Development and evaluation of a SYBR Green real-time RT-PCR assay for detection of avian hepatitis E virus.

Qin Zhao,Huiya Li,Yani Sun,En-Min Zhou,Xinjie Wang,Sha Xie,Jiming Gao,Baoyuan Liu,Shahid Faraz Syed,Lizhen Wang,Yiyang Chen,Gaiping Zhang

doi:10.1186/s12917-015-0507-5

Abstract

BackgroundAvian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease, as well as hepatitis-splenomegaly syndrome in chickens. To date, conventional reverse transcriptase polymerase chain reaction (RT-PCR) and nested RT-PCR methods have been used for the diagnosis of avian HEV infection in chickens. However, these assays are time consuming, inconvenient, and cannot detect the virus quantitatively. In this study, a rapid and sensitive SYBR Green real-time RT-PCR assay was developed to detect avian HEV RNA quantitatively in serum, liver, spleen, and fecal samples from chickens.ResultsBased on the sequence of the most conserved HEV gene, ORF3, the primers for the assay were designed, and the standard plasmid was constructed. The detection limit of the assay was shown to be 10 copies/μl of standard plasmid/reaction, with a corresponding cycle-threshold value of 29.3. The standard curve exhibited a dynamic linear range across at least 7 log units of DNA copy number. The specificity and reproducibility of this assay was high, showing that the assay detected avian HEV RNA specifically and with little variability. Compared to conventional RT-PCR, the current assay is more sensitive for detecting avian HEV in serum, liver, spleen, and fecal samples from chickens.ConclusionsA rapid, specific, and reproducible SYBR Green real-time RT-PCR assay was developed for the diagnosis of avian HEV infection in chickens. This assay can accurately detect avian HEV RNA in serum, liver, spleen, and fecal samples with more sensitivity than conventional RT-PCR.

Highlights

Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease, as well as hepatitis-splenomegaly syndrome in chickens
The aim of the present study was to develop a rapid, sensitive, and specific SYBR Green real time reverse transcriptase polymerase chain reaction (RT-PCR) assay for the identification and quantification of avian HEV RNA in chicken samples and to compare its sensitivity and specificity with the currently prevailing RT
Comparison of SYBR Green real-time RT-PCR and conventional RT-PCR for sensitivity in detecting positive clinical samples SYBR Green real-time RT-PCR and conventional RT-PCR were performed simultaneously to test 90 clinical samples (32 livers, 16 spleens, and 42 fecal samples), which were collected at different time intervals from birds showing a decrease in egg production in the Shaanxi province of Variation Different Ct values for the different dilution of dilutions standard plasmids

Summary

Introduction

Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease, as well as hepatitis-splenomegaly syndrome in chickens. Conventional reverse transcriptase polymerase chain reaction (RT-PCR) and nested RT-PCR methods have been used for the diagnosis of avian HEV infection in chickens. These assays are time consuming, inconvenient, and cannot detect the virus quantitatively. Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease, as well as hepatitissplenomegaly syndrome in chickens [1,2,3]. The genomes of the different genotypes share approximately 80 % nucleotide

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