Inhibitor resistance and in situ capability of nanoparticle based gene quantification

Abstract

We have demonstrated the preliminary results of the in situ monitoring capability of an inhibitor resistant gene quantification assay using magnetic bead (MB) and quantum dot (QD) nanoparticles (hereafter “MB–QD assay”) for the detection ofE. coli O157:H7 in environmental samples. The selectivity of the MB–QD assay was demonstrated via the discrimination of the target bacteria in the presence of nonspecific microbial populations. The effect of temperature on the assay was examined to evaluate the necessity of elevated temperature incubation. The reagents (i.e., particle complex and particle–DNA conjugate) were also shown to have a stability of at least 10 days without refrigeration, therefore enabling prior preparation and the subsequent storage of these reagents. In addition, it was found that the MB–QD assay was resistant to the presence of naturally occurring inhibitors (i.e., humic acids, Ca²⁺) and residual reagents from DNA extraction (i.e., surfactant, ethanol). Overall the results indicated that the MB–QD assay is potentially suitable for further development as an in situ bacteria monitoring method for working with inhibitor laden samples without requiring additional purification steps and elevated temperature processes.