Establishment and Application of a Fluorescence Quantitative RT-PCR for Akabane Disease Virus
In order to establish a rapid and sensitive nucleic acid detection method for Akabane disease virus(AKAV),a fluorescence quantitative RT-PCR assay for AKAV was developed targeting at AKAV S gene,through designing specific primers and Taq Man probe and optimizing reaction system and conditions,followed by evaluation on its sensitivity,specificity and reproducibility,then 80 clinical bovine and ovine blood samples were simultaneously detected using the established method and that recommended by AKAV quarantine industry standard.The results revealed that the established method was highly sensitive with the lowest detection limit of 13.4 copies/μL;good specificity was observed without cross-reaction with foot-and-mouth disease virus(FMDV),bovine coronavirus(BCV),lumpy skin disease virus(LSDV),bovine viral diarrhoea virus(BVDV),infectious bovine rhinotracheitis virus(IBRV),bluetongue virus(BTV)and bovine leukaemia virus(BLV);good reproducibility was represented by intra-and-inter group coefficients of variation of 0.93%-2.47%and 1.25%-2.76%,respectively;and 21 out of 80 clinical samples were positive as detected by the method,98.75%coincident with that by the recommended method by AKAV quarantine industry standard.In conclusion,the established method,with high sensitivity,good specificity and reproducibility,was appropriate to detect Akabane disease(AKD),technically supporting the development of pathogenetic monitoring and diagnostic technology standards for AKAV.
AKAVreal-time fluorescence quantitative RT-PCRdevelopment of methodapplication
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