title: Multiplex real-time PCR SYBR Green for detection and typing of group III Clostridium botulinum.
abstract: Clostridium botulinum type C and type D belonging to the group III organisms, are mainly responsible for animal botulism outbreaks. Clinical signs alone are often insufficient to make a diagnosis of botulism and a laboratory confirmation is required. Laboratory confirmation can be performed by demonstrating the presence of botulinum neurotoxins in serum, gastrointestinal contents, liver, wound of sick or dead animals, or by demonstrating the presence of C. botulinum in gastrointestinal contents, liver, and wound. Demonstration of spores in gastrointestinal contents or tissue of animals with clinical signs indicative of botulism reinforces the clinical diagnosis. With the aim of detecting and typing C. botulinum group III organisms, a multiplex real-time PCR SYBR Green was developed and in-house validated. Selectivity, limit of detection, relative accuracy, relative specificity, relative sensitivity, and repeatability of the method were investigated. The multiplex real-time PCR SYBR green used showed a 100% selectivity, 100% relative accuracy, 100% relative specificity, 100% relative sensitivity and a limit of detection of 277 and 580 DNA copies for C. botulinum type C and C. botulinum type D, respectively. The method reported here represents a suitable tool for laboratory diagnosis of type C and D botulism and for testing a large number of samples collected during the animal botulism surveillance and prevention activities.
authors: Istituto Superiore di Sanità, Department of Veterinary Public Health and Food Safety, National Reference Centre for Botulism, Viale Regina Elena, 299 - 00161 Rome, Italy.
authors: Fabrizio
authors: F
authors: Anniballi
authors: Bruna
authors: B
authors: Auricchio
authors: Elisabetta
authors: E
authors: Delibato
authors: Monia
authors: M
authors: Antonacci
authors: Dario
authors: D
authors: De Medici
authors: Lucia
authors: L
authors: Fenicia
mesh_terms: D000818:Animals; D015373:Bacterial Typing Techniques; D052160:Benzothiazoles; D003014:Clostridium botulinum / Q000145:classification / Q000235:genetics*; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I