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Development of a Cleaved Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of African Swine Fever Virus
African Swine Fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and lethal viral disease of pigs. However, commercial vaccines are not yet available, and neither are drugs to prevent or control ASF. Therefore, rapid, accurate on-site diagnosis is urgently needed for det...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2022
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9204333/ https://www.ncbi.nlm.nih.gov/pubmed/35719327 http://dx.doi.org/10.3389/fcimb.2022.884430 |
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author | Wang, Songqi Shen, Haiyan Lin, Qijie Huang, Jun Zhang, Chunhong Liu, Zhicheng Sun, Minhua Zhang, Jianfeng Liao, Ming Li, Yugu Zhang, Jianmin |
author_facet | Wang, Songqi Shen, Haiyan Lin, Qijie Huang, Jun Zhang, Chunhong Liu, Zhicheng Sun, Minhua Zhang, Jianfeng Liao, Ming Li, Yugu Zhang, Jianmin |
author_sort | Wang, Songqi |
collection | PubMed |
description | African Swine Fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and lethal viral disease of pigs. However, commercial vaccines are not yet available, and neither are drugs to prevent or control ASF. Therefore, rapid, accurate on-site diagnosis is urgently needed for detection during the early stages of ASFV infection. Herein, a cleaved probe-based loop-mediated isothermal amplification (CP-LAMP) detection method was established. Based on the original primer sets, we targeted the ASFV 9GL gene sequence to design a probe harboring a ribonucleotide insertion. Ribonuclease H2 (RNase H2) enzyme activity can only be activated when the probe is perfectly complementary, resulting in hydrolytic release of a quencher moiety, and consequent signal amplification. The method displayed robust sensitivity, with copy number detection as low as 13 copies/µL within 40 min at constant temperature (62°C). Visualization of the fluorescence product was employed using a self-designed 3D-printed visualization function cassette, and the CP-LAMP method achieved specific identification and visual detection of ASFV. Moreover, coupling the dual function cassette and smartphone quantitation makes the CP-LAMP assay first user-friendly, cost-effective, portable, rapid, and accurate point-of-care testing (POCT) platform for ASFV. |
format | Online Article Text |
id | pubmed-9204333 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-92043332022-06-18 Development of a Cleaved Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of African Swine Fever Virus Wang, Songqi Shen, Haiyan Lin, Qijie Huang, Jun Zhang, Chunhong Liu, Zhicheng Sun, Minhua Zhang, Jianfeng Liao, Ming Li, Yugu Zhang, Jianmin Front Cell Infect Microbiol Cellular and Infection Microbiology African Swine Fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and lethal viral disease of pigs. However, commercial vaccines are not yet available, and neither are drugs to prevent or control ASF. Therefore, rapid, accurate on-site diagnosis is urgently needed for detection during the early stages of ASFV infection. Herein, a cleaved probe-based loop-mediated isothermal amplification (CP-LAMP) detection method was established. Based on the original primer sets, we targeted the ASFV 9GL gene sequence to design a probe harboring a ribonucleotide insertion. Ribonuclease H2 (RNase H2) enzyme activity can only be activated when the probe is perfectly complementary, resulting in hydrolytic release of a quencher moiety, and consequent signal amplification. The method displayed robust sensitivity, with copy number detection as low as 13 copies/µL within 40 min at constant temperature (62°C). Visualization of the fluorescence product was employed using a self-designed 3D-printed visualization function cassette, and the CP-LAMP method achieved specific identification and visual detection of ASFV. Moreover, coupling the dual function cassette and smartphone quantitation makes the CP-LAMP assay first user-friendly, cost-effective, portable, rapid, and accurate point-of-care testing (POCT) platform for ASFV. Frontiers Media S.A. 2022-05-26 /pmc/articles/PMC9204333/ /pubmed/35719327 http://dx.doi.org/10.3389/fcimb.2022.884430 Text en Copyright © 2022 Wang, Shen, Lin, Huang, Zhang, Liu, Sun, Zhang, Liao, Li and Zhang 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 | Cellular and Infection Microbiology Wang, Songqi Shen, Haiyan Lin, Qijie Huang, Jun Zhang, Chunhong Liu, Zhicheng Sun, Minhua Zhang, Jianfeng Liao, Ming Li, Yugu Zhang, Jianmin Development of a Cleaved Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of African Swine Fever Virus |
title | Development of a Cleaved Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of African Swine Fever Virus |
title_full | Development of a Cleaved Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of African Swine Fever Virus |
title_fullStr | Development of a Cleaved Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of African Swine Fever Virus |
title_full_unstemmed | Development of a Cleaved Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of African Swine Fever Virus |
title_short | Development of a Cleaved Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of African Swine Fever Virus |
title_sort | development of a cleaved probe-based loop-mediated isothermal amplification assay for rapid detection of african swine fever virus |
topic | Cellular and Infection Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9204333/ https://www.ncbi.nlm.nih.gov/pubmed/35719327 http://dx.doi.org/10.3389/fcimb.2022.884430 |
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