EP1430148A2 - Method for determining the existence of animal or vegetable mixtures in organic substrates - Google Patents

Abstract

The invention concerns the use of a cloning method for determining the existence and the identification of a mixture of organic origin containing DNA of different animal species and/or different plant species and/or human individuals.

Description

 METHOD FOR DETERMINING THE EXISTENCE OF MIXTURES OF ANIMAL OR PLANT ORIGIN IN ORGANIC SUBSTRATES

The invention relates to a method for determining the existence of mixtures of animal or vegetable origins in organic substrates.

More particularly, the invention relates to a method for determining the existence of mixtures of species, populations or races of animal or plant origin or of human individuals in organic substrates.

Many fields currently use the amplification process (Polymerase Chain Reaction: PCR) in molecular biology to detect and identify species. Indeed, this technique is commonly used by molecular biology laboratories, veterinary control laboratories, microbiological food, environmental, pharmaceutical analysis laboratories, laboratories carrying out genetic fingerprints ... However, when several species, or populations , or races or individuals are present in the same sample, there is no known technique on the market which allows on the one hand, to determine if one is or not in the presence of a mixture and on the other hand, to detect the species, populations, races or individuals present in this mixture unless there are primers specific to all the species populations, races or individuals likely to be present in the mixture. Indeed, it very often happens that the samples analyzed are not composed from a single species, populations or races. For example, food preparations can be prepared from several species, populations or races (ready meals, soup, pâtés, terrine, ...). This mixture may be specified by the manufacturer, but may also be adulteration. Indeed, it may happen that this mixture is in favor of a species of lower economic value compared to another species which should have been found in a unique way in the preparation (example: brandade of cod prepared with a little common cod (Gadus morhua) and lots of cheaper Pacific cod (Gadus macrocephalus).

Given the lack of an adequate system for the detection and identification of mixtures of species, populations or animal and plant races in organic substrates, the Applicant endeavored to develop a sensitive and reliable method allowing to compensate for this absence.

The invention aims to provide a method for determining the existence of DNA mixtures of different species, populations or races of animals and / or plants and / or different human individuals.

The invention also aims to provide a method for detecting and / or identifying the DNA of different species populations or animal and / or plant races and / or different human individuals.

The possibility of determining the existence of mixtures of DNA from different human individuals is particularly interesting for forensic applications. Indeed, it is currently very difficult to reliably interpret a genetic fingerprint when several • individuals are present. However, the currently available DNA fingerprinting methods, based on the amplification of short repeating and variable sequences (CA repeats or microsatellites) of the nuclear genome, do not make it possible to clearly determine whether a mixture is present. The invention makes it possible, by PCR amplification of variable fragments of the human mitochondrial genome, to unambiguously determine whether the DNA of several individuals is present in a sample. Remarkably, the sensitivity of the methods developed in the invention makes it possible to detect mixtures of human DNA even when the proportions of each individual are very unbalanced (90% of one 10% of the other for example). In such a case, DNA fingerprinting methods often do not provide any indication that a mixture is present even though their results in terms of identification of an individual may be false, leading to misinterpretation. Sensitive and reliable detection of the existence of a mixture therefore provides crucial information for the interpretation of DNA data. In its most general form, the invention relates to the use of a cloning process for determining the existence of a mixture of organic origin containing mitochondrial or chloroplastic DNA of different species, populations or animal races and / or different species, populations or plant races and / or different human individuals. The method according to the invention for detecting a mixture of several species is simple and exhaustive.

The determination of the existence of the mixture of species is carried out by a cloning process. This cloning process is commonly used in molecular biology to obtain a series of identical molecules (nucleic acids) or isolated cells, unicellular beings (ex: bacteria) and multicellular beings (ex: mammal). Cloning has also made it possible to understand the role and functioning of many genes by manipulating them either to produce them in large numbers or by transferring them from one organism to another. The expression “DNA of different species, populations or animal races and / or different species, populations or plant races” designates the specific DNA of each of the species, populations or animal races, and / or plants present in the mixture.

This DNA represents any nucleotide sequence originating from the nuclear, mitochondrial or chloroplastic genome making it possible to distinguish reliably between the different species or populations or races studied or the different individuals studied. A species is defined as a group of living organisms genetically separated from other living organisms and capable of reproducing only between them.

A population is a group of individuals of a species which can be recognized from other individuals of the species by precise morphological and / or genetic characteristics. A species therefore often has several distinct populations. A breed is a group of individuals of a domestic species which have been obtained after a long selection to improve a specific trait (such as milk production for cows, or the quality of meat for pork) and which can be recognized by other individuals of this domestic species by precise morphological or genetic characteristics. The expression “DNA from different human individuals” designates any mitochondrial or nuclear DNA sequence of human origin making it possible to distinguish between two individuals. It is estimated that, to allow a reliable distinction, a sequence must contain at least 1 difference per 100 nucleotides.

By "mixture of organic origin" is meant a mixture containing the DNA of different species, populations or races from tissues from living beings, animals or plants. By organic matter is meant any solid or liquid matter which is assumed to have at least partially an organic origin.

According to an advantageous embodiment, the invention relates to the use as mentioned above, characterized in that the mitochondrial or chloroplastic DNA is degraded.

The degraded DNA is characterized by its appearance after extraction according to the protocol described above and deposit on a 1% agarose gel stained with ethidium bromide. We see that DNA from fresh tissue is present in the form of large molecules (ie around 20 kbp; see Figure 5 channel 1) while degraded DNA extracted from tissues that have undergone strong alterations such as cooking is either weakly visible in the form of a spread of small molecules (100 to 500 bp; see Figure 5 channel 3) or frankly invisible (see Figure 5, channels 4 and 5). It is only then the positive amplification by PCR which makes it possible to reveal the presence of this DNA. According to an advantageous embodiment, the invention relates to the use of a cloning process, for the detection and / or identification of the DNA of each of the different species, populations or animal races and / or each of the different species, populations or plant races and / or of each of the different human individuals present in a mixture of organic origin containing DNA of animal species and / or plant species and / or human individuals.

By “detection of the DNA of each of the species, populations or races or of each of the 'human' individuals”, is meant the fact of identifying the presence of specific DNA of each of the species or populations or races or individuals so as to be able to to appreciate the complexity of the initial mixture and to be able to identify, in a second time the various compounds of this mixture.

By "identification of the DNA of each of the different species populations, races or each of the individuals", is meant the fact, when the presence of species, has been detected by the presence of their specific DNA, to analyze the DNA of each of the species or populations or races or individuals so as to deduce therefrom the composition of the initial mixture. According to an advantageous embodiment of the invention, the use of the cloning process is such that each species, population or animal race and / or each species, population or plant race and / or each human individual is represented by at at least one DNA sequence or at least one DNA fragment, and in particular by a single DNA sequence or a single DNA fragment, each of said DNA sequences or each of said DNA fragments originating from DNA extract of the mixture of organic origin.

In the foregoing and in the following, the expression "at least one DNA sequence or fragment" means either that there are several copies of the same DNA fragment or sequence, or that it there are multiple copies of different DNA fragments or sequences. According to an advantageous embodiment of the invention, the use of the cloning process takes place for the detection and / or identification of the DNA of each of the different animal species present in a mixture of animal species, and in particular of the DNA of each of the subspecies, lines, races, varieties, strains and / or populations present in said mixture, each of said animal species, and in particular each of said subspecies species, races, varieties and / or strains being represented by at least one DNA sequence or at least one DNA fragment, and in particular by a single DNA sequence or a single DNA fragment.

By "species, subspecies, lines, races, varieties, strains and animal populations", we mean groups or groups of animals from the same species that are similar to each other on certain criteria. Subspecies, populations can be considered synonymous. A subspecies is a particularly identifiable population to which a specific Latin designation has been assigned. For example Otus megalotis everetti and Otus megalotis nigrorum are two distinct subspecies of the species Otus megalotis, a raptor bird. The terms breeds, varieties and strains are mainly used to designate domestic animals and can be considered synonymous.

According to another advantageous embodiment of the invention, the use of the cloning process takes place for the detection and / or identification of the DNA of each of the different plant species present in a mixture of plant species, and in particular of the DNA of each of the subspecies, lines, races, varieties, strains, grape varieties and / or populations present in said mixture, each of said plant species, and in particular each of said subspecies, races, varieties and / or strains being represented by at least one DNA sequence or at least one DNA fragment, and in particular by a single DNA sequence or a single DNA fragment. By "species, subspecies, lines, races, variety, strains, grape varieties, plant populations", we mean groups or sets of plants from the same species that are similar to each other on certain criteria. A grape variety is the term specifically used to designate a strain or a breed or variety of vine of the genus Vitis.

According to another advantageous embodiment of the invention, the use of the cloning process takes place for the detection and / or identification of the DNA of each of the different human individuals present in a mixture of DNA of different individuals humans, each of said human individuals being represented by at least one DNA sequence or at least one DNA fragment, and in particular by a unique DNA sequence or a unique DNA fragment. By "human individuals" is meant any one of the members of the species Homo sapiens.

In an advantageous embodiment of the invention, the DNA extracted from the mixture of organic origin is old DNA (or fossil), degraded DNA or modern DNA. By "old DNA" is meant DNA extracted from an organism after its death and having already undergone a process of degradation. Old DNA very often shows signs of degradation such as for example the presence of DNA molecules of small size, less than or equal to about 200 base pairs. By "degraded DNA", is meant DNA having undergone deteriorations which are related to transformation processes. Degraded DNA is usually found in small fragments (less than or equal to about 200 bp) and in small quantities.

By "modern DNA" is meant DNA extracted from a living individual or extracted quickly after death. Modern DNA is characterized by the presence of very large fragments of more than 20,000 base pairs.

In another advantageous embodiment of the invention, the DNA extracted from the mixture of organic origin is:

- undegraded DNA originating in particular from a fresh organic sample or,

- degraded DNA, in particular from a transformed organic sample, in particular cooked, lyophilized, dried, brined, canned or pasteurized.

By "non-degraded DNA" is meant DNA which has not undergone any deterioration and which is therefore intact. If the extraction conditions have been appropriate, such DNA is present in the form of molecules of at least 20,000 base pairs.

Examples in which DNA is found in degraded form are: - food (cooked, freeze-dried, dried, brined, canned, pasteurized, ...)

- fertilizers, flours, seeds (crushed, dried, fermented, ...)

- egg shells, bones, teeth, hair, hair, feathers, excrement (drying, action of time, temperature ...)

- alcohols (alcoholic, distilled, fermented, ...) - leathers, skins, furs, mummified fabrics (tanned, taxidermed, colored, ...)

- scrolls, papers, wood (time action, transformation process used in stationery, ...)

- ivory, amber (time action, ...)

- glues, natural pigments (paints), earths, sediments (transformation process used in these fields).

- Human or animal corpses and bones (action of time or the environment) According to another advantageous embodiment of the invention, the use of the cloning process takes place for the detection and possibly the identification of DNA of each of the different species, populations or animal races and / or of each of the different species, populations or races of plants and / or of each of the different human individuals present in the mixture of organic origin, in which at least one of said species, populations or races and / or at least one of said individuals is represented by a sequence of old DNA or degraded DNA or a fragment of old DNA or degraded DNA. According to another advantageous embodiment of the invention, the cloning process is preceded by a step of amplification of each of the DNA sequences or of each of the DNA fragments characteristic of each of the animal species, populations or races and / or each of the plant species, populations or races and / or each of the human individuals which it is desired to detect and / or identify, the DNA sequences or the amplified DNA fragments obtained ( e) s at the end of the amplification process being contained in a single amplification product.

Analysis of the genome using nucleic acid probes represents an effective approach to the identification of species, still underdeveloped at present. Work carried out on ancient DNA (or fossil DNA) since the early 1990s has shown that DNA is a very stable molecule after death, despite the action of time and the environment (Brown and Brown. , 1994, Bioessays 16: 719-26). However, when it remains in old substrates, this DNA is very degraded and present in small quantities, in the form of damaged and chemically modified molecules (Pàabo, 1989, Proc. Natl. Sci USA 86: 1939-1943). These characteristics are mainly due to the phenomena of hydrolysis and oxidation (Lindahl, 1993, Nature 362: 709-715). Thanks to the PCR technique (for Polymerase Chain Reaction) which constitutes a tool of remarkable analytical power, it is possible to multiply "in vitro" almost exponentially a given fragment of DNA. By amplifying DNA from a food or other preparation, which has undergone modifications such as cooking, smoking, etc., it will be possible to identify constituents of animal or vegetable origin. PCR has recently been used for the characterization of cooked pork (Meyer et al., 1994, Journal of the AOAC International 77 (3), 617-622), mutton or goat (Chikuni et al., 1994, Méat Science 37 (3) 337-345). Similarly, amplification of specific sequences of the Y chromosome has made it possible to determine the sex of beef carcasses of bovine and ovine origin (Apparao et al., 1995, Méat Science 39 (1), 123-126). The analysis of the sequences obtained is done by molecular phylogeny which makes it possible to identify different species (Wolf, 1999, J Agric Food Chem 47: 1350-1355).

The invention indeed consists in determining a mixture. This can be done technically at different levels. First, by "direct sequencing" of the product amplification, containing the DNA fragment (s), and observing the electropherograms obtained. The illegibility of the sequence can be interpreted:

- either as a degradation of DNA if it is in too bad a state to be analyzed or - or as a mixture of at least two species if we observe a superposition of sequences at the level of "indeterminate peaks" represented by the letter N.

Second, the determination of a mixture can be done once the amplification product has been cloned. If this amplification product contains only one DNA fragment, there is only one species, we will then obtain by taking, for example, 10 bacterial colonies, 10 identical sequences. On the other hand, if we are in the presence of a mixture of species, we will then have as many different sequences as there are species present in the sample.

Advantageously, the DNA sequence is of mitochondrial origin for animals and plants or chloroplastic for plants. Mitochondrial DNA (mtDNA) appears to be the most suitable molecule for this type of analysis (Kocher et al., 1989, Proc. Natl. Acad. Sci. USA 86: 6196-6200). From a technical point of view, it is easier to detect than genomic DNA because it is present from 100 to 1000 copies per cell against two copies for nuclear DNA. It can therefore be more surely detected in organic materials in which the DNA is subjected to various physical factors (temperature, pressure, ...) chemical or biochemical tending to its degradation. In addition it is an excellent species marker which is often used in phylogeny. Indeed, depending on the species being studied, certain regions of mtDNA make it possible to differentiate between species, others have a finer resolving power and make it possible to distinguish different populations (geographic races, species or even individuals): mtDNA replication control region, cytochrome b, cytochrome c oxidase or mitochondrial RNA (12S RNA or 16S RNA).

According to an advantageous embodiment of the invention, each DNA sequence or each amplified DNA fragment is of nuclear or mitochondrial origin when it is sought to detect and / or identify a mixture containing DNA from different animal species and / or different human individuals is of nuclear, mitochondrial or chloroplastic origin when it is sought to detect and / or identify a mixture containing DNA from different plant species.

The invention relates to a method for detecting and / or identifying the DNA of each of the different species, populations or animal races and / or of each of the different species, populations or races of plants and / or each of the different human individuals present in a mixture of organic origin, each of said species, populations or races of animals and / or each of said species, populations or races of plants and / or each of said individuals being represented by at least one DNA sequence or at least one DNA fragment, in particular a single DNA sequence or a single DNA fragment, each of said DNA sequences or each of said fragments of DNA from DNA extracted from said mixture, said method being characterized in that it comprises the following steps:

+ amplification of each DNA sequence or each of the DNA fragments characteristic of the animal species, populations or races, of the plant species, populations or races or of the human individual that one seeks to detect and / or identify, in particular by the polymerase chain reaction (PCR) method, in order to obtain a single amplification product containing the different DNA fragments or the different amplified DNA sequences, * cloning of the amplification product obtained ¹ at the end of the previous amplification step, in order to separate the different DNA sequences or the different DNA fragments present in the mixture of organic origin ,

Φ sequencing of each of the DNA sequences or of each of the DNA fragments thus separated from said mixture. The invention also relates to a detection and / or identification method as defined above, in which:

Φ the polymerase chain reaction (PCR) method involves repeating the cycle of the following steps:

heating the DNA extracted from the mixture of organic origin, so as to separate the DNA into two single-stranded strands,

- hybridization of the single-stranded DNA strands at an adequate temperature with the oligonucleotide primers suitable for amplifying the DNA of animal, plant or human species, populations or races which it is desired to detect and / or identify, - elongation of said oligonucleotide primers suitable by a polymerase at an adequate temperature, to obtain a single amplification product containing each of said DNA sequences or each of said DNA fragments characteristic of a given species, population or animal or plant race, or a human individual, 4 cloning of the amplification product obtained at the end of the preceding amplification step comprising the following steps:

- insertion, using a DNA ligase enzyme, of said unique amplification product containing the different DNA sequences or the different amplified DNA fragments characteristic of the DNA of the species, population or animal race, of the plant species, population or race or of the human individual which it is desired to detect and / or identify, in a previously linearized plasmid, in order to obtain several plasmids each containing a unique sequence of DNA or a single fragment of amplified DNA,

- incorporation of each plasmid obtained in the previous step, respectively into a bacterium, in particular Escherichia coli (E. coli),

- multiplication of each of the bacteria obtained in the previous step, by culturing said bacteria in an appropriate medium, in the presence of an antibiotic, in particular ampicillin, in order to select the bacteria which have incorporated a plasmid in which inserted a DNA fragment or a DNA sequence, - separation of each of the plasmids containing a unique DNA sequence or a unique DNA fragment, of each of the bacteria containing said plasmids, in order to recover all of the plasmids or “plasmid DNAs” thus multiplied, said plasmids each containing a unique DNA sequence or a unique DNA fragment,

- removal of a sufficient number of plasmids (or “plasmid DNA”) from all of the plasmids (or “plasmid DNA”) obtained at the end of the previous step, in order to detect and / or identify at at least two different DNA fragments or DNA sequences, each of said DNA sequences or DNA fragments being characteristic of a given species, population or animal or plant race, or of a human individual. The term "amplification product" is understood in the above and what follows, the DNA fragment (s) or the or. the amplified DNA sequences obtained at the end of the polymerization chain reaction (PCR). The amplification product contains several copies of different fragments or different sequences of amplified DNA when the sample of organic material to be analyzed contains a mixture of different DNA fragments from different species, populations or animal breeds or plants or different human individuals.

According to an advantageous embodiment of the invention, in the detection and / or identification method, each of the plasmid DNAs obtained after cloning is identified by sequencing, which makes it possible to identify the DNA characteristic of each of the species, animal populations or races and / or each of the plant species, populations or races and / or each of the human individuals present in the mixture of organic origin.

The cloning process has never been described as a technique for separating species, race populations or individuals present in a mixture. Molecular cloning is a basic molecular biology technique used to select a colony of cells containing particular DNA sequences. In this case, it is used to separate DNA fragments. These fragments are obtained by a global strategy as defined below. The choice of primers depends on the question asked. In general, we look for close species which are mixed with each other but with different commercial values. Thus, the primers used are the largest possible, that is to say that can amplify, an order, a family or a group of very specific species. The DNA extraction process is identical to that described in detail in Example 6 below. It is from amplification that the cloning process is used. We can then start from the amplification fragment obtained with the starting couple of the overall strategy and clone it. However, in the majority of cases, detection of the mixture is requested and no sequencing is therefore carried out before the cloning phase.

To perform cloning, you can use the Invitrogen cloning kit "TOPO TA Cloning", the principle of which is as follows. The DNA fragments obtained after amplification are purified and then introduced into a plasmid previously linearized by the supplier. The plasmid is a circular DNA whose replication within a cell takes place independently of the replication of the genome of this cell. Once the DNA fragment has been introduced into the plasmid using the DNA ligase enzyme, the plasmids are introduced into bacteria such as Escherichia Coli (by a process called "transformation" which involves osmotic shock and temperature shock or a electrical shock). These are then reproduced and selected according to the cloning technique. Plasmids often code for an enzyme capable of chemically degrading or altering an antibiotic substance, giving the bacteria into which they are introduced some resistance to an antibiotic (such as Pampicillin). This resistance is used in genetic engineering to select the bacteria containing the plasmid, in the presence of antibiotics. It should be noted that these antibiotic resistances are present naturally. The cells are then cultured on a dish in a nutrient medium for the production of bacteria, so that each cell leads, by replicating, to a clonal colony. Usually, the culture is carried out in the presence of an antibiotic whose plasmids have a resistance gene, which makes it possible to eliminate the clones which have not received a plasmid. Each colony contains only one type of plasmid. A visual system is used to represent the bacteria which have incorporated the plamisdes which themselves have incorporated a DNA fragment. On the agar plate blue colonies and other white colonies grow. Indeed, in the agar medium are introduced two reagents (L? TG and X-Gal) which in contact with β-galactosidase will produce a blue coloration. If there is synthesis (blue coloring), of this enzyme β-galactosidase, the DNA fragment has not ligated to the plasmid because when there is ligation, the DNA fragment must be positioned in the center of the gene coding for β-galactosidase thus blocking its synthesis, resulting in a white coloration of the bacterial colonies. These colonies are analyzed, and the one containing the desired plasmid is cultured and amplified. The plasmid is extracted from the bacteria and then sequenced.

The sequences obtained are compared with each other; if two sequences are different, then we are in the presence of two species. We realize that in most cases the removal of ten clones (white colonies) is sufficient to detect two species. Since the sample is taken at random, it may happen that out of the ten clones, only one species is obtained, while the mixture contains at least two. If more than two species are suspected in the mixture, then more clones (15, 20 or even 30) are taken. There are generally five clones taken at random to detect a species. In general, about ten plasmid DNAs are recovered, therefore coming from ten independent white bacterial colonies selected at random. These ten plasmid DNAs are therefore sequenced and ten sequences are obtained. These sequences are analyzed in order to identify if we have different profiles. If there is only one species in the DNA extracted from the sample of organic matter, we therefore obtain a single sequence profile for the ten sequenced clones and by comparing this sequence with the reference sequences, we can identify the species present. If on the contrary, we are in the presence of several species in the DNA extracted from the sample of organic matter, we therefore obtain the number of profiles corresponding to the number of species present in the sample. By comparing these sequences with the reference sequences, the species present can be identified. The number of clones selected can be increased if a large number of species to be identified are suspected. This technique can be applied to all organic substrates. Thus, according to an advantageous embodiment of the method of the invention, the sequencing of each of the different amplified DNA fragments is preceded by a cloning method when the sample of organic material comprises a mixture of different DNA fragments from of different animal or plant species or of different human individuals, said cloning method making it possible to separate from said mixture the different DNA fragments originating from different animal or plant species or from different human individuals.

The invention also relates to the oligonucleotides characterized in that they are chosen from those:

1) having a sequence identity of at least 80%, preferably 90% and advantageously 95% with an oligonucleotide consisting of a sequence of approximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides, included in the sequence SEQ ED N ° 21 following (position 16123 to 16144 of the mitochondrial DNA of the salmon - region of control of the replication-according to Hurst, et al, 1999. Gene 239: 237-242):

GCC GAATGT AAA GCATCT GG

2) or those having a sequence identity of at least 80%, preferably 90% and advantageously 95% with an oligonucleotide consisting of a sequence of approximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides, included in the sequence SEQ ED No. 22 following (position 16341 to 16361 of the mitochondrial DNA of the salmon - region of control of the replication-according to Hurst, et al., 1999. Gene 239: 237-242):

ACC TTA TGC ACT TGA TAT CC

3) or those having a sequence identity of at least 80%, preferably 90% and advantageously 95% with an oligonucleotide consisting of a sequence of approximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides, included in the sequence SEQ ED No. 24 following (position 14985 to 14996 of the mitochondrial DNA of salmon - cytochrome b-after Hurst, et al, 1999. Gene 239: 237-242):

ACC GGGTCT AAT AAC CCA GC

4) or those with a sequence identity of at least 80%, preferably

90% and advantageously 95% with an oligonucleotide consisting of a sequence of approximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides, included in the following sequence SEQ ED No. 25 (position 15409 to 15430 of the mitochondrial DNA of salmon - cytochrome b-after Hurst, et al, 1999. Gene 239: 237-242): ATG ATA ATG AAT GGG TGT TC

5) or those having a sequence identity of at least 80%, preferably

90% and advantageously 95% with an oligonucleotide consisting of a sequence of approximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides, included in the following sequence SEQ ED No. 28 (position 356 to 375 of the mitochondrial DNA of salmon - replication control region - after Albert, et al, 1992, Science 257 (5076), 1491-1495):

AAAGCT CTG CGC GCT CTA CG

6) or those with a sequence identity of at least 80%, preferably

90% and advantageously 95% with an oligonucleotide consisting of a sequence of approximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides, included in the following sequence SEQ ED No. 29 (position 539 to 558 of the mitochondrial DNA of salmon - replication control region - after Albert, et al, 1992, Science 257 (5076), 1491-1495):

CCG CGGAGA CAT TCA TAAAC

The invention also relates to the pairs of primers characterized in that they consist of:

- with the oligonucleotides SEQ ED N ° 21 and SEQ ED N ° 22

- with the oligonucleotides SEQ ED N ° 24 and SEQ ED N ° 25

- with the oligonucleotides SEQ ED N ° 28 and SEQ ED N ° 29

The invention also relates to a DNA fragment as amplified using the pairs of primers defined above, and comprising from approximately 100 to approximately 500 base pairs.

The invention also relates to a DNA fragment as defined above, characterized in that it has a sequence identity of at least 80%, preferably 90% and advantageously 95% with at least one of the sequences contained in: - the following SEQ ED N ° 23:

GCCBW HDWR VVRCAYSTKB BBM TRVWKH MRATCWYRWT GCSCGTTRMT CRCVRMRYCK

DBCRBYYTHT TRWVYRCHYM BGGKWSYYYT YH TKYWTYY TTWYCKYHYR GSKKGYMYDY

MCMRRTRSMA BYDMRRRRSK CYVRMRMBSK MRVMCYVKAY STYGMATTCC AGAGARYMYM

TGYVTY KVK YSM RYSHYA THCTMTHADK DATYRC YMH TKRGAYRKYH RARYATAHRG KBRAT where B is C, G or T, W is A or T, H is A, C or T, D is A, G or T, R is A or G, V is A, C or G, Y is C or T, S is C or G, K is G or T, M is A or C,

- the following SEQ ED N ° 26: WCVGGVTCHA AYAACCCMVY AGGHATYWMM TCMSAYKYHG AYAAAATYHC MTTYCACCCH

TACTWYWCM WYAARGACVY YYTNGGMTTN VYHSYYWTMC THMYYKBHMT RAYAYYMYTA

RYHCTRTTCK CMCCMRACCT CCTMGGVGAC CCRGAHAAYT WYACVCYWGC MAAYCCMYTM

RWHACHCCHC CHCA ATCAA RCCHGARTGA TAYTTYYTAT TYGCMTAYRC MATYYTHCGM

TCMRTYCYAA YAAACT GGM GGHGTMCTHG CCCTMKYMBY MTCNRTCCTV RTYCTHDYHV TMRTYCCYHT MCTMCAYAHM TCYAARCAAC RMRSMMTRAY MTTYCGMCCM CTM SCCAAW

BMYT TWYTG RVYYCTRGYM GCVRACMTHC TNAYHCTHAC MTGAATYGGR RGVM ACCHG TVRRMYACCC HT YAYYAYC ATYGG

where W is A or T, V is A, C or G, H is A, C or T, Y is C or T, M is A or C, S is C or G, - K is G or T, R is A or G, N is A, C, G or T, D is A, G or T, B is C, G or T,

- the following SEQ ED N ° 30:

AAAGCTCTGC GCGCTCTACG TCTARAGGAT CTGCGAATCC CCCTGCTTAT ACTAAAACTT

TCCAAGGCCC GCCTCATGGC ATCCAAGTTG AGAGAGATAA ATTGAACAAG TATGGTCGTC CCCTATTGGG ATGTACTATT AAACCTAAAT TGGGGTTATC CGCTAAGAAC TATGGTAGAG

CWGTTTATGA ATGTCTCCGC GG

where R is A or G, W is A or T.

Description of the figures

Figure 1: Figure 1 shows the cloning process used in the context of the invention to separate the different DNA fragments from different fish species in the same mixture. Part 1 of Figure 1 represents the plasmid pCR®2.1 - TOPO. The amplification product of the invention, which contains several copies of different DNA fragments, is brought into contact with the cut plasmid pCR ^® 2.1 - TOPO, using a DNA ligase enzyme.

Part 2 is a portion of each of the DNA fragments (initially contained in the amplification product) ligated into plasmid pCR ^® 2.1 - TOPO. Each DNA fragment corresponds to a plasmid.

Part 3 represents the Escherichia coli bacterial cells (E. coli).

In part 4, the E. coli bacteria are transformed by introduction of the plasmids represented in part 2. Each plasmid corresponds to each E. coli bacterium. Part 5 (symbolized by an arrow) represents the spreading of E. coli bacteria on a medium containing an antibiotic (for example ampicillin), in order to select the bacteria having incorporated a plasmid.

Part 6 represents an agar box on which blue colonies (symbolized by) and white colonies (symbolized by ₀ ) have grown. Blue colonies are characteristic of bacterial cells in which the DNA fragments have not ligated to the plasmid, while white colonies are characteristic of bacterial cells in which the DNA fragments have ligated to the plasmid.

Part 7 represents the individual sample of colonies of white bacteria (for example 10 bacterial colonies selected at random and symbolized by the letters A to J) which are cultured individually because, to each bacterial colony corresponds a plasmid and therefore a fragment of '' Specific DNA (symbolized respectively by the letters A,

B, C, D, E, F, G, H, I and J).

In part 8, the bacteria were eliminated in order to recover the plasmids and their DNA fragments (A to J). Sufficient plasmid DNA is thus obtained in order to sequence it.

Part 9 represents the result of the sequencing of the different DNA fragments A to J, carried out using two primers specific for the plasmid which frame the DNA fragment which it is desired to sequence. Of the ten sequence fragments, two different types of sequences are obtained. Thus, the mixture analyzed comprises two different species of gadiformes, in this case Gadus morhua (fragments A, B, D, E, F, G, I and J), and Merluccius hubbsi (fragments C and H).

FIG. 2: FIG. 2 represents the result of the sequencing of the different DNA fragments A to T, carried out using two primers specific for the plasmids which frame the DNA fragment which it is desired to sequence. Of the 20 sequence fragments, four different types of sequence are obtained. Thus, the mixture analyzed comprises four different species of vertebrates, in this case Bos taurus (the beef: fragments A, B, C, E, J and T), Ovis aries (the sheep: fragments D, I, K, O , P and S), Sus scrofa (pork: fragments L, M and R) and Gallus gallus (chicken: fragments F, G, H, N, N and Q). Figure 3: Figures 3-a and 3-b represent the results obtained for the detection and identification of a mixture of species in a sample from a box of pet food using two strategies species analysis.

In the figure 3-a, the extracted DNA was amplified using different pairs of primers specific to animal species and deposited on an agarose gel stained with bromide ethidium. A size marker of 100 base pairs (M) is deposited on the gel. Well 2 is the amplification product obtained using transvertebrate primers which migrates forming a band of 380 base pairs. Well 3 is the amplification product obtained using the specific primers of chicken which migrates by forming a strip of 160 base pairs. Well 4 is the amplification product obtained using specific primers for gadiforms which migrates forming a band of 340 base pairs. Well 5 is the amplification product obtained using the primers specific to Muscovy ducks which migrate forming a band of 360 base pairs. Well 6 is the amplification product obtained using the specific primers of beef which migrates by forming a band of 480 base pairs. Well 7 is the amplification product obtained using pig specific primers which migrate by forming a band of 407 base pairs. The specific primers of the sheep did not give an amplification product, there is no sheep in the sample (well 8). Well 9 is the amplification product obtained using specific primers for salmon which migrates by forming a band of 245 base pairs. The specific primers of the rat and the mouse did not give an amplification product, there are no rats and mice in the sample (well 10). Well 1 is a negative amplification control.

In the figure 3-b, the sequencing of the different DNA fragments A to AE was carried out using two primers specific for the plasmid which frame the DNA fragment which it is desired to sequence. Of the 30 sequence fragments, seven different types of sequence are obtained. Thus, the mixture analyzed comprises a mixture of seven species, in this case Bos taurus (the beef: fragments A, F, H, K, O, Q and V), Sus scrofa (the pig: fragments C, I, J and M)), Gallus gallus (chicken: fragments B, D, P, R, S, U and X), Cairina moschata (Muscovy duck: fragments: E, G, L, AA and AD), Salmo trutta (salmon: fragments: T, Z and AE) and Theragra chalcogramma (hake: N, Y, AB and AC). Figure 4: represents the alignment of part of the control region of human mitochondrial DNA allowing individual identification (Anderson et al. "Sequence and organization of the human mitochondrial genome" Nature 1981 vol 290, 457-465 ). SEQ ED N ° 33, SEQ ED N ° 34 and SEQ ED N ° 35 are sequences of different clones obtained after amplification for the oligonucleotides corresponding to SEQ ED N ° 31 and SEQ ED N ° 32. DIRECT corresponds to the direct sequence of the PCR product.

FIG. 5 represents an extraction of DNA, carried out from fresh beef DNA (channel 2), or from various substrates containing degraded DNA originating from "corned beef" (channel 3), from ravioli (channel 4 ) or Parmentier mince (channel 5). Channel 6 contains the blank extraction (DNA extraction carried out without substrate) and channel 1 contains the size marker.

FIGS. 6A, 6B and 6C represent the amplification of fragments of mitochondrial DNA (control region and cytochrome b) produced from mitochondrial DNA extracted from fresh beef samples (non-degraded mitochondrial DNA; channels 2), or substrates containing degraded mitochondrial DNA from ravioli (channels 6). Channels 1 contain the size marker and channels 3, 4 and 5 represent the PCRs carried out from the extraction blank so as to test the possible contamination of the extraction, amplification or environmental reagents. In FIG. 6 A, the oligonucleotides used, namely the sequences represented by

SEQ ED NO: 3 and SEQ ED NO: 12, make it possible to amplify a fragment of 258 bp.

In FIG. 6B, the oligonucleotides used, namely the sequences represented by SEQ ED NO: 3 and SEQ ED NO: 6, make it possible to amplify a 500 bp fragment. In FIG. 6C, the following oligonucleotides were used: H1: 5 'TCA TCT CCG GTT TAC AAG AC 3' and

L2: 5 'TGA TAT GAA AAA CCA TCG TTG 3'

These oligonucleotides make it possible to amplify a 1200 bp fragment. It is clearly seen that from the degraded mitochondrial DNA only the small fragments (258 and 500 bp) can be amplified while the non-degraded DNA makes it possible to amplify fragments up to 1200 bp.

FIGS. 7 A and 7B represent the amplification of fragments of nuclear DNA (gene of RNA 18S which is represented in the form of multiple copies in nuclear DNA) carried out from nuclear DNA extracted from samples of beef fresh (non-degraded nuclear DNA; channels 2), or substrates containing degraded nuclear DNA from ravioli (channels 6). Channels 1 contain the size marker and channels 3, 4 and 5 represent the PCRs carried out from the extraction blank so as to test the possible contamination of the extraction, amplification or environmental reagents. In FIG. 7A, the following oligonucleotides were used: 18S1: 5 'TAA CTG TGG TAA TTC TAG AG 3' 18S2: 5 'ACT CTA GAT AAC CTC GGG CC 3'

These oligonucleotides make it possible to amplify a fragment of 163 bp. In Figure 7B, the following oligonucleotides were used: 18S1: 5 'TAA CTG TGG TAA TTC TAG AG 3'

18S3: 5 'AGC TCC AAT AGC GTA TAT TA 3' These oligonucleotides make it possible to amplify a 1200 bp fragment. It is clearly seen that, whatever the size of the amplified fragment, the degraded nuclear DNA did not lead to any positive amplification whereas the non-degraded nuclear DNA gave specific products for the two sizes of amplified fragments. This is very different from what has been obtained with mitochondrial DNA.

FIG. 8 represents the amplification of fragments of nuclear DNA (gene coding for fetal β-globin which is represented in the form of a single copy in nuclear DNA) carried out from nuclear DNA extracted from samples of fresh beef ( Undegraded nuclear DNA; channel 2), or substrates containing degraded nuclear DNA from ravioli (channel 6). Channel 1 contains the size marker, and channels 3, 4 and 5 represent the PCRs carried out from the extraction blank so as to test the possible contamination of the extraction, amplification or environmental reagents. The following oligonucleotides were used:

BGLO1: 5 'GGA AGA GCT GGG CCA GCT GC 3' BGLO2: 5 'CAG GTA GGT ATC CCA CTT AC 3 *

These oligonucleotides make it possible to amplify a 180 bp fragment. It is clearly seen that the degraded nuclear DNA did not lead to any positive amplification while the undegraded nuclear DNA gave a specific product. This result is identical to that obtained on the multiple-copy nuclear gene, 18S RNA.

EXAMPLES

Cloning can be used as a means of identifying the animal and plant species present in a biological sample in different types of situations, the main ones of which are given below:

CASE 1: when the experimenter has no information on the biological product to be tested and simply wishes to list the species / populations / races / individuals present.

In this case, the mitochondrial DNA present is amplified using primers as general as possible, the PCR product obtained is cloned and the greatest number of clones are sequenced. Each different sequence therefore represents the sequence of a species, a population, a race or an individual. The more a large number of clones are sequenced, the more the list of the different species or populations or races or individuals present will be complete. CASE 2: the experimenter has an a priori idea of the type of species, populations, races or individuals present but wishes to identify them more precisely.

For example, we know that a food preparation contains fish, but we do not know the specific species present. In this case, the procedure is the same as for case 1 but by amplifying the mitochondrial DNA with primers specific to fish.

CASE 3: the experimenter knows precisely which species or populations or races or individuals are present but wishes to verify it.

It may then be advantageous to carry out a single amplification with generalist primers than to several independent amplifications with specific primers for each of the species. This is for example the case when the sample is only present in very small quantities.

CAS.4: we know a species / populations / races / individuals contained in the product and we want to know if a mixture exists.

In this case, amplify with general primers, clone, and the sequence of each clone makes it possible to know whether there are several 'species or populations or races or individuals, distinct or not, in the preparation. It should be noted that the invention is applicable, whether the nucleotide sequences of the amplified fragments of the species or populations or races or individuals are already known or not. So for example if we find in a food preparation two sequences unknown in databases, we know that two distinct species or populations or races or individuals are present. It then becomes possible by phylogenetic analysis to determine the zoological group to which these sequence differences belong. The analysis of control samples then makes it possible, in the vast majority of cases, to identify the species, population, race or individual in question, except in the case of a species or population or race or individual new to science. Even in the latter case, the invention makes it possible to be certain that several different species or populations or races or individuals are indeed present.

Example 1: detection and identification of a mixture of at least three species

There are primers that amplify all vertebrates (mammals, fish, birds, ...). These primers were defined by Kocher (Kocher, T. D., Thomas,

W.K., Meyer, A., Edwards, S.V., Paabo, S., Villablanca, F.X., and Wilson, A.C. 1989.

Dynamics of mitochondrial DNA evolution in animais: amplification and sequencing with conserved primers. Proc. Natl. Acad. Sci. USA. 86: 6196-6200) and give a fragment amplification of 360 bp (SEQ ED No. 27) of cytochrome b of mitochondrial DNA. These primers are called "transvertebrate primers". The primer sequences are as follows:

5 '- AAA CTG CAG CCC CTC AGA ATG ATA TTT GTC CTC A- 3' (SEQ ID N ° 1)

5 '- AAA AAG CTT CCA TCC AAC ATC TCA GCA TGA TGA AA- 3' (SEQ ID N ° 2)

The amplified sequence SEQ ED N ° 27 corresponds to the following formula:

TYCCHRCBCC VTCHAAYATY TCHKYHTGAT GRAAYTTYGG HTCHYTHYTD GSMVYNTGCY TNATNMYHCA RMTYHYMACH GGHYTAYTHY TAGCHATRCA CTAYWCMBCN GACRYMDMVM

YMGCHTTYTC MTCHRTHRYC CAYAYYWSYC GDRAYGTDMA HTAYGGCTGA MTHATYCGVW

AYMTNCAYGC HAAYGGHGCH TCHWTVTTYT TYATYTGY T HT YMTDCAY RTHGSVCGMG

GHHTMTAYTA YGGNTCHTWY VYHTWYNHRG ARACMTGAAA YAYHGGVRTH RTMCTHYTVY

TYDYARYHAT ARYMACMKCH TTYTYRTRGG HTAYGTHCTM CCVTGRGGMC AAATATCATT in which Y is C or T, H is A, C or T, R is A or G, V is A, C or G, K is G or T, D is A, G or T, S is C or G, M is A or C, N is A, C, G or T, W is A or T, B is

C, G or T.

These primers are used when a mixture between different animal groups is suspected, because this avoids carrying out several amplifications with pairs of primers which can amplify these different groups and therefore carrying out several cloning. Thus, one can for example detect a mixture of species in a meat between beef, pork, mutton, chicken, duck, ... In order to be able to develop a method of detection and identification of mixture of species by cloning, a DNA extraction must first be carried out followed by a genetic amplification of biological materials. - Extraction 1) DNA extraction by the phenol / chloroform method This method is intended for all types of samples likely to contain organic matter, such as fillet, soup, terrine, pâté, fat, flour, preparations based on fish, etc.

This method uses techniques described in the references HANNI et al., 1990, CR Acad. Sci. Paris., 310, 365-370 and HANNI et al., 1995, Nucl. Acids Res., 23, 881- 882, concerning the extraction of DNA from bones and teeth. An amount of approximately 1 to 2 g of sample of organic material is incubated for two hours at 37 ° C. in 400 μl of lysis buffer of the following composition:

- STE IX (lOOmM NaCl, Tris lOmM at pH 7.4, EDTA (ethylenediaminetetraacetic acid) ImM), - SDS 2%,

- proteinase K at 0.5 mg / ml.

Proteinase K breaks down proteins and releases nucleic acids. The lysate is then extracted twice with a volume of phenol / chloroform (1/1). Centrifuged for 15 minutes at 1000 g, the organic phase is eliminated, which makes it possible to get rid of the protein part of the lysate. The DNA is precipitated with 1/10 volume of 2M sodium acetate then with 2.5 volumes of isopropanol and centrifuged for 30 minutes at 10,000 g. The DNA is taken up in water: a DNA extract is thus obtained. The volume of water depends on the amount of DNA recovered.

The migration of DNA from the various samples gives streaks characteristic of degraded DNA, which is however perfectly amplifiable.

2) DNA extraction using the extraction kit or kit method

This method is carried out under the conditions described by the supplier Qiagen.

- Amplification:

The PCR is carried out with the pair of primers SEQ ED No. 2 and SEQ ED No. 8 as defined above. The amplifications are carried out in a total volume of 50 μl containing:

200 μg / ml of BSA (Bovine Albumin Serum),

250 mM of dNTP (deoxynucleotide Triphosphate), 300 ng of each primer,

1.5 mM magnesium chloride (MgC12), 10X PCR buffer (100 mM Tris-HCl pH 8.3; 500 mM KC1),

1 unit of Taq Polymerase, qs 50 μl of sterile distilled water, lμl of DNA extract. The reaction mixture is carried out in a sterile room in a horizontal flow hood, to avoid contamination as much as possible. The PCRs are carried out on an Ependorff PCR device. Each PCR is cut as follows:

- 1 initial cycle at a temperature of 94 ° C for 2 minutes then, - 40 cycles at a temperature of 94 ° C for 1 minute, at a temperature of 55 ° C at

63 ° C for 1 minute, at a temperature of 72 ° C for 2 minutes; in the last cycle, a terminal elongation is carried out at a temperature of 72 ° C. for 7 minutes.

The amplification products are analyzed by electrophoresis on 2% agarose gel under constant voltage of 100 V for 30 min, and using ethidium bromide to visualize the amplifications obtained.

The entire PCR amplification product (amplification product) can be purified directly using the "QIAquick PCR purification Kit" system from Qiagen according to the conditions announced. The entire PCR product is passed through a column of silica gel. The nucleic acids are retained on the column "while the other PCR residues are eluted by microcentrifugation. The impurities are eliminated and the DNA is eluted by Tris buffer or water. The DNA thus purified is visualized and quantified on 2% agarose gel.

- Direct sequence of the amplification product

Automatic sequencing is performed on the purified PCR products. The primers used are those which have served to amplify the DNA fragment or fragments which it is sought to characterize. The "Perkin-Elmer" Kit is used for the PCR reaction, which is carried out over 25 cycles under the conditions stated by the supplier. Each amplification product obtained at the end of the PCR reaction is precipitated with ethanol and deposited on a polyacrylamide gel. The sequences obtained are then compared with those of the banks or with the sequences already obtained.

The extraction and amplification of the DNA contained in a sample of organic matter containing a mixture of different vertebrate species is carried out as described above. A unique amplification product (containing at least one DNA fragment represented by a fragment of 360 base pairs), obtained using the primer pair (SEQ ID, is observed at the end of the PCR reaction. N ° 1, SEQ ID N ° 2) allowing to amplify all the vertebrates (see figure 3, well 2). Said amplification product is therefore capable of containing different DNA fragments characteristic of different vertebrate species. - Cloning

The amplification product is directly cloned, with or without purification, using the “TOPO TA cloning kit” system from Invitrogen according to the conditions announced, in order to isolate and obtain numerous identical copies of all the different fragments of DNA contained in the amplification product. In this regard, the amplification product is ligated into the plasmid "pCR®2.1-TOPO® 3.9 kb" sold by Invitrogen, (Figure 1-1). Each DNA fragment contained in the amplification product will be inserted into a plasmid "pCR®2.1-TOPO®" using the DNA ligase enzyme (Figure 1-2). Each DNA fragment therefore corresponds to a plasmid. All of the DNA fragments contained in the amplification product are thus separated during this step.

After ligation of each DNA fragment in a plasmid, each plasmid is introduced into a bacterium, for example Escherichia coli (E. coli) (Figure 1-4), by a process called "transformation" which involves an osmotic shock and a temperature shock or an electric shock. The E. coli bacteria thus obtained are cultured on agar in order to be multiplied, in the presence of an antibiotic (for example ampicillin).

The presence of the antibiotic makes it possible to select the E. coli bacteria which have incorporated a plasmid, since the plasmids naturally have a gene for resistance to an antibiotic.

Thus, E. coli bacteria which have not incorporated a plasmid will not be able to grow.

To select the E. coli bacteria which have incorporated a plasmid into which a DNA fragment has ligated, a visual system is used. Indeed, in the agar medium were introduced two reagents (EPTG and X-Gal) which, in contact with the enzyme β-galactosidase, will produce a blue coloring. The colonies of blue bacteria obtained are those which synthesize β-galactosidase, and which contain a plasmid in which a DNA fragment has not ligated. The colonies of white bacteria obtained are those which do not synthesize β-galactosidase, and which contain a plasmid in which a DNA fragment has ligated. This is linked to the fact that the DNA fragment is inserted into the plasmid in the middle of a gene coding for the β-galactosidase enzyme, the synthesis of which it blocks.

Each white bacterial colony obtained contains a single fragment or a single amplified DNA sequence, said fragment or said DNA sequence corresponding to one and the same species of gadiformes. The plasmid DNAs containing the DNA fragments must then be recovered, that is to say separated from the bacterial DNAs to be sequenced. In this example, we seek to detect at least 3 species of vertebrates. According to an advantageous embodiment of the method of the invention, about twenty plasmid DNA are recovered (symbolized respectively by the letters A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, and T in Figure 1) from 20 independent white bacterial colonies randomly selected. The plasmid DNAs (symbolized by the letters A to T in FIG. 2) thus recovered are then sequenced with the primers provided in the Invitrogen kit. The sequences obtained are analyzed in order to identify whether we have different profiles or not. The results obtained (Figure 2) indicate that the sample of organic matter comprises a mixture of four species, in this case Bos taurus (the beef: fragments A, B, C, E, J and T), Ovis aries (the sheep: fragments D, I, K, O, P and S), Sus scrofa (pig: fragments L, M and R) and Gallus gallus (chicken: fragments F, G, H, N, N and Q).

Example 2: detection and identification of a mixture of species: gadiformes

Certain food preparations can be prepared from several species of fish (ready meals, soup, pâtés, terrine, ...). "This mixture may be specified by the manufacturer, but may also be adulteration. Indeed, it may happen that this mixture is in favor of a species of fish of less economic value compared to another species which should have been found uniquely in the preparation (example: cod brandade prepared with a little cod (Gadus morhua) and a lot of cheaper Pacific cod (Gadus macrocephalus).

When it is not known whether the sample contains several species of gadiforms, the pairs of primers are used which make it possible to amplify them all.

The pair of primers SEQ ED N ° 3 and SEQ ED N ° 4 gives the fragment SEQ ED N ° 5 of 442 bp. SEQ ED N ° 3 answers the following formula:

AYC ARC AYY TRT TYT GRT TCT where Y is C or T, R is A or G.

SEQ ED N ° 4 responds to the following formula:

TAY GTW GTN GCN CAY TTY CA in which Y is C or T, W is A or T, N is A, C, G or T. SEQ ED N ° 5 has the following formula:

AYCARCAYYT RTTCTGATTC TKCGGNCAYC CYGAAGTHTA YATHCTNATY YTMCCHGGMT TCGGRATAAT YTCYCAYATY GTAGCVTAYT AYTCAGGNAA RMAAGARCCN TTYGGRYAYA TRGGHATRGT NTGAGCYATGGTGGTGY ACATRTTYAC AGTBGGRATR GAYGTDGAYA CMCGWGCHTA CTTYACATCY GCAACBATAA

TYATYGCYAT YCCRACAGGY GTWAAAGTYT TYAGYTGAYT AGCAACYYTV CAYGGRGGCT

CARTTAARTG RGAVACHCCB MTMCTBTGRG CCCTDGGYTT YATYTTYCTM TTYACMGTHG

GVGGMYT AC AGGNATYRTH YTRGCYAAYT CYTCYCTAGA YATYGTDCTY CAYGAYACRT AYTAMGTAGT MGCYCAYTTY CA

where Y is C or T, R is A or G, K is G or T, N is A, C, G or T, H is A, C or T, M is A or C, V is A, C or G, B is C, G or T, D is A, G or T, W is A or T.

The pair of primers SEQ ED No. 6 and SEQED No. 4 gives the fragment SEQ ED No. 7 of 328 bp.

SEQ ED N ° 6 responds to the following formula:

GGN YAY ATR GGN ATR GTN TGA GC in which N is A, C, G or T, Y is C or T, R is A or G.

SEQ ED N ° 7 responds to the following formula:

GGRYAYATRG GHATRGTNTG AGCYATRATR GCYATYGGMC TYCTYGGYTT TATYGTVTGR

GCYCAYCACA TRTTYACAGT BGGRATRGAY GTDGAYACMC G GCHTACTT YACATCYGCA

ACBATAATYA TYGCYATYCC RACAGGYGT AAAGTYTTYA GYTGAYTAGC ACYYTVCAYG

GRGGCTCART TAARTGRGAV ACHCCBMTMC TBTGRGCCCT DGGYTTYATY TTYCTMTTYA CMGTHGGVGG MYT ACAGGN ATYRTHYTRG CYAAYTCYTC YCTAGAYATY GTDCTYCAYG

AYACRTAYTA MGTAGTMGCY CAYTTYCA

where R is A or G, Y is C or T, K is G or T, N is A, C, G or T, H is A, C or T,

M is A or C, V is A, C or G, B is C, G or T, D is A, G or T, W is A or T.

When it is desired to detect a mixture of gadidae, it is possible to use the preceding primers which make it possible to identify all the gadiformes, but also the primers making it possible to amplify all the gadidae.

The cut of primers SEQ ED N ° 8 and SEQ ED N ° 9 gives the fragment SEQ ED N ° 10 of 386 bp.

SEQ ED N ° 8 answers the following formula:

ACC AAC ACT TAT TCT GAT TCT SEQ ED N ° 9 has the following formula:

GGC TTA ACA GGA ATT GTA CTA GCT SEQ ED N ° 10 answers the following formula:

AYCARCAYYT RTTCTGATTC TKCGGNCAYC CYGAAGTHTA YATHCTNATY YTMCCHGGMT

TCGGRATAAT YTCYCAYATY GTAGCVTAYT AYTCAGGNAA RMAAGARCCN TTYGGRYAYA

TRGGHATRGT NTGAGCYATR ATRGCYATYG GMCTYCTYGG YTTTATYGTV TGRGCYCAYC ACATRTTYAC AGTBGGRATR GAYGTDGAYA CMCGWGCHTA CTTYACATCY GCAACBATAA

TYATYGCYAT YCCRACAGGY GTWAAAGTYT TYAGYTGAYT AGCAACYYTV CAYGGRGGCT

CARTTAARTG RGAVACHCCB MTMCTBTGRG CCCTDGGYTT YATYTTYCTM TTYACMGTHG

GVGGMYTWAC AGGNATYRTH YTRGCY

where Y is C or T, R is A or G, K is G or T, N is A, C, G or T, H is A, C or T, M is A or C, V is A, C or G, B is C, G or T, D is A, G or T, W is A or T.

The pair of primers SEQ JD No. 11 and SEQ ED No. 9 gives the fragment SEQ ED No. 12 of 237 bp. - SEQ ED N ° l 1 corresponds to the following formula:

GGC CTC CTT GGC TTTATTGTA

SEQ ED N ° 12 responds to the following formula:

GGMCTYCTYG GYTTTATYGT VTGRGCYCAY CACATRTTYA CAGTBGGRAT RGAYGTDGAY ACMCGWGCHT ACTTYACATC YGCAACBATA ATYATYGCYA TYCCRACAGG YGTWAAAGTY

TTYAGYTGAY TAGCAACYYT VCAYGGRGGC TCARTTAART GRGAVACHCC BMTMCTBTGR

GCCCTDGGYT TYATYTTYCT MTTYACMGTH GGVGGMYTWA CAGGNATYRT HYTRGCY

where Y is C or T, R is A or G, K is G or T, N is A, C, G or T, H is A, C or T, M is A or C, V is A, C or G, B is C, G or T, D is A, G or T, W is A or T.

When it is desired to detect a mixture of merluccidae, one can use the preceding primers which make it possible to identify all the gadiformes, but also the primers making it possible to amplify all the merluccidae. The pair of primers SEQED N ° 13 and SEQ ED N ° 14 gives the fragment SEQ ED N ° 15 of

318 bp.

SEQ ED N ° 13 responds to the following formula:

TAA TYT CYC AYA TYG TAG CC in which Y is C or T. SEQ ED N ° 14 corresponds to the following formula:

ACT TAC AGG NAT YRT HCT RG in which N is A, C, G or T, Y is C or T, R is A or G, H is A, C or T. SEQ ED N ° 15 answers the following formula:

TAATYTCYCA YATYGTAGCV TAYTAYTCAG GNAARMAAGÀ RCCNTTYGGR YAYATRGGHA

TRGTNTGAGC YATRATRGCY ATYGGMCTYC TYGGYTTTAT YGTVTGRGCY CAYCACATRT

TYACAGTBGG RATRGAYGTD GAYACMCGWG CHTACTTYAC ATCYGCAACB ATAATYATYG CYATYCCRAC AGGYGTWAAA GTYTTYAGYT GAYTAGCAAC YYTVCAYGGR GGCTCARTTA

ARTGRGAVAC HCCBMTMCTB TGRGCCCTDG GYTTYATYTT YCTMTTYACM GTHGGVGGMY

TWACAGGNAT YRTHYTRG

where Y is C or T, R is A or G, K is G or T, N is A, C, G or T, H is A, C or T, M is A or C, V is A, C or G, B is C, G or T, D is A, G or T, W is A or T.

The pair of primers SEQ ED N ° 16 and SEQ ED N ° 14 gives the fragment SEQ ED N ° 17 of 189 bp.

SEQ ED N ° 16 answers the following formula:

GRA TRG AYG TDG AYA CMC GT in which R is A or G, Y is C or T, D is A, G or T, M is A or C. SEQ ED N ° 17 corresponds to the following formula:

GRATRGAYGT DGAYACMCGW GCHTACTTYA CATCYGCAAC BATAATYATY GCYATYCCRA

CAGGYGTWAA AGTYTTYAGY TGAYTAGCAA CYYTVCAYGG RGGCTCARTT AARTGRGAVA

CHCCBMTMCT BTGRGCCCTD GGYTTYATYT TYCTMTTYAC MGTHGGVGGM YTWACAGGNA TYRTHYTRG

where R is A or G, Y is C or T, D is A, G or T, M is A or C, W is A or T, B is C, G or T, V is A, C or G, H is A, C or T, N is A, C, G or T.

From this system, here are several examples of types of mixtures of species that can be identified.

The first is a mixture of gadiformes, for example, bobwhite quail (Theragra chalcogramma) which is a gadidae and Argentine hake (Merluccius hubbsi) which is a hake. The primer couple (SEQ ED N ° 6 and SEQ ED N ° 4) which makes it possible to amplify all the gadiformes and giving a fragment (SEQ JD N ° 7) of 328 bp is used. Two sequence profiles are observed on the ten clones analyzed. These two profiles when compared to reference sequences grouped in a sequence bank (Genebank for example), correspond to a species of gadidae: the bobwhite quail (Theragra chalcogramma) and a species of hake: the Argentine hake (Merluccius hubbsi) The second is a mixture between gadidae, for example common cod (Gadus morhua) and ling (Molva molva). The primer pair (SEQ ED No. 11 and SEQ ED No. 9) which makes it possible to amplify all the gadidae and giving a fragment (SEQ ED No. 12) of 237 bp is used. This fragment is cloned and then sequenced. Two sequence profiles are observed on the ten clones analyzed. These two profiles when compared to reference sequences grouped in a sequence bank (Genebank for example), correspond to two species of gadidae: cod (Gadus morhua) and ling (Molva molva). The third type of mixture is a mixture between merluccidae such as for example Cape hake (Merluccius capensis) and European hake (Merluccius merluccius). The primer couple (SEQ ED N ° 16 and SEQ ED N ° 14) which makes it possible to amplify all the merluccidae and giving a fragment (SEQ ED N ° 17) of 189 bp is used. This fragment is cloned and then sequenced. Two sequence profiles are observed on the ten clones analyzed. These two profiles when compared to reference sequences grouped in a sequence bank (Genebank for example), correspond to two species of hake: Cape hake (Merluccius capensis) and common hake (Merluccius merluccius).

Example 3: detection and identification of a mixture of species: anseriforms With regard to anseriforms, there may be different forms of mixture. The first concerns products such as foie gras where the goose can be largely replaced by duck of less commercial value and the duck foie gras can also be substituted by chicken in various proportions. These mixtures of species in foie gras mainly concern the blocks of foie gras which are livers reconstituted from several livers and not the whole fatty livers which them come from a single individual. The other types of mixes concern ready meals, prepared from different species.

The detection of mixtures of anseriform species is carried out by the process of cloning the fragment obtained by the primer couple defined in the patent for detecting anserforms. This primer pair (SEQ ED N ° 18 and SEQ ED N ° 19) sleeps a fragment (SEQ

ED N ° 20) of 203 bp. All species of anseriformes can then be detected in any mixture.

SEQ ED N ° 18 responds to the following formula:

ACT AGC TTC AGG CCC ATA CG SEQ ED N ° 19 has the following formula:

AAA AAT AAA AGG AAC CAG AG SEQ ED N ° 20 has the following formula:

ACYAGCTTCA GGCCCATACG TTCCCCCTAA ACCCCTCGCC CTCCTCACAT TTTTGCGCCT CTGGTTCCTC GGTCAGGGCC ATCMATTGGG TTCACTCACC YCYMYTYGCC YTTCAAAGTG GCATCTGTGG ANKACBTYCA CCWYYYCRRT GCGTWATCGC GGCATBYTYM ASYWTTTWSM CGCCTYTGGT TCYMYTTHTY TYT

where Y is C or T, M is A or C, N is A, C, G or T, K is G or T, B is C, G or T, W is A or T, R is A or G, S is C or G.

Example 4: detection and identification of a mixture of species: salmon

There is fraud between the only species of Atlantic salmon Salmo salar and the five species of Pacific salmon of the genus Oncorhynchus. In Europe, two species are raised industrially, Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). These species are generally smoked or once smoked, it is very difficult to distinguish between these two species both from a visual and tactile point of view on their texture. These two species are not sold at the same price since salmon is much more expensive than trout. There is therefore fraud for this product, when the salmon is not sold in the form of a fillet but in another packaging, especially during the holiday season.

Several pairs of primers have been defined in order to amplify all the salmonids. Two molecular markers were chosen.

For the mtDNA replication control region, the primer couple: SEQ ED N ° 21 corresponding to the following formula:

GCC GAA TGT AAA GCA TCT GG SEQ ED N ° 22 corresponding to the following formula:

ACC TTA TGC ACT TGA TAT CC gives a 245 bp fragment (SEQ ED N ° 23) SEQ ED N ° 23 corresponds to the following formula:

GCCBWWHDWR WRCAYSTKB BBMWTRVWKH MRATCWYRWT GCSCGTTRMT CRCVRMRYCK

DBCRBYYTHT TRWVYRCHYM BGGKWSYYYT YHWTKYWTYY TTWYCKYHYR GSKKGYMYDY

MCMRRTRSMA BYDMRRRRSK CYVRMRMBSK MRVMCYVKAY STYGMATTCC AGAGARYMYM

TGYVTYWKVK YSMWRYSHYA THCTMTHADK DATYRCWYMH TKRGAYRKYH RARYATAHRG KBRAT

where B is C, G or T, W is A or T, H is A, C or T, D is A, G or T, R is A or G, V is A, C or G, Y is C or T, S is C or G, K is G or T, M is A or C. For the cytochrome b of the mtDNA, the pair of primers: SEQ ED N ° 24 corresponding to the following formula:

ACC GGG TCT AAT AAC CCA GC SEQ ED N ° 25 corresponding to the following formula: ATG ATA ATG AAT GGG TGT TC gives a fragment of 442 bp (SEQ JD N ° 26) SEQ ED N ° 26 corresponds to the following formula:

WCVGGVTCHA AYAACCCMVY AGGHATYWMM TCMSAYKYHG AYAAAATYHC MTTYCACCCH

TACTWYWCMW WYAARGACVY YYTNGGMTTN VYHSYYWTMC THMYYKBHMT RAYAYYMYTA RYHCTRTTCK CMCCMRACCT CCTMGGVGAC CCRGAHAAYT WYACVCYWGC MAAYCCMYTM

RWHACHCCHC CHCAWATCAA RCCHGARTGA TAYTTYYTAT TYGCMTAYRC MATYYTHCGM

TCMRTYCYAA YAAACTWGGM GGHGTMCTHG CCCTMKYMBY MTCNRTCCTV RTYCTHDYHV

TMRTYCCYHT MCTMCAYAHM TCYAARCAAC RMRS MTRAY MTTYCGMCCM CTMWSCCAAW

BMYTWTWYTG RVYYCTRGYM GCVRACMTHC TNAYHCTHAC MTGAATYGGR RGVM ACCHG TVRRMYACCC HTWYAYYAYC ATYGG

where W is A or T, V is A, C or G, H is A, ^" C or T, Y is C or T, M is A or C, S is C or G, K is G or T, R is A or G, N is A, C, G or T, D is A, G or T, B is C, G or T.

The pair of primers defined from the control region is preferentially chosen because the fragment being smaller, it is more likely to obtain it for degraded foods.

Example 5: detection and identification of a mixture of individuals This type of detection can also be applied to a single species which is composed of several individuals. This is the case of the human species where it has been demonstrated that the region of control of its mtDNA makes it possible to distinguish between individuals according to the maternal line. This observation is very widely used in laboratories which carry out genetic fingerprints for the typing of individual DNA. Thus, when several human DNAs are found on a sample to be analyzed, the cloning process makes it possible to sort between the individuals whose DNA has been found and to independently identify each of the individuals by its DNA.

Such a situation can occur when the organic remains of several individuals are mixed (for example the case of several people who drank from the same glass, thus leaving several different types of traces). When one wishes to use mitochondrial DNA to carry out an identification in such a case, one obtains a sequence containing numerous indeterminacies making the identification of each person impossible. Cloning makes it possible to circumvent this problem by separating each sequence which makes it possible to obtain several distinct and specific signatures.

Cloning also makes it possible to detect the presence of several individuals while analysis of the direct sequence did not allow it to be predicted. So if we find old traces of organic remains (a blood stain several years old for example) and that these remains have been mixed with recent organic remains (hair dandruff of anyone who has intervened recently) we obtain an extract DNA containing very degraded old DNA and modern DNA in good condition. In such a case, modern DNA being preferably amplified, it is in the majority and a single visible signature will be obtained in direct sequence. However, if one clones and analyzes a large number of clones (for example 30) one can obtain a majority of clones coming from the contaminating modern DNA (for example 28) and a minority (for example 2) which comes from degraded DNA.

Cloning therefore makes it possible to reveal the presence of a mixture, whereas no conventional analysis suggested this. This method therefore makes it possible to carry out a much finer analysis of organic samples, and to detect and / or reveal the presence of unsuspected sequences corresponding to individuals.

In the example presented, bone fragments were extracted by the conventional methods used in molecular archeology (Anderson et al. "Sequence and organization of the human mitochondrial genome" Nature 1981 vol 290, 457-465). A 382 bp region of the mitochondrial DNA control region was amplified with the following oligonucleotides:

AAG CAG ATT TGG GTA CCA C-3 '(SEQ ED N ° 31)

GAT TTC ACG GAG GAT GGT G-3 '(SEQ ED N ° 32)

The amplified region corresponds to positions 16037 (SEQ ED No. 31) to 16419 (SEQ ED No. 32) of human mitochondrial DNA determined by Anderson in 1981 (Nature 1981).

This region is compared with the 3 sequences resulting from the cloning of this fragment (respectively SEQ ED N ° 33, SEQ ED N ° 34, and SEQ ED N ° 35) and with the direct sequence of the fragment resulting from PCR (DIRECT). We can clearly see that each difference between one of the 3 sequences corresponds to an N in the direct sequence. It is concluded that the bone fragments contain genetic material from 3 different individuals. Example 6: detection and identification of a mixture of vertebrates in a box of pet food:

We find the same steps as those defined in Example 1: extraction, amplification with the pair of transvertebrate primers, cloning, sequencing. What differs is the analysis of the results because in this example, in addition to having sequenced the amplification product obtained by the pair of transvertebrate primers (Figure 3 well 2), the results were confirmed using primers specific to vertebrate species which have been found after sequencing the plasmid DNAs.

"Transvertebrate" primers are used to amplify a cytochrome b fragment of mitochondrial DNA in a sample from a pet food box.

In this example, we are looking for a mixture of which we do not know the number of species. On the label is noted "minced in chicken and duck jelly", then ingredients (meat and animal by-products). It is therefore assumed that there are certainly more than two species in addition to chicken and duck such as pork and beef.

According to an advantageous embodiment of the method of the invention, about thirty plasmid DNA are recovered (symbolized respectively by the letters A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y, Z, AA, AB, AC, AD, AE in Figure 3 - b) from 30 colonies independent white bacteria selected at random. The plasmid DNAs (symbolized by the letters A to AE in FIG. 3 - b) thus recovered are then sequenced with the primers provided in the Envitiogen kit. The sequences obtained are analyzed in order to identify whether we have different profiles or not. The results obtained (Figure 3 - b) indicate that the sample of organic matter comprises a mixture of seven species, in this case Bos taurus (the beef: fragments A, F, H, K, O, Q and V), Sus scrofa (pork: fragments C, I, J and M)), Gallus gallus (chicken: fragments B, D, P, R, S, U and X), Cairina moschata (Muscovy duck: fragments: E , G, L, AA and AD), Salmo trutta (salmon: fragments: T, Z and AE) and Theragra chalcogramma (hake: N, Y, AB and AC).

Example 7: detection and identification of a mixture of plants:

A primer pair has been defined on the chloroplast genome to amplify the gene coding for the ribulose 1,5-bisphosphate carboxylase / oxygenase (rbcL) subunit present in all plants. The amplification product obtained makes it possible to detect and identify the different plant species. An example relates to a system of detection and identification of grape varieties since in general several grape varieties enter into the composition of the wine. The grape varieties, such as Cabernet Franc, Chardonnay, Chaselas, Gamay, Sauvignon, or Syrah, are all from the same species (Vitis vinifera). The primer sequences are as follows:

- AAA GCT CTG CGC GCT CTA CG (SEQ ED N ° 28)

- CCG CGG AGA CAT TCA TAA AC (SEQ ED N ° 29)

SEQ ED N ° 28 and SEQ ED N ° 29 give a fragment of 202 bp (SEQ ED N ° 30)

SEQ ED 30 has the following formula:

AAAGCTCTGC GCGCTCTACG TCTARAGGAT CTGCGAATCC CCCTGCTTAT ACTAAAACTT

TCCAAGGCCC GCCTCATGGC ATCCAAGTTG AGAGAGATAA ATTGAACAAG TATGGTCGTC

CCCTATTGGG ATGTACTATT AAACCTAAAT TGGGGTTATC CGCTAAGAAC TATGGTAGAG CWGTTTATGA ATGTCTCCGC GG

where R is A or G, W is A or T.

In order to be able to develop a method for detecting and identifying a varietal mixture by cloning, a DNA extraction must first be carried out followed by a genetic amplification of biological materials.

- CTAB extraction (cetyltrimethylammonium bromide).

This method described by MURRAY (Nucleic Acid Res. 8 (19), 4321-4325 (1980)) is preferably intended for plant samples. lg of sample is incubated for 3 hours at 56 ° C in 2.5 ml of lysis buffer, the composition of which is:

0.1 mM EDTA sodium dodecyl sulfate 1% proteinase K 100 μg / ml

450 μl of 5 M NaCl and 375 μl of CTAB 10% 0.7 M NaCl, preheated to 65 ° C, are added and the whole incubated for 20 min at 65 ° C. A volume of chloroform is then added. After stirring and centrifligation (10 min at 12,000 rpm at 4 ° C), the aqueous phase is recovered and reextracted a second time to make it clear. It is again extracted by a volume phenol / chloroform. The rest of the extraction is carried out as indicated in paragraph 1.

- Amplification: (see example 1 above) - Cloning (see example 1 above)

Example 8 Comparative Experiments Between Degraded Mitochondrial DNA and Degraded Nuclear DNA:

When comparing the amplification capacities of fragments of different sizes on mitochondrial DNA, it is very clear that if the non-degraded mitochondrial DNA from fresh substrate makes it possible to amplify fragments of small and large size ( up to 1200 bp), degraded mitochondrial DNA from transformed substrates (in the example of pure beef ravioli) only allows the amplification of small fragments (here 258 bp) while fragments of 500 and 1200 bp can be amplified. It is important to note that the amplification of the small fragment in degraded DNA clearly proves that even if by direct visualization no DNA was seen after extraction (see Figure 5, channel 4), an amplification product is detected after PCR (see Figure 6 A, channel 6). The various contamination controls carried out prove that the amplification product obtained does not result from contamination of the samples with exogenous DNA.

Unlike the results obtained with mitochondrial DNA, DNA amplifications carried out on nuclear DNA do not allow amplification to be obtained when degraded nuclear DNA is used as a substrate. While the amplification of small mitochondrial fragments made it possible to obtain an amplification with degraded nuclear DNA, this is not the case for the nuclear gene 18S, whatever the size of the fragment considered (FIGS. 7A and 7B) . This clearly shows that the amplification of small mitochondrial DNA fragments makes it possible to work on a whole range of substrates which is inaccessible to amplification if one is looking for nuclear genes. Quite similar results were obtained on a single copy nuclear gene, the fetal β-globin gene (Figure 8). Again, DNA can only be amplified from an undegraded substrate.

Claims

1. Use of a cloning process for determining the existence of a mixture of organic origin containing mitochondrial or chloroplastic DNA of different species, populations or animal races and / or different species, populations or plant races and / or different human individuals.

2. Use according to claim 1, characterized in that the mitochondrial or chloroplastic DNA is degraded.

3. Use of a cloning method according to claim 1 or 2 for the detection and / or identification of the DNA of each of the different species, populations or animal races and / or of each of the different species, populations or plant breeds and / or each of the different human individuals present in a mixture of organic origin containing the DNA of animal species, populations or races and / or plant species or populations and races and / or human individuals .

4. Use according to one of claims 1 to 3, in which each species, population or animal race and / or each species, population or plant race and / or each human individual is represented by at least one sequence of DNA or at least one DNA fragment, and in particular by a single DNA sequence or a single DNA fragment, each of said DNA sequences or each of said DNA fragments originating from DNA extracted from the mixture of organic origin.

5. Use according to one of claims 1 to 4 for the detection and / or identification of the DNA of each of the different animal species present in a mixture of animal species, and in particular of the DNA of each of the sub -species, lines, races, varieties and / or strains and / or populations present in said mixture.

6. Use according to one of claims 1 to 4 for the detection and / or identification of the DNA of each of the different plant species present in a mixture of plant species, and in particular of the DNA of each of the sub -species, lines, races, varieties and / or strains, grape varieties and / or populations present in said mixture, each of said plant species, and in particular each of said subspecies, races, varieties and / or strains being represented by at least one DNA sequence or at least one DNA fragment, and in particular by a single DNA sequence or a fragment unique DNA.

7. Use according to one of claims 1 to 4 for the detection and / or identification of the DNA of each of the different human individuals present in a mixture of DNA of different human individuals, each of said human individuals being represented by at least one DNA sequence or at least one DNA fragment, and in particular by a single DNA sequence or a single DNA fragment.

8. Use according to one of claims 1 to 7, wherein the DNA extracted from the mixture of organic origin is old DNA (or fossil), degraded DNA or modern DNA.

9. Use according to one of claims "1 to 8, in which the DNA extracted from the mixture of organic origin is:

- undegraded DNA originating in particular from a fresh organic sample or,

- degraded DNA, in particular from a transformed organic sample, in particular cooked, lyophilized, dried, brined, canning or pasteurized.

10. Use according to one of claims 1 to 9 for the detection and optionally the identification of the DNA of each of the different species, populations or animal races and / or of each of the different species, populations or plant races and / or of each of the different human individuals present in the mixture of organic origin, in which at least one of said species, populations or races and / or at least one of said individuals is represented by an ancient DNA sequence or an old DNA fragment.

11. Use according to one of claims 1 to 10, in which the cloning process is preceded by a step of amplification of each of the DNA sequences or of each of the DNA fragments characteristic of each of the species, populations or animal breeds and / or each of the plant species, populations or races and / or each of the human individuals sought to be detected and / or identified, the DNA sequences or the amplified DNA fragments s obtained at the end of the amplification process being contained in a single amplification product.

12. Use according to claim 11, in which each DNA sequence or each amplified DNA fragment is of nuclear or mitochondrial origin when it is sought to detect and / or identify a mixture containing DNA. of different animal species and / or different human individuals, is of nuclear, mitochondrial or chloroplastic origin when it is sought to detect and / or identify a mixture containing DNA of different plant species.

13. Method for detecting and / or identifying the DNA of each of the different species, populations or animal races and / or of each of the different species, populations or plant races and / or of each of the different human individuals present in a mixture of organic origin, each of said species, populations or animal races and / or each of said species, populations or plant races and / or each of said human individuals being represented by at least one DNA sequence or at least one fragment of DNA, in particular a unique DNA sequence or a unique DNA fragment, each of said DNA sequences or each of said DNA fragments originating from DNA extracted from said mixture, said method being characterized in that it comprises the following stages: amplification of each of the DNA sequences or of each of the DNA fragments characteristic of the animal species, population or race, of the plant species, population or race or of the human individual that is sought to be detected and / or identified, in particular by the polymerase chain reaction (PCR) method, in order to obtain a single amplification product containing the different DNA fragments or the different sequences of amplified DNA, cloning of the amplification product obtained at the end of the preceding amplification step, in order to separate the different DNA sequences or the different DNA fragments present in the mixture of organic origin,

4 sequencing of each of the DNA sequences or of each of the DNA fragments thus separated from said mixture.

14. Detection and / or identification method according to claim 13, in which:

4 the polymerase chain reaction (PCR) method involves repeating the cycle of the following steps: heating the DNA extracted from the mixture of organic origin, so as to separate the DNA into two single-stranded strands,

- hybridization of the single-stranded DNA strands at an adequate temperature with the appropriate oligonucleotide primers to amplify the DNA of animal, plant or human species, populations or races which one seeks to detect and / or identify,

- elongation of said appropriate oligonucleotide primers by a polymerase at an appropriate temperature, to obtain a single amplification product containing each of said DNA sequences or each of said DNA fragments characteristic of a given animal or plant species, or of a human individual,

4 cloning of the amplification product obtained at the end of the preceding amplification step comprising the following steps:

- insertion, using a DNA ligase enzyme, of said unique amplification product containing the different DNA sequences or the different amplified DNA fragments characteristic of the DNA of the species, pdpulation or animal race, of the plant species, population or race or of the human individual which it is desired to detect and / or identify, in a previously linearized plasmid, in order to obtain several plasmids each containing a unique sequence of DNA or a single fragment of amplified DNA,

- incorporation of each plasmid obtained in the previous step, respectively into a bacterium, in particular Escherichia coli (E. coli),

- multiplication of each of the bacteria obtained in the previous step, by culturing said bacteria in an appropriate medium, in the presence of an antibiotic, in particular ampicillin, in order to select the bacteria which have incorporated a plasmid in which inserted a DNA fragment or a DNA sequence, - separation of each of the plasmids containing a unique DNA sequence or a unique DNA fragment, of each of the bacteria containing said plasmids, in order to recover all of the plasmids or “plasmid DNAs” thus multiplied, said plasmids each containing a unique DNA sequence or a unique DNA fragment,

- removal of a sufficient number of plasmids (or “plasmid DNA”) from all of the plasmids (or “plasmid DNA”) obtained at the end of the previous step, in order to detect and / or identify at at least two different DNA fragments or DNA sequences, each of said DNA sequences or DNA fragments being characteristic of a given species, population or animal or plant race, or of a human individual.

15. A detection and / or identification method according to claim 14, in which each of the plasmid DNAs obtained after cloning is identified by sequencing, which makes it possible to identify the DNA characteristic of each of the species, animal populations or races and / or each of the plant species, populations or races and / or each of the human individuals present in the mixture of organic origin.

16. Oligonucleotides characterized in that they are chosen from those:

1) having a sequence identity of at least 80%, preferably 90% and advantageously 95% with an oligonucleotide consisting of a sequence of approximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides, included in the sequence SEQ ED N ° 21 following (position 16123 to 16144 according to Hurst, et al, 1999. Gene 239: 237-242):

GCC GAATGTAAAGCλTCT GG

2) or those having a sequence identity of at least 80%, preferably 90% and advantageously 95% with an oligonucleotide consisting of a sequence of approximately 15 to 25 nucleotides, especially 20 to 25 nucleotides, included in the sequence SEQ ED No. 22 following (position 16341 to 16361 after Hurst, et al, 1999. Gene 239: 237-242):

ACC TTATGC ACT TGATATCC

3) or those having a sequence identity of at least 80%, preferably 90% and advantageously 95% with an oligonucleotide consisting of a sequence of approximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides, included in the sequence SEQ ED No. 24 following (position 14985 to 14996 according to Hurst, et al., 1999. Gene 239: 237-242):

ACC GGGTCTAAT AAC CCAGC

4) or those having a sequence identity of at least 80%, preferably 90% and advantageously 95% with an oligonucleotide consisting of a sequence of approximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides, included in the sequence SEQ ED No. 25 following (position 15409 to 15430 according to Hurst, et al, 1999. Gene 239: 237-242): ATGATA ATGAAT GGG TGT TC

5) or those having a sequence identity of at least 80%, preferably 90% and advantageously 95% with an oligonucleotide consisting of a sequence of approximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides, included in the sequence SEQ ED No. 28 following (position 356 to 375 after Albert, et l., 1992, Science 257 (5076), 1491-1495):

AAAGCT CTGCGC GCTCTACG

6) or those with a sequence identity of at least 80%, preferably

90% and advantageously 95% with an oligonucleotide consisting of a sequence of approximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides, included in the following sequence SEQ ED No. 29 (position 539 to 558 according to Albert, et al , 1992, Science 257 (5076), 1491-1495):

CCG CGGAGA CAT TCATAAAC

17. Pair of primers characterized in that they consist of:

- by oligonucleotides SEQ ED N ° 21 and SEQ ED N ° 22 - by oligonucleotides SEQ ED N ° 24 and SEQ ED N ° 25

- with the oligonucleotides SEQ ED N ° 28 and SEQ ED N ° 29

18. DNA fragment as amplified using the primer pairs according to claim 17, and comprising approximately 100 to approximately 500 base pairs.

19. DNA fragment according to claim 18, characterized in that it has a sequence identity of at least 80% ", preferably 90%, and advantageously 95% with at least one of the sequences contained in:

- the following SEQ ED N ° 23:

GCCBWWHD R VVRCAYSTKB BBMWTRVWKH MRATC YR T GCSCGTTRMT CRCVRMRYCK

DBCRBYYTHT TR VYRCHYM BGGKWSYYYT YH TKY TYY TTWYCKYHYR GSKKGYMYDY

MCMRRTRSMA BYDMRRRRSK CYVRMRMBSK MRVMCYVKAY STYGMATTCC AGAGARYMYM

TGYVTY KVK YSM RYSHYA THCTMTHADK DATYRC YMH TKRGAYRKYH RARYATAHRG KBRAT where B is C, G or T, W is A or T, H is A, C or T, D is A, G or T, R is A or G, V is A, C or G, Y is C or T, S is C or G, K is G or T, M is A or C,

- or the following SEQ ED N ° 26:

CVGGVTCHA AYAACCCMVY AGGHATY MM TCMSAYKYHG AYAAAATYHC MTTYCACCCH

TACTWY CMW WYAARGACVY YYTNGGMTTN VYHSYYWTMC THMYYKBHMT RAYAYYMYTA

RYHCTRTTCK CMCCMRACCT CCTMGGVGAC CCRGAHAAYT YACVCY GC MAAYCCMYTM

RWHACHCCHC CHCA ATCAA RCCHGARTGA TAYTTYYTAT TYGCMTAYRC MATYYTHCGM TCMRTYCYAA YAAACTWGGM GGHGTMCTHG CCCTMKYMBY MTCNRTCCTV RTYCTHDYHV

TMRTYCCYHT MCTMCAYAHM TCYAARCAAC RMRSMMTRAY MTTYCGMCCM CTMWSCCAAW

BMYTWTWYTG RVYYCTRGYM GCVRACMTHC TNAYHCTHAC MTGAATYGGR RGVMWACCHG TVRRMYACCC HT YAYYAYC ATYGG

where W is A or T, V is A, C or G, H is A, C or T, Y is C or T, M is A or C, S is C or G, K is G or T, R is A or G, N is A, C, G or T, D is A, G or T, B is C, G or T,

- or the following SEQ ED N ° 30:

AAAGCTCTGC GCGCTCTACG TCTARAGGAT CTGCGAATCC CCCTGCTTAT ACTAAAACTT

TCCAAGGCCC GCCTCATGGC ATCCAAGTTG AGAGAGATAA ATTGAACAAG TATGGTCGTC

CCCTATTGGG ATGTACTATT AAACCTAAAT TGGGGTTATC CGCTAAGAAC TATGGTAGAG C GTTTATGA ATGTCTCCGC GG

where R is A or G, W is A or T.