| Neosporosis and Toxoplasmosis are reproductive barrier parasites diseases, respectively caused by Neospora caninum and Toxoplasma gondii parasitize a nuclear intracellular of mammals, they will lead to non-pregnancy, miscarriage, stillbirth, malformation of pregnancy females and newborn developmental delays. There is no effective drugs and vaccines to preventing this disease yet, thus it is very important to production significance for comprehensive prevention and control measures including establishing specific, sensitive, rapid and accurate detection method for early diagnosis, epidemiological surveillance, target elimination sick animals and screening treatment drug of those diseases.(1)This test designs specifically primers and TaqMan-MGB probes according to Neospora caninum Nc2 conserved sequences to establish TaqMan-MGB Real-time PCR detection method. The results showed that the positive template concentration had a good linear relationship with the Ct values. The result was negative for detecting Toxoplasma gondii, Theileria annulata, Babesia bovis and other parasites DNA of positive strains in Xinjiang. Determined by 3D digital PCR, the lowest effect detection was 6.41 copies/μL, sensitivity is 1000 times conventional PCR. Average coefficient variation within groups was 1.108% and between groups was 2.732%. This method was 100%(46/46) in accordance with “Technical specification of Neospora caninum’s quarantine”(SN/T 3499-2013).(2)According GRA7 gene conserved sequences design specific primers and TaqMan-MGB probe to establish Real-time PCR detection method. The results showed that only the Toxoplasma gondii was positive when when detected Toxoplasma gondii, Neospora caninum, Babesia bovis, Theileria annulata and Babesia caballi, positive DNA were negative; By 3D digital PCR determinate, the lowest effect detection was 4.58 copies/μL, and the coefficient variation of between groups and within groups were less than 5% determined by 3D digital PCR.(3)This study designs species-specific primers by Nc2 gene conservative regions to establish two-temperature PCR of detecting Neosporosis. The result showed that primers did not amplify negative DNA fragments such as Toxoplasma gondii etc, the minimum detection quantity was 32.5 fg/uL, and the circulation time was shortened about 38 minutes than traditional PCR method. The detection of positive rate was 10.90%(17/156), of which 83.33%(30/36) coincidence rate with “Technical specification of Neospora caninum’s quarantine”(SN/T 3499-2013), and the diversity is not remarkable(P>0.05). |
