Pentaplexed quantitative real-time PCR assay for the simultaneous detection and quantification of botulinum neurotoxin-producing clostridia in food and clinical samples

Kirchner, S. , Krämer, K. M., Schulze, M., Pauly, D., Jacob, D., Gessler, F., Nitsche, A., Dorner, B. G. and Dorner, M. B. (2010) Pentaplexed quantitative real-time PCR assay for the simultaneous detection and quantification of botulinum neurotoxin-producing clostridia in food and clinical samples. Applied and Environmental Microbiology, 76(13), pp. 4387-4395. (doi: 10.1128/AEM.02490-09) (PMID:20435756) (PMCID:PMC2897425)

Abstract

Botulinum neurotoxins are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven distinct serotypes (A to G) known to cause botulism in animals and humans. In this study, a multiplexed quantitative real-time PCR assay for the simultaneous detection of the human pathogenic C. botulinum serotypes A, B, E, and F was developed. Based on the TaqMan chemistry, we used five individual primer-probe sets within one PCR, combining both minor groove binder- and locked nucleic acid-containing probes. Each hydrolysis probe was individually labeled with distinguishable fluorochromes, thus enabling discrimination between the serotypes A, B, E, and F. To avoid false-negative results, we designed an internal amplification control, which was simultaneously amplified with the four target genes, thus yielding a pentaplexed PCR approach with 95% detection probabilities between 7 and 287 genome equivalents per PCR. In addition, we developed six individual singleplex real-time PCR assays based on the TaqMan chemistry for the detection of the C. botulinum serotypes A, B, C, D, E, and F. Upon analysis of 42 C. botulinum and 57 non-C. botulinum strains, the singleplex and multiplex PCR assays showed an excellent specificity. Using spiked food samples we were able to detect between 103 and 105 CFU/ml, respectively. Furthermore, we were able to detect C. botulinum in samples from several cases of botulism in Germany. Overall, the pentaplexed assay showed high sensitivity and specificity and allowed for the simultaneous screening and differentiation of specimens for C. botulinum A, B, E, and F.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Kirchner, Dr Sebastian
• Authors: Kirchner, S., Krämer, K. M., Schulze, M., Pauly, D., Jacob, D., Gessler, F., Nitsche, A., Dorner, B. G., and Dorner, M. B.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Journal Name: Applied and Environmental Microbiology
• Publisher: American Society for Microbiology
• ISSN: 0099-2240
• ISSN (Online): 1098-5336
• Published Online: 23 June 2010