Establishment And Application Of Multiplex PCR And Multiplex Fluorescence Quantitative PCR For Detection Of Main Pathogenic Bacteria Of Clinical Mastitis In Dairy Cows

Dairy mastitis has gradually become one of the most common infectious diseases in dairy farming,which has a great impact on the development of animal husbandry in China.Dairy mastitis usually refers to an inflammatory lesion caused by microbial infection,physical or chemical stimulation of dairy mammary tissue,which leads to a series of problems such as reduced milk yield and performance loss and brings significant economic losses to dairy farming industry.In order to timely and accurate diagnosis of dairy cow mastitis pathogenicity,reduce the cost of dairy farming in dairy cow mastitis,this study established the cow mastitis of six major pathogenic bacteria multiple PCR detection methods and multiple fluorescent PCR detection methods,as the main pathogenic bacteria of dairy cow mastitis cases detection,early diagnosis and treatment to provide technical support and epidemiological investigation.In order to detect six main pathogenic bacteria in dairy mastitis simultaneously,the present study targeted at NUC gene of Staphylococcus aureus,EF-TU gene of Streptococcus agalactiae,ETA gene of Pseudomonas aeruginosa,16srRNA gene of Streptococcus alactiae,Phoa gene of pathogenic Escherichia coli and OPP of Mycoplasma bovis Six pairs of specific primers for D/F gene were designed.Based on the establishment and verification of single PCR,a multiplex PCR reaction system was constructed,and its reaction system was optimized to verify the specificity and sensitivity of the method.The results showed that the multiplex PCR detection method had no specific amplification for other pathogens of cow mastitis.The minimum detection concentrations of Staphylococcus aureus,Streptococcus agalactiae,Pseudomonas aeruginosa,Streptococcus alactiae,pathogenic Escherichia coli and Mycoplasma bovis were 10-4ng/μL,10-3 ng/μL,10-2 ng/μL,10-4ng/μL,10-1ng/μL,10-4ng/μL,respectively.The results of milk samples collected from 50 clinical mastitis cases in Ningxia showed that:The detection results of Staphylococcus aureus,Streptococcus agalactiae,Pseudomonas aeruginosa,Streptococcus alactiae,pathogenic Escherichia coli and Mycoplasma bovis were 19.8%(45/227),18.1%(41/227),11.9%(27/227),respectively.,14.5%(33/227),14.1%(32/227),10.1%(10/227).To establish the identification of six main pathogenic bacteria in dairy mastitis.In this study,the NUC gene of Staphylococcus aureus,EF-TU gene of Streptococcus agalactiae,ETA gene of Pseudomonas aeruginosa,16srRNA gene of Streptococcus alactiae,Phoa gene of pathogenic Escherichia coli,OPP D/F gene of Mycoplasma bovis were respectively targeted at this experiment.The specific primers and TaqMan probe were designed,and the reaction conditions were optimized.Based on the establishment of single fluorescence PCR detection method,a multi-fluorescence quantitative PCR detection method was established.The results showed that the multi-fluorescence PCR detection method had good specificity.For pathogenic Escherichia coli,Staphylococcus aureus,Streptococcus agalactiae,Streptococcus alactiae,Pseudomonas aeruginosa and Mycoplasma bovis,the minimum final template concentration was 101 copies/μL.It shows that this method is highly sensitive.Tests on 227 milk samples collected in Ningxia showed that The detection results of Staphylococcus aureus,Streptococcus agalactiae,Pseudomonas aeruginosa,Streptococcus alactiae,Pathogenic Escherichia coli and Mycoplasma bovis were 25.6%(58/227),18.5%(42/227),10.6%(24/227),respectively.22.5%(51/227),15.9%(36/227),7.5%(17/227).The results showed that the multiplex PCR method and heavy fluorescence PCR method established in this study could be used for the detection and epidemiological investigation of six main pathogenic bacteria of cow mastitis.