Rapid detection of Mycoplasma hyopneumoniae by recombinase-aided amplification combined with the CRISPR/Cas12a system

Kaili Li; Tingyu Luo; Yu Zhang; Changwen Li; Hongyan Chen; Changyou Xia; Caixia Gao

doi:10.3389/fcimb.2024.1469558

Rapid detection of Mycoplasma hyopneumoniae by recombinase-aided amplification combined with the CRISPR/Cas12a system

Front Cell Infect Microbiol. 2024 Dec 20:14:1469558. doi: 10.3389/fcimb.2024.1469558. eCollection 2024.

Authors

Kaili Li^{1

2

3}, Tingyu Luo^{1

2

3}, Yu Zhang^{1

2

3}, Changwen Li^{1

2

3}, Hongyan Chen^{1

2

3}, Changyou Xia^{1

2

3}, Caixia Gao^{1

2

3}

Affiliations

¹ State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
² Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
³ National Poultry Laboratory Animal Resource Center, 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.

Keywords: CRISPR/Cas12a; Mycoplasma hyopneumoniae; rapid detection; recombinase-aided amplification; visualization.

MeSH terms

Animals
CRISPR-Cas Systems*
Clustered Regularly Interspaced Short Palindromic Repeats / genetics
Limit of Detection
Molecular Diagnostic Techniques / methods
Mycoplasma hyopneumoniae* / genetics
Mycoplasma hyopneumoniae* / isolation & purification
Nucleic Acid Amplification Techniques* / methods
Pneumonia of Swine, Mycoplasmal* / diagnosis
Pneumonia of Swine, Mycoplasmal* / microbiology
Recombinases* / genetics
Recombinases* / metabolism
Sensitivity and Specificity
Swine

Substances

Recombinases

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study was supported by National Key Research and Development Program of China (2021YFF0703000), National Center of Technology Innovation for Pigs (NCTIP-XD/C09), Central Public-interest Scientific Institution Basal Research Fund (1610302022018).