Carbon nanoparticles as detection labels in antibody microarrays.Detection of genes encoding virulence factors in Shiga toxin-producing Escherichia coli

The present study demonstrates that carbon nanoparticles (CNPs) can be used as labels in microarrays. CNPs were used in nucleic acid microarray immunoassays (NAMIAs) for the detection of di¿erent Shiga toxin-producing Escherichia coli (STEC) virulence factors: four genes speci¿c for STEC (vt1,vt2, eae, andehxA) and the gene forE. coli16S (hui). Optimization was performed using a Box Behnken design, andthe limit of detection for each virulence factor was established. Finally,this NAMIA using CNPs was tested with DNA from 48 ¿eld strains originating from cattle feces, and its performance was evaluated by comparing results with those achieved by the reference method q-PCR. All factors tested gave sensitivity and speci¿city values higher than 0.80 and e¿ciency values higher than 0.92. Kappa coe¿cients showed an almost perfect agreement (k > 0.8) between NAMIA and the reference method used forvt1, eae, and ehxA, and a perfect agreement (k = 1) forvt2 and hui. The excellent agreement between the developed NAMIA and q-PCR demonstrates that the proposed analytical procedure is indeed ¿t for purpose, i.e., it is valuable for fast screening of ampli¿ed genetic material such as E. coli virulence factors. This also proves the applicability of CNPs in microarrays.
This work was partially supported by the Generalitat Valenciana (BEST/2009/026), the Universidad Politecnica de Valencia (PAID-00-09-2837), and the Dutch Ministry of Agriculture, Nature and Food Quality (Strategic research program Food Safety, Monitoring and Detection KB-06-005). The authors thank Dr. Eva Moller Nielsen at the Danish Veterinary Institute (Copenhagen, Denmark) for providing E. coli control strains and Dr. Lutz Geue (Friedrich-Loeffler-Institut, Wusterhausen, Germany) and Dr. Dorte Dopfer (School of Veterinary Medicine, University of Wisconsin, Madison, WI) for field isolates.