Comparison of different approaches to quantify Staphylococcus aureus cells by real-time quantitative PCR and application of this technique for examination of cheese.
Two different real-time quantitative PCR (RTQ-PCR) approaches were applied for PCR-based quantification of Staphylococcus aureus cells by targeting the thermonuclease (nuc) gene. Purified DNA extracts from pure cultures of S. aureus were quantified in a LightCycler system using SYBR Green I. Quantification proved to be less sensitive (60 nuc gene copies/microl) than using a fluorigenic TaqMan probe (6 nuc gene copies/microl). Comparison of the LightCycler system and the well-established ABI Prism 7700 SDS with TaqMan probes revealed no statistically significant differences with respect to sensitivity and reproducibility. Application of the RTQ-PCR assay to quantify S. aureus cells in artificially contaminated cheeses of different types achieved sensitivities from 1.5 x 10(2) to 6.4 x 10(2) copies of the nuc gene/2 g, depending on the cheese matrix. The coefficients of correlation between log CFU and nuc gene copy numbers ranged from 0.979 to 0.998, thus enabling calculation of the number of CFU of S. aureus in cheese by performing RTQ-PCR.
Hein
I
I
Institute of Milk Hygiene, Milk Technology, and Food Science, University of Veterinary Medicine, 1210 Vienna, Austria. Ingeborg.Hein@vu-wien.ac.at
Lehner
A
A
Rieck
P
P
Klein
K
K
Brandl
E
E
Wagner
M
M
D052160:Benzothiazoles; D002611:Cheese / Q000382:microbiology*; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000032:analysis; D003959:Diamines; D005456:Fluorescent Dyes; D005470:Fluorometry; D018628:Gene Dosage; D006358:Hot Temperature; D008836:Micrococcal Nuclease / Q000235:genetics*; D009930:Organic Chemicals; D011804:Quinolines; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification*; D019914:Taq Polymerase / Q000378:metabolism
D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D019914:Taq Polymerase; D008836:Micrococcal Nuclease