A real-time immunoquantitative PCR (iqPCR) method for detection of Staphylococcus aureus enterotoxin B (SEB) was developed and evaluated using both pure cultures and foods. The assay consisted of immunocapture of SEB and real-time PCR amplification of the DNA probe linked to the detection antibody. iqPCR was compared to an in-house enzyme-linked immunosorbent assay (ELISA) using the same couple of capturedetection antibodies and to commercial kits for detection of S. aureus enterotoxins (SE). The iqPCR was approximately 1,000 times more sensitive (<10 pg ml ؊1 ) than the in-house ELISA and had a dynamic range of approximately 10 pg ml ؊1 to approximately 30,000 pg ml ؊1 . iqPCR was not inhibited by any of the foods tested and was able to detect SEB present in these foods. No cross-reactivity with SE other than SEB was observed. Application of iqPCR for detection of SEB in cultures of S. aureus revealed the onset of SEB production after 4 h of incubation at 22, 37, and 42°C, which was in the first half of the exponential growth phase. The total amounts of SEB produced by the two strains tested were larger at 42°C than at 37°C and were strain dependent.The reported detection limits of available commercial systems for detection of Staphylococcus aureus enterotoxins (SE) range from 0.5 to 2 ng enterotoxin per g of food (11,16,18,20,21). The sensitivity of detection methods and their ability to deliver quantitative information concerning the amounts of toxin present may require improvement, as it is possible that the established intoxication dose is underestimated due to constraining detection limits. Immuno-PCR is a detection method that combines the specificity of antibody-antigen recognition and the sensitivity of PCR. Immuno-PCR methods using endpoint detection with classical PCR and electrophoresis of amplicons for detection of Clostridium botulinum neurotoxin type A and E have been described (9,10,22). The immunoquantitative PCR (iqPCR) technology previously described in a patent (24) couples an antibody detection step, similar to an enzyme-linked immunosorbent assay (ELISA), with nucleic acid amplification of a DNA probe linked to the detection antibody by real-time PCR. Use of real-time PCR for quantitative amplification (13,14), unlike endpoint analysis, provides data required for quantification of the target DNA. The results can be expressed in absolute terms by reference to an external quantified standard or in relative terms by reference to another target sequence present in the sample (19).In the present work an iqPCR method for detection of S. aureus enterotoxin B (SEB) was developed and evaluated using both pure cultures and foods. The sensitivity of the iqPCR method was compared to the sensitivities of two commercially available systems (SET-RPLA [Oxoid, Basingstoke, United Kingdom] and VIDAS-SET2 [bioMérieux, Marcy-l'Etoile, France]), as well as to the sensitivity of an in-house ELISA that served as an internal reference for iqPCR. MATERIALS AND METHODSBacterial strains and culture conditions. All st...

The goal of the present study was twofold: (1) to detect the possible storage of dietary polyamines (PAs) in various tissues and (2) to investigate the role of dietary PAs in the differentiation of the pig intestinal epithelium. A first experimental series was designed to assess the accumulation of either milk PAs (mostly spermidine) or orally administered spermine (SPM) in piglet red blood cells (RBCs) and plasma, a preliminary stage in their distribution to growing and storage organs. Though PA concentrations of piglet RBCs and plasma were generally significantly higher than their sow counterparts, our experimental conditions failed to demonstrate that this increase could stem from ingested PAs. A second experimental series dealt with the determination of disaccharidase specific activities in proximal and distal parts of piglet gut on the 26th and 29th days after birth (preweaning time). In agreement with observations made previously on rat pups, we observed an increase in maltase specific activity (SA) at the end of the suckling period (the observed increase in sucrase SA was not significant). However, orally administered SPM did not affect this activity. Compared to the constant protein concentrations observed in both parts of the gut, the pancreatic protein content decreased sharply between the 26th and 29th postnatal days. At the same time pancreatic concentrations of spermidine (SPD) also decreased, suggesting that some pancreatic PAs were released as the organ secreted its proteins. In accordance with this hypothesis, we recorded SPM and SPD in pancreatic juice. The increases in PA concentrations seemed to follow the protein secretion pattern (i.e. PA concentrations reached a maximal value when the protein concentration was highest). The presence of PAs in pancreatic juice could be indicative of a control mechanism exerted by the pancreas on PA-induced growth and differentiation of porcine intestinal epithelium.

The goal of the present study was twofold: (1) to detect the possible storage of dietary polyamines (PAs) in various tissues and (2) to investigate the role of dietary PAs in the differentiation of the pig intestinal epithelium. A first experimental series was designed to assess the accumulation of either milk PAs (mostly spermidine) or orally administered spermine (SPM) in piglet red blood cells (RBCs) and plasma, a preliminary stage in their distribution to growing and storage organs. Though PA concentrations of piglet RBCs and plasma were generally significantly higher than their sow counterparts, our experimental conditions failed to demonstrate that this increase could stem from ingested PAs. A second experimental series dealt with the determination of disaccharidase specific activities in proximal and distal parts of piglet gut on the 26th and 29th days after birth (preweaning time). In agreement with observations made previously on rat pups, we observed an increase in maltase specific activity (SA) at the end of the suckling period (the observed increase in sucrase SA was not significant). However, orally administered SPM did not affect this activity. Compared to the constant protein concentrations observed in both parts of the gut, the pancreatic protein content decreased sharply between the 26th and 29th postnatal days. At the same time pancreatic concentrations of spermidine (SPD) also decreased, suggesting that some pancreatic PAs were released as the organ secreted its proteins. In accordance with this hypothesis, we recorded SPM and SPD in pancreatic juice. The increases in PA concentrations seemed to follow the protein secretion pattern (i.e. PA concentrations reached a maximal value when the protein concentration was highest). The presence of PAs in pancreatic juice could be indicative of a control mechanism exerted by the pancreas on PA‐induced growth and differentiation of porcine intestinal epithelium.