A Single-Stranded DNA Aptamer That Selectively Binds to Staphylococcus aureus Enterotoxin B

The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro, with affinity to staphylococcal enterotoxin B (SEB) that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APTSEB1, successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide

APT^SEB1 is selective for SEB but not other closely related enterotoxins.

<p>Aptamer-precipitation of SEB from a mixture of enterotoxins was visualized by 4–12% SDS-PAGE with silver stain. Dynabeads® M-270 Streptavidin magnetic beads coated with APT^SEB1 were incubated in 1000 µl PBS-T incurred with 1 µg each of SEA, SEC₁, SEC₂, SEC₃, SED, and SEE. The aptamer-precipitation was carried out either with (lane 3) or without (lane 4) 1 µg SEB present in the mixture. After washing the Dynabeads with PBS-T, the protein eluate (lanes 3–8) was loaded onto the SDS-PAGE gel. Lanes 1 and 2 contain 200 ng of standard BSA and SEB, respectively. The protein bands labeled as “SA” represent the monomer of streptavidin liberated by the elution protocol.</p

Values for the number of beads and incubation times used in each round.

<p>The values were modulated in later rounds to increase the stringency of the SELEX protocol.</p

APT^SEB1 is selective for SEB within a complex mixture.

<p>The toxin-rich cell-free culture supernatant from four <i>S. aureus</i> strains was assayed for the presence of SEB by aptamer-precipitation. Five microliters of each culture supernatant was loaded onto an 4–12% SDS-PAGE gel to determine the protein content (lanes 2, 4, 6, 8). Three milliliters of each culture supernatant was incubated with APT^SEB1-coated Dynabeads. After washing with PBS-T, the resultant protein eluate from the APT^SEB1-coated Dynabeads was analyzed (lanes 3, 5, 7, 9). By PCR and ELISA analysis (Sandra Tallent, personal communication) the four strains potentially express a total of 17 enterotoxins and toxic shock syndrome toxin. However, only strain BAA1747 contains the gene for SEB. The protein bands labeled as “SA” represent the monomer of streptavidin liberated by the elution protocol.</p

APT^SEB1 binds to SEB, but not BSA.

<p>Aptamer-precipitation of SEB from 10-fold excess of BSA using several DNA sequences was visualized by 4–12% SDS-PAGE with silver stain. Dynabeads® M-270 Streptavidin magnetic beads coated with APT^SEB1 (lane 3), random 78-base ssDNA (lanes 4–6), PCR forward primer (used in this study, lane 7), and nothing (lane 8), were incubated in 500 µl PBS-T incurred with 10 µg BSA and 1 µg SEB. After washing the Dynabeads with PBS-T, the protein eluate (lanes 3–8) was loaded onto the SDS-PAGE gel. Lanes 1 and 2 contain 50 ng of standard BSA and SEB, respectively. The protein bands labeled as “SA” represent the monomer of streptavidin liberated by the elution protocol.</p

The primer and library sequences used in this study as well as the sequences of the two aptamers discovered in this work. APT^SEB1 was reported in 48 out of 49 sequences.

<p>The primer and library sequences used in this study as well as the sequences of the two aptamers discovered in this work. APT^SEB1 was reported in 48 out of 49 sequences.</p