Clostridium spp. discrimination with a simple bead-based fluorescence assay

Abstract

C. chauvoei is the causative agent of blackleg, an endogenous bacterial infection which usually affects cattle and other ruminants. Due to the fact that the symptoms of this severe disease are very similar to the phenotype caused by an infection with C. septicum, a reliable differentiation of C. chauvoei from other Clostridium spp. is mandatory. Traditional microbiological detection methods are time consuming and the proper specification is hampered by the overgrowing tendency of swarming C. septicum colonies when both species are in the clinical sample. Thus, there is an urgent need to improve and simplify the specific detection of C. chauvoei and C. septicum. We report an easy and fast Clostridium spp. discrimination method via a magnetic bead-based fluorescence assay. To that end, the target DNA was amplified using 16S-23S rDNA spacer region specific primers. These PCR products were employed to generate single-stranded capture probe DNA, which was immobilized on magnetic beads. Functionalized magnetic particles exhibit numerous advantages, like their simple manipulation in combination with a huge binding capacity of biomolecules and make therefore excellent biosensors. In this context, the discrimination between C. chauvoei and C. septicum was realized by means of hybridization with complementary detection probe DNA. Finally, fluorescence spectroscopy allowed the signal readout. With this approach a precise discrimination between C. chauvoei, C. septicum and C. carnis was accomplished.