| Bovine mastitis has been widely reported as one of the common diseases in dairy farming and is prevalent in areas where dairy cows are raised.The occurrence of the disease is closely related to feeding management and environmental factors.Once it is ill,it is difficult to control its spread or radically eliminate the disease.Based on the DHI data of Hebei Province,this study monitored the somatic cell levels of dairy cows.The 175 samples of milk samples collected from large-scale dairy farms in seven areas including Handan,Hengshui,Shijiazhuang,Cangzhou,Baoding,Langfang and Zhangjiakou were Isolation culture,morphological observation,biochemical assay and gene sequencing.The results showed that four major pathogens were isolated from 175 milk samples.Among them,there are 93 strains of Staphylococcus aureus,accounting for 53.1%;85 strains of Streptococcus agalactiae,accounting for 48.6%;112 strains of Escherichia coli,accounting for 64%;28 strains of Pseudomonas aeruginosa,accounting for 16%.Most of the milk samples detected were mixed infections of Staphylococcus aureus,Streptococcus agalactiae and Escherichia coli,and 28strains of Pseudomonas aeruginosa were isolated from milk samples in Zhangjiakou area and Shijiazhuang area,and 12 of them showed a single infection.This assay designed and synthesized 4 pairs of specificity based on the nuc gene of Staphylococcus aureus,the ef-tu gene of Streptococcus agalactiae,the phoA gene of Escherichia coli and the eta gene of Pseudomonas aeruginosa,which are the main pathogens of cow mastitis.Primers,the amplified fragments of interest were 920 bp,515 bp,268 bp and 126 bp,respectively.The annealing temperature and primer concentration were optimized by four single PCR methods,and the optimal annealing temperature of single PCR was 54?,and the amount of upstream and downstream primers was set at 1?L.The specific assay showed that the results of the four single PCR methods were well,and has no other non-specific band amplification.The sensitivity test showed that the minimum detection limits of Staphylococcus aureus,Streptococcus agalactiae,Escherichia coli and Pseudomonas aeruginosa were respectively 5.97×10-55 ng/?L,3.6×10-6 ng/?L,1.37×10-7 ng/?L and1.2×10-6 ng/?L.On this basis,this study established a multiplex PCR assay for Staphylococcus aureus,Streptococcus agalactiae,Escherichia coli and Pseudomonas aeruginosa,and determined the annealing temperature to 54?by optimizing the reaction system and reaction conditions.The amount of the upstream and downstream primers added was 1?L.Specificity tests showed that the multiplex PCR assay did not cross-react with Klebsiella,Staphylococcus epidermidis,Pasteurella and water;sensitivity tests showed that the method was against Staphylococcus aureus,Streptococcus agalactiae,Escherichia coli and Pseudomonas aeruginosa the minimum detection limits of respectively were 5.97×10-2 ng/?L,3.6×10-3 ng/?L,1.37×10-3 ng/?L and 1.20×10-3 ng/?L.Using the multiplex PCR method established in this experiment,62 samples of mastitis were tested.The results showed that the positive rates of Staphylococcus aureus,Streptococcus agalactiae,Escherichia coli and Pseudomonas aeruginosa respectively were 43.5%?27/62?,41.9%?26/62?,62.9%?39/62?and 35.5%?22/62?,of which the mixed infection rate of E.coli was 40.3%?25/62?,and the co-infection rate of Pseudomonas aeruginosa was 20.9%?13/62?.The total coincidence rate between the method and the traditional bacterial isolation culture method was 90.3%for Staphylococcus aureus,95.2%for Streptococcus agalactiae,91.9%for E.coli and 93.5%for Pseudomonas aeruginosa.The above data indicates that Staphylococcus aureus and Escherichia coli are the dominant bacteria in bovine mastitis in mastitis samples.In summary,this study laid the foundation for the research of the pathogenic bacteria detection kit for dairy cow mastitis by the isolation and identification of pathogenic bacteria in the milk samples in Hebei and the establishment of multiplex PCR detection kits.And prevention and control have greater guiding significance. |
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