Real-time PCR method for quantification of Staphylococcus aureus in milk

• Published in: Journal of food protection Vol. 70; no. 1; pp. 90 - 96
• Main Authors: GOTO, M, TAKAHASHI, H, SEGAWA, Y, HAYASHIDANI, H, TAKATORI, K, HARA-KUDO, Y
• Format: Journal Article
• Language: English
• Published: Des Moines, IA International Association of Milk, Food and Environmental Sanitarians 2007
• Subjects: Animals; Base Sequence; Biological and medical sciences; Cattle; Colony Count, Microbial; Consumer Product Safety; DNA, Bacterial - analysis; Food Contamination - analysis; Food industries; Food microbiology; Fundamental and applied biological sciences. Psychology; Hot Temperature; Humans; Milk - microbiology; Milk and cheese industries. Ice creams; Polymerase Chain Reaction - methods; Sensitivity and Specificity; Species Specificity; Staphylococcus aureus; Staphylococcus aureus - isolation & purification; Time Factors; Polymerase chain reaction; Dairy product; Bacteria; Micrococcales; Micrococcaceae; Method; Molecular biology; Real time; Staphylococcus aureus; Quantitative analysis; Milk
• ISSN: 0362-028X; 1944-9097
• DOI: 10.4315/0362-028X-70.1.90

A reproducible real-time PCR method that targets the putative transcriptional regulator gene of Staphylococcus aureus was developed to quantify this microorganism in milk samples. On the basis of partial sequences of this gene determined from S. aureus strains, we designed the specific primers and probe for use in a quantitative PCR assay. These specificities were confirmed with 25 strains of S. aureus and 35 strains of other bacteria. A real-time PCR assay with serial 10-fold dilutions of purified DNA and pure culture was conducted. It was possible to construct standard curves with a high correlation coefficient (r2 = 0.99) in the range of 50 ng to 50 fg for purified DNA and 10(7) to 10(1) CFU/ml for a pure culture. The constructed standard curve for milk samples was similar to that for the pure culture, and the quantification of S. aureus in the range of 10(7) to 10(1) CFU/ml was possible. Moreover, to determine how our real-time PCR method would perform under actual analytical conditions, we quantified the DNA from S. aureus after two types of heat treatments were used for the pasteurization of milk. The amount of DNA found was affected after heat treatment at 63 degrees C for 30 min (low-temperature long-time method) but not at 72 degrees C for 15 s (high-temperature short-time method). The results indicate that the real-time PCR method developed in this study is effective for monitoring S. aureus contamination in milk because of its high specificity and sensitivity.