The Preliminary Application of PCR in Research of Clinical Diagnosis and Mechanisms of Immunity and Pathogeny of Duck Plague Virus (DPV)

According to the reference from GenBank,a pair of primers amplifying DNA of duck plague virus(DPV) was designed and synthesized.The forward primer was at the 246-266 site of DPV EcoR Ⅰ gene,and the reverse primer was at 727-744.A polymerase chain reaction(PCR) technique detecting DPV DNA was established after selecting the optimal reaction conditions.We had applied this technique to specifically amplify the 498bp DNA of the DPV attenuated CHa strain and the virulent SC1 strain.The sensitivity of the PCR was 10fg DNA of DPV attenuated CHa strain.Sequencing of the amplified products showed that the PCR method is specific.The negative results were achieved from control ducks,Marek's disease virus,infectious laryngotracheitis attenuated virus,duck hepatitis virus,Pasteurella Multocida,E.coli of serotypes O_1,O_8,O_(78) and O_(158),Salmonella anatamu,and Riemerella Anatipestifer of serotypes 1,2,4 and 5.The clinical tissue samples collected during 1990-2002 were detected for DPV by three methods,in which the PCR was more sensitive than,the virus isolation method and the Dot-ELISA.We applied PCR to detect heart,liver,spleen,lung,kidney,duodenum,rectum,bursa of Fabricius,thymus,pancreas,medulla oblongata,cerebrum,cerebellum,tongue,muscle bone marrow,excrement,esophagus and blood from one-day old ducklings vaccinated by DPV attenuated CHa strain in three different routes.The results indicated that ① heart,liver,spleen,kidney,bursa of Fabricius,thymus,pancreas,medulla oblongata,cerebrum,cerebellum were positive for DPV DNA 4h post vaccination(PV)subcutaneously,and all the samples were positive 8h PV;②tongue and esophagus were positive 4h PV orally,and heart,liver,spleen,kidney,thymus,pancreas,medulla oblongata,cerebrum,cerebellum,tongue,esophagus and blood were positive 8h PV;③no positive 4h PV nasally(nose dripping) and heart,liver,spleen,kidney,thymus,medulla oblongata,cerebrum,cerebellum,tongue,esophagus, blood were positive 8h PV;④all samples from three different vaccination routes were positive from 12h to 21d.Brain,liver,spleen,bursa of Fabricius,thymus from three-month old adult ducks were positive 2h post infection(PI) with DPV virulent SC1 strain,and heart,liver,spleen,lung,kidney,duodenum,rectum,bursa of Fabricius,thymus,pancreas,brain,brisket,esophagus,stomach,blood,tongue,buccal secretion,skin,bone marrow, medulla and excrement were positive 12h PI in live and dead ducks.The PCR provides a more sensitive,specific and reliable method for DPV rapid diagnosis,molecular biology epizootic study.The results will provide significant data for clarifying pathogeny of virulent DPV and immune mechanism of attenuated DPV.