Optimization of the PCR for detection of Staphylococcus aureus nuc gene inbovine milk

Staphylococcus aureus is an economically important and a major mastitis-cau sing pathogen that also poses food safety and antimicrobial resistance thre ats. Substances in mastitic milk inhibit the Tag DNA polymerase reaction (T aq PCR) making it of limited use for detecting S. aureus mastitis. In the s tudy reported here, a set of oligonucleotide primers of 21 and 24 bases was used in Taq-PCR to amplify DNA from S. aureus (isolates from bovine mastit is). A specific amplicon of 270 bp was generated as predicted. Replacing Ta g DNA polymerase with Thermus thermophilus (Tth) DNA polymerase alone (Tth- PCR) raised the sensitivity of S. aureus detection in milk from experimenta lly infected cows from 65 to 80%. Combining the use of Tth DNA polymerase a nd the purification of crude DNA extract using Chelex-100 before PCR raised the sensitivity to 100%. In a random survey involving 100 milk samples fro m cattle not infected with S. aureus, the test was 100% specific. With milk samples from clinical cases of bovine mastitis, 100% sensitivity and speci ficity were also observed. It is concluded that Tth-PCR on milk samples wit h the purification of crude DNA extracts using Chelex-100 is as sensitive a s but faster than conventional milk bacteriological culture techniques and is highly specific. The modified PCR correlates with elevated somatic cell counts, detects evidence of chronic and resolving infection based on S. aur eus-specific DNA and circumvents the endogenous inhibitory effects of milk.