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Development of a SYBR Green real-time RT-PCR assay for the detection of avian encephalomyelitis virus

•A highly sensitive SYBR Green real-time RT-PCR for detection of avian encephalomyelitis virus.•Quantitation assay examining at least 10 copies of the standard templates.•Highly specific for the isolated virus from infected chicken embryos.•Sensitively detectable in the 18 clinical chicken samples f...

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Published in:Journal of virological methods 2014-09, Vol.206, p.46-50
Main Authors: Liu, Qingtian, Yang, Zengqi, Hao, Huafang, Cheng, Shenli, Fan, Wentao, Du, Enqi, Xiao, Sa, Wang, Xinglong, Zhang, Shuxia
Format: Article
Language:English
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Summary:•A highly sensitive SYBR Green real-time RT-PCR for detection of avian encephalomyelitis virus.•Quantitation assay examining at least 10 copies of the standard templates.•Highly specific for the isolated virus from infected chicken embryos.•Sensitively detectable in the 18 clinical chicken samples for diagnosis. Avian encephalomyelitis virus (AEV) causes epidemic diseases in poultry worldwide. A SYBR Green real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay was developed for the rapid detection and quantitation of AEV in this study. A pair of specific primers was designed in the highly conserved VP1 gene of this virus. When comparing this assay with conventional RT-PCR, the rRT-PCR assay was 100 times more sensitive and could detect levels as low as 10 standard DNA copies of the AEV SX strain. The specificity of this technique was evaluated in five other avian pathogens. The AEV RNA was detected as early as three days post-infection in chicken embryos. All 18 clinical chicken brains collected from an AEV outbreak in Northwestern China were detected to be positive (100%) using the rRT-PCR assay. However, only 5 of the 18 samples were positive (28%) using the conventional RT-PCR. The results were confirmed by virus isolation in chicken embryos. This high sensitivity, specificity, and simplicity of the SYBR Green rRT-PCR approach can be a more effective method than the conventional one for AEV diagnosis and surveillance.
ISSN:0166-0934
1879-0984
DOI:10.1016/j.jviromet.2014.05.015