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      Survey and Visual Detection of Zaire ebolavirus in Clinical Samples Targeting the Nucleoprotein Gene in Sierra Leone

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          Abstract

          Ebola virus (EBOV) can lead to severe hemorrhagic fever with a high risk of death in humans and other primates. To guide treatment and prevent spread of the viral infection, a rapid and sensitive detection method is required for clinical samples. Here, we described and evaluated a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect Zaire ebolavirus using the nucleoprotein gene ( NP) as a target sequence. Two different techniques were used, a calcein/Mn 2+ complex chromogenic method and real-time turbidity monitoring. The RT-LAMP assay detected the NP target sequence with a limit of 4.56 copies/μL within 45 min under 61°C, a similar even or increase in sensitivity than that of real-time reverse transcription-polymerase chain reaction (RT-PCR). Additionally, all pseudoviral particles or non- Zaire EBOV genomes were negative for LAMP detection, indicating that the assay was highly specific for EBOV. To appraise the availability of the RT-LAMP method for use in clinical diagnosis of EBOV, of 417 blood or swab samples collected from patients with clinically suspected infections in Sierra Leone, 307 were identified for RT-LAMP-based surveillance of EBOV. Therefore, the highly specific and sensitive RT-LAMP method allows the rapid detection of EBOV, and is a suitable tool for clinical screening, diagnosis, and primary quarantine purposes.

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          Most cited references21

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          Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation.

          The loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that uses only one type of enzyme. One of the characteristics of the LAMP method is its ability to synthesize extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophosphate ion, is produced, yielding white precipitate of magnesium pyrophosphate in the reaction mixture. Judging the presence or absence of this white precipitate allows easy distinction of whether nucleic acid was amplified by the LAMP method. Since an increase in the turbidity of the reaction mixture according to the production of precipitate correlates with the amount of DNA synthesized, real-time monitoring of the LAMP reaction was achieved by real-time measurement of turbidity. Copyright 2001 Academic Press.
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            Ebola virus disease in West Africa--clinical manifestations and management.

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              ELISA for the detection of antibodies to Ebola viruses.

              EIAs for IgG and IgM antibodies directed against Ebola (EBO) viral antigens have been developed and evaluated using sera of animals and humans surviving infection with EBO viruses. The IgM capture assay detected anti-EBO (subtype Reston) antibodies in the sera of 5 of 5 experimentally infected animals at the time they succumbed to lethal infections. IgM antibodies were also detected in the serum of a human who was infected with EBO (subtype Reston) during a postmortem examination of an infected monkey. The antibody was detectable as early as day 6 after infection in experimentally infected animals and persisted for 400 days in 3 animals who survived infection, and it persisted for approximately 10 years after infection in the sera of 2 humans. Although these data are limited by the number of sera available for verification, the IgM assay seems to have great promise as a diagnostic tool. Furthermore the long-term persistence of the IgG antibodies measured by this test strongly suggests that the ELISA will be useful in field investigations of EBO virus.
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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
                01 December 2015
                2015
                : 6
                : 1332
                Affiliations
                Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China
                Author notes

                Edited by: Andres M. Perez, University of Minnesota, USA

                Reviewed by: Ilhem Messaoudi, Oregon Health and Science University, USA; Hideki Ebihara, National Institutes of Health, USA

                *Correspondence: Jing Yuan, yuanjing6216@ 123456163.com ; Chao Liu, liuchao9588@ 123456sina.com ; Xiangna Zhao, xnazhao@ 123456163.com

                These authors are co-first authors.

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

                Article
                10.3389/fmicb.2015.01332
                4664619
                26648918
                6f3c3bac-c13d-46d4-b79d-cf94f8949b50
                Copyright © 2015 Li, Wang, Liu, Wei, Lin, Li, Li, Dong, Cui, Hu, Li, Ma, Zhao, Liu and Yuan.

                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) or licensor 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
                : 02 August 2015
                : 12 November 2015
                Page count
                Figures: 4, Tables: 2, Equations: 0, References: 27, Pages: 7, Words: 0
                Funding
                Funded by: National Natural Science Foundation of China 10.13039/501100001809
                Award ID: 31370093
                Funded by: Mega-projects of Science and Technology Research of China
                Award ID: Grant 2011ZX10004-001
                Funded by: National High Technology Research and Development Program of China (863 Program)
                Award ID: SS2014AA022210
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                zaire ebov,rt-lamp,sensitivity,specificity,rapid detection,prevalence
                Microbiology & Virology
                zaire ebov, rt-lamp, sensitivity, specificity, rapid detection, prevalence

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