Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producing Clostridium botulinum, Clostridium baratii and Clostridium butyricum.
OBJECTIVE
To develop real-time PCR assays for tracking and tracing clostridia responsible for human botulism.


RESULTS
Real-time PCR assays based on the detection of the genes ntnh encoding the nontoxin-nonhaemagglutinin (NTNH) proteins or the most homologous regions of the botulinum neurotoxin (bont) genes have been developed together with four real-time PCR assays, each being specific of the genes bont/A, bont/B, bont/E, bont/F and enables a toxin type-specific identification. The specificity of the assays was demonstrated using a panel of botulinum toxin producing clostridia (29 strains), nonbotulinum toxin producing clostridia (21 strains) and various other bacterial strains. The toxin type-specific assays had a sensitivity of 100 fg-1000 fg of total DNA in the PCR tube (25-250 genome equivalents) which correspond to 10(3) to 10(4) cells ml(-1). After a 48 h enrichment in anaerobic conditions, these PCR assays allowed the detection of Clostridium botulinum type A in a naturally contaminated sample of 'foie gras' suspected in a C. botulinum outbreak.


CONCLUSIONS
These PCR tests are specific and reliable for detection of heterogeneous BoNT producing clostridia responsible for human botulism.


CONCLUSIONS
Adoption of these PCR assays is a step forward a reliable and rapid detection of these clostridia in food samples.
Fach
P
P
Agence Française de Sécurité Sanitaire des Aliments, Laboratoire d'Etudes et de Recherches sur la Qualité des Aliments et les Procédés Agro-alimentaires, Maisons-Alfort, France. p.fach@afssa.fr
Micheau
P
P
Mazuet
C
C
Perelle
S
S
Popoff
M
M
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001905:Botulinum Toxins / Q000235:genetics / Q000302:isolation & purification*; D003013:Clostridium / Q000235:genetics* / Q000302:isolation & purification; D003014:Clostridium botulinum; D046972:Clostridium butyricum; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D005234:Fatty Liver / Q000382:microbiology; D005777:Geese; D006388:Hemagglutinins / Q000032:analysis* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D006388:Hemagglutinins; D001905:Botulinum Toxins