Establishment And Preliminary Application Of Nested-PCR To Detect Infection Of Isospora Ohiensis In Dogs

Isospora ohioensis[Also known as Cystoisospora ohioensis]is a parasite that can invade intestinal epithelial cells in dogs, which can cause coccidiosis,especially in puppies. The main clinical symptom is anaemia,bloodystool,diarrhea,and even death,etc.Isospora ohioensisis one of the major pathogens of causing coccidiosis,itposes serious threat to the dog raising and pet health,so it's necessary to develope an effective detection tool for diagnosis of Isospora ohioensis.The traditionally used feces diagnosis,but they were in the face of difficulty in sample collection and treatment, and also need some specialized persons for morphological Discrimination.Here,We designed specific primers and established two Nested-PCR detection methods based on the ITS-1 and ITS-2 of Isospora ohioensis,also we tested fecal samples of dogs in Haerbin and Changchun.Establishment and Preliminary application of Nested-PCR based on ITS-1of Isospora ohioensis. We designed specific primers of Nested-PCR based on the ITS-1 of Isospora ohioensis, determined the optimal annealing temperatures for Nested-PCR by optimizing the Nested-PCR reaction system,also the sensitivity and specificity were detected,then we used the method to detect fecal samples in dogs of Changchun and Haerbin. The results appeared as shown below, the optimal annealing temperatures was 53?;After the test of the sensitivity,we concluded that the sensitivity of the Nested-PCR based on ITS-1 was 2.05×10^-12ng/?L,which was 1×10⁶ times than traditional PCR;Of course,it was necessary to have a test for the specificity, the Nested-PCR was no crossreaction when we used the method to detect Toxoplasma gondii, Neospora caninum,Giardia canis,C.parvum,Trichomonas vaginalis,confirming the specificity of the assay. We used the method to test 65 fecal samples of dogs in Changchun,44 fecal samples ofdogs in Haerbin, the results of samples showed the infection rate of 38.46% in Changchun,and 6.81% in Haerbin,confirming it was applicable for samples.Establishment and Preliminary application of Nested-PCR based on ITS-2of Isospora ohioensis.Here,We designed specific primers of Nested-PCR based on the ITS-2 of Isospora ohioensis, and the working conditions of the method were established by the optimization of the annealing temperature, the sensitivity test and the specificity test,then we applied the method to detect the fecal samples in dogs from Changchun and Haerbin.The optimal annealing temperatures of the second PCR was 59?. After testing the sensitivity of the Nested-PCR and traditional PCR at the same time,the sensitivity of the Nested-PCR based on ITS-1 was2.05×10^-12 ng/?L,which was 1×10⁵ times than traditional PCR. Of course,the specificity was tested,there was no crossreaction when we used the method to detect Toxoplasma gondii, Neospora caninum,Giardia canis,C.parvum,Trichomonas vaginalis,confirming the specificity of the assay. We used the method to 109 fecal samples of dogs in Changchun and Haerbin, the results of samples showed the infection rate of 38.46% in Changchun,and 6.81% in Haerbin,confirming it was applicable for samples.By testing the samples of the Changchun and Haerbin in different methods, we found that they got the same infection rate and all positive samples were one-to-one correspondence. Then 12 positive samples of Changchun and 3 positive samples of Haerbin were chosen randomly,sequenced, and analyzed by BLAST? basic local alignment search tool?with the ITS-1gene and the ITS-2 gene in NCBI,we found that the gene of the positive samples had the same mutation sites of the positive samples in Changchun and Haerbin,the results were: ITS-1, C>T in the site of 186, A>G in the site of 224, G>A in the site of 280;ITS-2, C>T in the site of 1107.For the ITS-1or ITS-2,the homology of the nucleotide sequence for the 15 positive samples selected in different rgion?Changchun and Haerbin?were over 99%, it proved that they were same species.In conclusion, we successfully constructed two specific Nested-PCR methods based on ITS-1 and ITS-2 of Isospora ohioensis,which were all applicable forsamples,and provided the effort for diagnosis and epidemiological investigation of Isospora ohioensisinfection in dogs.