title: Validation of an internally controlled multiplex real time RT-PCR for detection and typing of HEV genotype 3 and 4.
abstract: 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.
authors.affiliation: Zhejiang Entry-Exit Inspection and Quarantine Bureau, 126 Fuchun Road, Hangzhou 310016, China; Yiwu Entry-Exit Inspection and Quarantine Bureau, 299 Chengbei Road, Yiwu 322000, China. Electronic address: zxf@ziq.gov.cn.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Zhang
authors.forename: Aiyun
authors.initials: A
authors.lastname: Li
authors.forename: Jiangbing
authors.initials: J
authors.lastname: Shuai
authors.forename: Yuntong
authors.initials: Y
authors.lastname: Dai
authors.forename: Zhenjiang
authors.initials: Z
authors.lastname: Zhu
authors.forename: Shan
authors.initials: S
authors.lastname: Wu
authors.forename: Yongqiang
authors.initials: Y
authors.lastname: He
mesh_terms: D000818:Animals; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000235:genetics; D016751:Hepatitis E / Q000175:diagnosis* / Q000662:veterinary* / Q000821:virology; D016752:Hepatitis E virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Genotype; Hepatitis E virus; Internal control; Multiplex; Real time PCR