Optimization of the PCR for detection of Staphylococcus aureus nuc gene in bovine milk.
Staphylococcus aureus is an economically important and a major mastitis-causing pathogen that also poses food safety and antimicrobial resistance threats. Substances in mastitic milk inhibit the Taq DNA polymerase reaction (Taq PCR) making it of limited use for detecting S. aureus mastitis. In the study 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 mastitis). A specific amplicon of 270 bp was generated as predicted. Replacing Taq DNA polymerase with Thermus thermophilus (Tth) DNA polymerase alone (Tth-PCR) raised the sensitivity of S. aureus detection in milk from experimentally infected cows from 65 to 80%. Combining the use of Tth DNA polymerase and the purification of crude DNA extract using Chelex-100 before PCR raised the sensitivity to 100%. In a random survey involving 100 milk samples from cattle not infected with S. aureus, the test was 100% specific. With milk samples from clinical cases of bovine mastitis, 100% sensitivity and specificity were also observed. It is concluded that Tth-PCR on milk samples with the purification of crude DNA extracts using Chelex-100 is as sensitive as 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. aureus-specific DNA and circumvents the endogenous inhibitory effects of milk.
Kim
C H
CH
Department of Veterinary Pathobiology, University of Illinois, Urbana 61802, USA.
Khan
M
M
Morin
D E
DE
Hurley
W L
WL
Tripathy
D N
DN
Kehrli
M
M
Oluoch
A O
AO
Kakoma
I
I
D000818:Animals; D002417:Cattle; D004269:DNA, Bacterial / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D005260:Female; D005511:Food Handling; D008414:Mastitis, Bovine / Q000382:microbiology; D008892:Milk / Q000382:microbiology*; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification*
D004269:DNA, Bacterial