Development And Preliminary Application Of Real-time RT-PCR For Detection Of Avian Encephalomyelitis Virus

Avian encephalomyelitis (avian encephalomyelitis, AE) is primarily affecting thecentral nervous system of young chickens which is under4weeks old. It was firstdescribedn in United States in1930. Young chicks show neurological symptoms that arecharacterized by ataxia, paresis or paralysis, and rapid tremors of the head and neck. Noneurologic sign can be observed in adult laying birds, but the virus causes a slight reductionin egg production, AEV has caused considerable economic losses.The definitive methods are time consuming and labor intensive or are usuallyhampered by nonspecific reactions, cross-reactions, and having a high risk of contamination.Therefore, a rapid and highly sensitive technique is required for AEV diagnosis andsurveillance before the virus spreads widely. This study attempts to create a SYBER GreenrRT-PCR assay for the molecular diagnosis of AEV that is more sensitive and specific thanvirus isolation and serological assays to provide a reliable method for detection of AEV. Ourwork obtained following results:A pair of rRT-PCR primers based on the highly conserved VP1gene of AEV strains1143was designed. Gene fragment of VP1gene was amplified from AEV strain SX byRT-PCR and was inserted into the pMD-19T cloning vector to construct standard plasmids.The Real-time RT-PCR standard curve for detecting AEV were developed successfullyunder optimized conditions. The efficiency of the rRT-PCR assay was0.913, and thecorrelation coefficient (R2) of the standard curve was0.9977, with a slope value of thecurve of-3.5714. When comparing this assay with conventional RT-PCR, the rRT-PCRassay was100times more sensitive and could detect levels as low as10standard DNAcopies of the AEV. A rapid, sensitive, specific, and reproducible SYBR Green Real-timeRT-PCR was developed for the detection and quantification of AEV.The RNA of AEV was detected as early as three days post-infection in chickenembryos. All18clinical chicken brains collected from an AEV outbreak in NorthwesternChina were detected to be positive using the Real-time RT-PCR assay. However, only5ofthe18the samples were positive using the conventional RT-PCR. In conclusion, a rapid, sensitive, specific, and reproducible SYBR Green I Real-timeRT-PCR was developed, which can be a promising for the detection and quantification ofAEV.