PCR-based detection of genes encoding glyceraldehyde-3-phosphate dehydrogenase and 5-enolpyruvylshikimate-3-phosphate synthase in Staphylococcus aureus directly from mastitic milk of murrah buffaloes

The aim of present investigation was to characterize Staphylococcus aureus from milk samples of Murrah buffaloes infected with mastitis using genus specific polymerase chain reaction assay based on gap gene (encoding glyceraldehyde-3-phosphate dehydrogenase) with amplified product of size 933 bp and species specific polymerase chain reaction assay based on aroA gene (encoding 5enolpyruvylshikimate-3-phosphate synthase) with amplified product of size 1153 bp. For internal control, a set of universal primers with amplified product of size 210 bp were also optimized. Out of 628 milk samples, a total of 37.89% and 39.81% milk samples were found positive by bacteriological examination and universal primer based PCR directly on milk, respectively. As many as 67.20% and 56% samples were found positive for presence of Staphylococcus spp. and Staphylococcus aureus when subjected to genus and species specific PCR directly on milk, respectively whereas conventional bacteriological analysis could detect 65.55% and 53.78% samples, respectively. The assay could detect organisms even when they are not viable due to antibiotic therapy. Thus, PCR assay was found to be rapid, sensitive and specific assay for accurate identification of Staphylococcus aureus up to species level in milk from murrah buffaloes and can be used for screening large dairy herd for earlier and faster establishment of effective therapeutic measures against mastitis.