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A Novel, Cleaved Probe-Based Reverse Transcription Loop-Mediated Isothermal Amplification Method for Specific and Sensitive Detection of Porcine Deltacoronavirus

Porcine deltacoronavirus (PDCoV) causes watery diarrhea, vomiting, and 30–40% mortality in newborn piglets. A simple, rapid, and sensitive method for PDCoV detection is valuable in its surveillance and control. Here, we developed a novel, cleaved probe-based reverse transcription loop-mediated isoth...

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Autores principales: Shen, Haiyan, Wang, Songqi, Huang, Jun, Lin, Qijie, Zhang, Chunhong, Liu, Zhicheng, Zhang, Jianfeng, Liao, Ming
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9261778/
https://www.ncbi.nlm.nih.gov/pubmed/35812893
http://dx.doi.org/10.3389/fvets.2022.896416
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author Shen, Haiyan
Wang, Songqi
Huang, Jun
Lin, Qijie
Zhang, Chunhong
Liu, Zhicheng
Zhang, Jianfeng
Liao, Ming
author_facet Shen, Haiyan
Wang, Songqi
Huang, Jun
Lin, Qijie
Zhang, Chunhong
Liu, Zhicheng
Zhang, Jianfeng
Liao, Ming
author_sort Shen, Haiyan
collection PubMed
description Porcine deltacoronavirus (PDCoV) causes watery diarrhea, vomiting, and 30–40% mortality in newborn piglets. A simple, rapid, and sensitive method for PDCoV detection is valuable in its surveillance and control. Here, we developed a novel, cleaved probe-based reverse transcription loop-mediated isothermal amplification (CP-RT-LAMP) method for PDCoV detection. A cleaved probe with a ribonucleotide insertion that targeted the N gene of PDCoV was designed. During the reaction, the enzyme ribonuclease H2 is activated only when the cleaved probe is perfectly complementary to the template, leading to the hydrolytic release of a quencher moiety and signal output. This method can be easily used on a real-time fluorescence quantitative equipment or an on-site isothermal instrument combined with a smartphone. The specificity assay showed no cross-reactivity with other porcine enteric pathogens. This method had a detection limit of 25 copies/μL, suggesting comparable sensitivity with reverse transcription quantitative PCR (RT-qPCR). In detecting 100 clinical samples (48 fecal swab specimens and 52 intestinal specimens), the detection rate of the CP-RT-LAMP method (26%) was higher than that of RT-qPCR (17%). Thus, it is a highly specific and sensitive diagnostic method for PDCoV, with a great application potential for monitoring PDCoV in the laboratory or point-of-care testing in the field.
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spelling pubmed-92617782022-07-08 A Novel, Cleaved Probe-Based Reverse Transcription Loop-Mediated Isothermal Amplification Method for Specific and Sensitive Detection of Porcine Deltacoronavirus Shen, Haiyan Wang, Songqi Huang, Jun Lin, Qijie Zhang, Chunhong Liu, Zhicheng Zhang, Jianfeng Liao, Ming Front Vet Sci Veterinary Science Porcine deltacoronavirus (PDCoV) causes watery diarrhea, vomiting, and 30–40% mortality in newborn piglets. A simple, rapid, and sensitive method for PDCoV detection is valuable in its surveillance and control. Here, we developed a novel, cleaved probe-based reverse transcription loop-mediated isothermal amplification (CP-RT-LAMP) method for PDCoV detection. A cleaved probe with a ribonucleotide insertion that targeted the N gene of PDCoV was designed. During the reaction, the enzyme ribonuclease H2 is activated only when the cleaved probe is perfectly complementary to the template, leading to the hydrolytic release of a quencher moiety and signal output. This method can be easily used on a real-time fluorescence quantitative equipment or an on-site isothermal instrument combined with a smartphone. The specificity assay showed no cross-reactivity with other porcine enteric pathogens. This method had a detection limit of 25 copies/μL, suggesting comparable sensitivity with reverse transcription quantitative PCR (RT-qPCR). In detecting 100 clinical samples (48 fecal swab specimens and 52 intestinal specimens), the detection rate of the CP-RT-LAMP method (26%) was higher than that of RT-qPCR (17%). Thus, it is a highly specific and sensitive diagnostic method for PDCoV, with a great application potential for monitoring PDCoV in the laboratory or point-of-care testing in the field. Frontiers Media S.A. 2022-06-23 /pmc/articles/PMC9261778/ /pubmed/35812893 http://dx.doi.org/10.3389/fvets.2022.896416 Text en Copyright © 2022 Shen, Wang, Huang, Lin, Zhang, Liu, Zhang and Liao. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Veterinary Science
Shen, Haiyan
Wang, Songqi
Huang, Jun
Lin, Qijie
Zhang, Chunhong
Liu, Zhicheng
Zhang, Jianfeng
Liao, Ming
A Novel, Cleaved Probe-Based Reverse Transcription Loop-Mediated Isothermal Amplification Method for Specific and Sensitive Detection of Porcine Deltacoronavirus
title A Novel, Cleaved Probe-Based Reverse Transcription Loop-Mediated Isothermal Amplification Method for Specific and Sensitive Detection of Porcine Deltacoronavirus
title_full A Novel, Cleaved Probe-Based Reverse Transcription Loop-Mediated Isothermal Amplification Method for Specific and Sensitive Detection of Porcine Deltacoronavirus
title_fullStr A Novel, Cleaved Probe-Based Reverse Transcription Loop-Mediated Isothermal Amplification Method for Specific and Sensitive Detection of Porcine Deltacoronavirus
title_full_unstemmed A Novel, Cleaved Probe-Based Reverse Transcription Loop-Mediated Isothermal Amplification Method for Specific and Sensitive Detection of Porcine Deltacoronavirus
title_short A Novel, Cleaved Probe-Based Reverse Transcription Loop-Mediated Isothermal Amplification Method for Specific and Sensitive Detection of Porcine Deltacoronavirus
title_sort novel, cleaved probe-based reverse transcription loop-mediated isothermal amplification method for specific and sensitive detection of porcine deltacoronavirus
topic Veterinary Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9261778/
https://www.ncbi.nlm.nih.gov/pubmed/35812893
http://dx.doi.org/10.3389/fvets.2022.896416
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