Comparison of Different Approaches To Quantify Staphylococcus aureus Cells by Real-Time Quantitative PCR and Application of This Technique for Examination of Cheese

Abstract

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/l) than using a fluorigenic TaqMan probe (6 nuc gene copies/l). 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 ؋ 10 2 to 6.4 ؋ 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. Food poisoning caused by the enterotoxins produced byStaphylococcus aureus is of major concern in food hygiene. Many outbreaks involving contaminated milk and milk products have been reported (2,8,12,13,21). The natural habitat of S. aureus is warm-blooded animals, including humans. Ten to 40% of people are asymptomatic carriers of S. aureus, mostly at the mucosal membrane (20). Food handlers with infected skin lesions and asymptomatic carriers are often sources of food contamination, although with milk and milk products the raw milk itself can be contaminated with S. aureus.Once introduced into food-processing plants, bacteria can persist for a long time. CFU of S. aureus numbering 10 6 to 10 8 per g or ml of food must be present in order to produce sufficient amounts of toxins for poisoning (13). The bacteria are readily destroyed by heat, but the toxins are thermostable. Austrian national regulations concerning the presence of S. aureus in milk and milk products apply three-class plans that define m values to separate acceptable from marginally acceptable products and M values to separate marginally acceptable from defective-quality products; when numbers of bacteria are higher than M values, the presence of enterotoxins has to be investigated. The International Standardization Organization and International Dairy Federation methods (3, 4, 5, 6) apply conventional microbiological quantification techniques such as the plate count method and the most-probable-number (MPN) method. Some of these techniques require up to 6 days for quantification and detection, thus being time-consuming. There are rapid alternative methods for quantification of S. aureus cells. These comprise the application of MPN-PCR (17), a rather laborious procedure, and the quantification of the enzymatic activity of the bacterial phosphatase (10), which is not specific and can therefore be applied only to pure cultures. Another rapid alternative is real-time quantitative PCR (RTQ-PCR) (11), which quantifies DNA and th...

Quantification of Staphylococcus aureus in white cheese by the improved DNA extraction strategy combined with TaqMan and LNA probe-based qPCR

“…In another study, real time qPCR was applied to quantify S. aureus cells in artificially contaminated cheeses of different types. It was demonstrated that 1.5 × 10 2 to 6.4 × 10 3 copies of the nuc gene/2 g cheese could be detected based on the cheese matrix (Hein et al, 2001). Detection sensitivity improved approximately one log order by using the improved DNA extraction method in this study.…”

“…The procedure of Hein et al (2001) with slight modifications was applied. In summary, 2 g of cheese sample was mixed with 45 mL of digestion buffer containing 1 mg trypsin per mL and subsequently homogenized in a stomacher for 1 min, and incubated at 40°C for 3 h. After centrifugation at 5700 ×g for 15 min at 4°C, the fat layer and aqueous phases were discarded and the pellet was washed three times with TE buffer (10 mM Tris-HCl, 1 mM EDTA at pH 7.5) and once with the PCR buffer (50 mM KCl, 1.5 mM MgCl 2 , 10 mM Tris-HCl at pH 8.4).…”

“…Bacterial DNA extraction using trypsin and followed by food DNA kit method (Method 4) In this method, the food DNA extraction kit procedure described in Method 1 was combined with modified Hein et al (2001) procedure described in Method 3. The process steps described in Method 3 were followed until the pellet was resolved in 250 μL of PCR buffer and continued with the procedure described in Method 1.…”

“…The difference between qPCR and plate count results revealed that most of the bacteria could not be counted by classical plate counting method. The reason for this could be related to a few numbers of clusters formed by the bacteria causing less number of plate count results (Hein et al, 2001). Another reason for this difference was described by detection of dead and viable cells together (Mäntynen et al, 1997).…”

“…Several studies have been performed by qPCR to detect and quantify different bacterial cells in food products (D'Urso et al, 2009;Martínez-Blanch et al, 2009;Pennacchia et al, 2009). Several studies have been carried out on detection of S. aureus in food samples by qPCR method (Alarcón et al, 2006;Fusco et al, 2011;Hein et al, 2001;Poli et al, 2007). Cheese is a difficult matrix for direct S. aureus detection and quantification by qPCR method due to its high fat content (Ercolini et al, 2004).…”