A Study On Multiplex PCR And Microarray For Detecting Of Duck Plague Virus, Goose Parvovirus, Muscovy Duck Parvovirus, And Goose Paramyxovirus

The waterfowl breeding scale is expanding now,and the increase rate of the duck breeding is 10%-15%every year in China and the breeding amount of goose is over 70%all of the world.The waterfowl infectious diseases,such as Avian influenza(AI),Goose Paramyxovirosis(GPMVS),Duck plague(DP),Gosling plague(GP),Duck virus HepatitisⅠtype(DVH-Ⅰ),Muscovy duckling parvovirosis (MDPVS) and Pasteurellosis avian are very harm to the waterfowl breeding.In the study,we researched the multi-PCR and microarray detection technique on duck plague,gosling plague,goose paramyxovirosis and Muscovy duckling parvovirosis.The research contents were as follows:1 Study of multiplex PCR for detection of DPV and GPV,GPV and GPMV,GPV and MDPV1) Development of multiplex PCR for detections of GPV and DPVTwo pairs of primers were designed respectively according to sequences of VP3 gene(550bp) of GPV,and UL6 gene(376bp) of DPV published on GenBank.Perfectshot^TM Ex Taq was adopted,the reaction conditions was optimized,and a multiplex PCR assay was developed successfully for detection of the two virus.By the multiplex PCR assay,the specified fragments of 550 bp and 376 bp approximately in size were amplified respectively from the sample genomic DNA of GPV and DPV,but not from goose paramyxovirus,duck hepatitis virus,Salmonella anatamu,Pasteurella multocida and Riemerella anatigestifer; Sensitivity of the multiplex PCR reached 1.66 pg of GPV DNA and 0.166 pg of DPV DNA, respectively.Test of 20 samples gosling infected experimentally with GPV or /and DPV by the assay showed positive rate of 100%(5/5) for DPV and GPV,respectively.Co-infection rate 100%(10/10).These results showed that the PCR method is rapid,sensitive and specific for simultaneous diagnosis or differentiation of GPV and DPV infections.2)Development of multiplex PCR for detections of GPV and GPMVTwo pairs of primers were designed respectively according to sequences from GenBank.The cDNA of GPMV and DNA of GPV were mixed for amplification.Reaction conditions were optimized,a multiplex PCR assay was developed successfully for detection of the two virus.By the multiplex PCR assay,the specified fragments of 484 bp and 206 bp approximately in size were amplified respectively from cDNA of GPMV and DNA of GPV, but not from DPV,duck hepatitis virus,Salmonella anatamu,Pasteurella multocida and Riemerella anatigestifer;Sensitivity of the multiplex PCR reached 86 pg of GPMV RNA and 0.176 pg of GPV DNA,respectively.Test of 20 samples gosling infected experimentally with GPMV and GPV by the assay showed positive rate of GPMV 100%(5/5),GPV 100%(5/5).Co-infection rate 100%(10/10) of GPV and 80%(8/10) of GPMV.3) Development of multiplex PCR for detections of GPV and MDPVTwo primers GPV U/L and MDPV U/L were designed according to the genomic sequences in GenBank.Genomic DNA of GPV-GZ1 and MDPV strains were extracted respectively from allantoic fluids of goose and Muscovy duck embryo,uniplex PCR assay was developed successfully for detection of GPV and MDPV,respectively;Then the DNA samples were mixed for the amplification.Reaction conditions were optimized and a duplex PCR system was established successfully for detection of the two virus.By the uniplex PCR assay,only the specific fragments of 730 bp approximately in size was amplified from DNA samples of GPV-GZ1 and GPV-GZ2,only the specific fragments of 624 bp was amplified from DNA samples of MDPV,the specific fragments 730 bp and 624 bp were amplified from duplex PCR,but not from duck plague virus and goose paramyxovirus.Sensitivity of the uniplex PCR for GPV and MDPV DNA reached 0.144 pg and 28.8 pg,the duplex PCR reached 14.4 pg for GPV DNA and 28.8 pg for MDPV DNA.The results indicated that the PCR assay could be used to differentiate or diagnose of GPV and MDPV simultaneously. 2 Constructing and application of DPV-GPV-GPMV microarray1) The cloning and identification of target genes for microarray detectionAnalyzing the molecular biological characters and gene sequences of DPV,GPV and GPMV,primers of DPV,GPV and GPMV were designed by Array Designer software ver 4.2.The target genes of DPV,GPV and GPMV were amplified by PCR and RT-PCR and cloned using pMD18-T vector;13 recombinant plasmids,named as T/DPV DNA polymerase, T/DPV UL50,T/DPV TK,T/DPV UL6,T/DPV phosphoprotein,T/GPV NS,T/GPV VP3, T/GPV VP1-VP2,T/GPV VP2-VP3,T/GPMV HN,T/GPMV L,T/GPMV F,and T/GPMV NP were obtained and identified by restriction digestion,PCR and sequencing.Target gene were analyzed by MSM of DNAMAN software 5.0 and BLASTN and the results indicated the identity of target gene were above 88%for DPV,92%for GPV and GPMV.Target gene cloned and identified are positive and specific for corresponding virus and are avalible for constructing the microarray.2) Constructing of DPV-GPV-GPMV detection microarrayDPV-GPV-GPMV diagnostic microarray was developed by spotting all target genes including positive,location gene and blank control,which were diluted to 250 ng/μL with 50%DMSO spotting buffer,distributed onto the surface of aminated glass slide by Ominigrid 100 microarray printing system under condition of 25℃,65%relative humidity.The spotting parameters were spot diameter,100μm and center-to-center space,300μm,each row was 10 site;Target DNA on the array was fixed by UV cross-linking:After pre-hybridization,probes were labelled by PCR with Cy3-dCTP,and hybridized with the targets on the array.After washing,the array was scanned at 532 nm (Cy3) with a scanner.A threshold of SNRm≥2.0(based on quantification patterns of median intensity) is established as a measure of detectability.DPV-GPV-GPMV diagnostic microarray was evaluated by test samples.It is conclude that targets were specific and reliable,mini-variance with different microarrays of the same batch.3) Microarray detection for DPV,GPV and GPMVProbes were labeled with Cy3-dCTP by the optimal multi-PCR,Primers were devided into 4 groups:GroupⅠ(GPV NS,DPV DNApolymerase and GPMV F),GroupⅡ(DPV UL50,DPV phosphoprotein and GPMV HN),GroupⅢ(DPV TK,GPV VP1-VP2 and GPMV NP) and GroupⅣ(GPV VP3,DPV UL6 and GPMV L).The array can detect DPV, GPV and GPMV simultaneously,No cross-reaction was found with duck hepatitis virus, Salmonella anatamu,Pasteurella multocida,Riemerella anatigestifer and NS probe of MDPV,but cross- reaction was found with VP3 and VP1-VP2 probes of MDPV;The detecting sensitivity of the array can reache to 7.886 ng/L,higher than that of the agarose gel electrophoresis;Twenty samples,which were detected from organ samples of gosling infected experimentally with GPV,DPV and GPMV by the assay were detected.The results showed that the positive rate of DPV is 100%(20/20),GPV 85%(17/20),GPMV 80%(16/20).Co-infection rate 75%(15/20) of DPV,GPV and GPMV.These results showed that the DPV-GPV-GPMV diagnostic microarray is sensitive and specific and it is suitable for detection from multi-pathogen infection waterfowl at nucleic acid hybridization level.