Isolation And Identification Of Pathogenic Bacteria Of Bovine Mastitis And Establishment Of Multiplex PCR Detecting Method

To investigate infectious status of Staphylococcus aureus、 Escherichia Coli and Bacillus cereus in cow mastitis, six dairy farms were chosen in the Guangxi Zhuang Autonomous region. Cultivation and identification of above3pathogens were conducted from milk of dairy cows with mastitis. A multiplex PCR method was established to detect the three pathogenic bacteria.Part I:In total one hundred and fifty milk samples of mastitis were collected,62strains of pathogens were isolated from these samples, out of them Staphylococcus aureus was22strains (14.7%), Escherichia coli was34strains (22.7%), Bacillus cereus was6strains (4.0%). The detection rate of Escherichia coli is the highest in the five of six dairy farms. Mixed infection was dominant comparison with single pathogen infection; the mixed infection rate was77.4%.Part II:In order to detect above three germs which causing bovine mastitis, the specific primer of nuc gene of Staphylococcus aureus, the16S-23S rRNA gene of Escherichia coli, hblA gene of Bacillus cereus were designed for multiplex PCR by the software of Primer5.0and Oligo 7.0. Reaction components and parameters of multiplex PCR system were optimized to establish the method for the detection of Staphylococcus aureus, Escherichia coli and Bacillus cereus simultaneously. We used the multiplex PCR assay and compared it with the method of bacteriological cultivation. The resμLt of multiplex PCR’s optimization shows the most superior system which consist of Buffer2.5μL, dNTPs2.0μL, Mg2+1.5μL, Taq polymerase0.2μL, the primers of Staphylococcus aureus0.1μL, the primers of Escherichia coli0.3μL, the primers of Bacillus cereus0.5μL, mixed template3μL, and some water; the most superior annealing temperature was56.8℃; the most superior number of cycles was35. The multiplex PCR has high specificity. It was shown that249bp for Staphylococcus aureus,375bp for Escherichia coli and499bp for Bacillus cereus by this mμLtiplex PCR, no specific band was found from other pathogens of mastitis. The mμLtiplex PCR system can detect Staphylococcus aureus, Escherichia coli and Bacillus cereus with the sensitivity of10pg/μL DNA. DNA sequence homology between clinical samples and Staphylococcus aureus, Escherichia coli and Bacillus cereus in GeneBank were99%-100%. By the multiplex PCR, we examined fifty samples, and compared with the method of bacteriological cμLture. The results showed that the coincidence rate of two methods was92%, and detection rate of the multiplex PCR was higher than bacteriological culture.