title: Towards an international standard for detection and typing botulinum neurotoxin-producing Clostridia types A, B, E and F in food, feed and environmental samples: a European ring trial study to evaluate a real-time PCR assay.
abstract: A real-time PCR method for detection and typing of BoNT-producing Clostridia types A, B, E, and F was developed on the framework of the European Research Project "Biotracer". A primary evaluation was carried out using 104 strains and 17 clinical and food samples linked to botulism cases. Results showed 100% relative accuracy, 100% relative sensitivity, 100% relative specificity, and 100% selectivity (inclusivity on 73 strains and exclusivity on 31 strains) of the real-time PCR against the reference cultural method combined with the standard mouse bioassay. Furthermore, a ring trial study performed at four different European laboratories in Italy, France, the Netherlands, and Sweden was carried out using 47 strains, and 30 clinical and food samples linked to botulism cases. Results showed a concordance of 95.7% among the four laboratories. The reproducibility generated a relative standard deviation in the range of 2.18% to 13.61%. Considering the high level of agreement achieved between the laboratories, this real-time PCR is a suitable method for rapid detection and typing of BoNT-producing Clostridia in clinical, food and environmental samples and thus support the use of it as an international standard method.
authors.affiliation: Veterinary Public Health and Food Safety Department, Istituto Superiore di Sanità, National Reference Centre for Botulism, Viale Regina Elena 299, 00161 Rome, Italy. lucia.fenicia@iss.it
authors.forename: Lucia
authors.initials: L
authors.lastname: Fenicia
authors.forename: Patrick
authors.initials: P
authors.lastname: Fach
authors.forename: Bart J
authors.initials: BJ
authors.lastname: van Rotterdam
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Anniballi
authors.forename: Bo
authors.initials: B
authors.lastname: Segerman
authors.forename: Bruna
authors.initials: B
authors.lastname: Auricchio
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Delibato
authors.forename: Raditijo A
authors.initials: RA
authors.lastname: Hamidjaja
authors.forename: Peter R
authors.initials: PR
authors.lastname: Wielinga
authors.forename: Cedric
authors.initials: C
authors.lastname: Woudstra
authors.forename: Joakim
authors.initials: J
authors.lastname: Agren
authors.forename: Dario
authors.initials: D
authors.lastname: De Medici
authors.forename: Rickard
authors.initials: R
authors.lastname: Knutsson
mesh_terms: D000821:Animal Feed / Q000382:microbiology; D000818:Animals; D001905:Botulinum Toxins / Q000235:genetics; D001906:Botulism / Q000382:microbiology; D003014:Clostridium botulinum / Q000145:classification*; D047013:Clostridium botulinum type A / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D047014:Clostridium botulinum type B / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D047970:Clostridium botulinum type E / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D047971:Clostridium botulinum type F / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D004783:Environmental Microbiology; D005060:Europe; D005516:Food Microbiology / Q000379:methods / Q000592:standards; D006801:Humans; D051379:Mice; D058889:Molecular Typing / Q000379:methods* / Q000592:standards; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity
chemical_list: D001905:Botulinum Toxins