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      Rapid and Sensitive Recombinase Polymerase Amplification Combined With Lateral Flow Strip for Detecting African Swine Fever Virus

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          Abstract

          African swine fever virus (ASFV), the etiological agent of African swine fever (ASF), a hemorrhagic fever of domestic pigs, has devastating consequences for the pig farming industry. More than 1,000,000 pigs have been slaughtered since 3 August 2018 in China. However, vaccines or drugs for ASF have yet to be developed. As such, a rapid test that can accurately detect ASFV on-site is important to the timely implementation of control measures. In this study, we developed a rapid test that combines recombinase polymerase amplification (RPA) of the ASFV p72 gene with lateral flow detection (LFD). Results showed that the sensitivity of recombinase polymerase amplification with lateral flow dipstick (RPA-LFD) for ASFV was 150 copies per reaction within 10 min at 38°C. The assay was highly specific to ASFV and had no cross-reactions with other porcine viruses, including classical swine fever virus (CSFV). A total of 145 field samples were examined using our method, and the agreement of the positive rate between RPA-LFD (10/145) and real-time PCR (10/145) was 100%. Overall, RPA-LFD provides a novel alternative for the simple, sensitive, and specific identification of ASFV and showed potential for on-site ASFV detection.

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          Most cited references20

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          African Swine Fever Virus: A Review

          African swine fever (ASF) is a highly contagious viral disease of swine which causes high mortality, approaching 100%, in domestic pigs. ASF is caused by a large, double stranded DNA virus, ASF virus (ASFV), which replicates predominantly in the cytoplasm of macrophages and is the only member of the Asfarviridae family, genus Asfivirus. The natural hosts of this virus include wild suids and arthropod vectors of the Ornithodoros genus. The infection of ASFV in its reservoir hosts is usually asymptomatic and develops a persistent infection. In contrast, infection of domestic pigs leads to a lethal hemorrhagic fever for which there is no effective vaccine. Identification of ASFV genes involved in virulence and the characterization of mechanisms used by the virus to evade the immune response of the host are recognized as critical steps in the development of a vaccine. Moreover, the interplay of the viral products with host pathways, which are relevant for virus replication, provides the basic information needed for the identification of potential targets for the development of intervention strategies against this disease.
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            Highly sensitive PCR assay for routine diagnosis of African swine fever virus in clinical samples.

            This work provides a novel, highly sensitive, hot start PCR method for rapid and specific detection of African swine fever virus (ASFV) that can be used as a routine diagnostic test for ASFV in surveillance, control, and eradication programs. A confirmatory test of the specificity of this method based on restriction endonuclease analysis was also developed.
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              Preclinical diagnosis of African swine fever in contact-exposed swine by a real-time PCR assay.

              A fluorogenic probe hydrolysis (TaqMan) PCR assay for African swine fever virus (ASFV) was developed and evaluated in experimentally infected swine. This sensitive and specific one-step single-tube assay, which can be performed in 2 h or less, detected viral DNA in tonsil scraping samples 2 to 4 days prior to onset of clinical disease. Thus, the assay would have application for preclinical diagnosis of African swine fever and surveillance and/or emergency management of a disease outbreak.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                15 May 2019
                2019
                : 10
                : 1004
                Affiliations
                [1] 1Institute of Military Veterinary Medicine, Academy of Military Medical Science , Changchun, China
                [2] 2College of Life Science, Ningxia University , Yinchuan, China
                Author notes

                Edited by: Douglas P. Gladue, Plum Island Animal Disease Center (USDA-ARS), United States

                Reviewed by: Armanda Bastos, University of Pretoria, South Africa; Charles Masembe, Makerere University, Uganda

                These authors have contributed equally to this work

                This article was submitted to Virology, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2019.01004
                6530510
                31156571
                bb828eec-c670-4932-b648-2216dd1be98a
                Copyright © 2019 Miao, Zhang, Li, Chen, Wang, Zhang, Mi, Zhang, Wang, Wang, Zhou, Zhang, Li, Zhang and Hu.

                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.

                History
                : 28 February 2019
                : 18 April 2019
                Page count
                Figures: 5, Tables: 1, Equations: 0, References: 30, Pages: 7, Words: 0
                Categories
                Microbiology
                Methods

                Microbiology & Virology
                african swine virus,on site detection,recombinase polymerase amplification,lateral flow strip,rpa-lfd

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