Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1993-03-10
1997-04-08
Jones, W. Gary
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 911, 435 912, 4353201, 536 231, 536 237, 536 243, 536 2432, 536 2433, C07H 2102, C07H 2104, C12N 1570, C12Q 168
Patent
active
056186665
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The subject of the present invention is nucleic acid sequences derived from the genome of Salmonella Typhi, and their use, in particular, for the in vitro diagnosis of the presence of bacteria of the Salmonella genus in a biological sample likely to contain them, and more particularly in foodstuffs.
2. Discussion of the Background
At present, foodstuffs are of growing importance. For social reasons, transformed foodstuffs, ready for consumption, continue to create an increasing demand on the part of the buyers. The result of this is that the sources of production and display of these products have been modified considerably during the last fifteen years (mass manufacture in specialised factories; marketing in packaged units, usually in a plastic wrapping).
For reasons of public health, the manufacturing technologies and the products themselves are subjected to stricter and stricter measures of health supervision. In the framework of bacteriological controls, the bacteria responsible for toxic infections of foodstuffs are investigated systematically, and among them priority is given to the Salmonella. Moreover, the health standards are very clear for this bacterial genus: absence of Salmonella in 25 grams of product.
From the point of view of taxonomy, the Salmonella genus belongs to the family of the Enterobacteriaceae. This genus includes a single and unique species, S. enterica, which may be subdivided into even sub-species. Within each of these sub-species, it is possible to identify a large number of serotypes with the aid of sera directed against the polysaccharide 0 antigens and the flagellar H antigens. In 1989, 2267 serotypes of Salmonella were known.
The Salmonella are entero-invasive bacteria, which are pathogenic for man and animals. Their pathogenic potency is linked to their capacity to invade the intestinal epithelium. This stage can be limited to the mucosal membranes in the case of a toxic infection for example, or be followed by systemic dissemination (the case of typhoid fever). The contamination of the host by Salmonella occurs by the mouth in the very great majority of cases. This explains why the Salmonella are systematically investigated in the context of bacteriological controls of biological samples.
The standard method of systematic investigation of Salmonella, recommended by the Association Francaise de Normalisation (AFNOR) comprises: the placing in culture of the product to be analysed in two different enrichment media, incubated at two different temperatures and isolated on two different selective media (this step is often preceded by a "revivification" step in a nutrient broth); subculturing of at least 6 colonies on media for rapid identification; finally, serological typing of the strain. Even though the AFNOR protocol must be scrupulously followed during an official expert investigation, it can be imagined that it is difficult to apply during systematic controls on account of the time needed to carry out the investigation (at least one week) and its cost.
Novel methods of investigation of Salmonella are based either on enzymatic assays or on the use of nucleic acid probes. It must be emphasized that in the two cases a culture step on a rich medium and a subculture in an enrichment medium are recommended, and even essential.
The immuno-enzymatic assays which use monoclonal antibodies are easy to implement, give results in four to six hours and are available at a reasonable price. Their great disadvantage lies in the fact that they often give false positive reactions and sometimes false negative reactions. The consequences of such false reactions are considerable in both cases: if a false positive reaction is produced, there will be seizure of the product and the initiation by the analytical laboratory of a process to isolate a Salmonella which does not exist; if a false negative reaction is produced, the product will be marketed with the risks which that entails for public health.
A recent study involving 250 strains of
REFERENCES:
patent: 4689295 (1987-08-01), Taber et al.
Microbial Pathogenesis, Academic Press, Harcourt Jovanovich Publishers, vol. 6, Feb. 1989, Mroczenski-Wildey, M.J. et al.: "Invasion and Lysis of HeLa Cell Monolayers by Salmonella typhi: The Role of Lipopolysaccharide", pp. 143-152.
Proceedings of the National Academy of Sciences of USA, vol. 86, Aug. 1989, Washington, US Galan, J.E. et al: "Cloning and Molecular Characterization of Genes Whose Products Allow Salmonella typhimurium To Penetrate Tissue Culture", pp. 6383-6387.
Microbiology and Immunity, vol. 31, No. 1, 1987; Yokohama, H. et al: "An Ultrastructural Study of HeLa Cell Invasion with Salmonella typhi GIFU 10007", pp. 1-11.
Dion Michel
Popoff Michel Y.
Institut National de la Sante et de la Recherche Medicale
Institut Pasteur
Jones W. Gary
Tran Paul B.
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