Differentiation Of Sheeppox Virus And Goatpox Virus By Duplex PCR

Sheep pox and goat pox are caused by sheeppox virus (SPPV) and goatpox virus (GTPV), they are the member of capripoxvirus, they are highly contagious viral diseases of sheep and goats, typical characterised by fever, papules and pustules on exposed body surfaces. Sheep pox and goat pox are the most serious diseases of all animals poxvirus disease, the morbidity rate is50%-80%and the mortality rate is20%-80%, the mortality rate in young animals can be reach to100%. These diseases can cause abortion, reduced productivity, lower quality of wool and leather, significantly inflict by the heavy economic loss in sheep industry. At present, sheep pox and goat pox has all over the world, especially in Africa and Asia, also they are widely epidemic in our country, some areas were outbreaks and have a rising tendency. In addition, it has some public health security problems. It was once a common belief that the majority of SPPV and GTPV strains are considered as host specificity, but more researches indicated that the pathogenicity of some SPPV and GTPV strains was not limited to either sheep or goats. The diseases caused by either species are very similar with each other in clinical symptoms and pathogenesis, and they have same clinical symptoms to Parapoxviru, and some cases with other diseases mixed infection, so it’s difficult to distinguish between them by the clinical features. Virus isolation and electron microscope is specificity, but long time consuming, need professional equipment and technology, it’s not suitable for rapid diagnosis and the application requirements. Serological test methods are relatively simple and convenient, but the predominantly cell-mediated nature of immunity to capripoxvirus produce lower levels of neutralizing antibodies, and the common antigen and similar serotypes in SPPV, GTPV, LSDV and Parapoxviru, so those methods which have low specificity and senstitivity are not good for differential diagnosis them. Therefore, established a fast, simple and accurate method for detection and identification SPPV and GTPV has very important significance on the fields of early diagnosis, the epidemic situation monitoring, epidemiology and as soon as eliminate of these diseases.For the healthy development of sheep industry and speed up the modern animal husbandry pace, this study established a duplex PCR method to differential diagnosis SPPV and GTPV by molecular biology and based PCR as a major technique, it includes the following five main areas:1、Analysis the complete genome sequence of sheeppox virus and goatpox virus accessed in GenBank, two pair of PCR primers were designed by Primer Premier5.0biology software. The amplified PCR products were purified and then connected to the pMD19-T vector to construction plasmid pMD19-S and pMD19-G. The result of compare the homology of sequencing result and GeneBank login sequence showed that the two pair of PCR primers can specifically amplify target fragments of this two virus, respectively. The identity of pMD19-S and SPPV genome sequence is99%at least, and pMD19-G and GTPV genome sequence is96%.2、By optimizing the concentration of primers, Taq DNA polymerases and annealing temperature, two kinds of single PCR method for detecting SPPV and GTPV was established, respectively. The specificity test results showed that only target gene was amplified by each single PCR method, while no amplification of other pathogens or healthy tissue. The sensitivity test results showed that3.45X106copies/μL for SPPV and3.42X105copies/μL for GTPV could be detected by two sinlge PCR methods respectively.3、On the basis of the single PCR methods, established a duplex PCR method for simultaneously amplify SPPV and GTPV by optimization with primers dNTP and Mg-+concentration and annealing temperature in the PCR reaction system. The specificity test results showed that two specific fragments of177bp (SPPV) and222bp (GTPV) were amplified, while no product was amplified from other pathogens or healthy tissue. The sensitivity test results showed that this method with a detection limit of1.725×107copies/μL for SPPV and1.71X106copies/μL for GTPV.4、Respectively, virus isolation、SPPV and GTPV single PCR methods and duplex PCR method were used to test on50clinical samples from Gansu and Chongqing, and the positive detection rate of these samples were14%,4%s10%and14%. This indicated that duplex PCR assay can differential diagnosis of sheep pox and goat pox, with higher specificity than the virus isolation method. The oincidence rate of the two single PCR methods and duplex PCR method was100%, the singlet PCR can be used for the validation of the dual PCR results.In summary, this study established a double PCR for rapid detection of sheeppox virus and goatpox virus single or mixed infections of the virus samples. This method not only a scientific theory and technical support for the clinical detection of the sheep pox and goat pox, but also have great significance for the further study on the epidemiological investigation and prevention and control of this two diseases.