{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T02:27:03Z","timestamp":1789698423013,"version":"4.0.1"},"reference-count":44,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2022,4,17]],"date-time":"2022-04-17T00:00:00Z","timestamp":1650153600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"the Natural Science Foundation of China","award":["No. 32072685"],"award-info":[{"award-number":["No. 32072685"]}]},{"name":"the Sichuan Science and Technology Support Program","award":["No. SCSZTD-2021-09"],"award-info":[{"award-number":["No. SCSZTD-2021-09"]}]},{"name":"the earmarked fund for China Agriculture Research System","award":["No. CARS-36-05B"],"award-info":[{"award-number":["No. CARS-36-05B"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Porcine enteric coronaviruses have caused immense economic losses to the global pig industry, and pose a potential risk for cross-species transmission. The clinical symptoms of the porcine enteric coronaviruses (CoVs) are similar, making it difficult to distinguish between the specific pathogens by symptoms alone. Here, a multiplex nucleic acid detection platform based on CRISPR\/Cas12a and multiplex reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of four diarrhea CoVs: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV). With this strategy, we realized a visual colorimetric readout visible to the naked eye without specialized instrumentation by using a ROX-labeled single-stranded DNA-fluorescence-quenched (ssDNA-FQ) reporter. Our method achieved single-copy sensitivity with no cross-reactivity in the identification and detection of the target viruses. In addition, we successfully detected these four enteric CoVs from RNA of clinical samples. Thus, we established a rapid, sensitive, and on-site multiplex molecular differential diagnosis technology for porcine enteric CoVs.<\/jats:p>","DOI":"10.3390\/v14040833","type":"journal-article","created":{"date-parts":[[2022,4,20]],"date-time":"2022-04-20T02:04:22Z","timestamp":1650420262000},"page":"833","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":29,"title":["Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay"],"prefix":"10.3390","volume":"14","author":[{"given":"Jiajia","family":"Liu","sequence":"first","affiliation":[{"name":"College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dagang","family":"Tao","sequence":"additional","affiliation":[{"name":"Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education and Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinquan","family":"Chen","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6072-3268","authenticated-orcid":false,"given":"Linyuan","family":"Shen","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li","family":"Zhu","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bingrong","family":"Xu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education and Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hailong","family":"Liu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education and Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuhong","family":"Zhao","sequence":"additional","affiliation":[{"name":"Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education and Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"Hubei Hongshan Laboratory, Frontiers Science Center for Animal Breeding and Sustainable Production, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinyun","family":"Li","sequence":"additional","affiliation":[{"name":"Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education and Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiangdong","family":"Liu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education and Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8301-877X","authenticated-orcid":false,"given":"Shengsong","family":"Xie","sequence":"additional","affiliation":[{"name":"Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education and Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lili","family":"Niu","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,4,17]]},"reference":[{"key":"ref_1","first-page":"1","article-title":"Host Factors in Coronavirus Replication","volume":"419","author":"Snijder","year":"2018","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Haake, C., Cook, S., Pusterla, N., and Murphy, B. (2020). Coronavirus Infections in Companion Animals: Virology, Epidemiology, Clinical and Pathologic Features. Viruses, 12.","DOI":"10.3390\/v12091023"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1016\/j.virusres.2016.05.015","article-title":"Immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling","volume":"226","author":"Zhang","year":"2016","journal-title":"Virus Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"3995","DOI":"10.1128\/JVI.06540-11","article-title":"Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus","volume":"86","author":"Woo","year":"2012","journal-title":"J. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1007\/s11259-021-09808-0","article-title":"Porcine enteric coronaviruses: An updated overview of the pathogenesis, prevalence, and diagnosis","volume":"45","author":"Liu","year":"2021","journal-title":"Vet. Res. Commun."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1016\/j.coviro.2018.12.001","article-title":"Emerging and re-emerging coronaviruses in pigs","volume":"34","author":"Wang","year":"2019","journal-title":"Curr. Opin. Virol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"26915","DOI":"10.1073\/pnas.2001046117","article-title":"Swine acute diarrhea syndrome coronavirus replication in primary human cells reveals potential susceptibility to infection","volume":"117","author":"Edwards","year":"2020","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"198045","DOI":"10.1016\/j.virusres.2020.198045","article-title":"Porcine epidemic diarrhea virus (PEDV): An update on etiology, transmission, pathogenesis, and prevention and control","volume":"286","author":"Jung","year":"2020","journal-title":"Virus Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"113906","DOI":"10.1016\/j.jviromet.2020.113906","article-title":"Porcine epidemic diarrhea virus: Viral RNA detection and quantification using a validated one-step real time RT-PCR","volume":"283","author":"Bigault","year":"2020","journal-title":"J. Virol. Methods"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1016\/j.jviromet.2018.02.002","article-title":"Development of a TaqMan-based real-time RT-PCR assay for the detection of SADS-CoV associated with severe diarrhea disease in pigs","volume":"255","author":"Zhou","year":"2018","journal-title":"J. Virol. Methods"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"575","DOI":"10.1111\/tbed.12399","article-title":"Newly Emerged Porcine Deltacoronavirus Associated With Diarrhoea in Swine in China: Identification, Prevalence and Full-Length Genome Sequence Analysis","volume":"62","author":"Song","year":"2015","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"4943","DOI":"10.1007\/s00253-019-09835-7","article-title":"A TaqMan-probe-based multiplex real-time RT-qPCR for simultaneous detection of porcine enteric coronaviruses","volume":"103","author":"Huang","year":"2019","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1016\/j.virusres.2016.05.028","article-title":"Porcine deltacoronavirus: Overview of infection dynamics, diagnostic methods, prevalence and genetic evolution","volume":"226","author":"Zhang","year":"2016","journal-title":"Virus Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"678","DOI":"10.1111\/tbed.13385","article-title":"Development of a multiplex RT-PCR for the detection of major diarrhoeal viruses in pig herds in China","volume":"67","author":"Ding","year":"2020","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1016\/j.jviromet.2016.03.016","article-title":"Evaluation of two singleplex reverse transcription-Insulated isothermal PCR tests and a duplex real-time RT-PCR test for the detection of porcine epidemic diarrhea virus and porcine deltacoronavirus","volume":"234","author":"Zhang","year":"2016","journal-title":"J. Virol. Methods"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1014","DOI":"10.1111\/j.1365-2672.2012.05384.x","article-title":"PCR inhibitors\u2014Occurrence, properties and removal","volume":"113","author":"Schrader","year":"2012","journal-title":"J. Appl. Microbiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1007\/978-1-60327-353-4_2","article-title":"Overcoming inhibition in real-time diagnostic PCR","volume":"943","author":"Hedman","year":"2013","journal-title":"Methods Mol. Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1126\/science.aam9321","article-title":"Nucleic acid detection with CRISPR-Cas13a\/C2c2","volume":"356","author":"Gootenberg","year":"2017","journal-title":"Science"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"436","DOI":"10.1126\/science.aar6245","article-title":"CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity","volume":"360","author":"Chen","year":"2018","journal-title":"Science"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1038\/s41421-018-0028-z","article-title":"CRISPR-Cas12a-assisted nucleic acid detection","volume":"4","author":"Li","year":"2018","journal-title":"Cell Discov."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1186\/s13059-019-1742-z","article-title":"CDetection: CRISPR-Cas12b-based DNA detection with sub-attomolar sensitivity and single-base specificity","volume":"20","author":"Teng","year":"2019","journal-title":"Genome Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"839","DOI":"10.1126\/science.aav4294","article-title":"Programmed DNA destruction by miniature CRISPR-Cas14 enzymes","volume":"362","author":"Harrington","year":"2018","journal-title":"Science"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"12156","DOI":"10.1021\/acs.analchem.9b01526","article-title":"Cas12aVDet: A CRISPR\/Cas12a-Based Platform for Rapid and Visual Nucleic Acid Detection","volume":"91","author":"Wang","year":"2019","journal-title":"Anal. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"4711","DOI":"10.1038\/s41467-020-18575-6","article-title":"Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay","volume":"11","author":"Ding","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"112068","DOI":"10.1016\/j.bios.2020.112068","article-title":"High-throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system","volume":"154","author":"He","year":"2020","journal-title":"Biosens. Bioelectron."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"870","DOI":"10.1038\/s41587-020-0513-4","article-title":"CRISPR-Cas12-based detection of SARS-CoV-2","volume":"38","author":"Broughton","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"112766","DOI":"10.1016\/j.bios.2020.112766","article-title":"opvCRISPR: One-pot visual RT-LAMP-CRISPR platform for SARS-cov-2 detection","volume":"172","author":"Wang","year":"2021","journal-title":"Biosens. Bioelectron."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2339","DOI":"10.1021\/acssynbio.0c00057","article-title":"Application of CRISPR-Cas12a Enhanced Fluorescence Assay Coupled with Nucleic Acid Amplification for the Sensitive Detection of African Swine Fever Virus","volume":"9","author":"Tao","year":"2020","journal-title":"ACS Synth. Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1038\/s42003-020-0796-5","article-title":"CRISPR\/Cas12a technology combined with immunochromatographic strips for portable detection of African swine fever virus","volume":"3","author":"Wang","year":"2020","journal-title":"Commun. Biol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1470","DOI":"10.7150\/ijbs.21312","article-title":"CRISPR-offinder: A CRISPR guide RNA design and off-target searching tool for user-defined protospacer adjacent motif","volume":"13","author":"Zhao","year":"2017","journal-title":"Int. J. Biol. Sci."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"383","DOI":"10.1021\/acssynbio.1c00474","article-title":"Rapid Visual CRISPR Assay: A Naked-Eye Colorimetric Detection Method for Nucleic Acids Based on CRISPR\/Cas12a and a Convolutional Neural Network","volume":"11","author":"Xie","year":"2022","journal-title":"ACS Synth. Biol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"198024","DOI":"10.1016\/j.virusres.2020.198024","article-title":"Swine enteric alphacoronavirus (swine acute diarrhea syndrome coronavirus): An update three years after its discovery","volume":"285","author":"Yang","year":"2020","journal-title":"Virus Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"7521","DOI":"10.1007\/s00216-021-03716-7","article-title":"Reverse transcription-enzymatic recombinase amplification coupled with CRISPR-Cas12a for rapid detection and differentiation of PEDV wild-type strains and attenuated vaccine strains","volume":"413","author":"Yang","year":"2021","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"11510","DOI":"10.1021\/acs.analchem.9b03545","article-title":"Naked-Eye Detection of Grapevine Red-Blotch Viral Infection Using a Plasmonic CRISPR Cas12a Assay","volume":"91","author":"Li","year":"2019","journal-title":"Anal. Chem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"4029","DOI":"10.1021\/acs.analchem.9b05597","article-title":"Universal and Naked-Eye Gene Detection Platform Based on the Clustered Regularly Interspaced Short Palindromic Repeats\/Cas12a\/13a System","volume":"92","author":"Yuan","year":"2020","journal-title":"Anal. Chem."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"7506","DOI":"10.1021\/jacs.0c00217","article-title":"Single-Step, Salt-Aging-Free, and Thiol-Free Freezing Construction of AuNP-Based Bioprobes for Advancing CRISPR-Based Diagnostics","volume":"142","author":"Hu","year":"2020","journal-title":"J. Am. Chem. Soc."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"693","DOI":"10.1021\/acs.nanolett.0c04303","article-title":"CRISPR-Cas12a-Based Nucleic Acid Amplification-Free DNA Biosensor via Au Nanoparticle-Assisted Metal-Enhanced Fluorescence and Colorimetric Analysis","volume":"21","author":"Choi","year":"2021","journal-title":"Nano Lett."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"10167","DOI":"10.1021\/acs.analchem.1c01163","article-title":"CRISPR-\/Cas12a-Mediated Liposome-Amplified Strategy for the Surface-Enhanced Raman Scattering and Naked-Eye Detection of Nucleic Acid and Application to Food Authenticity Screening","volume":"93","author":"Liu","year":"2021","journal-title":"Anal. Chem."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"112642","DOI":"10.1016\/j.bios.2020.112642","article-title":"Contamination-free visual detection of SARS-CoV-2 with CRISPR\/Cas12a: A promising method in the point-of-care detection","volume":"169","author":"Chen","year":"2020","journal-title":"Biosens. Bioelectron."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"16204","DOI":"10.1021\/acs.analchem.0c04047","article-title":"Isothermal Amplification and Ambient Visualization in a Single Tube for the Detection of SARS-CoV-2 Using Loop-Mediated Amplification and CRISPR Technology","volume":"92","author":"Pang","year":"2020","journal-title":"Anal. Chem."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"444","DOI":"10.1126\/science.aas8836","article-title":"Field-deployable viral diagnostics using CRISPR-Cas13","volume":"360","author":"Myhrvold","year":"2018","journal-title":"Science"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"439","DOI":"10.1126\/science.aaq0179","article-title":"Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6","volume":"360","author":"Gootenberg","year":"2018","journal-title":"Science"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1038\/s41586-020-2279-8","article-title":"Massively multiplexed nucleic acid detection with Cas13","volume":"582","author":"Ackerman","year":"2020","journal-title":"Nature"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"394","DOI":"10.1111\/pbi.13474","article-title":"Field detection of multiple RNA viruses\/viroids in apple using a CRISPR\/Cas12a-based visual assay","volume":"19","author":"Jiao","year":"2021","journal-title":"Plant Biotechnol. J."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/4\/833\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:55:39Z","timestamp":1760136939000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/4\/833"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,4,17]]},"references-count":44,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2022,4]]}},"alternative-id":["v14040833"],"URL":"https:\/\/doi.org\/10.3390\/v14040833","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,4,17]]}}}