title: Multiplex PCR for detection of botulinum neurotoxin-producing clostridia in clinical, food, and environmental samples.
abstract: Botulinum neurotoxin (BoNT), the most toxic substance known, is produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, also by some strains of Clostridium butyricum and Clostridium baratii. The standard procedure for definitive detection of BoNT-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (SMB). The SMB is highly sensitive and specific, but it is expensive and time-consuming and there are ethical concerns due to use of laboratory animals. PCR provides a rapid alternative for initial screening for BoNT-producing clostridia. In this study, a previously described multiplex PCR assay was modified to detect all type A, B, E, and F neurotoxin genes in isolated strains and in clinical, food, environmental samples. This assay includes an internal amplification control. The effectiveness of the multiplex PCR method for detecting clostridia possessing type A, B, E, and F neurotoxin genes was evaluated by direct comparison with the SMB. This method showed 100% inclusivity and 100% exclusivity when 182 BoNT-producing clostridia and 21 other bacterial strains were used. The relative accuracy of the multiplex PCR and SMB was evaluated using 532 clinical, food, and environmental samples and was estimated to be 99.2%. The multiplex PCR was also used to investigate 110 freshly collected food and environmental samples, and 4 of the 110 samples (3.6%) were positive for BoNT-encoding genes.
authors.affiliation: Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. dario.demedici@iss.it
authors.forename: Dario
authors.initials: D
authors.lastname: De Medici
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Anniballi
authors.forename: Gary M
authors.initials: GM
authors.lastname: Wyatt
authors.forename: Miia
authors.initials: M
authors.lastname: Lindström
authors.forename: Ute
authors.initials: U
authors.lastname: Messelhäusser
authors.forename: Clare F
authors.initials: CF
authors.lastname: Aldus
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Delibato
authors.forename: Hannu
authors.initials: H
authors.lastname: Korkeala
authors.forename: Michael W
authors.initials: MW
authors.lastname: Peck
authors.forename: Lucia
authors.initials: L
authors.lastname: Fenicia
mesh_terms: D000818:Animals; D001483:Base Sequence; D001681:Biological Assay / Q000706:statistics & numerical data; D001905:Botulinum Toxins / Q000096:biosynthesis* / Q000235:genetics*; D003013:Clostridium / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D003014:Clostridium botulinum / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004783:Environmental Microbiology; D005516:Food Microbiology; D005798:Genes, Bacterial; D006801:Humans; D051379:Mice; D009498:Neurotoxins / Q000096:biosynthesis* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D009498:Neurotoxins; D001905:Botulinum Toxins
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