Establishment Of Fluorescent Quantitative PCR Detection Method For Lumpy Skin Disease Virus And Comparative Analysis Of Genomic Molecular Markers With Heterologous Attenuated Vaccine Goatpox Virus AV41

Lumpy skin disease(LSD),an acute or subacute infectious disease,is caused by lumpy skin disease virus(LSDV)in the genus of Capripoxvirus(CaPV),which is characterized as high fever,extensive nodular masses in skin,mucosal and organs.In August 2019,LSD was first confirmed in Xinjiang Yili Prefecture,China.Recently,24 cases were reported in Fujian,Jiangxi Prefecture,etc.LSD decreased production capacity and has led significant economic losses of domestic ruminates,threaten the global stockbreeding and international trade.LSDV was classified as notifiable animal diseases by the World Organization for Animal Health(OIE).As a new exotic animal disease,LSD should be further monitored by rapid and accurate molecular methods.1.Establishment of a specific Cycleave PCR method for distinguishing LSDVA specific Cycleave PCR method for LSDV was established,in which a pair of primer and a chimeric DNA-RNA-DNA probe were designed to amplify a part of LSDV ORF024 gene.This method was specific and did not react with viral nucleic acid samples in infectious diseases such as Goatpox(GTP),Sheeppox(SPP),Foot-and-mouth disease(FMD type O and type A),Bluetongue disease(BT),Infectious bovine rhinotracheitis(IBR)and Bovine viral diarrhea-mucosal disease(BVD-MD).The minimum detectable limitation of LSDV nucleic acid was 1.13 copies/?L.The Ct values showed good linearity with LSDV ORF024 plasmid in the range of 100?107 copies/?L,with a correlation coefficient of R2=0.999 for the standard curve;The coefficient of variation of inter-and intra-assay was less than 2%.Detecting 89 clinical samples,53 positive result were identified by Cycleave PCR method,and the positive detection rate of LSDV nucleic acid was 4.49%higher than that of the control method.All the above results indicated that this method was sensitive and specific,reproducible,and could provide technical reserves for the detection of LSDV in China.2.Establishment of a triple fluorescent qPCR method for distinguishing LSDV,GTPV and SPPV.A triple TaqMan MGB real-time PCR method was established for LSDV,Goatpox Virus(GTPV)and Sheeppox Virus(SPPV).This method was specific and had no Ct values for FMDV type O and type A,BTV,IBRV,BVDV,ORFV A good linear relationship was shown in the concentration range of 101?108 copies/×L,R2?0.998.The minimum limited detections for LSDV,SPPV and GTPV were 5.41 copies/?L,27.68 copies/?L and 17.28 copies/?L,respectively.The intra-and inter-assay coefficients of variation were all less than 2.5%.The method was applied to detect dual or triple artificial mixed infection samples good signal of each component and no cross reaction.123 samples were detected by this method,the number of positive samples for LSDV,GTPV,SPPV were 53,13,and 54,respectively.Compared with the CaPV triple Taq-Man MGB qPCR method,the detection rate of LSDV by this method was 8.9%higher than that of the control method,and that of GTPV and SPPV was the same.These results demonstrated that the method was sensitive and specific which can be applied to the detection of LSDV,GTPV,and SPPV clinical samples.3.Genomic molecular markers with heterologous attenuated vaccine Goatpox virus AV41To distinguish GTPV attenuated vaccine AV41 from wild-type LSDV(WT-LSDV)in China,genomic molecular markers of AV41 were primitively analyzed using bioinformatics method from complete genomic comparative analysis of LSDV,GTPV and SPPV strains published in GenBank.Nineteen molecular markers were successfully screened from genome of AV41.Thereafter,sequences with relevant molecular markers were verified by sequencing AV41 from different manufacturers and comparing with corresponding positions of WT-LSDV genome.Seven of these markers were specific to AV41 after being further confirmed,including DNA ligase gene 2433A2434?Kelch-like protein gene 1831A1832?GTPV_gp020 gene G203A?GTPV_gp021 gene C681T?DNA-binding viron core protein gene C47T?RNA polymerase subunit gene T64C and Myristylated protein gene C746A,which were not observed in WT-LSDV.Systematically,this study implied the distribution of AV41 specific genomic molecular markers,which can be utilized for distinguishing AV41 from WT-LSDV during the epidemic prevention and monitor of Lumpy skin disease in China.