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Visual and Rapid Detection of Porcine Epidemic Diarrhea Virus (PEDV) Using Reverse Transcription Loop-Mediated Isothermal Amplification Method

SIMPLE SUMMARY: Porcine epidemic diarrhea (PED) is a severe disease which has led to tremendous economic losses in the swine industry all over the world. The early detection of its pathogen (PEDV) is vital to prevent and cure this disease. Here, we report the development of a new visual diagnostic t...

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Autores principales: Li, Chunhua, Liang, Jieling, Yang, Dan, Zhang, Qi, Miao, Denian, He, Xizhong, Du, Yanan, Zhang, Wanjing, Ni, Jianping, Zhao, Kai
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9558507/
https://www.ncbi.nlm.nih.gov/pubmed/36230453
http://dx.doi.org/10.3390/ani12192712
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author Li, Chunhua
Liang, Jieling
Yang, Dan
Zhang, Qi
Miao, Denian
He, Xizhong
Du, Yanan
Zhang, Wanjing
Ni, Jianping
Zhao, Kai
author_facet Li, Chunhua
Liang, Jieling
Yang, Dan
Zhang, Qi
Miao, Denian
He, Xizhong
Du, Yanan
Zhang, Wanjing
Ni, Jianping
Zhao, Kai
author_sort Li, Chunhua
collection PubMed
description SIMPLE SUMMARY: Porcine epidemic diarrhea (PED) is a severe disease which has led to tremendous economic losses in the swine industry all over the world. The early detection of its pathogen (PEDV) is vital to prevent and cure this disease. Here, we report the development of a new visual diagnostic test for PEDV—the reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. This new assay proved to be specific and sensitive when applied to clinical specimens. ABSTRACT: Porcine epidemic diarrhea virus (PEDV) can cause severe infectious porcine epidemic diarrhea (PED) and infect different ages of pigs, resulting in sickness and death among suckling pigs. For PEDV detection, finding an effective and rapid method is a priority. In this study, we established an effective reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for PEDV detection. Three sets of primers, specific for eight different sequences of the PEDV N gene, were designed in this study. The optimized RT-LAMP amplification program was as follows: 59 min at 61.9 °C and 3 min at 80 °C. The RT-LAMP results were confirmed with the addition of SYBR Green I fluorescence dye and with the detection of a ladder-like band by conventional gel electrophoresis analysis, which demonstrated a significant agreement between the two methods. The LOD of PEDV by RT-LAMP was 0.0001 ng/μL. Compared with RT-LAMP, the traditional RT-PCR method is 100-fold less sensitive. The RT-LAMP results had no cross-reaction with porcine parvovirus (PPV), porcine circovirus type 1 (PCV1), porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), rotavirus (RV), transmissible gastroenteritis virus (TGEV) and porcine reproductive and respiratory syndrome virus (PRRSV). Consequently, the newly developed RT-LAMP method could provide an accurate and reliable tool for PEDV diagnosis.
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spelling pubmed-95585072022-10-14 Visual and Rapid Detection of Porcine Epidemic Diarrhea Virus (PEDV) Using Reverse Transcription Loop-Mediated Isothermal Amplification Method Li, Chunhua Liang, Jieling Yang, Dan Zhang, Qi Miao, Denian He, Xizhong Du, Yanan Zhang, Wanjing Ni, Jianping Zhao, Kai Animals (Basel) Article SIMPLE SUMMARY: Porcine epidemic diarrhea (PED) is a severe disease which has led to tremendous economic losses in the swine industry all over the world. The early detection of its pathogen (PEDV) is vital to prevent and cure this disease. Here, we report the development of a new visual diagnostic test for PEDV—the reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. This new assay proved to be specific and sensitive when applied to clinical specimens. ABSTRACT: Porcine epidemic diarrhea virus (PEDV) can cause severe infectious porcine epidemic diarrhea (PED) and infect different ages of pigs, resulting in sickness and death among suckling pigs. For PEDV detection, finding an effective and rapid method is a priority. In this study, we established an effective reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for PEDV detection. Three sets of primers, specific for eight different sequences of the PEDV N gene, were designed in this study. The optimized RT-LAMP amplification program was as follows: 59 min at 61.9 °C and 3 min at 80 °C. The RT-LAMP results were confirmed with the addition of SYBR Green I fluorescence dye and with the detection of a ladder-like band by conventional gel electrophoresis analysis, which demonstrated a significant agreement between the two methods. The LOD of PEDV by RT-LAMP was 0.0001 ng/μL. Compared with RT-LAMP, the traditional RT-PCR method is 100-fold less sensitive. The RT-LAMP results had no cross-reaction with porcine parvovirus (PPV), porcine circovirus type 1 (PCV1), porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), rotavirus (RV), transmissible gastroenteritis virus (TGEV) and porcine reproductive and respiratory syndrome virus (PRRSV). Consequently, the newly developed RT-LAMP method could provide an accurate and reliable tool for PEDV diagnosis. MDPI 2022-10-09 /pmc/articles/PMC9558507/ /pubmed/36230453 http://dx.doi.org/10.3390/ani12192712 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
Li, Chunhua
Liang, Jieling
Yang, Dan
Zhang, Qi
Miao, Denian
He, Xizhong
Du, Yanan
Zhang, Wanjing
Ni, Jianping
Zhao, Kai
Visual and Rapid Detection of Porcine Epidemic Diarrhea Virus (PEDV) Using Reverse Transcription Loop-Mediated Isothermal Amplification Method
title Visual and Rapid Detection of Porcine Epidemic Diarrhea Virus (PEDV) Using Reverse Transcription Loop-Mediated Isothermal Amplification Method
title_full Visual and Rapid Detection of Porcine Epidemic Diarrhea Virus (PEDV) Using Reverse Transcription Loop-Mediated Isothermal Amplification Method
title_fullStr Visual and Rapid Detection of Porcine Epidemic Diarrhea Virus (PEDV) Using Reverse Transcription Loop-Mediated Isothermal Amplification Method
title_full_unstemmed Visual and Rapid Detection of Porcine Epidemic Diarrhea Virus (PEDV) Using Reverse Transcription Loop-Mediated Isothermal Amplification Method
title_short Visual and Rapid Detection of Porcine Epidemic Diarrhea Virus (PEDV) Using Reverse Transcription Loop-Mediated Isothermal Amplification Method
title_sort visual and rapid detection of porcine epidemic diarrhea virus (pedv) using reverse transcription loop-mediated isothermal amplification method
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9558507/
https://www.ncbi.nlm.nih.gov/pubmed/36230453
http://dx.doi.org/10.3390/ani12192712
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