Kafkas Üniversitesi Veteriner Fakültesi Dergisi 2017 , Vol 23 , Issue 6
Development of A Multiplex PCR Method for Direct Detection of Common Mastitis Pathogens in Bovine Milk Samples
Recep KALIN, Murat KARAHAN, Mehmet Nuri ACIK, Bülent TASDEMIR, Burhan CETINKAYA
1Department of Microbiology, Faculty of Veterinary Medicine, Cumhuriyet University, TR-58140 Sivas - TURKEY
2Faculty of Veterinary Medicine, Kyrgyz-Turkish Manas University, KG-720044, Bishkek-KYRGYZSTAN
3Department of Microbiology, Faculty of Veterinary Medicine, Bingol University, TR-12000 Bingol - TURKEY
4Veterinary Control Institute, TR-23200, Elazig - TURKEY
5Department of Microbiology, Faculty of Veterinary Medicine, Firat University, TR-2311, Elazig - TURKEY
DOI : 10.9775/kvfd.2017.17995 The aim of this study was to evaluate a simple and rapid DNA extraction method combined with a multiplex polymerase chain reaction (mPCR) for the identification of the major mastitis pathogens (Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli and Mycoplasma bovis) from milk samples. Of the 200 California Mastitis Test (CMT) positive milk samples, 45 (22.5%), 21 (10.5%) and 11 (5.5%) were detected as positive for the presence of S. aureus, S. agalactiae and E. coli by culture, respectively. In mPCR by DNA isolation method optimised here, S. aureus, S. agalactiae and E. coli were detected in 26.5% (53/200), 12% (24/200) and 6% (12/200) of the milk samples, respectively. The abovementioned agents were observed in similar proportions when the samples were analysed by a commercial DNA isolation kit. On the other hand, M. bovis was not detected in any of the milk samples by either culture or mPCR methods. A significant difference was determined between the results of culture and mPCRs (P<0.001). Diagnostic sensitivity and specificity of the optimised mPCR were calculated as 100% and 89.2% respectively, when culture results were considered as reference. The results suggest that the mPCR 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 in bovine milk samples. Keywords : Mastitis, Major Pathogens, DNA isolation, Multiplex PCR