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Detection of Clostridium botulinum neurotoxin coding genes: analysis of PCR products by real time versus capillary gel electrophoresis methods

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Abstract

In this work, two PCR-based methods have been developed for the detection of Clostridium botulinum strains carrying the gene coding for C. botulinum neurotoxin C (BoNTC) responsible for avian botulism. Both methods are based on the same amplification primers designed using multiple sequence alignments between toxin C coding sequences from DNA sequence databases. The first is a real-time PCR method, using a Taqman-MGB probe. The second uses conventional end-point PCR, followed by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF). A comparison between both methods has been established for the individual and simultaneous detection of toxin C (BONTC) or bacterial 16S (BACT) sequences from C. botulinum. The results indicate that, in general, the same sensitivity was achieved by using RT-PCR and PCR-CGE-LIF allowing the detection of both C. botulinum amplicons from concentrations as low as 7 × 10−5 μg/ml of total genomic DNA. Some other features from RT-PCR and CGE-LIF are also critically discussed in this work, including quantification capability, size determination, analysis speed and identification strategies, to provide enough information to adequately select the best analytical technique in each case.

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Abbreviations

BACT:

Amplicon designed for the detection of bacterial 16S sequences

BoNTs:

Botulinum neurotoxins

BONTC:

Amplicon designed for the detection of Clostridium botulinum neurotoxin C coding sequences

CGE-LIF:

Capillary gel electrophoresis with laser induced fluorescence

C t :

Threshold cycle as used in RT-PCR

RT-PCR:

Real-time PCR

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Acknowledgments

Authors also thank Dr. Williamson (National Wildlife Health Center, Madison, WI, USA) and Drs. Rosario Muñoz and Blanca de las Rivas (Institute of Industrial Fermentations, CSIC, Madrid, Spain) for kindly providing DNA samples. This work was supported by grants from MEC (AGL2005-05320-C02-01), Comunidad de Madrid (S-505/AGR-0153-ALIBIRD) and Parques Nacionales (MMA, 99/2003).

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Authors and Affiliations

  1. Institute of Industrial Fermentations (CSIC), Juan de la Cierva 3, 28006, Madrid, Spain

    Laura Sánchez-Hernández, Alejandro Cifuentes & Ramón Gonzalez

  2. Institute of Organic Chemistry (CSIC), Juan de la Cierva 3, 28006, Madrid, Spain

    Begoña Jiménez

  3. Instituto de Investigación en Recursos Cinegéticos, IREC (CSIC, UCLM, JCCM), Ronda de Toledo s/n, 13005, Ciudad Real, Spain

    Rafael Mateo

Authors
  1. Laura Sánchez-Hernández
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  2. Alejandro Cifuentes
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  3. Begoña Jiménez
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  4. Rafael Mateo
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  5. Ramón Gonzalez
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Corresponding author

Correspondence to Ramón Gonzalez.

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Sánchez-Hernández, L., Cifuentes, A., Jiménez, B. et al. Detection of Clostridium botulinum neurotoxin coding genes: analysis of PCR products by real time versus capillary gel electrophoresis methods. Eur Food Res Technol 227, 495–502 (2008). https://doi.org/10.1007/s00217-007-0746-1

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  • DOI: https://doi.org/10.1007/s00217-007-0746-1

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