorgica in clinical samples. The Cas12a protein exhibited collateral cleavage activity following CRISPR RNA binding to specific targets, then indiscriminately cleaved nearby single-stranded DNA, and this was evaluated for diagnostic nucleic acid detection by measuring the fluorescence signal using a fluorescence reader. The assay takes only 2 h, including DNA extraction for 20 min, nucleic acid pre-amplification for 70 min, and fluorescence detection for 20 min. The limit of detection for N. cyriacigeorgica was 10-3 ng and the specificity was 100%. Thus, the N. cyriacigeorgica CRISPR-PCR assay is a rapid and specific method for detecting N. cyriacigeorgica, and the CRISPR-PCR fluorescence detection platform has great potential for detection of other pathogens.

Highlights

Nocardia bacteria are Gram-positive and partially acid-fast aerobic Actinomycetes (McTaggart et al, 2010)
More than 100 species belonging to the genus Nocardia have been recognised according to the National Center for Biotechnology Information (NCBI), and more than 50 species are of clinical significance (Conville et al, 2018)
We established the N. cyriacigeorgica CRISPR-PCR assay by combining the CRISPR-Cas12a system and PCR amplification to identify N. cyriacigeorgica DNA accurately and robustly, and the assay can be performed in routine laboratories

Summary

Introduction

Nocardia bacteria are Gram-positive and partially acid-fast aerobic Actinomycetes (McTaggart et al, 2010). The genus Nocardia is ubiquitous in environments associated with decaying vegetation and deposits from animals, and members are associated with water, soil and air (Brown-Elliott et al, 2006; Lebeaux et al, 2019). Nocardia can cause serious opportunistic infection in most organs, including traumatic infection, pulmonary disease and brain abscess (McTaggart et al, 2010; Huang et al, 2019; Lebeaux et al, 2019; Ji et al, 2020). The incidence of Nocardia infection is increased significantly in immunocompromised individuals (Minero et al, 2009; Coussement et al, 2016), including. CRISPR-Cas12a Detected Nocardia cyriacigeorgica advanced acquired immunodeficiency syndrome (AIDS) (Castro and Espinoza, 2007), patients undergoing organ transplantation (Coussement et al, 2016) and chronic lung disease. With an increase in immunocompromised patients and improvements in molecular identification of pathogens, the incidence of N. cyriacigeorgica infection is rising (Condas et al, 2013; Hashemi-Shahraki et al, 2015; Chen et al, 2017; Han et al, 2020)

Methods

Results

Conclusion