关键词: 新孢子虫; 新孢子虫病; 牛; PCR

Abstract: To screen applicable PCR diagnostic primers of Neosporacaninum based on Nc2 and Nc5 genes,two pairs of primer were designed according to the Nc2 and Nc5 genes sequences conserved region and"Quarantine Protocol For Neosporosis"(SN/T 3499-2013)were applied to verify the characteristic of primers.14 Holstein and 19 Simmental cattles blood DNA were detected using these primers by PCR to select specific primers which would be used to establish the new detection method and understand the neosporiasis infection rate of different local cattle farms.The results showed that 105,128 and 231 bp gene fragments were amplified using three pairs of primers,which were consistent with the expected fragment size.The F1/R1 and SN/T 3499 F/SN/T 3499 R minimum detectable amount both were 19.9 fg/ μL and the minimum amount of F2/R2 was 199 fg/ μL,indicating that F1/R1 and SN/T 3499 F/SN/T 3499 R were more sensitive than F2/R2.The bands were bright when used F1/R1,F2/R2 primers to amplify 19.9 pg/μL and 199 fg/μL DNA samples,proving two primers had good repeatability.6 positive samples were detected among 33 blood samples,and the positive rate was 21.43% and 15.79%,the recombination rate was 100%.It showed that F1/R1 and F2/R2 primer were both suitable to detect eneosporosis by PCR and the method was preliminary established.The condition of local cattle Neosporacaninum infections were learned.This study could provide a scientific basis for the effective prevention and control of neosporosis.

Key words: Neosporacaninum; neosporosis; cattle; PCR