<abstract xmlns="http://www.w3.org/1999/xhtml">
<p>A PCR-based procedure for detection of <italic>C. botulinum</italic> C and D in corn silage samples was validated. During the validation, method specificity, sensitivity, and accuracy were determined according to PN - EN ISO 16140:2004. Additionally, the specificity of the validated methods was proved by sequence analysis of PCR products obtained from examination of samples connected with botulism cases in cattle and mallard ducks. Limit of detection was estimated according to the Spearman - Kärber formula and expressed as LOD<sc>50.</sc> The obtained results showed that a 100% specificity was achieved. The sequencing of PCR products revealed 99% identity with sequences of <italic>bont/C</italic> and <italic>bont/D</italic> genes deposited in the GenBank. The sensitivity value ranged from 63.3% for <italic>C. botulinum</italic> type C to 75% for type D. The accuracy value varied from 72% for type C to 81.3% for type D. LOD<sc>50</sc> was estimated at the levels of 0.272 (0. D 188-0395) spore/g for type C and 0.17 (0.1-0.289) spore/g for type D. The described PCR-based procedure enabled detection of <italic>C. botulinum</italic> C and D at the stage of liquid culture. This makes examination of feed samples possible without isolation process. The presented procedure could support the diagnosis of botulism by faster and specific laboratory examination process.</p>
</abstract>

A PCR-based procedure for detection of C. botulinum C and D in corn silage samples was validated. During the validation, method specificity, sensitivity, and accuracy were determined according to PN - EN ISO 16140:2004. Additionally, the specificity of the validated methods was proved by sequence analysis of PCR products obtained from examination of samples connected with botulism cases in cattle and mallard ducks. Limit of detection was estimated according to the Spearman - Kärber formula and expressed as LOD50. The obtained results showed that a 100% specificity was achieved. The sequencing of PCR products revealed 99% identity with sequences of bont/C and bont/D genes deposited in the GenBank. The sensitivity value ranged from 63.3% for C. botulinum type C to 75% for type D. The accuracy value varied from 72% for type C to 81.3% for type D. LOD50 was estimated at the levels of 0.272 (0. D 188-0395) spore/g for type C and 0.17 (0.1-0.289) spore/g for type D. The described PCR-based procedure enabled detection of C. botulinum C and D at the stage of liquid culture. This makes examination of feed samples possible without isolation process. The presented procedure could support the diagnosis of botulism by faster and specific laboratory examination process.

In-house Validation of PCR Based Procedure for Specific Detection of <italic>Clostridium Botulinum</italic> Types C and D

Department of Hygiene of Animal Feedingstuffs

Journal of Veterinary Research

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

In-house Validation of PCR Based Procedure for Specific Detection of  Types C and D

A PCR-based procedure for detection of C. botulinum C and D in corn silage samples was validated. During the validation, method specificity, sensitivity, and...

In-house Validation of PCR Based Procedure for Specific Detection of Clostridium Botulinum Types C and D

Published Online: Oct 01, 2014

Page range: 393 - 398

Received: Apr 25, 2014

Accepted: Aug 24, 2014

DOI: https://doi.org/10.2478/bvip-2014-0061

Keywords
C and D toxins, feed, silage, validation

© 2014 National Veterinary Research Institute in Pulawy

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

In-house Validation of PCR Based Procedure for Specific Detection of Clostridium Botulinum Types C and D

Published Online: Oct 01, 2014

Page range: 393 - 398

Received: Apr 25, 2014

Accepted: Aug 24, 2014

DOI: https://doi.org/10.2478/bvip-2014-0061

KeywordsC and D toxins, feed, silage, validation

© 2014 National Veterinary Research Institute in Pulawy

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Keywords
C and D toxins, feed, silage, validation