Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-03-09
2000-09-12
Arthur, Lisa B.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 536 2344, C12Q 168, C12P 1934, C07H 2104
Patent
active
061176362
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a method for the rapid detection of heat-resistant eukaryotic microorganisms capable of contaminating certain products in the food sector, particularly fruit-based products.
BACKGROUND OF THE INVENTION
More particularly, the microorganisms studied are two filamentous fungi, Byssochlamys nivea and Neosartorya fischeri and a yeast, Zygosaccharomyces bailii.
These contaminants of food products can develop on the fruit-based products, giving them, particularly for Byssochlamys nivea, unpleasant notes of the plastic or antiseptic type; the growth of Neosartorya fischeri is responsible for spoilage and the appearance of mycotoxins, whereas as that of Zygosaccharomyces bailii causes a formation of gas.
These microorganisms particularly in the form of spores, exhibit a very high heat resistance which allows them to persist, even after conventional pasteurization treatments, which are applied in the food industry.
The existing tests allowing the detection of these microorganisms consist in altering the samples of a selective medium by taking advantage of, as a selection factor, the heat resistance of the filamentous fungi or the resistance of the yeast to certain preservatives conventionally used in the sector, such as benzoic acid. However, this type of detection involves cultures and, what is more, fairly long cultures, namely from several days to several weeks, which makes them practically unusable in the food sector since they could involve preserving the products, which may be contaminated, for a very long time.
The search is therefore underway for a detection test allowing the very rapid detection of these microorganisms, it being possible for the said test to be carried out in a few hours at the most, in order to find out rapidly if the sampled product can continue or otherwise to be treated in the subsequent stages.
DETAILED DESCRIPTION OF THE INVENTION
Accordingly, the present invention proposes a process allowing the detection of Byssochlamys nivea, Neosartorya fischeri and Zygosaccharomyces bailii by amplification of the genomic DNA of the targeted microorganisms using, as primers, a pair of sequences contained in the internal transcribed spacers (ITS) of the ribosomal unit:
In the ITS1 and ITS2 sequences, there will be chosen more particularly,
Among the amplification methods which can be used, the so-called PCR (Polymerase Chain Reaction) method will be used more particularly.
The tests, reported in particular in the examples, show that these primers are perfectly discriminating and make it possible to distinguish between the desired microorganisms and the microorganisms, which are even very close, and related.
The present invention also relates to a process for detecting these microorganisms in food products, particularly with fruit-based food products and more particularly strawberry-based food products, in which the sample containing the fruits is pretreated so as to liberate and concentrate the spores or the cells, to reduce or suppress the action of the inhibitors of Taq Polymerase and to extract the DNA from the spores or cells.
More particularly, the samples containing solid products will be treated with a mixture of cellulase/hemicellulase so as to liquefy them and then filtered and centrifuged under conditions which make it possible to obtain a product containing the cells from which the DNA can be extracted.
Since the samples contain most often inhibitors of Taq Polymerase, it is necessary to provide for a stage which makes it possible to reduce the activity of the inhibitors, either by dilution, or by treatment of the sample with phenol-chloroform and precipitation with alcohol.
Although the present invention is more particularly intended for the food industry, it is evident that it can also be used to detect the microorganisms involved in other samples which might not have any relationship with the food industry.
Finally, the present invention relates to primers, as defined above, as well as detection kits using the said primers
REFERENCES:
patent: 4683195 (1987-07-01), Mullis et al.
patent: 5324632 (1994-06-01), Weisburg et al.
Nazar R.N. et al (Physiological and Molecular Plant Pathology (1991)39, 1-11 Potential use of PCR--Amplified Ribosomal Intergenic Sequences in the Detection and Differentiation of Verticillium with Pathogens, 1991.
Berbee, Mary L. et al. (Mycologia (1995) 87(2), 210-211 is Penicillum monophyletic? An Evaluation of Phylogeny in the Family Trichocomaceae from 185, 5.85 and Its Ribosomal DNA Sequence Data, 1995.
Beuhat, L.R. (Journal of Food Science (1986) 51(6), 1506-1510 Extraordinary Heat Resistance of Talaromyces Flavus and Neosartorya fischeri Ascospores in Fruit Products, 1986.
Stubbs, S. et al. (Letters in Applied Microbiology (1994)) 19, 268-272 Differentiation of the Spoilage Yeast Zygosaccharomyces bailii From Other Zygosaccharomyces species using 18S rDNA as Target for a Non-Radioactive ligase detection reaction, 1994.
James, S.A. et al. (Yeast 10:871-881) Genetic Interrelationship Among Species of the Genus Zygosaccharomyces as Revealed by Small--Sub Unit rRNA Gene Sequences.
Aufrere Robert
Chrzavzez nee Taddei Emmanuelle
Arthur Lisa B.
Ultra Propre Nutrition Industrie Recherche
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