Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-03-11
1999-12-21
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 435 9152, 435 9151, 536 221, 536 2432, 536 2532, C12Q 168, C12P 1934, C07H 2104
Patent
active
06004747&
DESCRIPTION:
BRIEF SUMMARY
This invention relates to the detection and identification of Salmonella species.
The incidence of salmonellosis has increased significantly during the last two decades in several western countries. In general the human population is infected by Salmonella via contaminated foods and water, but transmission occurs, to a minor extent, by direct contact with infected animals. Standard culture methods are still widely used for detection of Salmonella in foods, but control of the infection depends increasingly on the availability of rapid and precise diagnostic tests for monitoring of the primary animal production, different food processing steps and of the final food products. For this purpose several rapid methods for Salmonella detection have been developed.
These methods include enzyme immuno assays using polyvalent somatic or flagellar antibodies (Krysinski, E. P. and Heimsch, R. C. (1977) Applied and Environmental Microbiology 33, 947-954; Minnich, S. A., Hartman, P. A. and Heimsch, R. C. (1982) Applied and Environmental Microbiology 43, 877-883; Rigby, C. E. (1984) Applied and Environmental Microbiology 47, 1327-1330); monoclonal antibodies (Mattingly, J. A. (1984) Journal of Immunological Methods 73, 147-156); DNA hybridization assays using DNA polynucleotide probes (Fitts, R., Diamond, M., Hamilton, C., and Neri, M. (1983) Applied and Environmental Microbiology 46, 1146-1151); Gopo, J. M., Melis, E., Filipska, E., Meneveri, R. and Filipski, J. (1988) Molecular and Cellular Probes 2, 271-279; Tsen, H. Y., Chen, M. H., Shieh, J. S., Wang, S. J. and Hu, N. T. (1989) Journal of Fermentation and Bioengenering 68, 1-6; Scholl, D. R., Kaufmann, C., Jollick J. D., York, C. K., Goodrom, G. R., and Charache, P. (1990) Journal of clinical microbiology 28, 237-241; Olsen J. E., Aabo, S., Nielsen, E. O., and Nielsen, B. B. (1991) APMIS 99, 114-120) and oligonucleotide probes from ribosomal RNA genes (Wilson, S. G., Chan, S., Deroo, M., Vera-Garcia, M., Jonson, A., Lane, D., and Halbert, D. N. (1990) Journal of Food Science 55, 1394-1398) or from single copy target sequences (Tsen, H. Y., Wang, S. J., Roe, B. A., Green, S. S. (1991) Applied Microbiology and Biotechnology 35, 339-347).
The polymerase chain reaction (PCR) has been used to detect gene alterations in connection with sickle cell anaemia and a number of reports have been published on PCR for detection of food borne pathogens e.g. Mycobacteria, Shigella, Verotoxin producing Escherichia coli, Yersinia and Listeria. A method for Salmonella specific detection, combining immunomagnetic separations (Lund, A., Hellemann, A. L. & Vartdal, F. (1988) Journal of Clinical Microbiology 26, 2572-2575) and PCR on pure cultures of bacteria has recently been published (Widjojoatmodjo, M. N., Fluit, A. C., Torensma, R., Keller, B. H. I., and Verhoef, J. (1991) European Journal of Clinical Microbiology and Infectious Diseases 10, 935-938).
The above 1991 publication of J. E. Olsen et al described a Salmonella specific DNA hybridisation probe comprising a 2.3 kb fragment of the Salmonella typhimurium LT2 chromosome. This fragment was produced by preparing a library of S.typhimurium LT2 DNA containing 6800 clones by shot-gun cloning of EcoRI/Hind III fragments. The sequence of a major fragment of the above 2.3 kb fragment is shown in FIG. 1 (SEQ I.D. NO. 1). This is the product of endonuclease restriction of the 2.3 kb fragment with Sau3A employing partial digestion. Certain regions of this provide primers and probes of use in identifying Salmonella species.
The present invention is based on using certain fragments of the above genomic DNA from Salmonella typhimurium LT2 (or corresponding nucleic acid fragments having the same sequence of bases, including RNA, PNA (peptide nucleic acid) etc.) as primers in PCR and other amplification systems, in particular certain fragments corresponding to regions of the genome which are highly conserved in Salmonella species. This enables target nucleic acid sequences from Salmonella to be selectively amplified and thus detected. Fragments
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S. Aabo et al., "Evaluation of a Salmonella-Specific DNA Probe by Colony Hybridization Using Non-Isotopic and Isotopic Labeling" APMIS 100:623-628 (1992).
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Ols
Aabo Soren
Olsen John Elmerdahl
Rasmussen Ole Feldballe
Rossen Lone
Aabo Soren
Bioteknologisk Institut
Horlick Kenneth R.
Olsen John Elmerdahl
Tung Joyce
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