Real-time PCR for Clostridium botulinum type C neurotoxin (BoNTC) gene, also covering a chimeric C/D sequence-Application on outbreaks of botulism in poultry

In recent years, botulism type C has become a serious problem in poultry flocks in Sweden. A real-time PCR assay for Clostridium botulinum (C. botulinum) type C neurotoxin (BoNTC) gene was developed as an alternative to the mouse bioassay for detection and identification of C. botulinum type C. The complete method consists of an optimized enrichment protocol followed by automated DNA extraction prior to real-time PCR. The sensitivity of the PCR assay was determined with purified DNA to approximately 50 copies per PCR reaction. The specificity of the PCR assay was evaluated on a panel of about thirty relevant bacteria and on samples of caecum from birds collected in connection with botulism outbreaks on Swedish poultry farms. The PCR assay also covers a previously reported chimeric C/D sequence of the gene. Caecum samples from the outbreaks were positive by real-time PCR. Some of these samples were also examined with a set of conventional PCR methods, to distinguish the gene for the chimeric form from the conserved type C gene. Interestingly, the caecum samples were found to be positive for the chimeric C/D sequence. This is the first study in Europe demonstrating the chimeric C/D sequence. When the toxin gene in two of the samples was sequenced, it was closely identical (99-100%) with several previously reported C/D chimeric sequences. DNA extraction and the real-time PCR assay were both performed in a 96-well format, facilitating for future large-scale detection in outbreak situations and prevalence studies. (C) 2010 Elsevier B.V. All rights reserved.