Development Of A Nested PCR Kit For Detecting Brucella In Milk

In order to detect Brucella in clinical milk samples, the nested PCR was established. According to the conserved genes of Brucella,15 sets of external primers were designed.The nested PCR reaction with these 15 sets of primers were optimized by orthogonal design.After screening the primers from the 15 sets of external primers, 4 pairs of internal primers were identified. The best internal primers were screened out by optimizing the PCR conditions. The internal and external primers PG3PG4 and PG1PG2 were determined. The external optimized PCR reaction condition were annealing temperature 54.4℃, Mg²⁺concentration 1.2 mM, the rTaq DNA polymerase 0.25μL,primer 0.45μM, dNTP concentration 0.2 mM. The internal PCR optimization condition were annealing temperature 46.4℃, Mg²⁺ concentration 2.1mM, the DNA polymerase 0.05μL, primer 0.4μM and dNTP concentration 0.3 mM. The extraction method of the genomic DNA of Brucella was improved and the process was standardized. The positive and negative controls in the kit were prepared. The nested PCR kit was assembled and the operation instructions was standardized. The validity of this kit is 6 months. The specificity test showed that the method was not cross reaction with Escherichia coli, Staphylococcus aureus, Streptococcus, Lactococcus lactis, Bacillus, Fetus fetus, Tox, Cryptozoite and Salmonella. Only the specific band of Brucella was amplified. The results demonstrated that the nested PCR was of excellent specificity. The sensitivity test was performed with the serial 2-fold dilution of the known bacterial count milk and the sensitivity of nested PCR was 8 CFU mL-1. The clinical milk and serum samples were collected from farms in order to detecting the antigen of Brucella. Compared with test tube agglutination test and the nested PCR kit, the sensitivity for the nested PCR kit was 100%, specificity was 96.24% and coincident rate was 96.62%. This nested PCR kit can be applied to detect the Brucella in milk.