Development of PCR primers and a DNA macroarray for the simultaneous detection of major Staphylococcus species using groESL gene.

Staphylococcus spp., including S. aureus, S. intermedius, S. hyicus, S. epidermidis, S. saprophyticus, S. haemolyticus, S. xylosus, and S. carnosus, are major bacterial species associated with food poisoning, and human and veterinary clinics. Traditional methods for the identification of these staphylococci are time-consuming, laborious, or inaccurate. Therefore, rapid and accurate diagnostic methods are needed. In this study, we designed the DNA probes and polymerase chain reaction (PCR) primers for the detection of the aforementioned Staphylococcus species. These primers were proved to be specific for the detection of their corresponding target strains. Furthermore, by using a consensus primer pair, we were able to co-amplify the intergenic region of groES-groEL for these staphylococci. Followed by a chromogenic macroarray system with the specific probes on the plastic chips, these staphylococci in milk products or clinical samples could be simultaneously detected. When the system was used for the inspection of milk or urine samples containing N × 10° target cells per milliliter of the sample, all these staphylococcal species could be identified after an 8-h pre-enrichment step. This system also allowed the adequate diagnosis of bacteremia, since N × 10° target cells per milliliter of the blood samples could be detected after a 12-h pre-enrichment. Compared to the multiplex PCR method, this approach has the additional advantage that it allowed the discrimination of more bacterial strains-even some bacterial strains that may generate PCR products with the same molecular sizes.

Quantitating staphylococcal enterotoxin B in diverse media using a portable fiber-optic biosensor.

A new, portable fiber-optic biosensor has been used to detect staphylococcal enterotoxin B, a causative agent of food poisoning, at levels as low as 0.5 ng/ml in buffer. The toxin (SEB) can also be detected and quantitated in other relevant media: human serum, urine, and aqueous extract of ham. The level of toxin, from 5 to 200 ng/ml, can be accurately predicted in these media by calibrating each fiber and by comparing results to a single standard curve based on toxin in buffer. The quantitative fluorescent sandwich immunoassay provides results in 45 min; qualitative results are provided in 15-20 min. Using a blender and a benchtop centrifuge, fast, simple aqueous extracts of contaminated ham samples were prepared and tested. Ham spiked with 5 or 40 micrograms SEB per 100 g food resulted in biosensor readings indicative of 11 or 69% recovery of the toxin, respectively. Finally, the SEB assay is highly specific; SEA and SED give only 2-3% of the signal at 5000 ng/ml as SEB gives at 1000 ng/ml. This specific, sensitive assay for SEB on the portable fiber-optic biosensor permits easy monitoring of clinical samples or on-site analysis of suspect food samples.