Quantification of Clostridium botulinum Toxin Gene Expression by Competitive Reverse Transcription-PCR

Abstract

Clostridium botulinum produces a characteristic botulinum neurotoxin which can cause an often fatal neuroparalytic condition known as botulism. Although food-borne botulism is rare, critical screening by food companies is necessary to ensure that food products are safe. At present, the food industry assesses the risks of botulinum neurotoxin production by challenge testing to check any new food products and to check the efficacy of new storage regimes. Challenge testing involves artificial introduction of defined strains of microorganisms into food, and microbial growth and possible toxin production are then monitored. Botulinum toxin is normally analyzed by using the mouse bioassay. However, the mouse bioassay is expensive, slow, and politically sensitive because of animal rights issues. In this paper we describe adaptation of a new assay, competitive reverse transcription-PCR (RT-PCR), to monitor botulinum neurotoxin production. This method accurately measures the level of toxin-encoding mRNA in C. botulinum cells. Measurement of mRNA should provide a good indication of gene expression as mRNA is turned over rapidly in bacterial cells. In addition, the method is rapid, specific, and sensitive. The competitive RT-PCR method was developed to examine C. botulinum E VH toxin gene expression and was used to investigate the level of toxin production by C. botulinum E VH when the organism was grown in two different types of broth. The results which we obtained with the competitive RT-PCR method demonstrated that this method is more rapid and more sensitive than the mouse bioassay.

Relative Neurotoxin Gene Expression inClostridium botulinumType B, Determined Using Quantitative Reverse Transcription-PCR

“…1 and 2). In four of the strains the maximum level of cntB mRNA was observed in the early stationary phase, whereas in the fifth strain, C. botulinum (21) and Clostridium difficile (9,16). The highest levels of neurotoxin expression in all growth phases were exhibited by the proteolytic strain C. botulinum ATCC 7949.…”

“…In recent years, in vitro methods have been developed for monitoring cnt expression in C. botulinum; these methods include a gene reporter system (7) and competitive reverse transcription (RT)-PCR (21). However, conventional PCR is not suitable for quantification as the final concentration of PCR products is not linearly related to the initial target nucleic acid concentration (23).…”

Expression of botulinum neurotoxins A and E, and associated non-toxin genes, during the transition phase and stability at high temperature: analysis by quantitative reverse transcription-PCR

“…In this study, we analysed by real-time RT-PCR the relative expression of the botulinum toxin locus genes versus the 16 rRNA gene as calibrator gene (Broussolle et al, 2002), in C. botulinum A and C. botulinum E strains, which in contrast to C. botulinum A contain no gene related to botR. Real-time RT-PCR is a very sensitive method to quantify gene expression and has been used to investigate various bacterial virulence genes and in particular to detect C. botulinum in food and clinical samples (Kimura et al, 2001) and to monitor expression of neurotoxin genes in C. botulinum B and C. botulinum E (Lövenklev et al, 2004a;McGrath et al, 2000).…”

Laboratory Diagnostics of Botulism

“…Another approach to ensure that only live cells are detected is reverse-transcription PCR (RT-PCR), where gene expression rather than the gene itself is detected. Reverse transcription-PCR protocols for the botB and botE genes have been described (136,137,146). In a case of a human botulism outbreak, however, laborious procedures to obtain high-quality RNA may prove to be too time-consuming.…”