Multiplex detection of antibiotic resistance genes using padlock probes.

Abstract: The elucidation of resistance mechanisms is of central importance to providing and maintaining efficient medical treatment. However, molecular detection methods covering the complete set of resistance genes with a single test are still missing. Here, we present a novel 100-plex assay based on padlock probes in combination with a microarray that allows the simultaneous large-scale identification of highly diverse β-lactamases. The specificity of the assay was performed using 70 clinical bacterial isolates, recovering 98% of the β-lactamase nucleotide sequences present. Additionally, the sensitivity was evaluated with PCR products and genomic bacterial DNA, revealing a detection limit of 104 DNA copies per reaction when using PCR products as the template. Pre-amplification of genomic DNA in a 25-multiplex PCR further facilitated the detection of β-lactamase genes in dilutions of 107 cells/mL. In summary, we present an efficient, highly specific, and highly sensitive multiplex detection method for any gene. [Copyright &y& Elsevier]