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Rapid detection of Mycoplasma hyopneumoniae by recombinase-aided amplification combined with the CRISPR/Cas12a system
Kaili Li1,2,3, Tingyu Luo1,2,3, Yu Zhang1,2,3
1State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Abstract:
Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the primary agents involved in porcine respiratory disease complex, and circulates in the swine industry worldwide. The prevention and control of M. hyopneumoniae is complicated. Thus, a recombinase-aided amplification (RAA) assay coupled with the clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas12a system was established for the detection of M. hyopneumoniae. The most suitable primer pairs and CRISPR RNA (crRNA) were screened and selected for the RAA-CRISPR/Cas12a detection system. We have achieved a detection limit of 1 copy/µL and 5 copies/µL per reaction for the RAA-CRISPR/Cas12a-fluorescence assay and RAA-CRISPR/Cas12a-lateral flow assay (LFA), respectively. Furthermore, the RAA-CRISPR/Cas12a system displayed no cross-reactivity with other respiratory pathogens. The performance of the RAA-CRISPR/Cas12a system was compared with PCR as recommended by the Chinese national standard (GB/T 35909-2018) and qPCR as recommended by the Chinese entry-exit inspection and quarantine industry standard (SN/T4104-2015) for clinical samples, and good consistency with these methods was observed. Above all, the methods shed a light on the convenient, portable, visual, highly sensitive and specific detection of M. hyopneumoniae, demonstrating a great application potential for on-site monitoring of M. hyopneumoniae in the field.
Insights
A new recombinase-aided amplification (RAA) assay combined with CRISPR/Cas12a technology offers a sensitive and specific method for detecting Mycoplasma hyopneumoniae. This assay shows great potential for convenient, on-site monitoring of this swine pathogen.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Biotechnology
Background:
- Mycoplasma hyopneumoniae is a major cause of porcine respiratory disease complex globally.
- Current control strategies for M. hyopneumoniae are often complicated, necessitating improved diagnostic tools.
Purpose of the Study:
- To develop and validate a novel detection system for Mycoplasma hyopneumoniae.
- To establish a convenient, portable, and highly sensitive diagnostic method for field applications.
Main Methods:
- Development of a recombinase-aided amplification (RAA) assay integrated with the CRISPR/Cas12a system.
- Screening and selection of optimal primer pairs and CRISPR RNA (crRNA) for the assay.
- Validation of the RAA-CRISPR/Cas12a system using fluorescence and lateral flow assays (LFA), and comparison with PCR and qPCR methods.
Main Results:
- The RAA-CRISPR/Cas12a assay achieved detection limits of 1 copy/µL (fluorescence) and 5 copies/µL (LFA).
- The system demonstrated high specificity, with no cross-reactivity observed against other common respiratory pathogens.
- The developed method showed good consistency with established PCR and qPCR diagnostic standards for clinical samples.
Conclusions:
- The RAA-CRISPR/Cas12a system provides a sensitive, specific, and visually detectable method for Mycoplasma hyopneumoniae.
- This technology holds significant promise for rapid, on-site diagnosis and monitoring of M. hyopneumoniae in swine populations.
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