Establishment And Application Of PCR Rapid Detection Technique Of Bovine Mastitis Pathogen

Dairy cow mastitis is an important disease in dairy-related industries,which seriously restricts the economic benefits of dairy farming and the development of dairy industry.Pathogen infection is the main cause of mastitis in dairy cows.Therefore,effective identification of infectious pathogens is important for the prevention and treatment of cow mastitis.Existing research shows that the main pathogens causing cow mastitis are Staphylococcus.sp,Streptococcus.sp and Enterobacteria.sp,among which Staphylococcus aureus,Escherichia coli and Streptococcus.sp occupy the main position in this process,and other pathogenic microorganisms such as gram-positive bacteria,fungi and other infections are also clinically visible.This study by Escherichia coli,Staphylococcus aureus,Staphylococcus epidermidis,Streptococcus agalactiae,Streptococcus dysgalactiae,Bacillus cereus and Candida albicans,a total of seven kinds of cow mastitis pathogens as the research object.Researcher first tested with different commercial kits,and studied the quality of genomic DNA extraction,the complexity of the extraction process,and the cost-effectiveness,screened out the best genomic DNA extraction kit;Later,Researcher selected specific genes of each strain,and comparing genes with better specificity through NCBI-Blast,The fragment was used as the target gene for the detection of the strain,and finally selected E.coli phoA gene,S.aureus nuc gene,S.epidermidis gyrB gene,S.agalactiae RDF₁058 tag,S.dysgalactis isp gene,B.cereus gyrB gene and C.albicans 18S rRNA gene sequence.The target genes of the strains were tested,and specific primers for each gene were designed,screened,and synthesized.The PCR reaction and conditions were studied,the optimal annealing temperature was 56?in the 20?L reaction system,and the addition amount of the upstream and downstream primers was 0.5?L each.The results of the primer specificity test showed good specificity and no amplification of non-specific bands.Based on the above experiments,a method for detecting pathogens in which seven reaction tubes were simultaneously reacted under uniform PCR conditions was established.The results showed that the minimum detection limit of E.coli was 1.04×10⁵ cfu/ml,S.aureus 1.04×10⁴ cfu/ml,and S.epidermidis 2.3×10⁴cfu/ml,S.agalactiae 9.4×10³ cfu/ml,S.dysgalactiae 9×10³ cfu/ml,B.cereus1.9×10² cfu/ml,and C.albicans 4.24 x 10³ cfu/ml.Finally,clinical milk samples and artificial simulated milk samples were tested to verify sensitivity and specificity,and the minimum detection limit was tested.The results showed that the specificity of the established PCR detection method was88.24%-100%,and the sensitivity was 92.73%-98%,which is suitable for rapid detection in dairy farms.