Genotyping of Clostridium perfringens toxins using multiple oligonucleotide microarray hybridization

Abstract

A microarray-based method for characterization of six Clostridium perfringens toxin genes: iA (iota toxin), cpa (alpha toxin), cpe (enterotoxin E), etxD (epsilon toxin), cpb1 (beta toxin 1),and cpb2 (beta toxin 2) was developed and evaluated using 17 C. perfringens isolates. Three individual oligonucleotide probes (oligoprobes), complementary to the unique sequences of each toxin gene, were designed and immobilized on a surface of aldehyde-coated glass slides. Multiplex PCR was used to simultaneously amplify DNA target regions of all six genes. Single-stranded DNA (ssDNA) samples for microarray analysis were prepared by following a primer extension of amplicons in the presence of one primer. Fluorescent moieties (Cy3) were incorporated into the ssDNA by chemical modification of guanine bases. The presence of toxin genes in C. perfringens was established by hybridization of the fluorescently labeled ssDNA representing different samples to the microarray gene-specific oligoprobes. Results of the study showed sensitivity and specificity of genotyping C. perfringens using multiple microarray-based assays.