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

• Published in: International journal of food microbiology 2011-03, Vol.145, p.S152-S157
• Main Authors: Fenicia, Lucia, Fach, Patrick, van Rotterdam, Bart J., Anniballi, Fabrizio, Segerman, Bo, Auricchio, Bruna, Delibato, Elisabetta, Hamidjaja, Raditijo A., Wielinga, Peter R., Woudstra, Cedric, Ågren, Joakim, De Medici, Dario, Knutsson, Rickard
• Format: Article
• Language: English
• Subjects: Animal Feed - microbiology; Animals; BoNT-producing Clostria; Botulinum Toxins - genetics; Botulism; Botulism - microbiology; Clostridium botulinum; Clostridium botulinum - classification; Clostridium botulinum type A - classification; Clostridium botulinum type A - genetics; Clostridium botulinum type A - isolation & purification; Clostridium botulinum type B - classification; Clostridium botulinum type B - genetics; Clostridium botulinum type B - isolation & purification; Clostridium botulinum type E - classification; Clostridium botulinum type E - genetics; Clostridium botulinum type E - isolation & purification; Clostridium botulinum type F - classification; Clostridium botulinum type F - genetics; Clostridium botulinum type F - isolation & purification; Environmental Microbiology; Europe; Food Microbiology - methods; Food Microbiology - standards; Humans; Mice; Molecular Typing - methods; Molecular Typing - standards; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - standards; Real time PCR; Ring trial; Sensitivity and Specificity
• ISSN: 0168-1605; 1879-3460
• DOI: 10.1016/j.ijfoodmicro.2011.02.001

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. ► We evaluated a real-time PCR method for detection and typing of BoNT-producing Clostridia types A, B, E, and F. ► Primary evaluation showed 100% relative accuracy, 100% relative sensitivity, 100% relative specificity, and 100% selectivity against the reference cultural method. ► A ring trial study performed at four different European laboratories was carried out. ► Results showed a concordance of 95.7% among the four laboratories. ► Results of ring trial support the use of this Real Time PCR as an international standard method.