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      A real-time PCR method for quantification of the total and major variant strains of the deformed wing virus

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      PLoS ONE
      Public Library of Science

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          Abstract

          European honey bees ( Apis mellifera) are critically important to global food production by virtue of their pollination services but are severely threatened by deformed wing virus (DWV) especially in the presence of the external parasite Varroa destructor. DWV exists as many viral strains with the two major variants (DWV-A and DWV-B) varying in virulence. A single plasmid standard was constructed containing three sections for the specific determination of DWV-A (VP2 capsid region), DWV-B (IRES) and a conserved region suitable for total DWV (helicase region). The assays were confirmed as specific and discriminatory with limits of detections of 25, 25 and 50 genome equivalents for DWV-A, DWV-B and total-DWV, respectively. The methods were successfully tested on Apis mellifera and V. destructor samples with varying DWV profiles. The new method determined a more accurate total DWV titre in samples with substantial DWV-B than the method currently described in the COLOSS Beebook. The proposed assays could be utilized for the screening of large quantities of bee material for both a total DWV load overview along with more detailed investigations into DWV-A and DWV-B profiles.

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          Most cited references42

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          Primer3Plus, an enhanced web interface to Primer3

          Here we present Primer3Plus, a new web interface to the popular Primer3 primer design program as an enhanced alternative for the CGI- scripts that come with Primer3. Primer3 consists of a command line program and a web interface. The web interface is one large form showing all of the possible options. This makes the interface powerful, but at the same time confusing for occasional users. Primer3Plus provides an intuitive user interface using present-day web technologies and has been developed in close collaboration with molecular biologists and technicians regularly designing primers. It focuses on the task at hand, and hides detailed settings from the user until these are needed. We also added functionality to automate specific tasks like designing primers for cloning or step-wise sequencing. Settings and designed primer sequences can be stored locally for later use. Primer3Plus supports a range of common sequence formats, such as FASTA. Finally, primers selected by Primer3Plus can be sent to an order form, allowing tight integration into laboratory ordering systems. Moreover, the open architecture of Primer3Plus allows easy expansion or integration of external software packages. The Primer3Plus Perl source code is available under GPL license from SourceForge. Primer3Plus is available at http://www.bioinformatics.nl/primer3plus.
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            GenBank

            GenBank® is a comprehensive database that contains publicly available DNA sequences for more than 165 000 named organisms, obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects. Most submissions are made using the web-based BankIt or standalone Sequin programs and accession numbers are assigned by GenBank staff upon receipt. Daily data exchange with the EMBL Data Library in the UK and the DNA Data Bank of Japan helps to ensure worldwide coverage. GenBank is accessible through NCBI's retrieval system, Entrez, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and the biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of the GenBank database are available by FTP. To access GenBank and its related retrieval and analysis services, go to the NCBI Homepage at http://www.ncbi.nlm.nih.gov.
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              The German bee monitoring project: a long term study to understand periodically high winter losses of honey bee colonies

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                Author and article information

                Contributors
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: Project administrationRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                19 December 2017
                2017
                : 12
                : 12
                : e0190017
                Affiliations
                [001]Institute of Biological and Environmental Sciences, School of Biological Sciences, University of Aberdeen, Aberdeen, United Kingdom
                University of North Carolina at Greensboro, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-0306-9033
                Article
                PONE-D-17-21936
                10.1371/journal.pone.0190017
                5736226
                29261772
                9c308142-ed57-48ae-9c1f-b5c70641a259
                © 2017 Bradford et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 8 June 2017
                : 6 December 2017
                Page count
                Figures: 7, Tables: 0, Pages: 19
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100000268, Biotechnology and Biological Sciences Research Council;
                Award ID: BB/M503526/1
                Award Recipient :
                Funded by: Scottish Government
                Award Recipient :
                Funded by: EARS
                Award Recipient :
                Funded by: European Union - Framework 7
                Award ID: 613960
                Award Recipient :
                Funded by: The Morley Agricultural Foundation
                Award Recipient :
                Funded by: Scottish Bee Keepers Association
                Award Recipient :
                ELB was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) EASTBIO Doctoral Training Partnership ( http://www.bbsrc.ac.uk) [grant number BB/J01446X/1] and an Eastern Association Regional Studentship (EARS) and The Morley Agricultural Foundation awarded to ASB. CRC was supported by a KTN BBSRC CASE studentship (BB/M503526/1) ( http://www.bbsrc.ac.uk), part-funded by the Scottish Beekeeping Association ( https://www.scottishbeekeepers.org.uk/) and the Animal Health - Disease Prevention, Scottish Government awarded to ASB CRC. This project received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no 613960 (SMARTBEES) ( http://www.smartbees-fp7.eu/) awarded to ASB. The funders had no role in study design, data collection and analysis decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Invertebrates
                Arthropoda
                Insects
                Hymenoptera
                Bees
                Honey Bees
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                DNA construction
                Plasmid Construction
                Research and analysis methods
                Molecular biology techniques
                DNA construction
                Plasmid Construction
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and analysis methods
                Extraction techniques
                RNA extraction
                Biology and Life Sciences
                Microbiology
                Virology
                Viral Transmission and Infection
                Viral Load
                Research and Analysis Methods
                Database and Informatics Methods
                Bioinformatics
                Sequence Analysis
                Sequence Alignment
                Biology and Life Sciences
                Microbiology
                Virology
                Viral Replication
                Internal Ribosome Entry Site
                Biology and Life Sciences
                Genetics
                Genomics
                Animal Genomics
                Invertebrate Genomics
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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                Uncategorized

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