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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...

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Autores principales: Wang, Songqi, Shen, Haiyan, Lin, Qijie, Huang, Jun, Zhang, Chunhong, Liu, Zhicheng, Sun, Minhua, Zhang, Jianfeng, Liao, Ming, Li, Yugu, Zhang, Jianmin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
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.
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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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