Development of an in vitro method for detection of Clostridium botulinum types A and E using real-time PCR

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2003-01-01
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Artin, Ingrid
Lövenklev, Maria
Rådström, Peter
Holst, Elisabet
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International Conference on the Epidemiology and Control of Biological, Chemical and Physical Hazards in Pigs and Pork
Iowa State University Conferences and Symposia

The SafePork conference series began in 1996 to bring together international researchers, industry, and government agencies to discuss current Salmonella research and identify research needs pertaining to both pig and pork production. In subsequent years topics of research presented at these conferences expanded to include other chemical and biological hazards to pig and pork production.

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Specific primers for C. botulinum types A and E neurotoxin genes were evaluated both from the literature and of own construction. Furthermore, a real-time system with specific hybridisation probes was designed for C. botulinum type E neurotoxin gene, and is under construction for type A. Sequencing of part of the neurotoxin gene for type E showed some differences with sequences reported in GenBank. The PCR methods were optimised regarding amplification efficiency, linear range and specificity. The detection limit for type E using real-time PCR is at least 0,1 ng/ml, corresponding to 0,4 pg of total DNA in the tube, and at least 0,5 ng/ml (2,5 pg of total DNA) for type A using conventional PCR. Quantitative reverse transcription PCR was used to study the relative expression of the neurotoxin gene in different growth phases.

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Wed Jan 01 00:00:00 UTC 2003