Sisay, Tesfaye (PhD)Assefa, Yared2019-05-212023-11-182019-05-212023-11-182018-02-02http://etd.aau.edu.et/handle/12345678/18282Mastitis is the most frequently occurring and economically one of the most important infectious disease in dairy cows. In most clinical laboratories, identification methods are based on microbiological culturing of milk and biochemical tests. However, there are many limitations associated with microbiological culture. Therefore, alternative, non-culture- based diagnostic methods are needed. This study was conducted with the objective of developing and assessing the use of multiplex PCR as a tool to identify bacterial species commonly associated with mastitis viz; Staphylococcus aureus, Escherchia coli, Streptococcus agalactiae and Streptococcus dysagalactiae. Conventional techniques based on biochemical and physiological characteristics of bacteria were utilized to identify the bacterial species. Then multiplex PCR assays were optimized using species specific primers for simultaneous detection of bacterial species. For rapid and accurate diagnosis, DNA was isolated from milk samples and subjected to multiplex PCR. Based on multiplex PCR results, S. aureus (60%) was found to be the predominant bacteria detected from milk followed by S. agalactiae (40 %) and E. coli (25%). The detection levels of S. aureus and S. agalactiae were higher when using mPCR compared to the culture-based method. However, S. dysagalactiae was not detected by multiplex PCR. Diagnostic sensitivity and specificity of the optimized multiplex PCR were calculated as 80% and 99.9% respectively. Furthermore, there was a substantial agreement between both diagnostic techniques (kappa result (K= 0.7)). Overall the results suggest that the multiplex PCR assay employed in this study could be used as an alternative routine diagnostic method for rapid, sensitive and specific simultaneous detection of major mastitis agents.enBacteriaBiochemical TestCulture-Based MethodsMastitisMultiplex- PCRThesis