The Development of a Multiplex PCR Assay for Fast and Cost-Effective Identification of the Five Most Significant Pathogenic Species.

A multiplex PCR system (m-PCR) has been developed to accurately differentiate the five most important pathogenic species, including the three species associated with infection in dairy cattle (, , and ) and the two species associated with human infections ( and ). The method is low-cost since it employs a simple "heat-shock" method in a TE buffer for DNA extraction. Furthermore, it requires only primers, a Taq polymerase, an agarose gel, and a molecular weight marker for identification. The method was based on published cytochrome B sequences and was evaluated using reference strains from each of the five species. The validity of the method was confirmed by identifying 50 strains isolated from milk samples. The specificity was tested in silico and with experimental PCR trials, showing no cross-reactions with other species, as well as with bacteria, fungi, cows, algae, animals, or humans. The method could detect mixed infections involving two or three species, providing a rapid test that delivers results within three hours.