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Frontiers in cellular and infection microbiology2025; 15; 1526516; doi: 10.3389/fcimb.2025.1526516

Integrated CRISPR-Cas12a and RAA one-pot visual strategy for the rapid identification of Streptococcus equi subspecies equi.

Abstract: Strangles, a highly contagious disease caused by subspecies (), significantly impacts horse populations worldwide, with Iceland as the only exception. This disease poses serious threats to equine health and results in considerable economic losses. Consequently, the accurate, sensitive, and rapid detection of from clinical samples is essential for early warning and effective disease management. This study introduces a novel detection method that integrates recombinase-aided amplification (RAA) with CRISPR/Cas12a technologies. We specifically designed RAA primers and CRISPR RNA to target the gene of , and we have carefully optimized the reaction systems for this purpose. The newly established visual diagnostic method has shown to be highly effective, demonstrating 97.14% specificity and 100% sensitivity, with the capability to detect as few as 5.6×10 copies of the target. This is the first study to propose the combined application of RAA and CRISPR/Cas12a for the on-site rapid detection of . This is the first study to propose the combined application of RAA and CRISPR/Cas12a for the on-site rapid detection of , which enables visual point-of-care diagnosis of Strangles.
Copyright © 2025 Zu, Sun, Li, Guo, Wang, Guo and Wang.
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Publication Date: 2025-08-21 PubMed ID: 40918251PubMed Central: PMC12408588DOI: 10.3389/fcimb.2025.1526516Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Integrated CRISPR-Cas12a and RAA technologies have been combined to develop a rapid, sensitive, and specific one-pot visual assay for detecting Streptococcus equi subspecies equi, the causative agent of the contagious equine disease strangles.

Background and Importance

  • Strangles is a highly contagious disease affecting horses globally, except Iceland.
  • It causes significant equine health issues and economic losses in the horse industry.
  • Rapid and accurate detection of Streptococcus equi subspecies equi is critical for early diagnosis, outbreak prevention, and disease management.

Objective of the Study

  • To develop a rapid, sensitive, and specific diagnostic test for the on-site detection of Streptococcus equi subspecies equi.
  • To integrate Recombinase-Aided Amplification (RAA) with CRISPR/Cas12a technology for a one-pot, visual, point-of-care diagnostic assay.

Methodology

  • Design of RAA primers and CRISPR RNA (crRNA) targeting the specific gene (eqbE gene) of Streptococcus equi subspecies equi.
  • Optimization of the combined RAA-CRISPR/Cas12a reaction system to enable simultaneous nucleic acid amplification and CRISPR-based detection.
  • Development of a one-pot assay enabling simultaneous amplification and detection without the need for separate steps or specialized equipment.
  • Visual detection facilitated by CRISPR/Cas12a’s collateral cleavage activity, which releases a fluorescent or colorimetric signal visible to the naked eye or simple detectors.

Performance and Validation

  • The assay demonstrated 97.14% specificity, effectively distinguishing Streptococcus equi subspecies equi from non-target strains.
  • Sensitivity reached 100%, detecting as low as 5.6×10 copies of the target bacteria’s DNA, showing high analytical sensitivity.
  • The one-pot assay enables rapid turnaround times suitable for on-site diagnosis.
  • This combined technology offers a straightforward and accessible approach, making it useful in field conditions without complex instrumentation.

Significance and Novelty

  • This is the first study to integrate RAA and CRISPR/Cas12a for the rapid, visual, point-of-care detection of Streptococcus equi subspecies equi.
  • The assay provides a valuable tool for veterinarians and equine health professionals to monitor and respond to strangles outbreaks efficiently.
  • By enabling early warning, it supports better control measures and reduces the overall impact of strangles on horse populations and the equine industry.

Conclusion

  • The integrated CRISPR-Cas12a and RAA one-pot visual detection method is a significant advancement in the diagnosis of strangles.
  • It combines high specificity, sensitivity, and ease of use to facilitate on-site testing, which can improve disease management and control strategies globally.

Cite This Article

APA
Zu H, Sun R, Li J, Guo X, Wang M, Guo W, Wang X. (2025). Integrated CRISPR-Cas12a and RAA one-pot visual strategy for the rapid identification of Streptococcus equi subspecies equi. Front Cell Infect Microbiol, 15, 1526516. https://doi.org/10.3389/fcimb.2025.1526516

Publication

Frontiers in cellular and infection microbiology
ISSN: 2235-2988
NlmUniqueID: 101585359
Country: Switzerland
Language: English
Volume: 15
Pages: 1526516
PII: 1526516

Researcher Affiliations

Zu, Haoyu
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Sun, Rongkuan
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
  • College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
Li, Jiaxin
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Guo, Xing
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Wang, Min
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Guo, Wei
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
  • Institute of Western Agriculture, Chinese Academy of Agricultural Sciences, Changji, China.
Wang, Xiaojun
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
  • Institute of Western Agriculture, Chinese Academy of Agricultural Sciences, Changji, China.

MeSH Terms

  • Streptococcus equi / genetics
  • Streptococcus equi / isolation & purification
  • Streptococcus equi / classification
  • Horses
  • CRISPR-Cas Systems
  • Animals
  • Streptococcal Infections / veterinary
  • Streptococcal Infections / diagnosis
  • Streptococcal Infections / microbiology
  • Horse Diseases / diagnosis
  • Horse Diseases / microbiology
  • Sensitivity and Specificity
  • Nucleic Acid Amplification Techniques / methods
  • Recombinases / metabolism
  • Recombinases / genetics
  • Molecular Diagnostic Techniques / methods
  • Iceland
  • Bacterial Proteins / genetics
  • Endodeoxyribonucleases
  • CRISPR-Associated Proteins
  • Streptococcus

Conflict of Interest Statement

The author(s) declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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