Development of an improved RT-qPCR Assay for detection of Japanese encephalitis virus (JEV) RNA including a systematic review and comprehensive comparison with published methods
| Autor: | Paul N. Newton, Viengmon Davong, Géraldine Piorkowski, Manivanh Vongsouvath, Onanong Sengvilaipaseuth, Phonepasith Panyanouvong, Audrey Dubot-Pérès, Malavanh Vongsouvath, Jeremy A. Garson, Tehmina Bharucha, Xavier de Lamballerie |
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| Přispěvatelé: | Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Mahidol University [Bangkok]-Mahosot Hospital, Departments of Infectious Diseases and Microbiology [London, UK], Royal Free Hospital [London, UK], Division of Infection and Immunity [London, UK], University College of London [London] (UCL), Unité des Virus Emergents (UVE), Institut de Recherche pour le Développement (IRD)-Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM), National Transfusion Microbiology Laboratories [London, UK], NHS Blood and Transplant [London, UK], Centre for Tropical Medicine and Global Health [Oxford, UK], Nuffield Department of Medicine [Oxford, UK] (Big Data Institute), University of Oxford [Oxford]-University of Oxford [Oxford], London School of Hygiene and Tropical Medicine (LSHTM), This study was supported by the Wellcome Trust of Great Britain, IRD to PNN and by the European Union’s Horizon 2020 research and innovation programme EVAg under grant agreement N˚ 653316 to XdL., European Project: 653316,H2020,H2020-INFRAIA-2014-2015,EVAg(2015), University of Oxford-University of Oxford, BUISINE, Soline, European Virus Archive goes global - EVAg - - H20202015-04-01 - 2019-03-31 - 653316 - VALID |
| Rok vydání: | 2018 |
| Předmět: |
RNA viruses
Central Nervous System 0301 basic medicine Serial dilution viruses lcsh:Medicine Artificial Gene Amplification and Extension Polymerase Chain Reaction Nervous System Biochemistry Database and Informatics Methods 0302 clinical medicine Genotype lcsh:Science Pathology and laboratory medicine [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology Encephalitis Virus Japanese Multidisciplinary Reverse Transcriptase Polymerase Chain Reaction Physics Medical microbiology Research Assessment 3. Good health Nucleic acids GenBank Physical Sciences Viruses Nucleic acid thermodynamics [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology RNA annealing Pathogens Anatomy West Nile virus Sequence Analysis Encephalitis Research Article Optimization Asia Systematic Reviews Bioinformatics In silico Annealing (genetics) 030231 tropical medicine Biophysics Biology Research and Analysis Methods Real-Time Polymerase Chain Reaction Microbiology Virus 03 medical and health sciences medicine Humans Molecular Biology Techniques Encephalitis Japanese Molecular Biology Medicine and health sciences Flaviviruses lcsh:R Organisms Viral pathogens Biology and Life Sciences RNA Reverse Transcriptase-Polymerase Chain Reaction Japanese encephalitis medicine.disease Virology Microbial pathogens 030104 developmental biology lcsh:Q Sequence Alignment Mathematics |
| Zdroj: | PLoS ONE PLoS ONE, Public Library of Science, 2018, 13 (3), pp.e0194412. ⟨10.1371/journal.pone.0194412⟩ PLoS ONE, 2018, 13 (3), pp.e0194412. ⟨10.1371/journal.pone.0194412⟩ PLoS ONE, Vol 13, Iss 3, p e0194412 (2018) |
| ISSN: | 1932-6203 |
| Popis: | Background Japanese encephalitis virus (JEV) is a major cause of encephalitis in Asia, and the commonest cause of mosquito-borne encephalitis worldwide. Detection of JEV RNA remains challenging due to the characteristic brief and low viraemia, with 0–25% of patients positive, and the mainstay of diagnosis remains detection of anti-JEV IgM antibody. Methods We performed a systematic review of published RT-PCR protocols, and evaluated them in silico and in vitro alongside new primers and probes designed using a multiple genome alignment of all JEV strains >9,000nt from GenBank, downloaded from the NCBI website (November 2016). The new assays included pan-genotype and genotype specific assays targeting genotypes 1 and 3. Results Ten RT-qPCR assays were compared, a pre-existing in-house assay, three published assays and six newly designed assays, using serial RNA dilutions. We selected three assays, one published and two novel assays, with the lowest limit of detection (LOD) for further optimisation and validation. One of the novel assays, detecting NS2A, showed the best results, with LOD approximately 4 copies/ reaction, and no cross-reaction on testing closely related viruses in the JEV serocomplex, West Nile Virus and St. Louis Virus. The optimised assays were validated in consecutive patients with central nervous system infections admitted to hospitals in Laos, testing paired CSF and serum samples. Conclusions We succeeded in developing a JEV specific RT-qPCR assay with at least 1 log10 improved sensitivity as compared to existing assays. Further evaluation is required, field-testing the assay in a larger group of patients. |
| Databáze: | OpenAIRE |
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