title: Development and validation of rapid magnetic particle based extraction protocols.
abstract: BACKGROUND
  In order to control and eradicate transboundary animal diseases, early diagnosis and reaction is essential for the implementation of control activities. Thus, mobile diagnostic units which allow analytical testing close to the site of occurrence could provide valuable support for centralized laboratories. Consequently, the availability of diagnostic tests using mobile amplification and detection technologies has been increasing over the past years. However, methods enabling rapid and simple nucleic acid extraction also under resource-limited settings are still scarce.
  
  
  METHODS
  In the present study rapid extraction protocols based on magnetic particle technology have been developed. For this purpose, the two open extraction platforms KingFisher™ Duo (Thermo Fisher Scientific) and BioSprint® 15 (Qiagen) as well as the fully automated EZ1® advanced XL instrument (Qiagen) were used. All protocols were validated in comparison to standard manual extraction using blood and serum samples from animals infected with Schmallenberg virus or bovine viral diarrhea virus.
  
  
  RESULTS
  All newly developed protocols allowed a complete extraction within 30 minutes of time. The fully automated EZ1-extraction yielded the highest reproducibility, whereas slightly higher intra- and inter-assay variations were observed using the open platforms. Compared to the manual procedure, the analytical sensitivity of all the rapid protocols was 1 log10 step reduced for extraction from blood samples. For sera a reduced dynamic range could only be observed using the maximally shortened BioSprint 15 protocol. Validation using clinical samples showed an excellent concordance of all the rapid extraction protocols to the standard manual extraction procedure, independent of sample materials and target viruses.
  
  
  CONCLUSIONS
  The results of this study show that the speed-optimized novel extraction protocols allow rapid and simple nucleic acid extractions for a variety of target viruses without significant loss of sensitivity compared to standard procedures. For this reason they represent valuable tools to accelerate magnetic particle based automated extraction.
authors.forename: Andrea
authors.initials: A
authors.lastname: Aebischer
authors.forename: Martin
authors.initials: M
authors.lastname: Beer
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Suedufer 10, 17493 Greifwald-Insel Riems, Germany. bernd.hoffmann@fli.bund.de.
authors.forename: Bernd
authors.initials: B
authors.lastname: Hoffmann
mesh_terms: D000818:Animals; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000175:diagnosis / Q000821:virology; D002417:Cattle; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics; D025202:Molecular Diagnostic Techniques / Q000295:instrumentation / Q000379:methods* / Q000592:standards*; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000302:isolation & purification; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic
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