Optimisation of a triplex real time RT-PCR for detection of hepatitis E virus RNA and validation on biological samples.
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/μl 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.
Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic. vasickova@vri.cz
Petra
P
Vasickova
Petr
P
Kralik
Iva
I
Slana
Ivo
I
Pavlik
D000818:Animals; D003670:Deer / Q000821:virology; D016751:Hepatitis E / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D016752:Hepatitis E virus / Q000235:genetics*; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics / Q000592:standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D012756:Sheep / Q000821:virology; D034421:Sus scrofa / Q000821:virology
D012367:RNA, Viral