Absolute Quantitation Of Marek's Disease Virus Ⅰ And Herpesvirus Of Turkeys Using Real-time Duplex PCR Of TaqMan-MGB Probe

Marek's disease (MD) is a highly contagious amalignant lymphoma of chickens, caused by Marek's disease virus (MDV) . Due to the crypticity of this cancer disease and lack of therapeutic tool, vaccine inoculation at 1 day old of chicken was regarded as one of main proventive measures. However, vaccine inoculation prevents death other than infection in infected chicken, it is very important to diagnose and monitor the status of MDV infection or contamination. TaqMan-MGB FQ-PCR characterized by quick, specificity, sensitivity provides a profitable method for detecing MDV and quantitating punctual virus copies.Based on sequences of UL36 and Bcl-2 of MDV-1 and MDV-3 in GenBank, two pair of specific primers and two labeled TaqMan-MGB probes were designed by Primer Express 3.0 software. By optimization of concentration of primer and probe, annealing temperature and reaction cycle parameter, the optimal reaction system was established. A duplex real-time fluorescent quantitative PCR was developed for MDV-1 and MDV-3 by the non-cross reaction of FQ-PCR. The results showed this assay was specific for MDV-1 and MDV-3, no band was amplified from herpesvirus of turkey, newcastle disease virus, laryngotracheitis virus, infectious bronchitis virus, infectious bursal disease virus and chick embryo fibroblast, and its sensitivity was 100 times more than that of regular PCR, the detection limit of 5.45 copies and 5.38 copies for MDV-1 and MDV-3 respectively; The coefficient of variation of intra-assay for same MDV-1 and MDV-3 DNA sample was <5%(n=5), the same standard plasmid was twice detected with one month interval, and the data from two experiments was not significant statistically. The MDV-l in feather tip was detected in all of samples(105-6copies/mL).The TaqMan-MGB Duplex real-time PCR could detect sensitivity MDV-1 by detecting experiment and clinical infected chicken. The number of MDV-1 was 103.5~6 copies/mL in different tissue sample of clinical infected chicken, the feather tip has the most number of MDV-1(105~6copies/mL),the FQ-PCR had a higher positive rate (100%)compared with AGP and regular PCR methods; HVT inhibited the replication level of MDV-1 in a certain degree by detecting the feather tip of challenged chicken with different time of, and delayed two weeks in the peak of elimination of MDV-1 in the feather tip. Compared with chicken challenged with BJ-1, the number of MDV-1 in feather tip chicken inoculated with HVT descented to 101~2 copies/mL; The number of MDV-3 in feather tip of chicken inoculated with HVT is more than chicken vaccinated with HVT, the replication level of MDV-3 in feather tip is limited.Our results demostrated that real-time Duplex PCR of TaqMan-MGB Probe provided a favourable technology for detecting, clinical diagnosis and etiopathogenesis of MDV, immunologic mechanism.