EP1212360A2 - Nucleotide sequences derived from genes coding for trimethylamine n-oxide reductase, uses thereof in particular for detecting bacteria - Google Patents

Abstract

The invention concerns the use of nucleotide sequences selected among those comprising a sequence coding for a protein of the trimethylamine N-oxide reductase (TMAO reductase) system in bacteria, or a fragment or a sequence derived from said sequence for implementing a method for detecting the presence of all bacteria involved in the process of decaying of aquatic animal flesh, in a host suspected of carrying such bacteria. The invention also concerns nucleotide sequences such as defined above, said detecting methods as well as kits for implementing said methods.

Description

NUCLEOTIDE SEQUENCES FROM GENES ENCODING TRIMETHYLAMINE N-OXIDE REDUCTASE, AND USES THEREOF, IN PARTICULAR FOR DETECTION OF BACTERIA.

The present invention relates to nucleotide sequences originating from genes coding for trimethylamine N-oxide reductase (TMAO reductase) and usable, in particular as primers, for the implementation of methods for detecting bacteria involved in the alteration the flesh of aquatic animals, and more particularly of marine animals. Trimethylamine N-oxide (TMAO) is one of the main components of small molecular weight in marine animals, where it can represent up to 1% of their dry weight. In anaerobic conditions, certain bacteria use TMAO as an exogenous electron acceptor for their respiration. The TMAO is then reduced to trimethylamine (TMA), a highly volatile compound, which is largely responsible for the foul odor of decomposing fish. The reduction of TMAO to TMA has been demonstrated in marine bacteria, such as those of the genus Shewanella, Photobacterium or Vibrio (1, 2), in photosynthetic bacteria isolated in brackish water, such as those of the genus Rhodobacter (3), but also in several enterobacteria, such as Escherichia coli, Salmonella typhimurium or Proteus vulgaris (4). The enzyme responsible for the reduction of TMAO to TMA, corresponds to trimethylamine Ν-oxide reductase (TMAO reductase), the properties of which have been studied in various organisms. For example, in E. coli there are two separate enzymes responsible for the reduction of TMAO: TMAO reductase and DMSO reductase. TMAO reductase, which is responsible for 90% of the reduction in TMAO, is a 90 kDa periplasmic molybdoenzyme, which can be found as a monomer or dimer (5). The TMAO reductases of the various bacteria studied are all 80-100 kDa molybdoenzymes which can be found in the form of a monomer, dimer or tetramer. These proteins are all inducible anaerobically by TMAO, but also by DMSO (dimethyl sulfoxide). Finally, except in P. vulgaris, the TMAO reductases are all localized in the periplasm of the bacteria.

The study of the TMAO reductase system at the genetic and molecular level was carried out in E. coli enterobacteria and those of the genus Rhodobacter. The two TMAO DMSO reductases from bacteria of the genus Rhodobacter and the inducible TMAO reductase from E. coli show homology at the level of their primary sequence (6, 7, 8). The overall structure of TMAO reductase from E. coli could be similar to those described for Rhodobacter sphaeroides or Rhodobacter capsulatus. In E. coli, the genes that code for the different components of the TMAO reductase system are organized into an operon: the torCAD operon, which codes for the three proteins TorC, TorD and TorA (6). In Rhodobacter, the genes which code for the various components of the TMAO / DMSO reductase system are also organized into operons: the operon dorCDA, which codes for three proteins DorC, DorD (also called DorB) and DorA, having homologies with respectively the proteins TorC, TorD and TorA from E. coli (9, 10). A phylogenetic study by sequencing of 16S rDNA was carried out on a bacterium isolated from a fish caught in the bay of Marseille, and kept at room temperature in filtered sea water. The results obtained show that this bacterium, which has a significant TMAO reductase activity, corresponds to a new species of the genus Shewanella, close to Shewanella putrefaciens. She was called Shewanella massilia. With the exception of the TorE protein, the products of the torECAD operon genes of S. massilia have strong homologies with the proteins involved in the TMAO reductase systems of E. coli and bacteria of the genus Rhodobacter (19). Likewise, the overall structure of the TMAO reductase of S. massilia exhibits homologies with the structures already described of the TMAO / DMSO reductases of bacteria of the genus Rhodobacter (20). The deterioration of fish flesh, like most foods, is mainly due to the metabolic activity of certain bacteria, which can represent up to 10 ⁸ cells per gram of product. The first post-mortem changes that occur in fish flesh are not necessarily due to bacteria, but to certain autolysis reactions. However, microbial development quickly becomes the major element of spoilage, the muscle tissue of the fish constituting a substrate for bacterial growth. Several factors intrinsic to fish flesh can explain their very high alterability, compared to other food products: significant hydration, a high post-mortem pH (> 6), a high content of non-protein nitrogenous substances, of which TMAO forms the essential part and, a low content of supporting proteins (11, 12). These different characteristics contribute to the development of a microbial flora of specific alteration of fish flesh. Interestingly, the diversity of fish species does not seem to affect the nature or number of bacteria initially present on Fish. In contrast, the bacterial microflora of fish seem to be directly linked to the natural environment in which they live. Thus, the contamination of water, which essentially depends on temperature, salinity or the concentration of dissolved oxygen, seems to play a decisive role. The origin of the bacteria found in fish flesh does not only come from the microflora of live fish: contaminating bacteria can in particular come from the cold chain (ice, containers containing the fish, processing steps, etc.). .). Among all the microbial flora found in fish flesh, only a part is made responsible for the putrefaction phenomenon. While bacterial contamination does not necessarily make fish flesh unfit for consumption, some bacteria quickly cause unpleasant changes in texture, flavor or odor (13).

Seafood therefore represents a highly perishable commodity, posing many conservation problems for fishing industry. The control of the freshness quality of these products is therefore an increasingly important concern for the entire fishing sector (producers, processors or distributors), leading for a few years manufacturers to seek new techniques making it possible to account the state of freshness of the fish in a very short time.

Currently, the determination of the state of freshness of seafood products is essentially based on a sensory assessment (appearance, odor, flavor) which allows at best to conclude with a fresh product, moderately fresh or unfit for consumption. This organoleptic method is however only applicable to whole products, and more reliable laboratory analyzes are often essential.

The methods based on a bacteriological analysis (total bacterial flora) applicable to processed products are used more and more to assess the state of freshness of the fish. Direct estimation of the number of cultivable bacteria on a fish sample remains the most commonly used method: after growth on different media (for example iron citrate medium), each cell will form a visible colony on petri dishes, allowing thus a count. Although this technique is easy to use and quite sensitive, it still requires a significant incubation time for growth (3 to 5 days approximately). In addition, the composition of the growth medium, or the temperatures used for these tests (20-30 ° C), do not always make it possible to detect all of the spoilage bacteria. Thus, the current regulations which advocate a temperature of 30 ° C to carry out these tests (decree of 1979), does not make it possible to be able to detect certain bacteria of deterioration like Photobacterium phosphoreum which do not grow at this temperature. Thus, even if the non-specific measurement of the total microbial load constitutes a good indicator of the state of hygiene of the fish, it does not however make it possible to assess the sensory quality of this type of product. Microscopic examination of food is a quick technique to estimate the level of bacterial contamination. The cells stained with acridine orange are visualized by a fluorescence microscopy technique; the main drawback of this method, however, remains its low sensitivity (between 10 ⁴ and 10 ⁵ cells). Degradation of the cellular components of fish leads to the production of many substances which can serve as an indirect indicator of bacterial contamination. An alternative to the methods listed above therefore consisted in following, by chemical techniques, different degradation products of fish flesh (14). The determination of total volatile basic nitrogen (ABVT), of which ammonia (NH ₃ ) and trimethylamine (TMA) form the essential part, is a widely used technique today to estimate the degree of decomposition of the flesh. of fish. The assay of ABVT has the advantage of being simple, rapid and of a very low cost price. The determination of the rate of TMA in the ABVT, makes it possible to bring an additional qualitative element to the criterion ABVT. This criterion seems in fact less dependent on variations which generally affect the rate of TMA or ABVT (the species of fish, its fat content etc ...). The same methodology as for ABVT can be used to measure TMA. Other colorimetric, enzymatic or chromatographic techniques have also been described to quantify the TMA (15, 16). During the reaction of reduction of TMAO to TMA by bacteria, the flesh of decomposing fish undergoes various changes: decrease in the redox potential, an increase in pH, in electrical conductivity. Several studies have therefore also used these different indicators to attempt to indirectly monitor the release of TMA. However, all of these studies have never made it possible to establish a definitive correlation between the level of TMA released and the freshness quality of the fish samples. Many other degradation products have also been tested in order to be able to determine the level of fish spoilage by chemical assays: hydrogen sulfide, hypoxanthine, indol, histamine and amino acids. However, these different dosages are only significant in certain fish species, or at a very advanced level of spoilage.

The polymerase chain reaction (PCR) is a molecular technique that amplifies large amounts of a target DNA sequence in order to visualize it. In recent years, the PCR technique has been used more and more for the detection and identification of microorganisms, particularly in the field of clinical bacteriology or food safety (17, 18). Currently, PCR tests applied to food products mainly allow the detection of well-defined pathogenic bacteria (Shigella, Salmonella, Listeria ...). The present invention follows from the discovery by the inventors of the fact that the DNA sequences coding for a protein of the TMAO reductase system have sufficient homology within the various bacteria, in particular within the bacteria responsible for the alteration of the tissues of aquatic organisms, and more particularly within marine bacteria, to allow the implementation of methods for detecting all bacteria responsible for the alteration of tissues by their TMAO reductase activity, said methods being applicable to any aquatic animal, and especially on all marine animals.

One of the aims of the present invention is to provide a test of the freshness quality of aquatic products, and more particularly of seafood, which is both rapid, inexpensive, and offering high reliability in terms of specificity and sensitivity. . One of the other aims of the present invention is to provide a test for the detection, not of a bacterial type, but of all of the bacteria responsible for the deterioration of the tissues of aquatic organisms, and in particular the putrefaction of fish flesh, namely both marine bacteria, enterobacteria or bacteria isolated from brackish waters. Another object of the present invention is to provide a test for the detection of bacteria responsible for the alteration of the tissues of marine organisms, applicable to different species of fish from different geographic regions.

Another object of the present invention is to provide a test for detecting bacteria responsible for the alteration of the tissues of marine organisms, sensitive enough to detect early stages of the alteration.

The subject of the present invention is the use of nucleotide sequences chosen from those comprising a sequence coding for a protein of the trimethylamine N-oxide system. reductase (TMAO reductase) in bacteria, or a fragment of this sequence such as a probe or a primer of about 15 to about 25 nucleotides, or a sequence derived by addition, deletion, and / or substitution of one or more nucleotides of this sequence coding for a protein of the TMAO reductase system or of a fragment of the latter, said fragment and said derived sequence being capable of hybridizing, in particular under the hybridization conditions defined below, with said coding sequence for a protein of the TMAO reductase system, for the implementation of a method for detecting the presence of all bacteria involved in the process of degradation of the flesh of aquatic animals, in a host capable of carrying such bacteria. Advantageously, the nucleotide sequences coding for a protein of the system

TMAO reductase mentioned above, are chosen from those corresponding to the torCAD operon or the DA gold operon mentioned above, of the TMAO reductase system in bacteria.

Preferably, the sequence coding for a protein of the aforementioned TMAO reductase system is chosen from the sequences coding for TMAO reductase in bacteria, also designated protein TorA or DorA, or coding for a cytochrome type c in bacteria, also designated TorC or DorC protein.

According to a particularly advantageous embodiment of the invention, the nucleotide sequences coding for a protein of the TMAO reductase system are chosen from those of the following bacteria: - marine bacteria, such as those of the genus Shewanella, Photobacterium or Vibrio, said sequences being chosen in particular among the following:

* the sequence SEQ ID NO: 1 coding for the TorA protein of Shewanella massilia represented in FIG. 1,

* the sequence SEQ ID NO: 2 coding for the TorA protein of Shewanella putrefaciens represented in FIG. 1,

* the sequence SEQ ID NO: 3 coding for the protein TorA of Shewanella c represented in FIG. 2,

* the partial sequence SEQ LO NO: 4 coding for the TorA protein of Photobacterium phosphoreum represented in FIG. 3, * the sequence coding for the TorC protein SEQ ID NO: 5 of Shewanella massilia represented in FIG. 14,

- bacteria from brackish water, such as those of the genus Rhodobacter, or Roseobacter, said sequences being chosen in particular from the following:

* the sequence SEQ ID NO: 6 coding for the DorA protein of Rhodobacter sphaeroides represented in FIG. 4,

* the sequence SEQ ID NO: 7 coding for the DorA protein of Rhodobacter capsulatus represented in FIG. 4,

* the sequence coding for the DorC protein SEQ ED NO: 8 of Rhodobacter sphaeroides represented in FIG. 14,

- enterobacteria, such as those of the genus Escherichia, or Salmonella, said sequences being chosen in particular from the following: * the sequence SEQ ID NO: 9 coding for the protein TorA of Escherichia coli shown in FIG. 4,

* the partial sequence SEQ ID NO: 10 coding for the TorA protein of Salmonella typhimurium represented in FIG. 5,

* the sequence coding for the protein TorC SEQ ID NO: 11 from Escherichia coli represented in FIG. 14.

A more particular subject of the invention is the abovementioned use of nucleotide sequences chosen from the fragments of the sequences defined above, or from the sequences derived from these fragments, said fragments or derived sequences comprising approximately 15 to 25 nucleotides, and are used in pairs to form pairs of primers allowing the amplification of fragments of genes coding for a protein of the TMAO-reductase system of bacteria involved in the degradation of the flesh of aquatic animals, according to the PCR technique.

Advantageously, the abovementioned nucleotide sequences are used in the form of primer pairs chosen from any one of the following three primer groups: (1) the “DDN” primer group amplifying DNA fragments of the torA gene, this group comprising:

* the following “DDN +” nucleotide sequence compositions:

- DDN1 + (SEQ ID NO: 12): 5 'CGG vGA yTA CTC bAC hGG TGC 3': mixture of 54 nucleotide sequences, - DDN5 + (SEQ ID NO: 13): 5 'ATy GAT GCG ATy CTC GAA CC 3': mixture of 4 nucleotide sequences,

* the following “DDN-” nucleotide sequence compositions: - DDN2- (SEQ ID NO: 14): 5'CGT Amw sGT CGA kAT CGT TrC GCT C 3 ': mixture of 32 nucleotide sequences,

- DDN3- (SEQ ID NO: 15): 5 'GAC TCA CAy Awy TGy GAG TG 3': mixture of 16 nucleotide sequences, - DDN4- (SEQ ID NO: 16): 5 'TGr CCd CGr kCG TTA AAG AC 3 ': mixture of 24 nucleotide sequences,

- DDN5- (SEQ ID NO: 17): 5 'CCv GGT TCG AGr ATC GCA TC 3': mixture of 6 nucleotide sequences,

(2) the group of primers "BN" amplifying DNA fragments of the torA gene, this group comprising:

* the following “BN +” nucleotide sequence compositions:

- BN1 + (SEQ ID NO: 18): 5 'C bGA yAT CsT rCT GCC 3': mixture of 16 nucleotide sequences,

- BN3 + (SEQ ID NO: 19): 5 'GGm GAy TAy TCb ACm GGy GC 3': mixture of 96 nucleotide sequences,

- BN6 + (SEQ ID NO: 20): 5 'Twy GAr CGy AAC GAy mTC GA 3': mixture of 64 nucleotide sequences,

* the following “BN-” nucleotide sequence compositions:

- BN2- (SEQ ID NO: 21): 5 'GG vyC rTA CCA bsC vCC TTC 3': mixture of 216 nucleotide sequences,

- BN4- (SEQ ID NO: 22): 5 'ATC Arr CCn swv GGC GTG CC 3': mixture of 192 nucleotide sequences,

- BN5- (SEQ ID NO: 23): 5 'GbC ACr TCd GTy TGy GG 3': mixture of 72 nucleotide sequences, (3) the group of primers "BC" amplifying DNA fragments of the torC gene, this group comprising:

* the following “BC +” nucleotide sequence compositions:

- BC1 + (SEQ ID NO: 24): 5 'ACn CCn GAr AAr TTy GAr GC 3': mixture of 256 nucleotide sequences, - BC2 + (SEQ ID NO: 25): 5 'TGy ATh GAy TGy CAy AAr GG 3': mixture of 96 nucleotide sequences, the following “BC-” nucleotide sequence compositions: - BC2- (SEQ ID NO: 26): 5 'CCy TTr TGr CAr TCd ATr CA 3': mixture of 96 nucleotide sequences,

- BC3- (SEQ ID NO: 27): 5 'TTn GCr TCr AAr TGn GC 3': mixture of 128 nucleotide sequences, in which n = (A, C, G, T), y = (C, T), r = (A, G), h = (A, C, T), d = (G, A, T), m = (A, C), w = (A, T), b = (G, T , C), s = (G, C), v = (G, A, C), and k = (G, T), the pairs of primers being chosen such that one of the primers d 'a pair corresponds to one of the above-mentioned compositions of nucleotide sequences DDN +, BN + or BC +, while the other primer corresponds respectively to one of the above-mentioned compositions of nucleotide sequences DDN-, BN- or BC-, said pairs d primers being chosen in particular from any one of the following four pairs:

(a) the DDN1 + / DDN5- couple, leading to the amplification of fragments of the gene coding for the protein TorA in bacteria, with a size of approximately 820 base pairs (bp), and in particular to the amplification of an 821 bp fragment of the gene coding for the TorA protein in bacteria of the genus Shewanella, such as the 821 bp fragment delimited by the nucleotides located at positions 620 to 1450 of the torA gene of S. massilia represented in FIG. 4,

(b) the BN6 + / BN2- pair, leading to the amplification of fragments of the gene coding for the protein TorA in bacteria, of a size of approximately 710 bp, and in particular to the amplification of a fragment of 727 bp of the gene coding for the protein TorA in bacteria of the genus Shewanella, such as the fragment of 727 bp delimited by the nucleotides located at positions 1657 to 2403 of the torA gene of S. massilia represented in FIG. 4,

(c) the BN6 + / BN4- pair, leading to the amplification of fragments of the gene coding for the protein TorA in bacteria, of a size of approximately 360 bp, and in particular to the amplification of a fragment of 355 bp of the gene coding for the protein TorA in bacteria of the genus Shewanella, such as the fragment of 355 bp delimited by the nucleotides located at positions 1657 to 2023 of the torA gene of S. massilia represented in FIG. 4,

(d) the couple BC1 + / BC2-, leading to the amplification of fragments of the gene coding for the protein TorC in bacteria, of a size of approximately 170 bp, and in particular to the amplification of a fragment of 197 bp of the gene coding for the TorC protein in bacteria of the genus Shewanella, such as the 197 bp fragment coding for the polypeptide fragment delimited by the amino acids located at positions 114 to 179 of the TorC protein of S. massilia shown in Figure 14.

The hosts likely to be carriers of bacteria involved in the process of degradation of the flesh of aquatic animals, as described above, are aquatic organisms, in particular marine organisms such as fish and crustaceans, and more particularly the Atlantic fish such as sole, cod, or Mediterranean sea fish such as red mullet, sea bream, as well as some fresh or brackish water animals.

A more particular subject of the invention is the aforementioned use of sequences, nucleotides described above, for the implementation of a method for detecting the presence of any bacteria involved in the degradation of the flesh of aquatic animals, in as part of a process for evaluating the state of freshness of the aquatic animals from which the test sample is taken, when the latter are extracted from their natural environment.

The invention also relates to any nucleotide sequence corresponding to one of the following sequences:

- DDN1 +: 5 'CGG vGA yTA CTC bAC hGG TGC 3',

- DDN5 +: 5 'ATy GAT GCG ATy CTC GAA CC 3',

- DDN2-: 5 'CGT Amw sGT CGA kAT CGT TrC GCT C 3',

- DDN3-: 5 'GAC TCA CAy Awy TGy GAG TG 3', - DDN4-: 5 'TGr CCd CGr kCG TTA AAG AC 3',

- DDN5-: 5 'CCv GGT TCG AGr ATC GCA TC 3',

- BN1 +: 5 'C bGA yAT CsT rCT GCC 3',

- BN3 +: 5 'GGm GAy TAy TCb ACm GGy GC 3',

- BN6 +: 5 'Twy GAr CGy AAC GAy mTC GA 3', - BN2-: 5 'GG vyC rTA CCA bsC vCC TTC 3',

- BN4-: 5 'ATC Arr CCn swv GGC GTG CC 3',

- BN5-: 5 'GbC ACr TCd GTy TGy GG 3',

- BC1 +: 5 'ACn CCn GAr AAr TTy GAr GC 3',

- BC2 +: 5 'TGy ATh GAy TGy CAy AAr GG 3', - BC2-: 5 'CCy TTr TGr CAr TCd ATr CA 3',

- BC3-: 5 'TTn GCr TCr AAr TGn GC 3', in which n = (A, C, G, T), y = (C, T), r = (A, G), h = (A, C, T), d = (G, A, T), m = (A, C), w = (A, T), b = (G, T, C), s = (G, C), v ≈ (G, A, C), and k = (G, T).

The subject of the invention is also any composition of nucleotide sequences in mixture, corresponding to one of the following compositions:

* the following “DDN +” nucleotide sequence compositions: - DDN1 +: 5 'CGG vGA yTA CTC bAC hGG TGC 5': mixture of 54 nucleotide sequences,

- DDN5 +: 5 'ATy GAT GCG ATy CTC GAA CC 3': mixture of 4 nucleotide sequences,

* the following “DDN-” nucleotide sequence compositions: - DDN2-: 5 'CGT Amw sGT CGA kAT CGT TrC GCT C i': mixture of 32 nucleotide sequences,

- DDN3-: 5 'GAC TCA CAy Awy TGy GAG TG 3': mixture of 16 nucleotide sequences,

- DDN4-: 5 'TGr CCd CGr kCG TTA AAG AC': mixture of 24 nucleotide sequences,

- DDN5-: 5 'CCv GGT TCG AGr ATC GCA TC 3': mixture of 6 nucleotide sequences, the following “BN +” nucleotide sequence compositions:

- BN1 +: 5 'C bGA yAT CsT rCT GCC 3': mixture of 16 nucleotide sequences, - BN3 +: 5 'GGm GAy TAy TCb ACm GGy GC 5': mixture of 96 nucleotide sequences,

- BN6 +: 5 'Twy GAr CGy AAC GAy mTC GA 3': mixture of 64 nucleotide sequences,

* the following “BN-” nucleotide sequence compositions: - BN2-: 5 'GG vyC rTA CCA bsC vCC TTC 3': mixture of 216 nucleotide sequences,

- BN4-: 5 'ATC Air CCn swv GGC GTG CC 5': mixture of 192 nucleotide sequences,

- BN5-: 5 'GbC ACr TCd GTy TGy GG 3': mixture of 72 nucleotide sequences, * the following "BC +" nucleotide sequence compositions:

- BC1 +: 5 'ACn CCn GAr AAr TTy GAr GC 3': mixture of 256 nucleotide sequences, - BC2 +: 5 'TGy ATh GAy TGy CAy AAr GG 5': mixture of 96 nucleotide sequences, the following compositions of “BC-” nucleotide sequences:

- BC2-: 5 'CCy TTr TGr CAr TCd ATr CA 3': mixture of 96 nucleotide sequences, - BC3-: 5 'TTn GCr TCr AAr TGn GC 3': mixture of 128 nucleotide sequences, in which n = (A, C, G, T), y = (C, T), r = (A, G), h = (A, C, T), d = (G, A, T), m = (A, C) , w = (A, T), b = (GTC), s = (G, C), v = (G, A, C), and k = (G, T).

The invention also relates to the pairs of primers chosen from one of the following groups of primers: (1) the “DDN” group of primers comprising:

* the following “DDN +” nucleotide sequence compositions:

- DDN1 +: 5 'CGG vGA yTA CTC bAC hGG TGC 3': mixture of 54 nucleotide sequences,

- DDN5 +: 5 'ATy GAT GCG ATy CTC GAA CC': mixture of 4 nucleotide sequences, the following “DDN-” nucleotide sequence compositions:

- DDN2-: 5 'CGT Amw sGT CGA kAT CGT TrC GCT C i': mixture of 32 nucleotide sequences,

- DDN3-: 5 'GAC TCA CAy Awy TGy GAG TG i': mixture of 16 nucleotide sequences,

- DDN4-: 5 'TGr CCd CGr kCG TTA AAG AC i': mixture of 24 nucleotide sequences,

- DDN5-: 5 'CCv GGT TCG AGr ATC GCA TC i': mixture of 6 nucleotide sequences, (2) the group of primers "BN" comprising:

* the following “BN +” nucleotide sequence compositions:

- BN1 +: 5 'C bGA yAT CsT rCT GCC i': mixture of 16 nucleotide sequences,

- BN3 +: J 'GGm GAy TAy TCb ACm GGy GC i': mixture of 96 nucleotide sequences, - BN6 +: 5 'Twy GAr CGy AAC GAy mTC GA i': mixture of 64 nucleotide sequences, the nucleotide sequence compositions "BN- »Following: - BN2-: 5 'GG vyC rTA CCA bsC vCC TTC i': mixture of 216 nucleotide sequences,

- BN4-: 5 'ATC Arr CCn swv GGC GTG CC i': mixture of 192 nucleotide sequences, - BN5-: 5 'GbC ACr TCd GTy TGy GG i': mixture of 72 nucleotide sequences,

(3) the group of primers "BC" comprising: the following compositions of nucleotide sequences "BC +":

- BC1 +: 5 'ACn CCn GAr AAr TTy GAr GC 3': mixture of 256 nucleotide sequences, - BC2 +: 5 'TGy ATh GAy TGy CAy AAr GG i': mixture of 96 nucleotide sequences,

* the following “BC-” nucleotide sequence compositions:

- BC2-: 5 'CCy TTr TGr CAr TCd ATr CA i': mixture of 96 nucleotide sequences,

- BC3-: 5 'TTn GCr TCr AAr TGn GC 3': mixture of 128 nucleotide sequences, in which n = (A, C, G, T), y = (C, T), r - (A, G) , h = (A, C, T), d = (G, A, T), m = (A, C), w = (A, T), b = (G, T, C), s = ( G, C), v = (G AC), and k = (G, T), the pairs of primers being chosen such that one of the primers of a pair corresponds to one of the compositions of above-mentioned DDN +, BN + or BC + nucleotide sequences, while the other primer corresponds respectively to one of the above-mentioned compositions of DDN-, BN- or BC- nucleotide sequences, said pairs of primers being chosen in particular from any one of the following four couples:

(a) the DDN1 + / DDN5- couple, leading to the amplification of fragments of the gene coding for the protein TorA in bacteria, with a size of approximately 820 base pairs (bp), and in particular to the amplification of an 821 bp fragment of the gene coding for the TorA protein in bacteria of the genus Shewanella, such as the 821 bp fragment delimited by the nucleotides located at positions 620 to 1450 of the torA gene of S. massilia represented in FIG. 4,

(b) the BN6 + / BN2- pair, leading to the amplification of fragments of the gene coding for the protein TorA in bacteria, of a size of approximately 710 bp, and in particular to the amplification of a fragment of 727 bp of the gene coding for the protein TorA in bacteria of the genus Shewanella, such as the fragment of 727 bp delimited by the nucleotides located at positions 1657 to 2403 of the torA gene of S. massilia represented in FIG. 4, (c) the BN6 + / BN4- pair, leading to the amplification of fragments of the gene coding for the protein TorA in bacteria, of a size of approximately 360 bp, and in particular to the amplification of a fragment of 355 bp of the gene coding for the protein TorA in bacteria of the genus Shewanella, such as the fragment of 355 bp delimited by the nucleotides located at positions 1657 to 2023 of the torA gene of S. massilia represented in FIG. 4,

(d) the couple BC1 + / BC2-, leading to the amplification of fragments of the gene coding for the protein TorC in bacteria, of a size of approximately 170 bp, and in particular to the amplification of a fragment of 197 bp of the gene coding for the TorC protein in bacteria of the genus Shewanella, such as the fragment of 197 bp coding for the polypeptide fragment delimited by the amino acids located at positions 114 to 179 of the TorC protein of S. massilia represented in the figure 14.

The subject of the invention is also a method of detecting all bacteria involved in the degradation of the flesh of aquatic animals in a host capable of carrying such bacteria, said method being carried out from a biological sample taken from this host, this biological sample corresponding in particular to a subcutaneous fragment of the flesh of the aquatic animal in question, and being characterized in that it comprises a step of hybridization of at least one nucleotide sequence as defined above , with fragments of genes coding for a protein of the TMAO-reductase system of bacteria involved in the degradation of the flesh of aquatic animals likely to be present in the biological sample taken from said host, followed by a revelation step, in particular by electrophoresis, of the possible presence in said sample of genes coding for a protein of the system TMAO-reductase, or fragments of these genes, the copy number of which has been amplified if necessary.

The invention more particularly relates to a detection method as defined above, characterized in that it comprises the following steps:

the treatment of a biological sample taken from this host in order to extract the total DNA of this host and to make the genome of these bacteria accessible to the nucleotide sequences or primers defined above, this treatment being carried out in particular at using a rapid DNA extraction technique based on the attachment of nucleic acids to silica beads, - the amplification of the number of copies of genes coding for the proteins of the system

TMAO-reductase from bacteria involved in the degradation of the flesh of aquatic animals, or fragments of these genes, which may be present in this sample, using nucleotide sequences or primers mentioned above,

- the detection of the possible presence of an amplified number of copies of genes coding for a protein of the TMAO-reductase system of the above-mentioned bacteria, or of fragments of these genes, and therefore of the presence of such bacteria in the biological sample studied . Advantageously, the detection methods according to the invention are characterized in that the amplification of the number of genes coding for the proteins of the TMAO-reductase system comprises the following steps:

the predenaturation of the total double-stranded DNA of the host into single-stranded DNA, preferably in a buffer made up of 10 mM Tris-HCl pH 8.3, 50 mM KC1, 1.5 mMgCl ₂ , 0.01% of gelatin, of the 4 constitutive DNA deoxynucleotides (dCTP, dATP, dGTP, dTTP) at a concentration of 100 μM each, and pairs of primers, as defined above, by heating between approximately 90 ° C. and approximately 100 ° C, advantageously at 94 ° C., for approximately 1.5 minutes,

the actual amplification by addition to the medium obtained in the preceding stage of DNA polymerase, for example Taq polymerase, heating at approximately 94 ° C. for approximately 30 seconds, which corresponds to the denaturation stage proper,

* then heating between approximately 35 ° C to approximately 60 ° C, and in particular to approximately 45 ° C or 55 ° C, for approximately 30 seconds, which corresponds to the stage of hybridization of the primers with the genes coding for the proteins of the TMAO-reductase system of bacteria, or fragments of these genes, which may be present in the biological sample studied, and finally, heating at 72 ° C., for approximately 45 seconds, which corresponds to the step of elongation of the primers, hybridized in the preceding step, towards one another, thus producing nucleotide sequences complementary to fragments of the genes coding for the proteins of the TMAO-reductase system of bacteria, the latter sequences being delimited by the nucleotides s '' hybridizing with the aforementioned primers,

- repetition of the previous amplification step between approximately 15 and approximately 35 times, advantageously approximately 30 times.

The invention also relates to the kits for the implementation of a detection method mentioned above, characterized in that they comprise:

one or more nucleotide sequences or primers as defined above,

- a DNA polymerase, a reaction medium advantageously consisting of 10 mM Tris-HCl pH 8.3, 50 mM KC1, 1.5 mM MgCl ₂ , 0.01% of gelatin, of the 4 deoxynucleotides constituting DNA (dCTP, dATP, dGTP, dTTP) at a concentration of 100 μM each.

The invention also relates to any nucleotide sequence comprising: the sequence represented in FIG. 2 of the torA gene coding for the protein TorA of the marine bacterium Shewanella C,

- or any sequence derived from the above-mentioned sequence by degeneration of the genetic code, and coding for the protein TorA of Shewanella C, the peptide sequence of which is shown in FIG. 6, - or any sequence derived from the above-mentioned nucleotide sequence, in particular by substitution , deletion or addition of one or more nucleotides, said derived sequence preferably having a homology of approximately 35% to 100% with the abovementioned nucleotide sequence represented in FIG. 2,

- or any fragment of the above-mentioned nucleotide sequence, or of a sequence derived from the latter as defined above, said fragment preferably consisting of at least about 15 nucleotides.

The subject of the invention is also any peptide sequence encoded by the above-mentioned nucleotide sequence shown in FIG. 2, and comprising:

the amino acid sequence represented in FIG. 6 of the TorA protein of Shewanella c,

- or a sequence derived from the above-mentioned peptide sequence, in particular by substitution, deletion or addition of one or more amino acids, said derived sequence preferably having a homology of approximately 35% to 100% with the above-mentioned peptide sequence shown in the FIG. 6, - or a fragment of the above-mentioned peptide sequence, or of a sequence derived from the latter as defined above, said fragment preferably consisting of at least about 5 amino acids.

The invention also relates to any nucleotide sequence comprising:

the partial sequence represented in FIG. 3 of the gene coding for the protein TorA of the marine bacterium Photobacterium phosphoreum,

- or any sequence derived from the above-mentioned sequence by degeneration of the genetic code, and coding for the fragment of the protein TorA of Photobacterium phosphoreum whose peptide sequence is represented in FIG. 7,

- or any sequence derived from the abovementioned nucleotide sequence, in particular by substitution, deletion or addition of one or more nucleotides, said derived sequence preferably having a homology of approximately 35% to 100% with the abovementioned nucleotide sequence shown in the figure 3

- or any fragment of the above-mentioned nucleotide sequence, or of a sequence derived from the latter as defined above, said fragment preferably consisting of at least about 15 nucleotides.

The invention also relates to any peptide sequence encoded by the above-mentioned nucleotide sequence represented in FIG. 3, and comprising:

- the partial amino acid sequence shown in FIG. 7 of the TorA protein of Photobacterium phosphoreum,

- or a sequence derived from the above-mentioned peptide sequence, in particular by substitution, deletion or addition of one or more amino acids, said derived sequence preferably having a homology of approximately 35% to 100% with the above-mentioned peptide sequence shown in the figure 7,

- Or a fragment of the above-mentioned peptide sequence, or of a sequence derived from the latter as defined above, said fragment preferably consisting of at least about 5 amino acids. The subject of the invention is also any nucleotide sequence comprising:

the partial sequence represented in FIG. 5 of the gene coding for the protein TorA of the marine bacterium Salmonella typhimurium,

- or any sequence derived from the above-mentioned sequence by degeneration of the genetic code, and coding for the TorA protein of Salmonella typhimurium, the peptide sequence of which is shown in FIG. 8,

- or any sequence derived from the abovementioned nucleotide sequence, in particular by substitution, deletion or addition of one or more nucleotides, said derived sequence preferably having a homology of approximately 35% to 100% with the abovementioned nucleotide sequence shown in the figure 5, - or any fragment of the above-mentioned nucleotide sequence, or of a sequence derived from the latter as defined above, said fragment preferably consisting of at least about 15 nucleotides. The invention also relates to any peptide sequence encoded by the above-mentioned nucleotide sequence shown in FIG. 5, and comprising:

- the amino acid sequence shown in FIG. 8 of the TorA protein of Salmonella typhimurium, - or a sequence derived from the above-mentioned peptide sequence, in particular by substitution, deletion or addition of one or more amino acids, said derived sequence having preferably a homology of about 35% to 100% with the above-mentioned peptide sequence represented in FIG. 8,

- Or a fragment of the above-mentioned peptide sequence, or of a sequence derived from the latter as defined above, said fragment preferably consisting of at least about 5 amino acids.

The invention will be further illustrated with the aid of the detailed description which follows of obtaining primers of the invention, and of their use for the implementation of a detection method according to the invention. Description of the figures

- Figure 1 shows the alignment of the nucleotide sequences of the torA genes of Shewanella massilia (torA / S.m.), Shewanella c (torA / S.c), and Shewanella putrefaciens (torA / S.p.).

- Figure 2 shows the complete nucleotide sequence of the torA gene encoding the TMAO reductase from Shewanella c.

FIG. 3 represents the partial nucleotide sequence of the torA gene coding for the TMAO reductase of Photobacterium phosphoreum.

FIG. 4 represents the alignment of the nucleotide sequences of the torA genes of Shewanella massilia (torA / Sm), of E. coli (torA / Ε. c), of Rhodobacter sphaeroides (TorA / Rs), and Rhodobacter capsulatus (TorA / rc). The arrows indicate the position of the different primers on the torA gene of the bacterium Shewanella massilia, developed from the conserved regions. More particularly, the positions of the various primers "DDN" and "BN" on the torA gene of S. massilia are as follows:

DDN1 +: 620 BN1 +: 1628 DDN5 +: 1428 BN3 +: 621

DDN2-: 1684 BN6 +: 1657

DDN3-: 2201 BN2-: 2403 DDN4-: 2325 BN4-: 2023

DDN5-: 1450 BN5-: 946

- Figure 5 shows the partial nucleotide sequence of the torA gene coding for the TMAO reductase of Salmonella typhimurium. - Figure 6 shows the complete peptide sequence of TMAO reductase from

Shewanella c.

FIG. 7 represents the alignment of the peptide sequences of the fragment of the protein TorA of Photobacterium phosphoreum (TorA / Pp), deduced from the DNA fragment amplified in P. phosphoreum using the molecular primers DDN1 + / DDN5-, with the corresponding protein regions of the TorA proteins of Shewanella massilia (TorA / Sm), of E. coli (TorA / Ε.c), and of Rhodobacter sphaeroides (DorA / Rs).

FIG. 8 represents the alignment of the peptide sequences of the fragment of the TorA protein of Salmonella typhimurium (TorA / St), deduced from the DNA fragment amplified in S. typhimurium using the molecular primers DDN1 + / DDN5-, with the corresponding protein regions of the E. coli TorA protein (TorA / Ε. c), of the DorA protein of Rhodobacter sphaeroides (DorA / Rs), and of the TorA protein of Shewanella massilia (TorA / Sm).

FIG. 9 represents the alignment of nucleic acid sequences of a highly conserved region of the gene which codes for the TMAO reductase of bacteria of the genus Shewanella and of E. coli, used for the development of one of the molecular primers "DDN" according to the invention.

(a) approximate position and orientation of the different DDN primers on the torA gene coding for the TMAO reductase of different bacteria of the genus Shewanella and of E. coli,

(b) Example of a strategy for developing one of the DDN primers (DDN1 +); the boxed zone corresponds to a very conserved nucleic region of the gene which codes for the TMAO reductase of bacteria of the genus Shewanella [Shewanella massilia (torA / S. massilia), Shewanella putrefaciens (torA / S. putrefaciens), Shewanella c (torA / S c)] and E. coli.

FIG. 10 represents the electrophoresis on agarose gel of the PCR products when the amplification is carried out from the chromosomal DNA of Shewanella massilia (tracks 1, 2, 3 and 4), of Shewanella c (tracks 5, 6, 7 and 8) and of Shewanella putrefaciens MR-1 (tracks 9, 10, 11 and 12) using the molecular primers DDN1 + / DDN5- (tracks 1, 5 and 9; expected size: 820 bp), DDN5 + / DDN2- (tracks 2, 6 and 10; expected size: 234 bp), DDN5 + / DDN3- (tracks 3, 7 and 11; expected size: 740 bp), DDN5 + / DDN4- (tracks 4, 8 and 12; size expected: 866 bp). Tracks M: Size markers.

FIG. 11 represents the electrophoresis on agarose gel of the PCR products when the amplification is carried out from the chromosomal DNA of Photobacterium phosphoreum using the pairs of primers DDN1 + / DDN5- (lane 1; size expected: 820 bp), DDN5 + / DDN2- (track 2; expected size: 234 bp), DDN5 + / DDN3- (track 3; expected size: 740 bp) and DDN5 + / DDN4- (track 4: expected size: 866 bp) . Tracks M: size markers.

- Figure 12 shows the agarose gel electrophoresis of PCR products when the amplification is carried out from the chromosomal DNA of S. massilia (lanes 1, 4, 7 and 10), of E. coli (tracks 2, 5, 8 and 11) and S. typhimurium (tracks 3, 6 and 9), using the pairs of molecular primers DDN1 + / DDN5- (tracks 1 to 3; expected size: 820 bp), DDN5 + / DDN2- (tracks 4 to 6; expected size: 234 bp), DDN5 + / DDN3- (tracks 7 to 9; expected size: 740 bp), DDN5 + / DDN4- (tracks 10 and 11; expected size: 866 bp). Tracks M: Size markers.

FIG. 13 represents the alignment of nucleic acid sequences of a very conserved region of the gene which codes for the TMAO reductase of bacteria of the genus Shewanella,

Rhodobacter and E. coli, used for the preparation of one of the molecular primers "BN" according to the invention.

(a) Approximate position and orientation of the different BN primers on the torA gene coding for the TMAO reductase of different bacteria of the genus Shewanella, Rhodobacter and E. coli,

(b) Example of a strategy for developing one of the BN primers (BN3 +); the boxed zone corresponds to a very conserved nucleic region of the gene which codes for the TMAO reductase of the bacteria Shewanella massilia (torA / S. massilia), of E. coli, of Rhodocbacter sphaeroides (dorAJR. sphaeroides), and of Rhodocbacter capsulatus (dorA / R. Capsulatus). FIG. 14 represents the alignment of protein sequences of the cytochrome TorC of S. massilia (TorC / Sm), of E. coli (TorC / Ec) and of the cytochrome DorC of R. sphaeroides (DorC / Rs). The arrows indicate the position of the different primers on the torC gene of the bacterium Shewanella massilia, developed from the conserved regions. By way of example, the couple BC1 + / BC2- leads to the amplification of the fragment with a size of 197 bp of the gene coding for the protein TorC in S. massilia, this fragment coding for the polypeptide of 66 amino acids delimited by the amino acids located at positions 114 and 179 of the peptide sequence of TorC of S. massilia. The couple BC1 + / BC3- leads to the amplification of the fragment of a size of 719 bp of the gene coding for the protein TorC in S. massilia, this fragment coding for the polypeptide of 240 amino acids delimited by the amino acids located at positions 114 and 353 of the peptide sequence of TorC of S. massilia. The couple BC2 + / BC3- leads to the amplification of the fragment with a size of 542 bp of the gene coding for the protein TorC in S. massilia, this fragment coding for the polypeptide of 181 amino acids delimited by the amino acids located at the positions 173 and 353 of the peptide sequence of TorC from S. massilia.

FIG. 15 represents the electrophoresis on agarose gel (2%) of the PCR products when the amplification is carried out from the chromosomal DNA of E. coli (lane 1), of S. typhimurium (lane 2), P. phosphoreum (lane 3), S. massilia (lane 4), R. sphaeroides (lane 5), or dΕrwinia chrysanthemi (lane 6) using the molecular primers BC1 + and BC2-. The size of the expected DNA fragment (177 base pairs) is indicated by an arrow. Tracks M: Size markers.

- Figure 16 shows the influence of fish chromosomal DNA on the specificity of the molecular primers according to the invention. The PCR amplification is carried out using the molecular primers DDN1 + and DDN5- in the presence of 2.5 μg (tracks 1 and 4), 5 μg (tracks 2 and 5), or 10 μg (tracks 3 and 6) d Purified chromosomal DNA from Herring. Lanes 4, 5 and 6 correspond to the tests carried out by adding 2.5 μl of Shewanella massilia cell suspension to the reaction mixture. Tracks M: size markers.

- Figure 17 shows the agarose gel electrophoresis (2%) of the PCR products when the amplification is carried out from total DNA extracted from decomposing fish flesh (cod: track 1 to 3 ; sole: track 4 to 6; sea bream: track 7 to 9 and red mullet: track 10 to 12) using primers BN6 + / BN4- (tracks 1, 4, 7 and 10, expected size: 364 bp) , primers BN6 + BN2- (tracks 2, 5, 8 and 11, expected size: 711 bp), primers DDN1 + / DDN5- (tracks 3, 6, 9 and 12, expected size: 820 bp). M slopes: size standard (1 Kb BRL scale).

FIG. 18 represents the electrophoresis on agarose gel (2%) of the PCR products when the amplification is carried out using the molecular primers BC1 + / BC2- on two different preparations of total DNA of a red mullet in the process of putrefaction (track 1 and 2). The arrow indicates the amplified DNA fragment of 177 base pairs. FIG. 19 represents the alignment of the nucleotide sequences of the torA genes of

Photobacterium phosphoreum (torA Pp), Shewanella putrefaciens (torA / Sp), Shewanella massilia (torA / Sm), Shewanella c (torA / S. C), and Salmonella typhimurium (torA / S. t.).

A) METHODS OF STUDY

Development of specific primers from the gene coding for the enzyme TMAO reductase: molecular probes "DDN", "BN" and "BC".

The first stage of the study below relates to the development of molecular primers (or probes) usable for a PCR reaction, and to the study of their specificity for different bacteria involved in the alteration of fish flesh. The second step concerns the application of the PCR technique on fish flesh.

I) Development and validation of molecular primers used for the PCR reaction.

The choice of nucleotide primers is a critical step for the success of a PCR test. These primers must be produced from highly conserved regions of the gene which codes for TMAO reductase in all of the bacteria responsible for the degradation of fish flesh. Several strategies were used to develop these primers.

(1) Molecular primers developed from regions of the torA gene conserved in bacteria of the genus Shewanella and in E. coli: primers "DDN".

The development of primers with very little degeneration from conserved protein regions is very difficult because of the degeneracy of the genetic code.

The molecular primers, called “DDN”, were defined from the alignment of nucleic sequences of the torA gene from different bacteria of the genus Shewanella and from E. coli (Figure 9) ^" . Several DNA regions of the torA gene in these bacteria exhibit a high degree of sequence identity with very few mismatches. The molecular primers DDN are the molecular primers DDN1 +, DDN2-, DDN3 -

DDN4-, DDN5- and DDN5 + as defined above.

For all of the PCR reactions, the pairing temperature is brought to 55 ° C. in order to make the amplification more specific and therefore to limit the appearance of contaminating DNA bands. This is made possible by the slight degeneration of the newly synthesized primers. PCR amplification involves 30 successive reaction cycles. Each cycle includes a denanitation step at 94 ° C for 30 s, a hybridization step at 45 ° C for 30 s and an elongation step at 72 ° C for 45 s. a) Use of the molecular primers DDN for the detection of the TMAO reductase system of bacteria of the genus Shewanella.

The first series of experiments, using the pairs of primers DDN1 + / DDN5-,

DDN5 + / DDN2-, DDN5 + / DDN3-, DDN5 + / DDN4-, was carried out on the chromosomal DNA of the strain Shewanella massilia (S. massilia). The pairs of primers DDN1 + / DDN2-,

DDN1 + / DDN3- and DDN1 + / DDN4- were deliberately discarded due to the remoteness of their positions on the torA gene.

The results, grouped in FIG. 10 (tracks 1 to 4), reveal for the majority of the pairs of molecular primers used, the amplification of a single DNA fragment specific to the expected size. Although for the pair of primers DDN5 + / DDN2- (lane 2), an additional non-specific DNA fragment of approximately 300 base pairs is amplified, the molecular primers DDN appear to be specific for the TMAO reductase system of S. massilia.

The pairs of DDN primers were then tested on other bacteria belonging to the genus Shewanella. The same series of experiments was carried out using as chromosomal DNA the strain Shewanella c and S. putrefaciens MR-1. The set of primer pairs makes it possible to amplify a DNA fragment specific to the size expected for these two bacteria (FIG. 10).

The different pairs of DDN primers therefore make it possible to detect all of the Shewanella bacteria tested. b) Use of the molecular primers DDN for the detection of the gene coding for TMAO reductase in the marine bacterium Photobacterium phosphoreum, and for the implementation of the sequencing of said gene.

Photobacterium phosphoreum (P. phosphoreum) is a bacterium which, like S. putrefaciens, is in a certain number of cases made responsible for the deterioration of the flesh of marine fishes (12, 21). It belongs to the Vibrionaceae family, and has an optimal growth temperature of 15 ° C. It is a very little studied bacterium, whose putative TMAO reductase system is absolutely unknown to date.

In order to verify the specificity of the molecular primers according to the invention on bacteria distinct from the Shewanella group, and liable to contaminate fish flesh, a collection P. phosphoreum strain was obtained by the ATCC (American Type Culture

Collection No. 11040). The first growth tests carried out on this bacteria on medium marine (Difco) at 15 ° C have shown that the addition of TMAO to the culture medium significantly improves its growth rate. The optical density (OD ₆₀₀ ) obtained after 24 hours of growth is 0.28 for the strain cultivated in the absence of TMAO, and 0.47 for the strain cultivated in the presence of TMAO. This result is in agreement with the presence of a respiratory TMAO reductase in P. phosphoreum. Gene amplification was carried out from the chromosomal DNA of this bacterium (prepared from 10 ml of culture in a marine environment) using all the pairs of molecular primers DDN.

The results, presented in FIG. 11, show that the DDN primers allow in all cases the amplification of a specific DNA fragment. The different sizes of DNA fragments are in agreement with those obtained for the amplification carried out on the chromosome of bacteria of the genus Shewanella.

In order to confirm that the DNA fragment amplified in P. phosphoreum corresponds well to a part of the gene coding for the TMAO reductase of P. phosphoreum, the sequencing of said fragment was carried out. Thus, the sequencing of approximately 600 nucleotides of the fragment amplified from the pair of molecular primers DDN1 + / DDN5- was carried out. The product

PCR directly purified by Geneclean (BiolOl) was sequenced by the chain termination technique (22) using the molecular primer DDN1 +. The protein sequence deduced from this nucleic sequence was aligned with those of various TMAO reductases (FIG. 7).

This protein sequence has approximately 60% identity with a region of the TMAO reductase of S. massilia. The homologies observed are high enough to conclude that the sequenced DNA fragment corresponds well to a part of the gene which codes for the TMAO reductase of P. phosphoreum.

These results demonstrate the presence of a TMAO reductase system in the bacteria P. phosphoreum, and validate the detection method according to the invention by the PCR test, since the latter makes it possible to detect the TMAO-reductase system in bacteria d alteration relatively distant from a phylogenetic point of view. c) Use of the molecular primers DDN for the detection of genes coding for TMAO reductase of enterobacteria, and for the implementation of the sequencing of said genes. In order to verify that the PCR test is valid for the detection of enterobacteria such as E. coli or certain pathogenic bacteria such as Salmonella typhimurium, the same experiment

PCR as that described above was carried out, with DNA as DNA template chromosome of E. coli and S. typhimurium.

In S. typhimurium, the promoter region of the TMAO reductase system could be demonstrated by a PCR approach (23).

The results obtained are grouped in FIG. 12. The four pairs of molecular primers DDN1 + / DDN5-, DDN5 + / DDN2-, DDN5 + / DDN3- and DDN5 + / DDN4- allow the amplification of a specific DNA fragment in E coli (track 2, 6, 10 and 16). In the case of S. typhimurium, the pair of primers DDN1 + / DDN5- also allows the amplification of a DNA fragment, of 820 base pairs, compatible with a specific fragment of the system.

TMAO reductase (lane 3). In order to confirm that the DNA fragment amplified in S. typhimurium corresponds well to a part of the gene coding for the TMAO reductase of S. typhimurium, the sequencing of said fragment was carried out. The protein sequence, deduced from a part of this DNA fragment (477 nucleotides), has significant homologies with the protein sequence of various TMAO reductases (FIG. 8). It also has more than 88% identity with the sequence of TMAO reductase from E. coli and 45% with that of the enzyme in S. massilia. The DNA fragment amplified using the primers DDN1 + / DDN5- therefore probably corresponds to a part of the gene which codes for the TMAO reductase of S. typhimurium. d) Conclusion

As previously described, the pair of primers DDN1 + / DDN5- makes it possible to detect the torA gene of marine bacteria (Shewanella and P. phosphoreum), but also of enterobacteria (E. coli and S. typhimurium).

The same PCR tests were carried out on bacteria isolated in brackish water (Rhodobacter) which can possibly be found in fish flesh. In order to carry out these tests, samples of chromosomal DNA from the bacterium R. sphaeroides were used as template DNA.

The bacterium R. sphaeroides has a DMSO / TMAO reductase system similar to that described in E. coli (9, 10). Although the terminal enzyme of this bacterium can reduce TMAO and DMSO indifferently, it has numerous protein sequence homologies with TMAO reductase from E. coli or S. massilia (respectively 47.5% and 45% identity).

However, the PCR tests carried out with the DDN molecular probes did not make it possible to amplify a DNA fragment corresponding to the TMAO reductase gene of this bacterium. (2) Molecular primers developed from regions of the torA gene conserved in all of the bacteria studied: primers "BN".

In order to further broaden the detection spectrum of the molecular primers of the PCR test, the nucleic sequence of the TMAO / DMSO reductase gene from bacteria of the genus Rhodobacter (R. sphaeroides and R. capsulatus) was integrated into the alignment carried out at from the sequences of the torA gene of bacteria of the genus Shewanella and of E. coli.

A new series of primers, called “BN”, was thus developed (Figure 13).

For all of the experiments, the PCR amplification is carried out with a hybridization temperature of 55 ° C. At this temperature, only a small part of the primers present in the mixture will be able to hybridize specifically to the template DNA. For this reason, a larger quantity of molecular primers is added to the reaction mixture.

(approximately 100 pmoles in 50 μl final).

The primers BN obtained are the primers BN1 +, BN2-, BN3 +, BN4-, BN5-, BN6 + as defined above. Table 1 below represents the results obtained with regard to the application of the BN molecular primers to the DNA of different bacteria.

The amplification is carried out using 5 ng of bacterial chromosomal DNA. The PCR reaction involves 30 successive reaction cycles.

+: A DNA fragment specific to the expected size is amplified -: No amplification

Table 1

Two pairs of primers: BN6 + / BN2- and BN6 + / BN4-, make it possible to amplify a specific DNA fragment in all of the bacteria tested, and in particular in R. sphaeroides.

BN primers therefore prove to be perfectly suitable for the detection of bacteria which have a TMAO reductase system, and which are capable of contaminating fish flesh.

On the other hand, the use during PCR of a high hybridization temperature (55 ° C.), and a larger quantity of degenerate molecular primers, has made it possible to significantly improve the specificity of amplification.

(3) Molecular primers developed from the gene coding for the cytochrome TorC of the TMAO reductase system: “BC” primers.

The study of the TMAO reductase system in different bacteria has shown that the electron transfer chain to the terminal enzyme TorA in all cases involves a pentahemic type c cytochrome, cytochrome TorC (or DorC) (6, 9, 10, 19).

In order to demonstrate the TMAO reductase system in spoilage bacteria, nucleic primers directed against the gene coding for this cytochrome have been developed.

The multi-alignment of protein sequences, presented in FIG. 14, reveals numerous protein regions conserved between the cytochromes of the TMAO reductase system of different bacteria: S. massilia, E. coli, R. sphaeroides. Some of these conserved regions correspond to the heme binding sites of type c (CxxCH motif) and are therefore found in a large number of tetrahemic cytochromes of NapC / NirT class involved in other respiratory systems (24). The cytochrome TorC nevertheless has the particularity of containing an additional carboxy-terminal domain which fixes a fifth heme (6, 19).

The BC molecular primers are the primers BC1 +, BC2 +, BC2- and BC3- as defined above. To develop said primers, the conserved protein regions were searched for among all of the NapC / NirT class cytochromes (probes BC2 + and BC2-), but also only among the TorC cytochromes (probes BC1 + and BC3-). The position and orientation of the four degenerate molecular primers BC are shown in Figure 14.

The BC molecular primers are capable of hybridizing to the gene coding for the cytochrome TorC of the TMAO reductase system. The PCR reaction is carried out under the same conditions as those used with the BN molecular primers. It involves 30 successive reaction cycles. Each cycle consists of a denaturation step at 94 ° C for 30 s, a hybridization step at 55 ° C for 30 s and an elongation step for 45 s. About 100 pmol of each molecular primers are added to the reaction mixture (final volume 50 μl). Among the three pairs of molecular primers tested (BC1 + / BC2-, BC1 + / BC3- and BC2 + / BC3-), the couple BC1 + / BC2- made it possible to amplify a DNA fragment to the expected size (177 pairs of bases) for all the bacteria previously tested (Figure 15; tracks 1 to 5). This result allows us to consider the use of the pair of molecular primers BC1 + / BC2- for the PCR test.

In parallel, the same PCR experiment was carried out with the pair of primers

BC1 + / BC2- from the chromosome of the bacterium Erwinia chrysanthemi, a phytopathogenic enterobacterium which is said to lack a TMAO reductase system (4). In this case, no DNA fragment of a size compatible with that expected for amplification from the torC gene is observed (FIG. 15, lane 6).

The pair of primers BC1 + / BC2- therefore seems to specifically recognize the cytochrome TorC of the TMAO reductase system.

(4) Application of the PCR technique to whole cells For carrying out a PCR test intended to be used directly from fish samples, it is important to do without the preparation stage. Chromosomal DNA from bacteria. For this reason, the PCR experiments using the pairs of primers DDN, BN and BC ^" were reproduced from whole bacteria of S. massilia. The bacterial colonies isolated on dish are resuspended in 200 μl of distilled water sterile (OD ₆₀₀ between 0.1 and 0.5) The PCR reaction is then carried out under the conditions described above with 2.5 μl of cell suspension as matrix (in 50 μl of final volume). PCR reactions performed (with primers DDN, BN and BC), the result obtained is equivalent to that observed with purified chromosomal DNA. Bacteria are therefore well lysed during the first stage of PCR which corresponds to a incubation of the samples at 94 ° C. for 1.5 min This positive result makes it possible to envisage using the PCR test directly from the fish flesh. II) Application of the PCR-TMAO test to fish flesh

After having verified the specificity of the molecular primers according to the invention on bacteria responsible for the alteration of seafood, the next step therefore consists in the direct application of the PCR test to fish flesh. The four pairs of primers DDN1 + / DDN5-, BN6 + / BN4-, BN6 + / BN2- and BC1 + / BC2- were chosen to carry out these tests because they allow the amplification of a specific DNA fragment for the vast majority previously tested bacteria.

(1) Influence of chromosomal DNA from fish during PCR amplification of the TMAO reductase gene from bacteria.

For the use of the PCR test on fish flesh samples, it must first be verified that the chromosomal DNA of the fish does not compete with the bacterial DNA, during the hybridization of the molecular primers.

In order to test the influence of chromosomal DNA from fish on the specificity of the molecular primers according to the invention, variable concentrations of chromosomal DNA purified from herring sperm were added to the PCR reaction mixture containing or not 2, 5 μl of cell suspension of the bacteria S. massilia.

FIG. 16 obtained with the pair of primers DDN1 + / DDN5- shows that no DNA fragment is amplified in the absence of bacteria added to the reaction mixture (lane 1 to 3), regardless of the concentration of Fish chromosomal DNA. If the bacteria S. massilia is added to the reaction mixture (lanes 4 to 6) a specific DNA fragment is amplified. The same results were obtained for the pairs of primers BN6 + / BN2-, BN6 + / BN4- or BC1 + / BC2-, and thus make it possible to conclude that the presence of foreign DNA in the reaction mixture of the PCR does not interfere with the specificity of the molecular primers according to the invention.

These important results therefore make it possible to exclude the possibility of a non-specific amplification which would be due to the chromosomal DNA of the fish.

(2) Extraction of total DNA from fish flesh and validation of the PCR technique.

Traditionally, microorganism detection tests, during microbiological food control, require a first step of pre-enrichment of bacteria on a selective medium.

In the perspective of a rapid freshness test, the PCR technique was applied directly to fish samples. Different conditions for extracting total DNA from fish flesh undergoing alteration (bacterial DNA + fish DNA) were therefore tested.

First, a rapid technique for extracting DNA from cells was tested by treating fish samples with NaOH / SDS (25). This technique allows cell lysis and therefore the extraction of total chromosomal DNA. The preparation is made from approximately 25 mg of flesh taken from a fish (red mullet) kept 12 hours at room temperature. After 10 min of incubation at 95 ° C, 5 μl of the mixture of homogenized fish flesh are used directly in the reaction mixture of the PCR.

However, by this rapid technique of extracting total DNA, all of the PCR reactions carried out with the various molecular primers did not make it possible to amplify a specific DNA fragment. The presence of several PCR-inhibiting substances in fish flesh could explain this result. Indeed, compounds present in large quantities in certain food products (fats, proteins, etc.) can have an influence on the effectiveness of PCR (26). In order to verify this hypothesis, the same PCR tests were carried out by adding 5 ng of chromosomal DNA from S. massilia to the fish flesh samples. However, as before, no DNA fragment was amplified by PCR, whatever the molecular primers used.

These results allow us to conclude that the PCR reaction is inhibited by certain compounds contained in fish flesh.

The technique for purifying total chromosomal DNA from fish flesh, used according to the present invention, must therefore make it possible to remove all of the PCR inhibitor compounds.

A rapid DNA extraction kit (approximately 1 hour), based on the attachment of nucleic acids to silica beads, was thus tested (kit marketed under the name

"High Pure PCR Template Preparation Kit" by Boehringer Mannheim / Roche). After DNA fixation, this technique makes it possible to remove salts, proteins and other cellular impurities by a simple washing step.

The extraction of total DNA (bacterial DNA + fish DNA) was carried out on four different fish species in the process of decomposition (kept 12 h at room temperature): 2 species from the Mediterranean Sea (red mullet and sea bream), and 2 species from the Atlantic (sole and cod). From approximately 50 mg of flesh of these fish (taken from under the skin), 1 to 4 μg of total chromosomal DNA were thus purified. About 25 ng of this DNA are then added to the PCR reaction mixture (final volume 50 μl). For all the PCR reactions, the hybridization temperature is brought to 55 ° C., and 100 pmol of each molecular primers are added to the reaction mixture.

FIG. 17 groups together the results obtained using the pairs of primers DDN1 + / DDN5-, BN6 + / BN2- and BN6 + / BN4-. For all of the fish species tested, a single DNA fragment is amplified, and this with each of the primer pairs used. The size of the amplified DNA fragment corresponds to the size expected for amplification from the torA gene. This result indicates that the different DNA fragments were indeed amplified from the torA gene of bacteria present on fish flesh.

Likewise, tests carried out on two other species of fish in the process of rotting (7 days under melting ice), mackerel (fatty fish) and whiting, made it possible to highlight the torA gene of spoilage bacteria.

Thus, the use of the pairs of molecular primers DDN1 + / DDN5-, BN6 + / BN2- and BN6 + BN4- can be envisaged for the application of the PCR test to fish flesh.

In addition, the use of a silica bead filter is an adequate method to purify the total DNA contained in fish flesh. This technique is very fast (about an hour), and very easy to implement compared to an isoamylalcohol / chloroform extraction which requires several steps to remove the proteins contained in fish flesh (28).

The amplification carried out under the same conditions with the pair of primers BC1 + / BC2- also gave encouraging results on the total DNA of flesh of a red mullet in the process of putrefaction (FIG. 18). Indeed, despite a significant background noise, the agarose gel electrophoresis of the PCR products reveals the presence of the fragment of 177 base pairs.

The application of the technique to monitoring of spoilage of cod has shown that the technique is in direct correlation with the freshness quality of the fish meat during its conservation over time.

III) Conclusion The pairs of molecular primers according to the invention therefore make it possible to detect the torA gene in marine bacteria, but also in bacteria that are relatively phylogenetically distant, namely enterobacteria and bacteria from brackish waters.

The application of the PCR test to fish flesh gives encouraging results since the molecular primers according to the invention make it possible to detect the torA gene of bacteria present on the fish in the process of putrefaction. Since PCR amplification cannot be applied directly to samples of fish flesh, it is necessary to extract the total DNA from this flesh.

In addition, the technique is directly linked to the freshness quality of the fish flesh and is more sensitive than conventional techniques such as ABVT.

B) MATERIALS AND METHODS

1) Isolation of the bacterial strains, media and growth conditions The fish used for the isolation of the bacterial strains Shewanella c and Shewanella massilia corresponds to a red mullet (Mullus surmuletus) caught in the Mediterranean Sea off Marseille, and kept at temperature ambient for 48 hours in a sterile tube containing sea water. The sea water was previously sterilized through a millipore filter (0.45 μM). The Shewanella strains are cultured anaerobically or aerobically at 30 ° C in LB medium (“Luria Broth”: yeast extract 10 g / 1, bacto-peptone 5g / l and NaCl 5g / l). The E. coli and Salmonella typhimurium bacteria are cultured at 37 ° C. on LB medium. The Photobacterium phosphoreum strain was obtained by ATCC (n ° 11040) and does not grow on conventional media such as LB medium. It is cultivated at 15 ° C on a marine environment (marine broth; Difco).

2) PCR techniques a) Standard PCR

Standard PCR amplifications are carried out under the conditions described in reference (6). The reaction mixture (50 μl) contains 10 ng of chromosomal DNA, 0.2 μg of each of the oligonucleotide probes, 100 μM of each of the four deoxyribonucleotide triphosphates (dXTPs), 10 mM of Tris / HCl (pH 8.3), 1.5 mM MgCl ₂ , 50 mM KC1, 0.01 % gelatin and one unit of DNA polymerase (Taq polymerase, Boehringer Mannheim Roche). The reaction mixture is finally covered with 50 μl of mineral oil in order to prevent evaporation during the PCR process.

The amplification, carried out in a hermocycling device (MJ Research), involves 30 reaction cycles, a cycle comprising a denaturation step at 94 ° C for 30 seconds, a hybridization step at 55 ° C for 30 seconds and an elongation step at 72 ° C for about 45 seconds (lkb / min). The DNA is denatured beforehand for 1.5 min at 94 ° C. before starting the first cycle. 10 μl of each of the amplification products are then analyzed by electrophoresis on an agarose gel.

b) PCR conditions with degenerate molecular primers according to the invention

The molecular primers according to the invention, in particular the primers called “DDN”, “BN” or “BC”, are as defined above.

The reaction mixture (50 μl) contains either 10 ng of chromosomal DNA of the bacteria, or 5 μl of cell suspension (OD ₆₀₀ = 0.5-1) or 25 ng of total DNA extracted from fish, 100 μM each of the four deoxyribonucleotide triphosphates (dXTPs),

0.8 μg of each of the primer mixtures according to the invention, 10 mM Tris-HCl (pH 8.3), 1.5 mM

MgCl ₂ , 50 mM KC1, 0.01% gelatin and one unit of Taq polymerase.

The PCR reaction involves 30 successive reaction cycles consisting of a denaturation step at 94 ° C for 30 seconds followed by a hybridization step at 55 ° C or 45 ° C for 30 seconds, then a step d elongation at 72 ° C for 45 seconds. The DNA is denatured beforehand for 1.5 min at 94 ° C. before starting the first cycle.

3) Preparation of chromosomal DNA of bacteria The preparation of chromosomal DNA of bacteria is carried out from 10 ml of culture (OD ₆₀₀ > 1). After centrifugation of the cells at 10,000 rpm for 10 min, the pellet is resuspended in 1 ml of 10 mM EDTA (pH 8). Lysis of the cells is obtained by addition of SDS (0.5% final). This solution is mixed with an equal volume of isoamyl alcohol / chloroform (1:24). After centrifugation for 20 min at 7,000 rpm, the upper phase is recovered. This operation is repeated several times in order to remove the largest amount of residual proteins. The DNA is finally precipitated by adding NaCl (0.3 M final) and ethanol (2.2 Vol). 4) Preparation of total chromosomal DNA from putrefying fish flesh for PCR a) Rapid preparation using a kit based on the attachment of DNA to sillice beads (High pure PCR template Preparation Kit, Boehringer Mannheim / Roche) 25 mg of fish flesh are incubated 45 min at 55 ° C in 200 μl of lysis buffer

(4 M urea, 200 mM Tris, 20 mM NaCl and 200 mM EDTA, pH 7.4) in the presence of 40 μl of proteinase K (0.8 mg). In order to facilitate cell lysis, the sample is previously ground using a scalpel. 200 μl of fixing buffer (guanidine-HCl 6 M, urea 10 mM, Tris-HCl 10 mM and Triton 20% v / v, pH 4.4) are added to the sample which is then incubated for 10 min at 72 ° C. This solution is mixed with 100 μl of isopropanol and then centrifuged at 8,000 m for 1 min through a filter based on sillice beads (High Pure Filter tube + collecting tube). Residual impurities are removed by two washing steps (washing buffer: 20 mM NaCl, 2 mM Tris-HCl, 80% v / v ethanol, pH 7.5). The DNA is then eluted in an elution buffer (10 mM Tris, pH 8.5). b) DNA extraction from cells by NaOH / SDS treatment

25 mg of fish flesh are taken from a putrefying fish (after 16 hours of incubation in a sterile tube kept at room temperature), and placed in 50 μl of a 0.05 M NaOH solution, SDS 0 , 25%. After grinding the cells, the sample is incubated for 15 min at 95 ° C. 450 μl of sterile water are added to the mixture and the cellular debris is then removed by a centrifugation step of 2 min at 8,000 g. 5 μl of the supernatant obtained are directly used for the PCR test. c) Isoamyl alcohol chloroform extraction

The preparation of total chromosomal DNA by isoamyl alcohol / chloroform extraction is carried out from 25 mg of contaminated fish flesh as described above for bacterial DNA. On the other hand, the number of extraction stages is increased in order to be able to remove the large quantities of impurities contained in the fish flesh samples. Bibliographic references

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(2) Easter M.C., Gibson D.M. and Ward F.B. (1983) The induction and location of trimethylamine N-oxide reductase in Alteromonas sp. ΝCMB 400. J.Gen.Microbiol. 129, 3689-3696.

(3) McEwan A.G., Ferguson SJ. and Jackson J.B. (1983) Electron flow to dimethylsulphoxide or trimethylamine N-oxide generâtes a membrane potential in

Rhodopseudomonas capsulata. Arch.Microbiol. 136, 300-305.

(4) Barrett E.L. and Kwan H.S. (1985) Bacterial reduction of trimethylamine oxide. Annu.Rev.Microbiol. 39, 131-149.

(5) Silvestro A., Pommier J. and Giordano G. (1988) The inducible trimethylamine N- oxide reductase of Escherichia coli Kl 2: biochemical and immunological studies.

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(6) Méjean V., Iobbi-Νivol C, Lepelletier M., Giordano G., Chippaux M. and Pascal M.C. (1994) TMAO anaerobic respiration in Escherichia coli: involvement of the tor operon. Mol.Microbiol. 11, 1169-1179. (7) Yamamoto L, Wada Ν., Ujiiye T., Tachibana M., Matsuzaki M., Kajiwara H.,

Watanabe Y., Hirano H., Okubo A. and Satoh T. (1995) Cloning and nucleotide sequence of the gene encoding dimethyl sulfoxide reductase from Rhodobacter sphaeroides f sp. denitriβcans. Biosci.Biotechnol.Biochem. 59, 1850-1855.

(8) Knablein J., Mann K., Ehlert S., Fonstein M., Huber R. and Schneider F. (1996) Isolation, cloning, sequence analysis and localization of the operon encoding dimethyl sulfoxide / trimethylamine Ν-oxide reductase from Rhodobacter capsulatus. J. Mol. Biol. 263, 40-52.

(9) Ujiiye T., Yamamoto I., Νakama H., Okubo A., Yamazaki S. and Satoh T. (1996) Nucleotide sequence of the genes, encoding the pentaheme cytochrome (dmsC) and the transmembrane protein (dmsB), involved in dimethyl sulfoxide respiration from Rhodobacter sphaeroides f. sp. denitrificans. Biochim.Biophys.Acta 1277, 1-5. (10) Mouncey NJ, Choudhary M. and Kaplan S. (1997) Characterization of genes encoding dimethyl sulfoxide reductase of Rhodobacter sphaeroides 2.4.1T: an essential metabolic gene function encoded on chromosome IL J. Bacteriol. 179, 7617-7624.

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(12) Gram L. and Huss H.H. (1996) Microbiological spoilage of fish and fish products. Int.J. Food Microbiol. 33, 121-137.

(13) Dalgaard P., Gram L. and Huss H.H. (1993) Spoilage and shelf-life of cod fillets packed in vacuum or modified atmospheres. Int.J. Food Microbiol. 19, 283-294. (14) Malle P. and Poumeyrol M. (1989) A new chemical criterion for the quality control of fish: Trimethylamine / Total Volatile Basic Nitrogen (%). J.FoodProt. 52, 419-423.

(15) Large PJ. and MacDougall H. (1975) An enzymatic method for the microestimation of trimethylamine. Anal.Biochem. 64, 304-310.

(16) Ritskes T.M. (1975) Gas chromatographic determination of trimethylamine and dimethylamine in fish, fishery products, and other foods. J. Food Technol. 10, 221-228.

(17) Innis M.A., Gelfand D.H., Sninsky J.J. and White TJ. (1990) PCR Protocols. Academy Press, Inc., San Diego.

(18) Lee H., Morse S. and Olsvik O. (1997) Nucleic Acid Amplification Technologies: Application to Disease Diagnosis. Eaton Publishing, Boston. (19) Dos Santos J.P., Iobbi-Nivol C, Couillault C, Giordano G. and Méjean V. (1998)

Molecular analysis of the trimethylamine N-oxide (TMAO) reductase respiratory System from a Shewanella species. J. Mol. Biol. 284, 421-433.

(20) Czjzek M., Dos Santos J.P., Pommier J., Giordano G., Méjean V. and Haser R. (1998) Crystal structure of oxidized trimethylamine N-oxide reductase from Shewanella massilia at 2.5 À Resolution. J. Mol. Biol. 284, 434-447.

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Conservation of cis-acting elements within the tor regulatory region among different Enterobacteriaceace. Uncomfortable. 152, 53-57. (24) Roldan MD, Sears HJ, Cheesman MR, Ferguson SJ, Thomson AJ, Berks BC and Richardson DJ. (1998). Spectroscopic characterization of a novel multiheme ç-type cytochrome widely implicated in bacterial electron transport. J. Biol. Chem. 273, 28785-28790.

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(26) Rossen L., Norskov P., Holmstrom K. and Rasmussen O.F. (1992) Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions. Int.J. Food Microbiol. 17, 37-45. (27) Laemmli U.K. (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nαtwre 227, 680-685.

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762-768.

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Claims

1. Use of nucleotide sequences chosen from those comprising a sequence coding for a protein of the trimethylamine N-oxide reductase system (TMAO reductase) in bacteria, or a fragment of this sequence such as a probe or a primer of approximately 15 to approximately 25 nucleotides, or a sequence derived by addition, deletion, and / or substitution of one or more nucleotides of this sequence coding for a protein of the TMAO reductase system or of a fragment of the latter, said fragment and said derived sequence being capable of hybridizing with said sequence coding for a protein of the TMAO reductase system, for the implementation of a method for detecting the presence of all bacteria involved in the process of degradation of the flesh of aquatic animals, in a host likely to carry such bacteria.

2. Use according to claim 1, characterized in that the sequence coding for a protein of the TMAO reductase system is chosen from the sequences coding for TMAO reductase in bacteria, also designated protein TorA or DorA, or coding for a cytochrome of the type c, also known as TorC or DorC protein.

3. Use according to claim 1 or claim 2, characterized in that the nucleotide sequences coding for a protein of the TMAO reductase system are chosen from those of the following bacteria:

- marine bacteria, such as those of the genus Shewanella, Photobacterium or Vibrio, said sequences being chosen in particular from the following:

* the sequence coding for the TorA protein of Shewanella massilia represented in FIG. 1,

* the sequence coding for the TorA protein of Shewanella putrefaciens represented in FIG. 1,

* the sequence coding for the protein TorA of Shewanella c represented in FIG. 2, * the partial sequence coding for the protein TorA of Photobacterium phosphoreum represented in FIG. 3,

* the sequence coding for the TorC protein of Shewanella massilia represented in FIG. 14, bacteria from brackish water, such as those of the genus Rhodobacter or Roseobacter, said sequences being chosen in particular from the following:

* the sequence coding for the DorA protein of Rhodobacter sphaeroides represented in FIG. 4, * the sequence coding for the DorA protein of Rhodobacter capsulatus represented in FIG. 4,

* the sequence coding for the DorC protein of Rhodobacter sphaeroides represented in FIG. 14,

- Enterobacteriaceae, such as those of the genus Escherichia, or Salmonella, said sequences being chosen in particular from the following:

* the sequence coding for the protein TorA of Escherichia coli represented in FIG. 4,

* the partial sequence coding for the TorA protein of Salmonella typhimurium represented in FIG. 5, * the sequence coding for the TorC protein of Escherichia coli represented in FIG. 14.

4. Use according to any one of claims 1 to 3, characterized in that the nucleotide sequences are used in the form of pairs of primers chosen from any one of the following three groups of primers:

(1) the group of primers "DDN" comprising:

* the following “DDN +” nucleotide sequence compositions:

- DDN1 +: 5 'CGG vGA yTA CTC bAC hGG TGC i': mixture of 54 nucleotide sequences, - DDN5 +: 5 'ATy GAT GCG ATy CTC GAA CC i': mixture of 4 nucleotide sequences,

* the following “DDN-” nucleotide sequence compositions:

- DDN2-: 5 'CGT Amw sGT CGA kAT CGT TrC GCT C i': mixture of 32 nucleotide sequences, - DDN3-: 5 'GAC TCA CAy Awy TGy GAG TG i': mixture of 16 nucleotide sequences, - DDN4-: 5 'TGr CCd CGr kCG TTA AAG AC i': mixture of 24 nucleotide sequences,

- DDN5-: 5 'CCv GGT TCG AGr ATC GCA TC': mixture of 6 nucleotide sequences, (2) the group of primers "BN" comprising: the following compositions of nucleotide sequences "BN +":

- BN1 +: 5 'C bGA yAT CsT rCT GCC i': mixture of 16 nucleotide sequences,

- BN3 +: 5 'GGm GAy TAy TCb ACm GGy GC': mixture of 96 nucleotide sequences, - BN6 +: 5 'Twy GAr CGy AAC GAy mTC GA 3': mixture of 64 nucleotide sequences,

* the following “BN-” nucleotide sequence compositions:

- BN2-: 5 'GG vyC rTA CCA bsC vCC TTC i': mixture of 216 nucleotide sequences, - BN4-: 5 'ATC Arr CCn swv GGC GTG CC i': mixture of 192 nucleotide sequences,

- BN5-: 5 'GbC ACr TCd GTy TGy GG i': mixture of 72 nucleotide sequences, (3) the group of primers "BC" comprising:

* the following “BC +” nucleotide sequence compositions: - BC1 +: 5 'ACn CCn GAr AAr TTy GAr GC i': mixture of 256 nucleotide sequences,

- BC2 +: 5 'TGy ATh GAy TGy CAy AAr GG i': mixture of 96 nucleotide sequences, the following “BC-” nucleotide sequence compositions: - BC2-: 5 'CCy TTr TGr CAr TCd ATr CA i': mixture of 96 nucleotide sequences,

- BC3-: 5 'TTn GCr TCr AAr TGn GC 3': mixture of 128 nucleotide sequences, in which n = (A, C, G, T), y = (C, T), r = (A, G) , h = (A, C, T), d = (G, A, T), m = (A, C), w = (A, T), b = (G, T, C), s = ( G, C), v = (G, A, C), and k = (G, T), the pairs of primers being chosen such that one of the primers of a pair corresponds to the one of the above-mentioned compositions of nucleotide sequences DDN +, BN + or BC +, while the other primer corresponds respectively to one of the compositions of nucleotide sequences DDN-, BN- or BC- above, said pairs of primers being chosen in particular from any one of the following four couples:

(a) the DDN1 + / DDN5- couple, leading to the amplification of fragments of the gene coding for the protein TorA in bacteria, with a size of approximately 820 base pairs (bp), and in particular to the amplification of an 821 bp fragment of the gene coding for the TorA protein in bacteria of the genus Shewanella, such as the 821 bp fragment delimited by the nucleotides located at positions 620 to 1450 of the torA gene of S. massilia represented in FIG. 4,

(b) the BN6 + / BN2- pair, leading to the amplification of fragments of the gene coding for the protein TorA in bacteria, of a size of approximately 710 bp, and in particular to the amplification of a fragment of 727 bp of the gene coding for the protein TorA in bacteria of the genus Shewanella, such as the fragment of 727 bp delimited by the nucleotides located at positions 1657 to 2403 of the torA gene of S. massilia represented in FIG. 4,

(c) the BN6 + / BN4- pair, leading to the amplification of fragments of the gene coding for the protein TorA in bacteria, of a size of approximately 360 bp, and in particular to the amplification of a fragment of 355 bp of the gene coding for the protein TorA in bacteria of the genus Shewanella, such as the fragment of 355 bp delimited by the nucleotides located at positions 1657 to 2023 of the torA gene of S. massilia represented in FIG. 4,

(d) the couple BC1 + / BC2-, leading to the amplification of fragments of the gene coding for the protein TorC in bacteria, of a size of approximately 170 bp, and in particular to the amplification of a fragment of 197 bp of the gene coding for the TorC protein in bacteria of the genus Shewanella, such as the fragment of 197 bp coding for the polypeptide fragment delimited by the amino acids located at positions 114 to 179 of the TorC protein of S. massilia represented in the figure 14.

5. Use according to one of claims 1 to 4, characterized in that the hosts likely to be carriers of bacteria involved in the process of degradation of the flesh of aquatic animals, as described in claim 3, are organisms aquatic, in particular marine organisms such as fish and crustaceans, and more particularly Atlantic fish such as sole, cod, or Mediterranean sea fish such as red mullet, sea bream, as well as certain fresh or brackish water animals.

6. Use according to any one of claims 1 to 5, for the implementation of a method for detecting the presence of all bacteria involved in the degradation of the flesh of aquatic animals, in the context of a process d evaluation of the state of freshness of the aquatic animals from which the test sample is taken, when the latter are extracted from their natural environment.

7. Nucleotide sequence corresponding to one of the following sequences:

- DDN1 +: 5 'CGG vGA yTA CTC bAC hGG TGC i',

- DDN5 +: 5 'ATy GAT GCG ATy CTC GAA CC 3', - DDN2- 5 'CGT Amw sGT CGA kAT CGT TrC GCT C 3',

- DDN3- 5 'GAC TCA CAy Awy TGy GAG TG i',

- DDN4- 5 'TGr CCd CGr kCG TTA AAG AC i',

- DDN5- 5 ^• CCv GGT TCG AGr ATC GCA TC i ',

- BN1 +: 5 'C bGA yAT CsT rCT GCC i', - BN3 +: 5 'GGm GAy TAy TCb ACm GGy GC i',

- BN6 +: 5 'Twy GAr CGy AAC GAy mTC GA i',

- BN2-: 5 'GG vyC rTA CCA bsC vCC TTC i',

- BN4-: 5 'ATC Arr CCn swv GGC GTG CC i',

- BN5-: 5 'GbC ACr TCd GTy TGy GG i', - BC1 +: 5 'ACn CCn GAr AAr TTy GAr GC i',

- BC2 +: 5 'TGy ATh GAy TGy CAy AAr GG i',

- BC2-: 5 'CCy TTr TGr CAr TCd ATr CA i',

- BC3-: 5 'TTn GCr TCr AAr TGn GC i', in which n = (A, C, G, T), y = (C, T), r = (A, G), h = (A, C, T), d = (GAT), m = (A, C), w = (A, T), b = (G, T, C), s = (G, C), v = (GAC) , and k = (G, T).

8. Composition of mixed nucleotide sequences, corresponding to one of the following compositions:

* the following “DDN +” nucleotide sequence compositions: - DDN1 +: 5 'CGG vGA yTA CTC bAC hGG TGC i': mixture of 54 nucleotide sequences, - DDN5 +: 5 'ATy GAT GCG ATy CTC GAA CC i': mixture of 4 nucleotide sequences, the following “DDN-” nucleotide sequence compositions:

- DDN2-: 5 'CGT Amw sGT CGA kAT CGT TrC GCT C i': mixture of 32 nucleotide sequences,

- DDN3-: 5 'GAC TCA CAy Awy TGy GAG TG i': mixture of 16 nucleotide sequences,

- DDN4-: 5 'TGr CCd CGr kCG TTA AAG AC i': mixture of 24 nucleotide sequences, - DDN5-: 5 'CCv GGT TCG AGr ATC GCA TC i': mixture of 6 nucleotide sequences,

* the following “BN +” nucleotide sequence compositions:

- BN1 +: 5 'C bGA yAT CsT rCT GCC i': mixture of 16 nucleotide sequences,

- BN3 +: 5 'GGm GAy TAy TCb ACm GGy GC i': mixture of 96 nucleotide sequences,

- BN6 +: 5 'Twy GAr CGy AAC GAy mTC GA i': mixture of 64 nucleotide sequences, the following “BN-” nucleotide sequence compositions:

- BN2-: 5 'GG vyC rTA CCA bsC vCC TTC i': mixture of 216 nucleotide sequences,

- BN4-: 5 'ATC Arr CCn swv GGC GTG CC i': mixture of 192 nucleotide sequences,

- BN5-: 5 'GbC ACr TCd GTy TGy GG i': mixture of 72 nucleotide sequences, the following “BC +” nucleotide sequence compositions: - BC1 +: 5 'ACn CCn GAr AAr TTy GAr GC 3': mixture of 256 sequences nucleotide,

- BC2 +: 5 'TGy ATh GAy TGy CAy AAr GG i': mixture of 96 nucleotide sequences, the following “BC-” nucleotide sequence compositions: - BC2-: 5 'CCy TTr TGr CAr TCd ATr CA i': mixture of 96 nucleotide sequences,

- BC3-: 5 'TTn GCr TCr AAr TGn GC 3': mixture of 128 nucleotide sequences, where n - (A, C, G, T), y - (C, T), r = (A, G), h = (A, C, T), d = (G, A, T), m = (A, C), w = (A, T), b = (G, T, C), s = (G, C), v = (G, A, C), and k = (G, T).

9. A pair of primers characterized in that it is chosen from one of the following groups of primers:

(1) the group of primers "DDN" comprising:

* the following “DDN +” nucleotide sequence compositions:

- DDN1 +: 5 'CGG vGA yTA CTC bAC hGG TGCi': mixture of 54 nucleotide sequences, - DDN5 +: 5 'ATy GAT GCG ATy CTC GAA CCi': mixture of 4 nucleotide sequences,

* the following “DDN-” nucleotide sequence compositions:

- DDN2-: 5 'CGT Amw sGT CGA kAT CGT TrC GCT Ci': mixture of 32 nucleotide sequences, - DDN3-: 5 'GAC TCA CAy Awy TGy GAG TG i': mixture of 16 nucleotide sequences,

- DDN4-: 5 'TGr CCd CGr kCG TTA AAG AC': mixture of 24 nucleotide sequences,

- DDN5-: 5 'CCv GGT TCG AGr ATC GCA TCi': mixture of 6 nucleotide sequences,

(2) the group of primers "BN" comprising: the following compositions of nucleotide sequences "BN +":

- BN1 +: 5 'C bGA yAT CsT rCT GCC i': mixture of 16 nucleotide sequences,

- BN3 +: 5 'GGm GAy TAy TCb ACm GGy GCi': mixture of 96 nucleotide sequences,

- BN6 +: 5 'Twy GAr CGy AAC GAy mTC GA 3': mixture of 64 nucleotide sequences,

* the following “BN-” nucleotide sequence compositions:

- BN2-: 5 'GG vyC rTA CCA bsC vCC TTC i': mixture of 216 nucleotide sequences,

- BN4-: 5 'ATC Arr CCn swv GGC GTG CCi': mixture of 192 nucleotide sequences, - BN5-: 5 'GbC ACr TCd GTy TGy GG i': mixture of 72 nucleotide sequences, (3) the group of primers "BC" comprising: the following compositions of nucleotide sequences "BC +":

- BC1 +: 5 'ACn CCn GAr AAr TTy GAr GC i': mixture of 256 nucleotide sequences,

- BC2 +: 5 'TGy ATh GAy TGy CAy AAr GG i': mixture of 96 nucleotide sequences,

* the following “BC-” nucleotide sequence compositions:

- BC2-: 5 'CCy TTr TGr CAr TCd ATr CA i': mixture of 96 nucleotide sequences, - BC3-: 5 'TTn GCr TCr AAr TGn GC i': mixture of 128 nucleotide sequences, in which n = (A, C, G, T), y = (C, T), r ≈ (A, G), h = (A, C, T), d = (G, A, T), m = (A, C) , w = (A, T), b = (G, T, C), s = (G, C), v = (GAC), and k = (G, T), the pairs of primers being chosen from such that one of the primers of a pair corresponds to one of the compositions of nucleotide sequence DDN +, BN + or BC + above, while the other primer corresponds respectively to one of the compositions of nucleotide sequence DDN- , BN- or BC- mentioned above, said pairs of primers being chosen in particular from any one of the following four pairs:

(a) the DDN1 + / DDN5- couple, leading to the amplification of fragments of the gene coding for the protein TorA in bacteria, with a size of approximately 820 base pairs (bp), and in particular to the amplification of an 821 bp fragment of the gene coding for the TorA protein in bacteria of the genus Shewanella, such as the 821 bp fragment delimited by the nucleotides located at positions 620 to 1450 of the torA gene of S. massilia represented in FIG. 4,

(b) the BN6 + / BN2- pair, leading to the amplification of fragments of the gene coding for the protein TorA in bacteria, of a size of approximately 710 bp, and in particular to the amplification of a fragment of 727 bp of the gene coding for the protein TorA in bacteria of the genus Shewanella, such as the fragment of 727 bp delimited by the nucleotides located at positions 1657 to 2403 of the torA gene of S. massilia represented in FIG. 4,

(c) the BN6 + / BN4- pair, leading to the amplification of fragments of the gene coding for the protein TorA in bacteria, of a size of approximately 360 bp, and in particular to the amplification of a fragment of 355 bp of the gene coding for the protein TorA in bacteria of the genus Shewanella, such as the fragment of 355 bp delimited by the nucleotides located at positions 1657 to 2023 of the torA gene of S. massilia represented in FIG. 4,

(d) the couple BC1 + / BC2-, leading to the amplification of fragments of the gene coding for the protein TorC in bacteria, of a size of approximately 170 bp, and in particular to the amplification of a fragment of 197 bp of the gene coding for the TorC protein in bacteria of the genus Shewanella, such as the fragment of 197 bp coding for the polypeptide fragment delimited by the amino acids located at positions 114 to 179 of the TorC protein of S. massilia represented in the figure 14.

10. Method for detecting all bacteria involved in the degradation of the flesh of aquatic animals in a host capable of carrying such bacteria, said method being carried out using a biological sample taken from this host, this corresponding biological sample in particular to a subcutaneous fragment of flesh of the aquatic animal in question, and being characterized in that it comprises a step of hybridization of at least one nucleotide sequence as defined in any one of claims 1 to 9, with fragments of the genes coding for a protein of the TMAO-reductase system of bacteria involved in the degradation of the flesh of aquatic animals likely to be present in the biological sample taken from said host, followed by a revelation step , in particular by electrophoresis, of the possible presence in said sample of genes coding for a protein of the system e TMAO-reductase, or fragments of these genes, the number of copies of which has been amplified if necessary.

11. Detection method according to claim 10, characterized in that it comprises the following steps:

the treatment of a biological sample taken from this host in order to extract the total DNA from this host and to make the genome of these bacteria accessible to the nucleotide sequences or primers defined according to any one of claims 1 to 9, treatment being carried out in particular using a rapid DNA extraction technique based on the attachment of nucleic acids to silica beads,

- the amplification of the number of copies of genes coding for the proteins of the TMAO-reductase system of bacteria involved in the degradation of the flesh of aquatic animals, or of fragments of these genes, likely to be present in this sample, at 1 using the abovementioned nucleotide sequences or primers hybridizing with the genes or fragments of aforementioned genes,

- the detection of the possible presence of an amplified number of copies of genes coding for a protein of the TMAO-reductase system of the above-mentioned bacteria, or of fragments of these genes, and therefore of the presence of such bacteria in the biological sample studied .

12. Detection method according to claim 11, characterized in that the amplification of the number of genes coding for TMAO-reductase comprises the following steps:

the predenaturation of the total double-stranded DNA of the host into single-stranded DNA, preferably in a buffer made up of 10 mM Tris-HCl pH 8.3, 50 mM KC1, 1.5 mMgCl ₂ , 0.01% of gelatin, of the 4 constitutive DNA deoxynucleotides (dCTP, dATP, dGTP, dTTP) at a concentration of 100 μM each, and pairs of primers, as defined in claims 1 to 9, by heating between approximately 90 ° C and about 100 ° C, advantageously at 94 ° C, for about 1.5 minutes,

the actual amplification by addition to the medium obtained in the preceding stage of DNA polymerase, for example Taq polymerase, heating at approximately 94 ° C. for approximately 30 seconds, which corresponds to the denaturation stage proper,

* then heating between approximately 35 ° C to approximately 60 ° C, and in particular to approximately 45 ° C or 55 ° C, for approximately 30 seconds, which corresponds to the stage of hybridization of the primers with the genes coding for the proteins of the TMAO-reductase system of bacteria, or fragments of these genes, which may be present in the biological sample studied, and finally, heating at 72 ° C., for approximately 45 seconds, which corresponds to the step of elongation of the primers, hybridized in the preceding step, towards each other, thus producing nucleotide sequences complementary to fragments of genes coding for proteins of the TMAO-reductase system of bacteria, the latter sequences being delimited by the nucleotides s '' hybridizing with the aforementioned primers,

- repetition of the previous amplification step between approximately 15 and approximately 35 times, advantageously approximately 30 times.

13. Kit for implementing a detection method according to one of claims

10 to 12, characterized in that it comprises:

- one or more nucleotide sequences or primers defined in one of claims 1 to 9,

- a DNA polymerase,

a reaction medium advantageously consisting of 10 mM Tris-HCl pH 8.3, 50 mM KC1, 1.5 mM MgCl ₂ , 0.01% of gelatin, of the 4 deoxynucleotides constituting DNA (dCTP, dATP, dGTP, dTTP) at a concentration of 100 μM each.

14. Nucleotide sequence comprising:

the sequence represented in FIG. 2 of the torA gene coding for the TorA protein of the marine bacterium Shewanella c, * or any sequence derived from the above-mentioned sequence by degeneration of the genetic code, and coding for the protein TorA of Shewanella c, including the peptide sequence is shown in FIG. 6,

* or any sequence derived from the above-mentioned nucleotide sequence, in particular by substitution, deletion or addition of one or more nucleotides, said derived sequence preferably having a homology of approximately 35% to 100% with the above-mentioned nucleotide sequence shown in the figure 2

* or any fragment of the abovementioned nucleotide sequence, or of a sequence derived from the latter as defined above, said fragment preferably consisting of at least approximately 15 nucleotides, - the partial sequence represented in FIG. 3 of gene coding for the protein TorA of the marine bacterium Photobacterium phosphoreum,

* or any sequence derived from the above-mentioned sequence by degeneration of the genetic code, and coding for the protein TorA of Photobacterium phosphoreum whose peptide sequence is represented in FIG. 7, * or any sequence derived from the above-mentioned nucleotide sequence, in particular by substitution, deletion or addition of one or more nucleotides, said derived sequence preferably having a homology of approximately 35% to 100% with the above-mentioned nucleotide sequence shown in FIG. 3,

* or any fragment of the above-mentioned nucleotide sequence, or of a sequence derived from the latter as defined above, said fragment preferably consisting of at least about 15 nucleotides, the partial sequence represented in FIG. 5 of the gene coding for the protein TorA of the marine bacterium Salmonella typhimurium,

* or any sequence derived from the above-mentioned sequence by degeneration of the genetic code, and coding for the TorA protein of Salmonella typhimurium, the peptide sequence of which is represented in FIG. 8,

* or any sequence derived from the above-mentioned nucleotide sequence, in particular by substitution, deletion or addition of one or more nucleotides, said derived sequence preferably having a homology of approximately 35% to 100% with the above-mentioned nucleotide sequence shown in the figure 5, * or any fragment of the above-mentioned nucleotide sequence, or of a sequence derived from the latter as defined above, said fragment preferably consisting of at least about 15 nucleotides.

15. Peptide sequence encoded by a nucleotide sequence according to claim 14, and comprising:

the amino acid sequence represented in FIG. 6 of the TorA protein of Shewanella c,

* or a sequence derived from the above-mentioned peptide sequence, in particular by substitution, deletion or addition of one or more amino acids, said derived sequence preferably having a homology of approximately 35% to 100% with the above-mentioned peptide sequence shown in the figure 6,

* or a fragment of the above-mentioned peptide sequence, or of a sequence derived from the latter as defined above, said fragment preferably consisting of at least about 5 amino acids, - the partial amino acid sequence shown in Figure 7 of the TorA protein of

Photobacterium phosphoreum,

* or a sequence derived from the above-mentioned peptide sequence, in particular by substitution, deletion or addition of one or more amino acids, said derived sequence preferably having a homology of approximately 35% to 100% with the above-mentioned peptide sequence shown in the figure 7,

* or a fragment of the above-mentioned peptide sequence, or of a sequence derived from the latter as defined above, said fragment preferably consisting of at least minus about 5 amino acids,

- the partial amino acid sequence shown in FIG. 8 of the TorA protein of Salmonella typhimurium,

* or a sequence derived from the above-mentioned peptide sequence, in particular by substitution, deletion or addition of one or more amino acids, said derived sequence preferably having a homology of approximately 35% to 100% with the above-mentioned peptide sequence shown in the figure 8,

* or a fragment of the above-mentioned peptide sequence, or of a sequence derived from the latter as defined above, said fragment preferably consisting of at least about 5 amino acids.