| Peste des petits ruminants (PPR) is a fatal disease caused by Peste des Petitis Ruminants Virus (PPRV), which occurred in small ruminants, especially in goats. In our country, it is classified as A class of animal disease.and it is on the FAO / OIE noticed list.The PPR is an acute, highly contagious disease characterized by fever, anorexia, necrotic stomatitis, diarrhea, purulent ocular and nasal discharges, and respiratory distress.Nowadays, our country have't commercialized ELISA kit used to detect PPR. Therefore, base on the preliminary established indirect ELISA method ,we do further verification and optimization, to improve the stability, repeatability of the method, it make sure that the result are scientific and effective,which can be applied to clinical practice. we have done some work. As fellow:1. Identified the PET-PPRV-N recombinant plasmid and standardized the protein expression conditionsThe recombinant plasmid was identified by PCR and restriction enzyme digestion. In order to determine the optimal expression conditions, we tried with different time and concentration of IPTG. According to standardized conditions, we expressed a large number of PPRV-N protein and purified the target protein with His-tag, which used as coating antigen for indirect ELISA.2. Optimized the Indirect ELISA methodsOur research was based on predecessor's work. We compared with different conditions to choose an optimum concentration of coating antigen, dilution rate of serum, the blocking time, the blocking solution choice, serum dilution, serum reaction time, as well as substrate, which reduced the negative background values, and enhanced the positive value. It is in favor of determining the experiment results. In addition, according to the ELISA procedure, we compared two different batches of the ELISA. It shows that the two batches are consistent with each other, which is up to 95%. We used the PPRV ELISA kit to detected positive serum of other sheep disease. The result is negative. It proved that our kit has good specificity.3. Determine the threshold value by compared with the competition ELISA KitWe Chose 244 sheep serum from different sources, and differentiated them from positive and negative by competitive ELISA. Then detected the serum by indirect ELISA. We analyzed the distribution of OD values of the positive and negative serum and used the TG - ROC software analysis to determine the threshold under the selection for the 95% degree of confidence. The threshold is 0.227. The sensitivity and specify of indirect ELISA is up to 85.4%,98.6%4. The application of the PPR ELISA method in the clinical diagnosisThe indirect ELISA and competitive ELISA kit are used to detected Tibet, Qinghai, Xinjiang and other places of sheep serum samples. Then we analyzed both of test results. it is turn out that two methods have highly consistent .The ELISA antibody titers of natural infection and vaccine sheep serum was tested, and found that there were obvious different. The antibody of infected group is between 1:800 and 1:1600, but the vaccine group is 1:20-1:400. According to this, it is possible to distinguish between infected group and immunity group.5. The epidemiological study of PPR in Ngari ,TibetThe first outbreak of peste des petits ruminants in Tibet was confirmed in July 2007. In order to determine the geographical distribution, host range, and prevalence of this outbreak,we carried out a systemic epidemiological investigation by etiology , serology and field survey. The result shows that the PPR has invaded into Longmengka, Rutu and caused a large number of sheep death in winter 2005. Afterwards,the mixed group, grazing together, and the further breed introduction led the epidemic spreading over. In September 2007, it presented in Rutu, Gaize, Geji and Zhada County. After taking quick and scientific measures, the epidemic has been effectively under-control.
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