Quantification of Clostridium botulinum toxin gene expression by competitive reverse transcription-PCR.

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.

Cloning of a DNA sequence unique to Clostridium botulinum group I by selective hybridization.

Nucleic acid sequences were isolated from a strain of Clostridium botulinum type A by a selective hybridization method known as deletion enrichment. Nontoxigenic C. sporogenes was used to produce a C. botulinum type A sequence-enriched library. A probe, pCBM44, which showed specific hybridization to a 4.0-kb HindIII fragment present in all of the C. botulinum type A strains tested was isolated, and there was no hybridization to any strains of C. sporogenes. Upon further investigation, pCBM44 was found to hybridize to all of the group I proteolytic C. botulinum strains tested (toxin types A, B, and F) but not to hybridize to groups II, III, and IV (toxin types B, C, D, or E). The probe did not cross-react with nine other Clostridium spp. Such a probe, which differentiates between nontoxigenic C. sporogenes and neurotoxigenic C. botulinum group I strains, should prove extremely useful.

Characterisation of the LdsRNA encoded mRNA of yeast.

Radioactive messenger RNA (mRNA) was isolated from a Saccharomyces cerevisiae strain containing the L double stranded RNA (dsRNA) species. This mRNA was hybridised to LdsRNA isolated from the same strain. Analysis of the hybrid formed shows it to be of a similar size to the LdsRNA. It is concluded from this result that the mRNA complementary to the -ve strand of the LdsRNA is polycistronic.When the total mRNA is fractionated on oligo (dT) cellulose, RNA complementary to the -ve strand of LdsRNA is found only in the non-binding fraction. When LdsRNA is fractionated on oligo (dT) cellulose the majority is found in the non-binding fraction. It is concluded that neither the LdsRNA genome nor the mRNA it encodes have poly(A) tails of a significant length.