A CRISPR/Cas12a-assisted rapid detection platform by biosensing the apxIVA of Actinobacillus pleuropneumoniae
Actinobacillus pleuropneumoniae is an important respiratory pig pathogen that causes substantial losses in the worldwide swine industry. Chronic or subclinical infection with no apparent clinical symptoms poses a challenge for preventing transmission between herds. Rapid diagnostics is important for...
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2022-09-01
|
| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.928307/full |
| _version_ | 1798002129273618432 |
|---|---|
| author | Tian Luan Lu Wang Jiyu Zhao Hui Luan Yueling Zhang Chunlai Wang Paul R. Langford Siguo Liu Wanjiang Zhang Gang Li |
| author_facet | Tian Luan Lu Wang Jiyu Zhao Hui Luan Yueling Zhang Chunlai Wang Paul R. Langford Siguo Liu Wanjiang Zhang Gang Li |
| author_sort | Tian Luan |
| collection | DOAJ |
| description | Actinobacillus pleuropneumoniae is an important respiratory pig pathogen that causes substantial losses in the worldwide swine industry. Chronic or subclinical infection with no apparent clinical symptoms poses a challenge for preventing transmission between herds. Rapid diagnostics is important for the control of epidemic diseases. In this study, we formulated an A. pleuropneumoniae species-specific apxIVA-based CRISPR/Cas12a-assisted rapid detection platform (Card) that combines recombinase polymerase amplification (RPA) of target DNA and subsequent Cas12a ssDNase activation. Card has a detection limit of 10 CFUs of A. pleuropneumoniae, and there is no cross-reactivity with other common swine pathogens. The detection process can be completed in 1 h, and there was 100% agreement between the conventional apxIVA-based PCR and Card in detecting A. pleuropneumoniae in lung samples. Microplate fluorescence readout enables high-throughput use in diagnostic laboratories, and naked eye and lateral flow test readouts enable use at the point of care. We conclude that Card is a versatile, rapid, accurate molecular diagnostic platform suitable for use in both laboratory and low-resource settings. |
| first_indexed | 2024-04-11T11:47:14Z |
| format | Article |
| id | doaj.art-0b295e17db39417498701cfe66bee76d |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-11T11:47:14Z |
| publishDate | 2022-09-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-0b295e17db39417498701cfe66bee76d2022-12-22T04:25:29ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2022-09-011310.3389/fmicb.2022.928307928307A CRISPR/Cas12a-assisted rapid detection platform by biosensing the apxIVA of Actinobacillus pleuropneumoniaeTian Luan0Lu Wang1Jiyu Zhao2Hui Luan3Yueling Zhang4Chunlai Wang5Paul R. Langford6Siguo Liu7Wanjiang Zhang8Gang Li9State Key Laboratory of Veterinary Biotechnology, Division of Bacterial Diseases, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaCollege of Veterinary Medicine, Xinjiang Agricultural University, Xinjiang, ChinaCollege of Veterinary Medicine, Northeast Agricultural University, Harbin, ChinaState Key Laboratory of Veterinary Biotechnology, Division of Bacterial Diseases, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaState Key Laboratory of Veterinary Biotechnology, Division of Bacterial Diseases, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaState Key Laboratory of Veterinary Biotechnology, Division of Bacterial Diseases, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaSection of Paediatric Infectious Disease, Department of Infectious Disease, Imperial College London, St. Mary’s Campus, London, United KingdomState Key Laboratory of Veterinary Biotechnology, Division of Bacterial Diseases, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaState Key Laboratory of Veterinary Biotechnology, Division of Bacterial Diseases, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaState Key Laboratory of Veterinary Biotechnology, Division of Bacterial Diseases, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaActinobacillus pleuropneumoniae is an important respiratory pig pathogen that causes substantial losses in the worldwide swine industry. Chronic or subclinical infection with no apparent clinical symptoms poses a challenge for preventing transmission between herds. Rapid diagnostics is important for the control of epidemic diseases. In this study, we formulated an A. pleuropneumoniae species-specific apxIVA-based CRISPR/Cas12a-assisted rapid detection platform (Card) that combines recombinase polymerase amplification (RPA) of target DNA and subsequent Cas12a ssDNase activation. Card has a detection limit of 10 CFUs of A. pleuropneumoniae, and there is no cross-reactivity with other common swine pathogens. The detection process can be completed in 1 h, and there was 100% agreement between the conventional apxIVA-based PCR and Card in detecting A. pleuropneumoniae in lung samples. Microplate fluorescence readout enables high-throughput use in diagnostic laboratories, and naked eye and lateral flow test readouts enable use at the point of care. We conclude that Card is a versatile, rapid, accurate molecular diagnostic platform suitable for use in both laboratory and low-resource settings.https://www.frontiersin.org/articles/10.3389/fmicb.2022.928307/fullCRISPRCas12acrRNAActinobacillus pleuropneumoniaediagnostics |
| spellingShingle | Tian Luan Lu Wang Jiyu Zhao Hui Luan Yueling Zhang Chunlai Wang Paul R. Langford Siguo Liu Wanjiang Zhang Gang Li A CRISPR/Cas12a-assisted rapid detection platform by biosensing the apxIVA of Actinobacillus pleuropneumoniae Frontiers in Microbiology CRISPR Cas12a crRNA Actinobacillus pleuropneumoniae diagnostics |
| title | A CRISPR/Cas12a-assisted rapid detection platform by biosensing the apxIVA of Actinobacillus pleuropneumoniae |
| title_full | A CRISPR/Cas12a-assisted rapid detection platform by biosensing the apxIVA of Actinobacillus pleuropneumoniae |
| title_fullStr | A CRISPR/Cas12a-assisted rapid detection platform by biosensing the apxIVA of Actinobacillus pleuropneumoniae |
| title_full_unstemmed | A CRISPR/Cas12a-assisted rapid detection platform by biosensing the apxIVA of Actinobacillus pleuropneumoniae |
| title_short | A CRISPR/Cas12a-assisted rapid detection platform by biosensing the apxIVA of Actinobacillus pleuropneumoniae |
| title_sort | crispr cas12a assisted rapid detection platform by biosensing the apxiva of actinobacillus pleuropneumoniae |
| topic | CRISPR Cas12a crRNA Actinobacillus pleuropneumoniae diagnostics |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.928307/full |
| work_keys_str_mv | AT tianluan acrisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae AT luwang acrisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae AT jiyuzhao acrisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae AT huiluan acrisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae AT yuelingzhang acrisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae AT chunlaiwang acrisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae AT paulrlangford acrisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae AT siguoliu acrisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae AT wanjiangzhang acrisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae AT gangli acrisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae AT tianluan crisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae AT luwang crisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae AT jiyuzhao crisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae AT huiluan crisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae AT yuelingzhang crisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae AT chunlaiwang crisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae AT paulrlangford crisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae AT siguoliu crisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae AT wanjiangzhang crisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae AT gangli crisprcas12aassistedrapiddetectionplatformbybiosensingtheapxivaofactinobacilluspleuropneumoniae |