An innovative molecular detection tool for tracking and tracing Clostridium botulinum types A, B, E, F and other botulinum neurotoxin producing Clostridia based on the GeneDisc cycler.
Rapid and specific detection of botulinum neurotoxin (BoNT) producing Clostridia is a priority for public health authorities, in case of both natural and intentional botulism outbreaks. This study reports on the evaluation of a detection system based on the GeneDisc Cycler designed for simultaneously testing the bont/A, bont/B, bont/E and bont/F genes encoding for the botulinum neurotoxins types A, B, E and F. BoNT-producing Clostridia (n = 102) and non-BoNT-producing bacteria (n = 52) isolated from clinical, food and environmental samples were tested using this macro-array and results were compared to the reference lethality test on mice. The bont genes were correctly detected in all C. botulinum type A, B, E and F strains available, as well as in toxigenic C. baratii type F and toxigenic C. butyricum type E. No cross reactivity was observed with non human-toxigenic bacteria, C. botulinum types C, D and G. The identification of the bont genotype using the macro-array was correlated to toxino-typing of the BoNTs as determined by the mouse bioassay. An "evaluation trial" of the GeneDisc array performed blind in four European laboratories with 77 BoNT-producing Clostridia as well as 10 food and clinical samples showed that the developed macro-array is specific and reliable for identifying BoNT/A-, BoNT/B-, BoNT/E- and BoNT/F-producing clostridial strains and for screening naturally contaminated food and fecal samples. The test is robust, has a low detection limit (c.a. 5 to 50 genome copies in the PCR reaction microwell) and is promising for monitoring BoNT-producing Clostridia in different kinds of samples including food and clinical samples.
Fach
P
P
French Food Safety Agency, Laboratory for Study and Research on Food Quality and Processes, 23 Av du Général De Gaulle, Fr-94706 Maisons-Alfort, France. p.fach@afssa.fr
Fenicia
L
L
Knutsson
R
R
Wielinga
P R
PR
Anniballi
F
F
Delibato
E
E
Auricchio
B
B
Woudstra
C
C
Agren
J
J
Segerman
B
B
de Medici
D
D
van Rotterdam
B J
BJ
D000818:Animals; D001905:Botulinum Toxins / Q000235:genetics*; D019274:Botulinum Toxins, Type A / Q000235:genetics; D003014:Clostridium botulinum / Q000235:genetics / Q000302:isolation & purification*; D047013:Clostridium botulinum type A / Q000235:genetics / Q000302:isolation & purification; D047014:Clostridium botulinum type B / Q000235:genetics / Q000302:isolation & purification; D047970:Clostridium botulinum type E / Q000235:genetics / Q000302:isolation & purification; D047971:Clostridium botulinum type F / Q000235:genetics / Q000302:isolation & purification; D005243:Feces / Q000382:microbiology; D005516:Food Microbiology / Q000379:methods*; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction
C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096326:botulinum toxin type E; C096321:botulinum toxin type F