Rapid visualization of Clostridioides difficile toxins A and B by multiplex RPA combined with CRISPR-Cas12a

Tong Jiang; Xinyi Hu; Chunhui Lin; Zhaoxin Xia; Wensu Yang; Yi Zhu; Huaming Xu; Hao Tang; Jilu Shen

doi:10.3389/fmicb.2023.1119395

Rapid visualization of Clostridioides difficile toxins A and B by multiplex RPA combined with CRISPR-Cas12a

Front Microbiol. 2023 Mar 8:14:1119395. doi: 10.3389/fmicb.2023.1119395. eCollection 2023.

Authors

Tong Jiang^{1

2}, Xinyi Hu^{1

2}, Chunhui Lin^{1

2}, Zhaoxin Xia^{1

2}, Wensu Yang^{1

2}, Yi Zhu^{1

2}, Huaming Xu^{1

2}, Hao Tang^{1

2}, Jilu Shen^{1

2}

Affiliations

¹ The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China.
² Anhui Public Health Clinical Center, Hefei, Anhui, China.

Abstract

Purpose: Clostridioides difficile (C. difficile) infection is the most common cause of nosocomial infection, which is a severe challenge in modern medical care. Currently, many laboratory diagnostic methods for C. difficile are available, such as PCR, culture-based tests, and antigen-based tests. However, these methods are not suitable for rapid point-of-care testing (POCT). Therefore, it is of great significance to develop a rapid, sensitive, and cost-effective method to detect C. difficile toxin genes.

Methods: Recently, the development of clustered regularly interspaced short palindromic repeats (CRISPR) technology has emerged as a promising tool for rapid POCT. In this study, we developed a rapid and specific detection platform for dual C. difficile toxins by combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a.

Results: The platform includes multiplex RPA-cas12a-fluorescence assay and multiplex RPA-cas12a-LFS (Lateral flow strip) assay, through which the detection limit for tcdA and tcdB was 10 copies/μL and 1 copy/μL, respectively. The results can be more clearly distinguished using a violet flashlight, which realized a portable visual readout. The platform can be tested within 50 min. Furthermore, our method did not cross-react with other pathogens that cause intestinal diarrhea. The results of 10 clinical samples using our method was 100% consistent with those from real-time PCR detection.

Conclusion: In conclusion, the CRISPR-based double toxin gene detection platform for C. difficile is an effective, specific, and sensitive detection method, which can be used as a powerful on-site detection tool for POCT in the future.

Keywords: CRISPR/Cas12a; Clostridioides difficile; lateral flow strip; point-of-care testing; recombinase polymerase amplification; toxin gene.