Establishment Of A Duplex Fluorescence Quantitative RT-PCR Detection System For Velogenic And Lentogenic Strains Of NDV And The Assembly Of Detection Kit

Newcastle Disease(ND) is caused by Newcastle disease virus, which is an acute,feverous,septic and highly contagious disease and can seriously harm the health of avian. It has a high morbidity and high mortality. The incidence and prevalence of Newcastle disease increase these years and the mixed infection of velogenic and lentogenic strains of NDV becomes more common. Thus differentiation the velogenic and lentogenic strains of NDV receives more attention in diagnosis and prevention of NDV. Conventional laboratory tests for Newcastle Disease detection are time- consuming and can’t meet the demand, such as chicken embryos mean death time(MDT), 6-week-old chicken intravenous pathogenicity index(IVPI) and one day-old chicks intracerebral pathogenicity index(ICPI), etc. Despite the differentiation of velogenic and lentogenic strains of NDV by Fluorescence Quantitative RT-PCR abroad in recent years, there are some differences between the epidemic genotypes of domestic NDV velogenic strains and the strains abroad. In addition, there has not been any detection kit of velogenic and lentogenic strains of NDV by fluorescence quantitative RT-PCR system appeared in domestic market. So it is urgent to develop the method to differentiate velogenic and lentogenic strains of NDV by Fluorescence Quantitative RT-PCR and assemble the detection kit.In this study, F gene sequence of velogenic and lentogenic strains of NDV were compared.Probes and primers were designed according to the differences in the F0 cleavage site and the conserved sequences of F gene. NDV-F & NDV-R were selected as the best primer sets and NDV-V-Probe & NDV-L-Probe were selected as the best probe sets after screening groups of probes and primers. The optimal reaction conditions and the reaction system were determined.Wherein the optimal annealing temperature was 56 ℃, NDV-F and NDV-R final concentration were 0.6μM, NDV-V-Probe and NDV-L-Probe final concentrations were 0.6μM and 0.2μM respectively. Finally the method was established to detect velogenic and lentogenic strains of NDV by a duplex fluorescence quantitative RT-PCR system and the detection kit was assembled.The kit’s specificity, sensitivity, reproducibility and other parameters were tested. The shelf life and storage conditions of the kit were determined.The minimum detectable quantity for the standard of virulent strains of NDV of established method and kit in this study are 102 copies/μL. The minimum detectable quantity for the standard of lentogenic strains of NDV are 101 copies/μL. All of them can meet the detection need. The kit was adopted to detect clinical samples and the positive detection rate of it was higher than that of ordinary PCR, which showed the application value of this detection kit.