Development and validation of a multiplex real-time PCR for detection of Clostridium chauvoei and Clostridium septicum

Abstract

Clostridium chauvoei is the causative agent of blackleg in cattle and sheep. The clinical symptoms of this severe disease are very similar to that of malignant edema ( Clostridium septicum), infections of other Clostridium species belonging to the gas edema complex, and anthrax ( Bacillus anthracis). C. chauvoei and C. septicum are closely related taxa and share many phenotypic properties hampering diagnosis by using traditional microbiological methods. Thus, there is a need for a fast and reliable identification method for specific detection of both species in clinical samples. The multiplex real-time PCR assay presented here is based on the detection of the spo0A gene and enables the simultaneous identification of C. chauvoei and C. septicum. The assay design includes an amplification control DNA template for the recognition of PCR-inhibitors. Assay validation was performed using a collection of 29 C. chauvoei, 38 C. septicum strains and 26 strains of other Clostridium species. Furthermore, the real-time PCR assay was successfully tested on tissue samples from 19 clinical blackleg cases. The assay allowed the reliable detection of one picogram DNA which represents approximate 239 genome equivalents.