A proposed validation method for automated nucleic acid extraction and RT-qPCR analysis: an example using Bluetongue virus

Journal of Virological Methods
Elise VandemeulebrouckeFrank Vandenbussche

Abstract

This study proposes a validation strategy for an automated extraction procedure, followed by RT-qPCR analysis. To avoid false-negative results, a triplex RT-qPCR was used which detects the target viral RNA, an internal and an external control. The methods to determine the validation parameters such as linearity, efficiency, analytical sensitivity, analytical specificity and intra- and interrun variability are described in detail. Special attention is given to the analytical sensitivity, which is determined by probit analysis. The limit of detection was set at the input concentration resulting in a positive result in 95% of the repeats. The intra- and interrun variability was analysed profoundly by testing samples covering a broad range of viral loads, from strong positive to weak positive. To increase the diagnostic capacity, the extraction protocol was automated with a JANUS Automated Workstation (PerkinElmer, Waltham, MA), which can extract 186 samples in 2h and 30 min. The automation of the extraction protocol implied some additional validation parameters to be determined such as position-effect, absence of cross-contamination and comparison with the manual protocol. These parameters give essential information about the perf...Continue Reading

References

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Citations

May 25, 2011·Transboundary and Emerging Diseases·M GariglianyK De Clercq
Aug 3, 2010·Journal of Virological Methods·Tine VanbinstKris De Clercq
Nov 24, 2012·Journal of Virological Methods·Françoise PolMarie-Frédérique Le Potier
Apr 1, 2014·Journal of Virological Methods·Melina FischerBernd Hoffmann
Feb 18, 2014·Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc·Xingnian GuPeter D Kirkland
Sep 25, 2019·Archives of Virology·Felipe Augusto SouzaAntônio Augusto Fonseca Júnior
Nov 8, 2020·Archives of Virology·Gabriel Augusto de Oliveira LopesJenner Karlisson Pimenta Dos Reis

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