Establishment Of Multiple PCR Method For Three Main Pathogenic Bacteria In Bovine Mastitis

The increase of milk demand which drove by the improvement of people's living standards directly promote the vigorous development of dairy farming,but bovine mastitis is still one of the important reasons that affect its rapid development.Staphylococcus aureus,Escherichia coli,and Mycoplasma bovis are the main causes of bovine mastitis in dairy cows.In this study,three pairs of specific primers were designed and synthesized based on Staphylococcus aureus nuc gene,Escherichia coli 16S-23 Sr RNA gene and Mycoplasma bovis OPPD/F gene.Multiple PCR assays for three pathogenic bacteria were established by optimizing the PCR reaction system and reaction conditions,and then the sensitivity and specificity of this method were tested.The traditional bacterial culturing method and the multiplex PCR method established in this study were used to detect pathogens in 13 bovine mastitis positive milk samples and 56 normal milk samples,to compare the applicability of the two methods.The experimental results showed that the multiple PCR method established in this paper has good specificity and sensitivity.This method could amplify Staphylococcus aureus,Escherichia coli,and Mycoplasma specific DNA bands,size of the amplified bands are 237 bp,634bp,and 333 bp,respectively,matchs the expected size.No DNA bands were amplified from the control strains.Sensitivity experiments showed that the minimum detection concentration of Escherichia coli was 1 pg/?L and that of M.bovis and S.aureus were both 10 pg/?L.The results of clinical samples showed that the multiplex PCR assay established in this study was superior to the traditional culturing method.A total of 61 pathogenic bacteria-positive milk samples were detected by PCR and the detection rate was 29.5%.Staphylococcus aureus positive milk was detected.24 samples,27 E.coli-positive milk samples and 10 samples of M.bovis positive milk samples;40 samples of pathogen-positive milk samples were detected in the traditional culture method,and the detection rate was 19.3%.Staphylococcus aureus positive milk samples 17 Parts,E.coli positive milk sample 19,M.bovis positive milk sample 4 copies.The experimental results show that the multiplex PCR method established in this paper has a good application value.