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Development of a pan-Simbu real-time reverse transcriptase PCR for the detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR systems

BACKGROUND: Schmallenberg virus (SBV), a novel orthobunyavirus of the Simbu serogroup, was first identified in October 2011 in dairy cattle in Germany, where it caused fever, diarrhea and a drop in milk yield. Since then, SBV additionally has been detected in adult sheep and goats. Although symptoms...

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Autores principales: Fischer, Melina, Schirrmeier, Horst, Wernike, Kerstin, Wegelt, Anne, Beer, Martin, Hoffmann, Bernd
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4228258/
https://www.ncbi.nlm.nih.gov/pubmed/24188175
http://dx.doi.org/10.1186/1743-422X-10-327
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author Fischer, Melina
Schirrmeier, Horst
Wernike, Kerstin
Wegelt, Anne
Beer, Martin
Hoffmann, Bernd
author_facet Fischer, Melina
Schirrmeier, Horst
Wernike, Kerstin
Wegelt, Anne
Beer, Martin
Hoffmann, Bernd
author_sort Fischer, Melina
collection PubMed
description BACKGROUND: Schmallenberg virus (SBV), a novel orthobunyavirus of the Simbu serogroup, was first identified in October 2011 in dairy cattle in Germany, where it caused fever, diarrhea and a drop in milk yield. Since then, SBV additionally has been detected in adult sheep and goats. Although symptoms of acute infection were not observed, infection during a vulnerable phase of pregnancy caused congenital malformations and stillbirths. In view of the current situation and the possible emergence of further Simbu serogroup members, a pan-Simbu real-time reverse transcriptase (RT) PCR system for the reliable detection of Simbu serogroup viruses should be developed. METHODS: In this study a pan-Simbu real-time RT-PCR system was established and compared to several SBV real-time RT-PCR assays. All PCR-systems were tested using a panel of different Simbu serogroup viruses as well as several field samples from diseased cattle, sheep and goats originating from all over Germany. Several pan-Simbu real-time RT-PCR products were sequenced via Sanger sequencing. Furthermore, in silico analyses were performed to investigate suitability for the detection of further orthobunyaviruses. RESULTS: All tested members of the Simbu serogroup (n = 14) as well as most of the field samples were successfully detected by the pan-Simbu real-time RT-PCR system. The comparison of this intercalating dye assay with different TaqMan probe-based assays developed for SBV diagnostics confirmed the functionality of the pan-Simbu assay for screening purposes. However, the SBV-TaqMan-assay SBV-S3 delivered the highest analytical sensitivity of less than ten copies per reaction for duplex systems including an internal control. In addition, for confirmation of SBV-genome detection the highly specific SBV-M1 assay was established. CONCLUSION: The pan-Simbu real-time RT-PCR system was able to detect all tested members of the Simbu serogroup, most of the SBV field samples as well as three tested Bunyamwera serogroup viruses with a suitable sensitivity. According to in silico analyses, this system seems to be able to detect a broad orthobunyavirus spectrum. As an additional feature of the pan-Simbu real-time RT-PCR system, subsequent species classification via sequencing is feasible. Regarding SBV diagnostics, the performance of the S-segment targeting SBV-S3 assay was superior with respect to the analytical sensitivity.
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spelling pubmed-42282582014-11-13 Development of a pan-Simbu real-time reverse transcriptase PCR for the detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR systems Fischer, Melina Schirrmeier, Horst Wernike, Kerstin Wegelt, Anne Beer, Martin Hoffmann, Bernd Virol J Research BACKGROUND: Schmallenberg virus (SBV), a novel orthobunyavirus of the Simbu serogroup, was first identified in October 2011 in dairy cattle in Germany, where it caused fever, diarrhea and a drop in milk yield. Since then, SBV additionally has been detected in adult sheep and goats. Although symptoms of acute infection were not observed, infection during a vulnerable phase of pregnancy caused congenital malformations and stillbirths. In view of the current situation and the possible emergence of further Simbu serogroup members, a pan-Simbu real-time reverse transcriptase (RT) PCR system for the reliable detection of Simbu serogroup viruses should be developed. METHODS: In this study a pan-Simbu real-time RT-PCR system was established and compared to several SBV real-time RT-PCR assays. All PCR-systems were tested using a panel of different Simbu serogroup viruses as well as several field samples from diseased cattle, sheep and goats originating from all over Germany. Several pan-Simbu real-time RT-PCR products were sequenced via Sanger sequencing. Furthermore, in silico analyses were performed to investigate suitability for the detection of further orthobunyaviruses. RESULTS: All tested members of the Simbu serogroup (n = 14) as well as most of the field samples were successfully detected by the pan-Simbu real-time RT-PCR system. The comparison of this intercalating dye assay with different TaqMan probe-based assays developed for SBV diagnostics confirmed the functionality of the pan-Simbu assay for screening purposes. However, the SBV-TaqMan-assay SBV-S3 delivered the highest analytical sensitivity of less than ten copies per reaction for duplex systems including an internal control. In addition, for confirmation of SBV-genome detection the highly specific SBV-M1 assay was established. CONCLUSION: The pan-Simbu real-time RT-PCR system was able to detect all tested members of the Simbu serogroup, most of the SBV field samples as well as three tested Bunyamwera serogroup viruses with a suitable sensitivity. According to in silico analyses, this system seems to be able to detect a broad orthobunyavirus spectrum. As an additional feature of the pan-Simbu real-time RT-PCR system, subsequent species classification via sequencing is feasible. Regarding SBV diagnostics, the performance of the S-segment targeting SBV-S3 assay was superior with respect to the analytical sensitivity. BioMed Central 2013-11-05 /pmc/articles/PMC4228258/ /pubmed/24188175 http://dx.doi.org/10.1186/1743-422X-10-327 Text en Copyright © 2013 Fischer et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research
Fischer, Melina
Schirrmeier, Horst
Wernike, Kerstin
Wegelt, Anne
Beer, Martin
Hoffmann, Bernd
Development of a pan-Simbu real-time reverse transcriptase PCR for the detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR systems
title Development of a pan-Simbu real-time reverse transcriptase PCR for the detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR systems
title_full Development of a pan-Simbu real-time reverse transcriptase PCR for the detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR systems
title_fullStr Development of a pan-Simbu real-time reverse transcriptase PCR for the detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR systems
title_full_unstemmed Development of a pan-Simbu real-time reverse transcriptase PCR for the detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR systems
title_short Development of a pan-Simbu real-time reverse transcriptase PCR for the detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR systems
title_sort development of a pan-simbu real-time reverse transcriptase pcr for the detection of simbu serogroup viruses and comparison with sbv diagnostic pcr systems
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4228258/
https://www.ncbi.nlm.nih.gov/pubmed/24188175
http://dx.doi.org/10.1186/1743-422X-10-327
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