In-House Validation of Multiplex PCR Method for Detection of Clostridium Botulinum in Food and Feed

Tomasz Grenda 1 , Elżbieta Kukier 1 , Zbigniew Sieradzki 1 , Magdalena Goldsztejn 1  and Krzysztof Kwiatek 1
  • 1 Department of Hygiene of Animal Feedingstuffs, National Veterinary Research Institute. 24-100 Pulawy, Poland


The aim of this study was to perform an in-house validation of multiplex PCR method for C. botulinum detection in food and feed samples. The study was carried out on food and feed matrixes artificially contaminated by spores of C. botulinum reference strains. The following characteristic parameters for qualitative detection were estimated: limit of detection expressed as LOD50 according to the Spearman-Kärber formula, specificity, sensitivity, and accuracy according to the PN-EN ISO 16140:2004. The validated method showed high specificity. Specific PCR products were revealed only for DNA obtained from samples contaminated with C. botulinum spores. PCR inhibition was observed, especially during examination of contaminated feed. The calculated LOD50 for feed was nearly 10 times higher than for food. The implemented method enables to obtain test results during 3 d without time- consuming process of isolation and proving the ability of strains to produce botulinum toxins.

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