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Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay

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...

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Autores principales: Liu, Jiajia, Tao, Dagang, Chen, Xinquan, Shen, Linyuan, Zhu, Li, Xu, Bingrong, Liu, Hailong, Zhao, Shuhong, Li, Xinyun, Liu, Xiangdong, Xie, Shengsong, Niu, Lili
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9032155/
https://www.ncbi.nlm.nih.gov/pubmed/35458562
http://dx.doi.org/10.3390/v14040833
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author Liu, Jiajia
Tao, Dagang
Chen, Xinquan
Shen, Linyuan
Zhu, Li
Xu, Bingrong
Liu, Hailong
Zhao, Shuhong
Li, Xinyun
Liu, Xiangdong
Xie, Shengsong
Niu, Lili
author_facet Liu, Jiajia
Tao, Dagang
Chen, Xinquan
Shen, Linyuan
Zhu, Li
Xu, Bingrong
Liu, Hailong
Zhao, Shuhong
Li, Xinyun
Liu, Xiangdong
Xie, Shengsong
Niu, Lili
author_sort Liu, Jiajia
collection PubMed
description 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.
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spelling pubmed-90321552022-04-23 Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay Liu, Jiajia Tao, Dagang Chen, Xinquan Shen, Linyuan Zhu, Li Xu, Bingrong Liu, Hailong Zhao, Shuhong Li, Xinyun Liu, Xiangdong Xie, Shengsong Niu, Lili Viruses Article 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. MDPI 2022-04-17 /pmc/articles/PMC9032155/ /pubmed/35458562 http://dx.doi.org/10.3390/v14040833 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Liu, Jiajia
Tao, Dagang
Chen, Xinquan
Shen, Linyuan
Zhu, Li
Xu, Bingrong
Liu, Hailong
Zhao, Shuhong
Li, Xinyun
Liu, Xiangdong
Xie, Shengsong
Niu, Lili
Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay
title Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay
title_full Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay
title_fullStr Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay
title_full_unstemmed Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay
title_short Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay
title_sort detection of four porcine enteric coronaviruses using crispr-cas12a combined with multiplex reverse transcriptase loop-mediated isothermal amplification assay
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9032155/
https://www.ncbi.nlm.nih.gov/pubmed/35458562
http://dx.doi.org/10.3390/v14040833
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