Application Of Multiplex-PCR For Detection Of Four Pathogens Cause Bovine Mastitis

Bovine mastitis is a kind of infectious disease,mostly caused by lots of pathogenic microorganism.subclinical bovine mastitis has high incidence rate and no clinical symptoms during the infection process,so preventing and curing diseases has great difficulties.This disease not only caused a huge loss of milk yield,reduced the economic benefits of the breeding industry,but also caused a great impact on animal and human health.In order to find an efficient,sensitive,and specific detection method to improve the diagnosis of bovine mastitis and pathogenic microorganisms and detect above four germs which causing bovine mastitis,the specific primer of16S-23S rRNA gene of E.coil,Staphylococcus aureus,Strepotococcus agalactiae,18S rRNA gene of Candida albicans,Miparameters of multiplex PCR system were optimized to establish the method for the detection of E.coli,Staphylococcus aureus,Strepotococcus agalactiae,Candida albicans.We used the multiplex PCR assay and compared it with the method of bacteriological cultivation,and Multiplex PCR assays for the four major pathogenic strains.At the same time,this experiment collected bovine mastitis clinical milk samples.The pathogens were isolated,cultured and identified to verify the feasibility of this method.?1?In total forty milk samples of mastitis wewe collected,out of them Staphylococcus aureus was 4 strains?10%?,Streptococcus agalactiae was7strains?17.5%?,Candida albicans was 3 strains?7.5%?,Escherichia coli was 5strains?12.5%?.The detection rate of Streptococcus agalactiae is the highest in the four kind of bacteria.Mixed infection was dominant comparison with single pathogen infection.?2?The result of multiplex PCR's optimization shows the most superior system which consist of Taq 2X Master MiX 10?L,;the primers of E.coil 0.3?L,the primers of Staphylococcus aureus 0.5?L,the primers of Candida albicans 0.2?L,the primers of Strepotococcus agalactiae 0.5?L;the template of E.coil 0.6?L,the template of Staphylococcus aureus 1?L,the template of Candida albicans 0.4?L,the template of Strepotococcus agalactiae 1?L,and some ddH₂O;the most superior annealing temperature was 53?.?3?The most superior number of cycles was 35.The multiplex PCR has high specificity.It was shown that 1319bp for Staphylococcus aureus,730bp for Candida albicans,663bp for E.coil and 406bp for Strepotococcus agalactiae by the multiplex PCR.?4?The multiplex PCR system can setect E.coil,Staphylococcus aureus,Strepotococcus agalactiae,Candida albicans with the sensiticity of 10²CFU/mL?10⁴CFU/m L?10⁵ CFU/mL?10² CFU/mL.?5?By the multiplex PCR,we examined forty samples,and compared with method of bacteriological culture.The resules showed that the coincidence rate of two methods was high compliance rate,and detection rate of the multiplex PCR was higher than bacteriological culture.