The Establishment And Application Of A Fluorescent Quantitative PCR Method Based On Duck Plague Virus ICP4 And UL48 Genes

Duck plague virus DPV also known as duck herpes simplex virus type 1,which is filterable virus belonging to the herpes virus family herpes virus genus.Plague gives ducks and geese acute febrile,septic diseases,which is a highly lethal infectious disease.It is very important to detect the duck plague rapidly and accurately in the latent period of infection.In this experiment,the ICP4 gene of DPV were identified by drug inhibition test.A fluorescent quantitative PCR(FQ-PCR)detection method was established for the detection of ICP4 and UL48 genes.The method was used to detect the clinical samples of 24 duck blood samples and compared with the PCR method which has been constructed in the laboratory.Duck plague latent infection model was constructed by artificial infection.On the basis of that,the expression of duck plague virus gene ICP4?UL48 in the trigeminal nerve of latent infection duck were detected by FQ-PCR method.Specific work:? Using drugs inhibit test on the ICP4 gene type determination;? Constructed fluorescence quantitative detection method based on the ICP4,UL48 gene;?The FQ-PCR method was used to detect the clinical samples of the suspected duck plague,and compared with the traditional PCR method in the literature.?Constructed duck plague latent infection model by infected DPV virulent strain CHv,then using quantitative PCR to detect mRNA levels of ICP4?UL48?TK and gC genes in trigeminal.Specific studies are as follows:1?Determination of two IE geneUsing ganciclovir drugs to inhibit DNA replication and cycloheximide drugs to inhibit protein synthesis to identify the gene types of DPV ICP4.And the immediate early gene ICP27,the early gene dUTPase and the late gene US2 were set as control.In the control group,the early gene ICP27,the early gene dUTPase and the late gene US2 were consistent with the results reported in the literature.Indicating that the test results are correct.Ganciclovir and actinomycetone drug groups can amplified ICP4 gene-specific band.Indicating that the two drugs can not inhibit ICP4 gene transcription.It can be concluded that the ICP4 gene is an immediate early gene.2?The establishment of the FQ-PCR method based on the four genesAccording to the nucleotide sequence of ICP4 gene of DPV CHv strain,the primers of ICP4 whole gene amplification were designed by Oligo7.0,and the amplified fragment size was 4881 bp.ICP4 full-length gene has been connect to pMD19T vector.PCR and sequencing showed pMD19T-ICP4 plasmid was successfully constructed.The analysis show UL48 andICP4 gene sequence are highly conserved and primers were designed in its conserved region.The results of NCBI Primer BLASTshowed that the primers of ICP4 and UL48 FQ-PCR could not only match the DPV CHV strain but also the other DPV strains.Use the laboratory preserved pMD18T-?-actin,pET32a-UL48 and pMD19T-ICP4plasmid to construct FQ-PCR method.Standard curve correlation coefficients were:?-actin,R2=0.996;ICP4,R2=0.998;UL48,R2=0.997.Standard curve shows the Ct values and the starting template concentration are highly correlated with high sensitivity.According to the following formula can do quantitative detection and copy number calculation of the expression level of duck plague virus ICP4?UL48 genes:?-actin,Y=-3.427X+41.191;ICP4,Y=-3.224X+40.218;UL48,Y=-3.480X+44.79(Y = Ct value,the value of X = the logarithmic number of copies).Detection of UL48,ICP4FQ-PCR method only specific detection of viruses containing ICP4 and UL48 genes.Samples with a minimum of1.75×102 copies/ul and 1.80×102 copies/ul concentrations can be detected.Reproducibility tests showed a standard deviation(SD)are both less than 0.5,indicating good reproducibility of the two FQ-PCR methods.At the same time,the DNA or cDNA of DPV virulent Chv strain,DPV attenuated Cha strain,Duck hepatitis A virus,New gosling type viral enteritis virus,Avian influenza H5N1 virus,Duck swollen head septicemia virus,Muscovy duck parvovrius,Tambusa virus,DuckCircovirus,GooseParvovirus,Duck hepatitis B Virus,Salmonella enteritidis(No.50338),Escherichia coli(078),Riemerella anatipestifer and Pasteurellawere used as templates to amplification.The results showed that both of the primers specific amplified DPV virulent Chv strain,DPV attenuated Cha strain virus.This indicated that the two FQ-PCR methods have high specificity and can be used for the clinical diagnosis of duck plague virus3?Clinical detection application by ICP4 and UL48 gene FQ-PCR methodSelect 24 2-mouth-old ducks in a suspected DPV infection area to collection jugular vein blood and DNA extraction.24 samples were detected by DPV ICP4 and UL48 gene FQ-PCR method.At the same time,compared with the traditional PCR method constructed in the literature.Results show that the number ofDPVDNA positive duck from ICP4 FQ-PCR method was 9,positive rate was 38%;the UL48 FQ-PCR method was 9,the positive rate was 38%;the traditional PCR methods was5,the positive rate was 21%?The number of positive duck numbers detected by the two FQ-PCR methods and the number corresponding to the positive number of the traditional PCR method were detected.It can be determined that the duck farm is infected with duck plague virus?DPV DNA negative samples can be detected by DPV ICP4 and UL48 gene FQ-PCR method in the detection of the traditional PCR method.The results show that the PQ-PCR method is more sensitive,and the detection effect is more accurate.4?Study on the latent infection of DPV by ICP4 and UL48 gene FQ-PCR methodStandard curves were drawn using laboratory-stored pMD18T-TK,pMD18T-gC plasmids.The correlation coefficients of the standard curve were:TK,R2=0.999;gC,R2=0.999.Standard curve shows the Ct values and the starting template concentration are highly correlated with high sensitivity.According to the following formula can do quantitative detection and copy number calculation of the expression level of duck plague virus TK and gC genes:TK,Y=-3.386X+38.891;gC,Y=-3.222X+37.511.Use DPV CHv virulent strain to infect 25 1-month-old duckswhich identified pathogens negative.Measure daily duck rectal temperature within 20 days after infection and detect the cloacal swabs virus content of ducklings at dpi5,dpi 10,dpi 15,dpi20,dpi25(dpi:days post infection).Promptly eliminated dead duck.Respectively and randomly selected three ducks after dpi26,dpi32,dpi35,dpi39,dpi42,dpi46,dpi49 then take their trigeminal nerve sterile for quantitative detection.The plague virus test results of cloacal swabs in dpi25 and blood from 21 recover ducks showed negative,indicating the recover duck has stopped excrete the virus and the latent infection was successfully constructed.The results showed that ICP4 and UL48 mRNA can be detected in all the ducks trigeminal nerve when the TK and gC gene mRNA can be detected in part ducks.ICP4 and UL48 gene expression was consistently detected at all sampling time points.The amount of gene expression of TK and gC was significantly lower than UL48 and ICP4.