Rapid methods for the detection of toxigenic Clostridium perfringens

Abstract

Clostridium perfringens may be the most widely occurring bacterial pathogen and is responsible for a variety of diseases in both humans and animals. The virulence of this organism is associated with the ability to produce an estimated 17 potential exotoxins. The production of one or more of the five major toxins (α,β,ε, and ι) is the basis for placing isolates into five toxigenic types, A through E. Enterotoxin (CPE), is not used in typing but is considered a major virulence factor. A multiplex PCR genotyping assay was developed, utilizing primers derived from sequences of cpa, cpb, etx, iA, and cpe, yielding products of 324, 196, 655, 446, and 233 bp, respectively. Template for this assay was derived from individual colonies suspended in 200 μl of HPLC-grade water, boiled for 20 min or heated in a microwave oven for 10 min at 700 W. Included in the 50 μl reaction volume was 10 μl of template, 0.15 to 0.7 μM of each primer, 0.1 mM dNTPs, 2 mM MgCl₂, and 2 units of Taq DNA polymerase. The PCR products were examined by electrophoresis in a 1.5% agarose gel stained with EtBr. Correlation of genotype with toxin phenotype in strains examined by mouse inoculation was excellent, and it was possible to provide results rapidly, usually in < 4 h. An ELISA procedure was established for detection of β toxin produced by C. perfringens types B and C. The ELISA was used to differentiate Cpb⁺ from Cpb⁻ isolates grown in overnight broth cultures and to measure β toxin in commercial fermentations of type C organisms. In addition to the above assays, preliminary work was initiated on the development of a PCR procedure for quantitation of C. perfringens in clinical or environmental samples, and involved the construction of a 233 bp homologous, competitive mimic from a restriction digest of a 323 bp PCR product generated from cpa.