LAMP Coupled CRISPR-Cas12a Module for Rapid, Sensitive and Visual Detection of Porcine Circovirus 2
Porcine circovirus 2 (PCV2) is the main pathogen of porcine circovirus-associated disease (PCVAD), which can cause considerable economic loss to the pig industry. The diagnosis of PCVAD is complicated and requires a series of clinical, pathological, and virological methods. Therefore, a rapid, highl...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-09-01
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| Online Access: | https://www.mdpi.com/2076-2615/12/18/2413 |
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| author | Lei Lei Fan Liao Lei Tan Deyong Duan Yang Zhan Naidong Wang Yuge Wang Xiaoye Peng Kaixin Wang Xiaojiu Huang Yi Yang Aibing Wang |
| author_facet | Lei Lei Fan Liao Lei Tan Deyong Duan Yang Zhan Naidong Wang Yuge Wang Xiaoye Peng Kaixin Wang Xiaojiu Huang Yi Yang Aibing Wang |
| author_sort | Lei Lei |
| collection | DOAJ |
| description | Porcine circovirus 2 (PCV2) is the main pathogen of porcine circovirus-associated disease (PCVAD), which can cause considerable economic loss to the pig industry. The diagnosis of PCVAD is complicated and requires a series of clinical, pathological, and virological methods. Therefore, a rapid, highly sensitive, on-site, and visual diagnostic approach would facilitate dealing with the spread of PCV2. In this study, we intended to establish a new and effective PCV2 detection method through combining the no specific equipment requirement advantage of loop-mediated isothermal amplification (LAMP) with the property of clustered regular interspaced short palindromic repeats (CRISPR)/Cas12a system possessing the huLbCas12a collateral cleavage activity able to cleave single-stranded DNA fluorophore quencher probe sensor (designed as LAPM-CRISPR). Following a series of optimizations of its reaction conditions, this LAMP-CRISPR-based PCV2 detection could be conducted in constant temperature equipment, with the result reflected in a direct visual readout way. This established PCV2 detection approach presented fine sensitivity, rapidity, specificity, and reliability, as demonstrated by a low detectable limit of 1 copy/μL, completed within an hour, no cross-reaction with main porcine DNA or RNA viruses like PCV1, PCV3, and PEDV, and a 100% coincidence rate with that of the quantitative PCR (qPCR) method in the evaluation of 30 clinical blood samples, respectively. Therefore, this novel method makes rapid, on-site, visual, highly sensitive, and specific detection of PCV2 possible, facilitating the prevention of this pathogen in the field. |
| first_indexed | 2024-03-10T00:56:38Z |
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| id | doaj.art-56fb98baf4094d5d9da0968d218b6237 |
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| issn | 2076-2615 |
| language | English |
| last_indexed | 2024-03-10T00:56:38Z |
| publishDate | 2022-09-01 |
| publisher | MDPI AG |
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| series | Animals |
| spelling | doaj.art-56fb98baf4094d5d9da0968d218b62372023-11-23T14:42:29ZengMDPI AGAnimals2076-26152022-09-011218241310.3390/ani12182413LAMP Coupled CRISPR-Cas12a Module for Rapid, Sensitive and Visual Detection of Porcine Circovirus 2Lei Lei0Fan Liao1Lei Tan2Deyong Duan3Yang Zhan4Naidong Wang5Yuge Wang6Xiaoye Peng7Kaixin Wang8Xiaojiu Huang9Yi Yang10Aibing Wang11Hunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), Changsha 410128, ChinaHunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), Changsha 410128, ChinaHunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), Changsha 410128, ChinaHunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), Changsha 410128, ChinaHunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), Changsha 410128, ChinaHunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), Changsha 410128, ChinaHunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), Changsha 410128, ChinaHunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), Changsha 410128, ChinaHunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), Changsha 410128, ChinaHunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), Changsha 410128, ChinaHunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), Changsha 410128, ChinaHunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), Changsha 410128, ChinaPorcine circovirus 2 (PCV2) is the main pathogen of porcine circovirus-associated disease (PCVAD), which can cause considerable economic loss to the pig industry. The diagnosis of PCVAD is complicated and requires a series of clinical, pathological, and virological methods. Therefore, a rapid, highly sensitive, on-site, and visual diagnostic approach would facilitate dealing with the spread of PCV2. In this study, we intended to establish a new and effective PCV2 detection method through combining the no specific equipment requirement advantage of loop-mediated isothermal amplification (LAMP) with the property of clustered regular interspaced short palindromic repeats (CRISPR)/Cas12a system possessing the huLbCas12a collateral cleavage activity able to cleave single-stranded DNA fluorophore quencher probe sensor (designed as LAPM-CRISPR). Following a series of optimizations of its reaction conditions, this LAMP-CRISPR-based PCV2 detection could be conducted in constant temperature equipment, with the result reflected in a direct visual readout way. This established PCV2 detection approach presented fine sensitivity, rapidity, specificity, and reliability, as demonstrated by a low detectable limit of 1 copy/μL, completed within an hour, no cross-reaction with main porcine DNA or RNA viruses like PCV1, PCV3, and PEDV, and a 100% coincidence rate with that of the quantitative PCR (qPCR) method in the evaluation of 30 clinical blood samples, respectively. Therefore, this novel method makes rapid, on-site, visual, highly sensitive, and specific detection of PCV2 possible, facilitating the prevention of this pathogen in the field.https://www.mdpi.com/2076-2615/12/18/2413porcine circovirus 2CRISPR/Cas12acrRNALAMPvisual detectionqPCR |
| spellingShingle | Lei Lei Fan Liao Lei Tan Deyong Duan Yang Zhan Naidong Wang Yuge Wang Xiaoye Peng Kaixin Wang Xiaojiu Huang Yi Yang Aibing Wang LAMP Coupled CRISPR-Cas12a Module for Rapid, Sensitive and Visual Detection of Porcine Circovirus 2 Animals porcine circovirus 2 CRISPR/Cas12a crRNA LAMP visual detection qPCR |
| title | LAMP Coupled CRISPR-Cas12a Module for Rapid, Sensitive and Visual Detection of Porcine Circovirus 2 |
| title_full | LAMP Coupled CRISPR-Cas12a Module for Rapid, Sensitive and Visual Detection of Porcine Circovirus 2 |
| title_fullStr | LAMP Coupled CRISPR-Cas12a Module for Rapid, Sensitive and Visual Detection of Porcine Circovirus 2 |
| title_full_unstemmed | LAMP Coupled CRISPR-Cas12a Module for Rapid, Sensitive and Visual Detection of Porcine Circovirus 2 |
| title_short | LAMP Coupled CRISPR-Cas12a Module for Rapid, Sensitive and Visual Detection of Porcine Circovirus 2 |
| title_sort | lamp coupled crispr cas12a module for rapid sensitive and visual detection of porcine circovirus 2 |
| topic | porcine circovirus 2 CRISPR/Cas12a crRNA LAMP visual detection qPCR |
| url | https://www.mdpi.com/2076-2615/12/18/2413 |
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