Rapid detection of Mycoplasma hyopneumoniae by recombinase-aided amplification combined with the CRISPR/Cas12a system
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 th...
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| Format: | Article |
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Frontiers Media S.A.
2024-12-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2024.1469558/full |
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| author | Kaili Li Kaili Li Kaili Li Tingyu Luo Tingyu Luo Tingyu Luo Yu Zhang Yu Zhang Yu Zhang Changwen Li Changwen Li Changwen Li Hongyan Chen Hongyan Chen Hongyan Chen Changyou Xia Changyou Xia Changyou Xia Caixia Gao Caixia Gao Caixia Gao |
| author_facet | Kaili Li Kaili Li Kaili Li Tingyu Luo Tingyu Luo Tingyu Luo Yu Zhang Yu Zhang Yu Zhang Changwen Li Changwen Li Changwen Li Hongyan Chen Hongyan Chen Hongyan Chen Changyou Xia Changyou Xia Changyou Xia Caixia Gao Caixia Gao Caixia Gao |
| author_sort | Kaili Li |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-bc49a22b87cd43989f95cffaaa9c84b5 |
| institution | OA Journals |
| issn | 2235-2988 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj-art-bc49a22b87cd43989f95cffaaa9c84b52025-08-20T01:58:38ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882024-12-011410.3389/fcimb.2024.14695581469558Rapid detection of Mycoplasma hyopneumoniae by recombinase-aided amplification combined with the CRISPR/Cas12a systemKaili Li0Kaili Li1Kaili Li2Tingyu Luo3Tingyu Luo4Tingyu Luo5Yu Zhang6Yu Zhang7Yu Zhang8Changwen Li9Changwen Li10Changwen Li11Hongyan Chen12Hongyan Chen13Hongyan Chen14Changyou Xia15Changyou Xia16Changyou Xia17Caixia Gao18Caixia Gao19Caixia Gao20State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaHeilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaNational Poultry Laboratory Animal Resource Center, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaState Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaHeilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaNational Poultry Laboratory Animal Resource Center, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaState Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaHeilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaNational Poultry Laboratory Animal Resource Center, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaState Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaHeilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaNational Poultry Laboratory Animal Resource Center, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaState Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaHeilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaNational Poultry Laboratory Animal Resource Center, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaState Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaHeilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaNational Poultry Laboratory Animal Resource Center, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaState Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaHeilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaNational Poultry Laboratory Animal Resource Center, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaMycoplasma 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.https://www.frontiersin.org/articles/10.3389/fcimb.2024.1469558/fullMycoplasma hyopneumoniaeCRISPR/Cas12arecombinase-aided amplificationvisualizationrapid detection |
| spellingShingle | Kaili Li Kaili Li Kaili Li Tingyu Luo Tingyu Luo Tingyu Luo Yu Zhang Yu Zhang Yu Zhang Changwen Li Changwen Li Changwen Li Hongyan Chen Hongyan Chen Hongyan Chen Changyou Xia Changyou Xia Changyou Xia Caixia Gao Caixia Gao Caixia Gao Rapid detection of Mycoplasma hyopneumoniae by recombinase-aided amplification combined with the CRISPR/Cas12a system Frontiers in Cellular and Infection Microbiology Mycoplasma hyopneumoniae CRISPR/Cas12a recombinase-aided amplification visualization rapid detection |
| title | Rapid detection of Mycoplasma hyopneumoniae by recombinase-aided amplification combined with the CRISPR/Cas12a system |
| title_full | Rapid detection of Mycoplasma hyopneumoniae by recombinase-aided amplification combined with the CRISPR/Cas12a system |
| title_fullStr | Rapid detection of Mycoplasma hyopneumoniae by recombinase-aided amplification combined with the CRISPR/Cas12a system |
| title_full_unstemmed | Rapid detection of Mycoplasma hyopneumoniae by recombinase-aided amplification combined with the CRISPR/Cas12a system |
| title_short | Rapid detection of Mycoplasma hyopneumoniae by recombinase-aided amplification combined with the CRISPR/Cas12a system |
| title_sort | rapid detection of mycoplasma hyopneumoniae by recombinase aided amplification combined with the crispr cas12a system |
| topic | Mycoplasma hyopneumoniae CRISPR/Cas12a recombinase-aided amplification visualization rapid detection |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2024.1469558/full |
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