Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-03-02
2004-06-29
Fredman, Jeffrey (Department: 1634)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024300, C435S006120, C435S091200
Reexamination Certificate
active
06756495
ABSTRACT:
The present invention relates to a process for detection of the presence of biological matters of bovine origin in a sample of organic matter.
It also relates to oligonucleotides for carrying out this process.
In 1986, bovine spongiform encephalopathy (BSE), or “mad cow” disease, showed up for the first time in British bovine livestock. Since then, more than 100,000 clinical cases have been identified in cattle. This disease, which is present in Great Britain, has as endemic evolution. It has also been observed in various European countries: Ireland, Switzerland, France, Denmark, Germany—where it evolves sporadically.
The clinical picture of the disease is known. The infectious agent responsible, called “prion” is characterized by, among other properties, an extreme resistance to conventional decontaminating agents, such as heat, radiation or detergents. Recent studies furthermore have demonstrated the very high resistance of the infectious agent under “natural” conditions, and in particular its persistence in pastures. The infectiousness can therefore persist in the ground for at least 3 years.
One of the modes of transmission of the infectious agent is ingestion of contaminated foods, and this transmission can furthermore be from one animal species to another.
Analysis of the epidemiological data has enabled the origin of the English epidemic to be discovered, this being due to the infectious agent contaminating the meat meals and bone meals (MBM) used to manufacture food additives intended for feeding dairy cows. These meals are the by-products of quartering shops arising from treatment of carcasses and waste originating from abattoirs.
The structures of “prion” is not known to this day, and there is no test for detecting it.
It therefore important to determine whether organic matter contains biological matters of bovine origin and is capable of containing “prions”.
In the agricultural food field, the characterization of animal species initially used biochemical techniques for analysis of proteins (BARA et al. 1992, Trends in Food Science and Technology, 3, 69-72; SOTELO et al. 1993, Trends in Food Science and Technology, 4, 395-401; Hernandez et al., Food and Agricultural Immunology, 6, 95-104). However, these methods are not very specific (electrophoresis), or are incompatible with denaturation of the samples to be analysed (immunoanalysis). They are now progressively being replaced by the techniques of DNA analysis, this molecule being less sensitive than proteins to denaturing physico-chemical conditions.
Identification of the main animal species of interest was first carried out by the technique of hybridization of nucleic probes (BUNTJER et al. 1995, Zeitschrift fuer Lebensmittel Untersuchung und Forschung 201 (6): 577-582; MEYER et al., 1994, Fleischwirtschaft 74 (11) 1237-1238; TSUMURA et al. 1992, Journal of Japanese Society of Food Science and Technology 39 (1) 60-63; EBBEHOJ and THOMSEN, 1991, Meat Science 30 (4): 359-366; BAUER et al., 1987, Archiv fuer Lebensmittelhygiene 38 (6): 172-174; EBBEHOJ and THOMSEN, 1991, Meat Science 30 (3): 221-234).
This technology, which is tricky to use, has now been superseded by the PCR (polymerase chain reaction) method, which has been used to characterize biological matters originating from various animal species.
The only methods for amplification by PCR described in beef (
Bos taurus
) use amplification of the mitochondrial DNA (mtDNA) region which codes for a cytochrome by means of PCR primers which recognize sequences conserved in species of vertebrates, and then their characterization by means of restriction enzymes (RFLP) or by sequencing (MEYER et al. 1995, Journal of AOAC—International 78 (6): 1542-1551; CHIKUNI et al. 1994, Animal Science and Technology, 65 (6): 571-579; GUGLICH at al. 1994, J. Forensic. Sci. 39 (2): 353-61).
The organization and complete sequence of bovine mitochondrial DNA (mtDNA) are known (ANDERSON et al. 1982, J. Mol. Biol. 156 (4): 683-717). On the basis of these data, several works have been dedicated to the study of the genetic variability of the mitochondrial genome of domestic Bovidae by analysis of the restriction polymorphism (CHEN et al. 1995, Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 111 (4): 643-649; KIKKAWA et al. 1995, Biochem. Genet. 33; (1-2): 51-60; BRADLEY et al. 1994, Anim. Genet (4): 265-271; AMANO et al. 1994, Anim. Genet. 25 (1): 29-36; SUZUKI et al. 1993, Anim. Genet 24 (5): 339-343: Lan et al. 1993, I. Chuan. Hsueh Pao 20 (5): 419-425; BHAT et al. 1990, Biochem. Genet 28 (7-8): 311-318; LOFTUS et al., 1994, Anim. Genet. 25:265-271) or by sequencing (LOFTUS et al. 1994, Proc. Natl. Sci. USA 91 (7): 2757-2761; RON et al. 1993, Anim. Genet. 24 (3): 183-186; BRADLEY et al., 1996, Proc. Natl. Acad. Sci. USA 93: 5131-5135; BAILEY et al., 1996, Proc. R Soc. Lond. B, 263:1467-1473).
Partial sequencing of the control region of bovine mtDNA has furthermore been carried out for various European, African and Indian bovine breeds (LOFTUS et al., 1994 Proc. Natl. Sci. USA 91 (7): 2757-2761; BRADLEY et al., 1996, Proc. Natl. Acad. Sci. USA 93: 5131-5135; BAILEY et al., 1996, Proc. R. Soc. Lond. B, 263:1467-1473).
It thus emerges from studying the prior art that works on analyses of the DNA of certain species and bovine breeds have already been carried out. Nevertheless, none of the documents of the prior art describes a specific and sensitive method for amplification of bovine DNA which enables traces of biological matters of bovine origin to be identified and can be used for all bovine breeds and in organic matter having widely varying compositions.
In fact, the known techniques for identification of bovine DNA (for example analysis of genomes by RFLP or PCR-RFLP carried out on portions of sequences which vary little) have disadvantages. By these methods, which are not very specific, it is often difficult to analyse mixtures of DNA originating from different species because of the large number of bands and the difficulties in interpretation associated with this characteristic.
The low sensitivity of some of these techniques does not enable the presence of organic matter of bovine origin in widely diverse organic substrates to be demonstrated in a reliable manner. These methods cannot be used if the DNA present in the sample is degraded in the form of small fragments having a size of less than about 500 base pairs.
The problem of identification of organic matter originating from any of the bovine breed is particularly crucial, since bovine spongiform encephalitis is not limited to European bovine breeds, but extends to African and Indian breeds (
Bos taurus
and
Bos indicus
).
The Applicant has thus concerned himself with solving these problems.
It has been found that the presence of biological matter of bovine origin could be detected in a specific and simple manner and with a high sensitivity, whatever the bovine breed (
Bos taurus
and
Bos indicus
), in samples of organic matter by amplifying a defined sequence of the bovine genome in a specific manner.
In its most general form, the present invention thus relates to a process for obtaining a fragment of bovine DNA which has a defined size and sequence and is specific to bovines, and in particular the species
Bos taurus
and
Bos indicus
, from a sample of organic matter, and a process by which a defined sequence of the bovine genome present in bovine genomes but absent from the genomes of other animal species is amplified by a polymerase chain reaction.
The present invention also relates to a process for detection and identification of the presence of biological matter of bovine origin in a sample of organic matter, characterized in that the presence of DNA of bovine origin is determined in the said organic matter by amplification of a specific DNA sequence of the bovine genome.
Organic matter is understood as meaning any solid or liquid matter which is assumed to have at least partly a biological origin.
The DNA sequence is advantageously of mitochondrial origin. The choice of a mitochondrial sequence is particularly advantag
Grangette Corine
Hanni Catherine
Lange Marc
Laudet Vincent
Pasteau Stephane
Centre National de la Recherche Scientifique
Fredman Jeffrey
Switzer Juliet C.
Young & Thompson
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