Dataset Information

Rapid, quantitative PCR monitoring of growth of Clostridium botulinum type E in modified-atmosphere-packaged fish.

ABSTRACT: A rapid, quantitative PCR assay (TaqMan assay) which quantifies Clostridium botulinum type E by amplifying a 280-bp sequence from the botulinum neurotoxin type E (BoNT/E) gene is described. With this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during PCR by using the ABI Prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the C. botulinum type E toxin gene. The sensitivity and specificity of the assay were verified by using 6 strains of C. botulinum type E and 18 genera of 42 non-C. botulinum type E strains, including strains of C. botulinum types A, B, C, D, F, and G. In both pure cultures and modified-atmosphere-packaged fish samples (jack mackerel), the increase in amounts of C. botulinum DNA could be monitored (the quantifiable range was 10(2) to 10(8) CFU/ml or g) much earlier than toxin could be detected by mouse assay. The method was applied to a variety of seafood samples with a DNA extraction protocol using guanidine isothiocyanate. Overall, an efficient recovery of C. botulinum cells was obtained from all of the samples tested. These results suggested that quantification of BoNT/E DNA by the rapid, quantitative PCR method was a good method for the sensitive assessment of botulinal risk in the seafood samples tested.

SUBMITTER: Kimura B

PROVIDER: S-EPMC92548 | biostudies-literature | 2001 Jan

REPOSITORIES: biostudies-literature

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Rapid, quantitative PCR monitoring of growth of Clostridium botulinum type E in modified-atmosphere-packaged fish.

Kimura B B Kawasaki S S Nakano H H Fujii T T

Applied and environmental microbiology 20010101 1

A rapid, quantitative PCR assay (TaqMan assay) which quantifies Clostridium botulinum type E by amplifying a 280-bp sequence from the botulinum neurotoxin type E (BoNT/E) gene is described. With this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during PCR by using the ABI Prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the C. botulinum type E ...[more]

PMID: 11133447

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Project description:Oxygen and carbon dioxide are common protective gases used in modified atmosphere packaging (MAP) of meat. Within the package, they selectively suppress members of the spoilage microbiome, reshaping it to adapted species concomitantly growing upon MAP. Thus, this species must exhibit adaptation mechanisms to withstand the inhibitory effect of carbon dioxide and oxygen, and cope with selective nutrition on MAP meat. In order to uncover these mechanisms, the typical representative meat-spoiling bacteria Brochothrix (B.) thermosphacta TMW2.2101 and four lactic acid bacteria (LAB) Carnobacterium (C.) divergens TMW2.1577, C. maltaromaticum TMW2.1581, Leuconostoc (L.) gelidum subsp. gelidum TMW2.1618 and L. gelidum subsp. gasicomitatum TMW2.1619 were grown in a meat simulation medium under a controlled, sterile environment, aerated constantly with either air, 100%_N2, 30%_CO2/70%_O2 or 30%_CO2/70%_N2. Growth dynamics were monitored and a label-free quantitative mass spectrometric approach was employed to determine changes within the bacterial proteomes in response to the different gas atmospheres. Revealed bacterial tolerance to modified atmospheres (MA) comprise two possible scenarios: Either bacteria were intrinsically adapted to MA, exhibiting no proteomic regulation of enzymes (L. gelidum subsp. gelidum and gasicomitatum) or, tolerance was provided by varying specific metabolic adaptation (B. thermosphacta, C. divergens, C. maltaromaticum). In detail, metabolic adaptation mechanisms to oxygen comprised an enhanced oxidative stress reduction response, adjustment of the pyruvate metabolism and catabolic oxygen consuming reactions. Adaptation to carbon dioxide was characterized by an upregulation of proteins involved in intracellular pH homeostasis, maintenance of osmotic balance and alteration of the fatty acid composition of the cell membrane. We furthermore predict species-specific strategies for different and preferential carbon source utilization enabling a non-competitive coexistence on meat and resulting in a synergistic spoilage. We conclude that a gas atmosphere containing 30%_CO2/70%_O2 has no inhibitory effect on the analyzed prominent meat-spoiling bacteria whereas 30%_CO2/70%_N2 predictively inhibits C. divergens TMW21577 and B. thermosphacta TMW2.2101 but not the other three species. This gives a mechanistically explanation of their acknowledged status as typical spoilage organisms on packaged meats.

Dataset Information

Rapid, quantitative PCR monitoring of growth of Clostridium botulinum type E in modified-atmosphere-packaged fish.

Publications

Rapid, quantitative PCR monitoring of growth of Clostridium botulinum type E in modified-atmosphere-packaged fish.

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