Isolation And Identification Of Bovine Mastitis Pathogenic Bacteria And The Development And Application Of Rapid Diagnostic Kit

In this study,75milk samples from5different dairy farms in Guangxi were collected. According to the result of bacteria identification, we developed the method of multiple-PCR for mastitis diagnosis, which targeting at Streptococcus agalactiae, staphylococcus aureus, escherichia coli and bacillus cereus. By means of optimizing and assessment, we developed a rapid diagnostic kit based on multiple-PCR which could be clinically used in milk samples and milk products. The experiment results were organized as follows:1. In total135strains of bacteria were found by isolation and identification of75milk samples. Among those, we got24strains of staphylococcus aureus,17.87%.8strains of staphylococcus epidermidis,5.93%.5strains of staphylococcus saprophyticus,3.70%.17strains of Streptococcus agalactiae,12.59%.9strains of streptococcus uberis,6.67%.7strains of Streptococcus lactis,5.19%.5strains of streptococcus dysgalactiae,5.19%.5strains of streptococcus faecium,3.70%.12strains of Candida albicans,8.89%.6strains of saccharomycetes,4.44%.17strains of escherichia coli,12.59%.6strains of Salmonella enteritidis,4.44%.2strains of Shiga bacillus,1.48%.2strains of Corynebacterium pyogenes.1.48%.10strains of bacillus cereus,7.42%. The above data indicated that Streptococcus agalactiae, staphylococcus aureus, escherichia coli and bacillus cereus were the primary pathogenic bacteria. Bovine mastitis in investigated farms were occurred mainly by the mixed Infection form and the mixed infection rate was88%.2. We designed4pairs of primers which specific to the gene sip of Streptococcus agalactiae, the gene nuc of staphylococcus aureus, the gene rrnB of escherichia coli and the gene hblA of bacillus cereus. Based on these primers, we established a rapid diagnosic method by multiple-PCR. Result showed that the amplified band of Streptococcus agalactiae, staphylococcus aureus, escherichia coli and bacillus cereus were252bp,464bp,722bp and995bp respectively. Accuracy was assured by gene sequencing and blast comparison. The minimum detectable amount of Streptococcus agalactiae, staphylococcus aureus, and bacillus cereus was0.1pg/uL, escherichia coli was0.01pg/uL.The data demonstrated this method possessed a very good sensitivity and specificity.Also we tested50milk samples with multiple-PCR and traditional biochemical detection. It turned out that concidence rate was more than97%, by using this method the work efficiency was increased.3. We successfully assembled the multiple-pcr rapid diagnosic kit targeting at Streptococcus agalactiae, staphylococcus aureus, escherichia coli and bacillus cereus in milk. Assessment of quality showed the kit was very stable and well-preserved in a conventional way. In the state of0℃,expiration date was60days. In the state of-20℃or-80℃, expiration date was half year long. Nevertheless, multigelation had a bad effect on kit quality, we suggested users choose a rational way for perservation based on the reality.