Journal
JOURNAL OF VIROLOGICAL METHODS
Volume 180, Issue 1-2, Pages 38-42Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jviromet.2011.12.007
Keywords
Internal amplification control; RNA quantitation; Zoonosis
Funding
- Ministry of Agriculture [MZE0002716202]
- Ministry of Education, Youth and Sports of Czech Republic [0008045, AdmireVet CZ 1.05/2.1.00/01.0006-ED0006/01/01]
- EC [929]
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The aim of this study was to optimise a two-tube reverse transcription triplex quantitative real time PCR (qRT-PCR) combining amplification of two loci with an internal amplification control (IAC) for detection and quantitation of hepatitis E virus (HEV) RNA and to validate its performance on a pool of biological samples. Optimisation was performed on serially diluted home-made RNA standards. The limit of detection was determined experimentally as 10 copies/mu l of the RNA standard for both amplification targets. The qRT-PCR was validated on a cohort of samples originating from 48 wild boars (Sus scrofa), 17 fallow deer (Dama dama) and 28 mouflons (Ovis musimon), with nested RT-PCR used as a reference method. qRT-PCR was found to be more specific for the detection of HEV RNA in examined samples. HEV RNA was detected in samples of five more animals (one wild boar and four mouflons) in comparison with nested RT-PCR. (C) 2011 Elsevier B.V. All rights reserved.
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