Novel Real-Time PCR Assay for Simultaneous Detection and Differentiation of Clostridium chauvoei and Clostridium septicum in Clostridial Myonecrosis

Abstract

ABSTRACT A real-time PCR assay based on the 16S rRNA gene sequence was designed for differentiation of blackleg-causing Clostridium chauvoei and Clostridium septicum , a phylogenetically closely related bacterium responsible for malignant edema. In order to exclude false-negative results, an internal amplification control was included in the assay. A set of three probes, one specific for C. chauvoei , one specific for C. septicum , and one specific for both species, permitted unequivocal detection of C. chauvoei in tests of 32 Clostridium sp. strains and 10 non- Clostridium strains. The assay proved to be sensitive, detecting one genome of C. chauvoei or C. septicum per PCR and 1.79 × 10 3 C. chauvoei cells/g artificially contaminated muscle tissue. In tests of 11 clinical specimens, the real-time PCR assay yielded the same results as an established conventional PCR method.