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

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• 作者：Melina Fischer (1)
  Horst Schirrmeier (1)
  Kerstin Wernike (1)
  Anne Wegelt (1)
  Martin Beer (1)
  Bernd Hoffmann (1)
  
• 关键词：Orthobunyavirus ; Simbu serogroup ; Schmallenberg virus ; Real ; time RT ; PCR
• 刊名：Virology Journal
• 出版年：2013
• 出版时间：December 2013
• 年：2013
• 卷：10
• 期：1
• 全文大小：898 KB
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• 作者单位：Melina Fischer (1)
  Horst Schirrmeier (1)
  Kerstin Wernike (1)
  Anne Wegelt (1)
  Martin Beer (1)
  Bernd Hoffmann (1)
  
  1. Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, D-17493, Greifswald-Insel Riems, Germany
  
• ISSN：1743-422X

文摘

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--4) 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.