Multiplex PCR Assay For Detecting Four Major Mastitis-causing Pathogens In Milk

Bovine mastitis is the most important source of loss for the dairy loss all around the world. The major cause of bovine mastitis is the infection of the udder by pathogenic bacteria of which S. aureus, S. agalactiae, Mycoplasma bovis and E. coli are the most common.In this research, the bacterial DNA was extracted directly from milk using Chelex-100 method. The four common mastitis pathogens were artificially inoculated to milk samples. Then the sensitivities of the PCR assay using the Chelex-100 DNA extraction method were detected. Four sets of primers which had been proved to have high specificity were selected, a multiplex PCR assay was developed by step-by-step optimization for the simultaneous detection of the four major causes of bovine mastitis. The sensitivity of the multiplex PCR was compared with cultural method, which provides the "gold standard", on milk samples.The results showed that the Chelex-100 DNA extraction method was rapid, economic and efficient for the detection of pathogens by PCR. The multiplex PCR assay showed specificity and sensitivity in detecting the four common pathogens. The sensitivity of the PCR assay using the Chelex-100 DNA extraction method in detecting S.aureus and S. agalactiae were both 1.25×103CFU/mL, and in dectecting M. bovis and E. coli were both 6.25×1022 CFU/mL. The sensitivities of culture and PCR for detection of S. aureus, S. agalactiae and E. coli were not significantly different. However, multiplex PCR was more sensitive than culture for detection of M. bovis.The multiplex PCR assay was economic, rapid and efficient for detecting four major mastitis-causing pathogens in milk. Undoubtedly, this assay will make great sense for controlling bovine mastitis in practice.