Establishment and Application of the Multiplex PCR Method for Detecting Five Pathogenic Bacteria in Cows with Mastitis
To develop a multiplex PCR assay for the rapid detection of the main pathogens of dairy mastitis(E.coli,S.agalactiae,K.pneumoniae,S.aureus and S.enterica),in this study,specific primers were designed according to the phoA gene of E.coli,ef-tu gene of S.agalactiae,Khe gene of K.pneumoniae,Nuc gene of S.aureus and invA gene of S.enterica.PCR reaction system and reaction procedure were optimized and specific tests were performed.At the same time,pMD-19T recombinant plasmids containing pathogenic bacteria genes were constructed for sensitivity tests.The results showed that the optimal working concentration of primers was 0.8 μmol/L,the optimal annealing temperature was 59.6℃,the optimal extension time was 40 s,and the optimal number of cycles was 30.The optimized multiplex PCR system can amplify the specific bands of five target pathogens.The PCR sensitivity of the five pathogens was 5 copies/µL.The multiplex PCR system was further used to detect 108 samples of milk from cows with mastitis.The re-sults showed that the positive detection rate of 108 samples was 77.8％.The detection rates of E.coli,S.agalactiae,S.aureus,K.pneumoniae and S.enterica were 23.1％,8.3％,34.3％,8.3％and 5.6％,respectively.This study established a multiplex PCR detection method with good specificity and sensitivity,which can be used to rapidly identify the patho-genic bacteria of dairy cow mastitis in clinical production practice,and provide a methodological basis for the diagnosis,prevention and control of dairy cow mastitis and epidemiological investigation.
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