Validation of an internally controlled multiplex real time RT-PCR for detection and typing of HEV genotype 3 and 4

Hepatitis E virus (HEV) genotypes 1 and 2 are restricted to humans, whereas genotypes 3 (HEV 3) and genotype 4 (HEV 4) infect humans and a variety of animal species. Cross-species infections by animal strains raise potential public health concerns for zoonotic HEV transmission. Therefore, a real-time reverse transcription polymerase chain reaction (RT-qPCR) combining the HEV 3-tpye specific RT-qPCR assay with the HEV 4-tpye specific assay was developed. Furthermore, a heterologous RNA, an in vitro transcript of the enhanced green fluorescent protein (EGFP) gene, was introduced as an internal control. The data showed that EGFP gene provided a very reliable and simple way of monitoring both the sample manipulation and amplification procedures. The final multiplex RT-qPCR assay showed a high analytical sensitivity of less than 50 copies RNA per reaction for both HEV genotypes. The specificity and amplification efficiency of the multiplex assay for the respective HEV were confirmed by co-amplification of the other target. By comparing with the results of mono-specific assay and nested PCR as well as sequencing, HEV infection in a panel of clinical samples was reliably detected and typed, which indicated that the novel multiplex RT-qPCR assay could be used for sensitive detection and rapid differentiation of zoonotic HEV genotype 3 and 4.