Establishment And Application Of Duplex Real-Time PCR Method For Sheep Contagious Pustular Dermatitis Virus And Sheep Pox Virus

Sheep infectious pustules and sheep pox exist worldwide;they have become a kind of common and frequently-occurring diseases.Both of sheep contagious pustular dermatitis virus and sheep pox virus belong to the poxviridae family.They are the double-stranded DNA viruses,both of them are the acute and highly contagious diseases.These viruses spread quickly,and have great destructive impact on the development of sheep husbandry and animal husbandry economy resulting to a massive loss.Sheep infectious pustules and sheep pox on the pathogenesis of clinical features are similar so that cross reaction may occur in the serological test.That is difficult to identify,therefore,this study established a fast and convenient duplex real-time PCRdetection method for Sheep infectious pustules and sheep pox to identify and diagnose effectively and accurately.This will make contributions to the sheep husbandry development of our country.This is divided into the following five parts:1.The MegAlign software in DNAStar was used to arrange,analyze and compare the sequences with reference to the whole ORFV gene sequence(NC005336)in GenBank.To choose ORFV059,a highly conserved fragment of sheep infectious impetigo virus,and using Primer Express3.0.1 to design the primers for rt PCR and probes,and the amplified fragment is 64 bp,to build response template according to the gene sequence of Goat pox virus Iraq61 isolates(GU119941)OPR30 protein in the GenBank.To design primers and probes according to the ?OIE terrestrial animal diagnostic laboratory and vaccine manual?,and through the Blast assessment and comparison to ensure its specificity,and the amplified fragment is 102 bp.FAM fluorescein was labeled at the 5' end of the probe for detecting ORFV,TAMRA fluorescein was labeled at the 3' end,HEX fluorescein was labeled at the 5' end of the probe for detecting GTPV,and TAMRA fluorescein was labeled at the 3' end.2.Extracting the nucleic acid of the virus is as a reaction template for dual rt PCR3.Optimizing the concentration and amount of primers and probes for duplex rt quantitative PCR to select the optimal concentration ratio of primers and probes.4.Amplifying the target fragment,and ligated it into the PMD18-T vector,transformed it with DH5-?.The plasmid was extracted to establish a standard in the end.5.To determine the specificity,sensitivity and repeatability of the duplex rt quantitative PCR assay.The results showed that only the primers and templates that were designed with specific fragments could amplify the target fragments.The detection of small ruminant diseases,foot-and-mouth disease,brucellosis,pasteurellosis in sheep and colibacillosis in sheep were all negative which indicated that the specificity was good.ThereforeA minimum of 10 copies of ORFV and GTPV were detected in gradient dilution of viral positive particles.Furthermore A total of 256 suspected samples were collected from some cities in Jilin Province,whereby out of 20 ORFV positive samples and 39 GTPV positive samples were determined using dual rt quantitative PCR.The positive relevance radio was in line with the national industry standards.This proves that this method is feasible.