Optimisation of a triplex real time RT-PCR for detection of hepatitis E virus RNA and validation on biological samples

► Reverse transcription triplex quantitative real time PCR assay for detection and quantitation of hepatitis E virus (HEV) RNA was optimised. ► The assay was able to detect at least 10 copies of the HEV genome/μl of isolated RNA and allows quantitation based on RNA standards. ► Application on biological samples revealed that amplification of two HEV loci increased the likelihood of HEV RNA detection. ► The use of RNA internal amplification control facilitated monitoring of false negative results.