FR2867485A1 - Detecting presence, or risk of developing, encephalopathy, especially bovine spongiform encephalopathy, based on determining specific nucleic acid or protein markers - Google Patents

Abstract

Method for detecting the presence, or risk of developing, an encephalopathy in a mammal by determining, in a biological sample, specific target molecules (A). (A) are (a) any of 26 nucleic acids (I), sequences reproduced, or their fragments of at least 5, preferably 6-10, consecutive bases; (b) the complements of (a); (c) a functional analog of (a) or (b), derived from another species or a natural variant; or (d) a polypeptide (II) encoded by (a)-(c). Independent claims are also included for the following: (1) product comprising a support on which is/are immobilized at least one (I) or (II); and (2) kit comprising, in a compartment or container, at least one (I).

Description

Identification of diagnostic markers

  The present application relates to biological markers of transmissible spongiform subacute encephalopathies and their uses in diagnostic methods. It also relates to tools and / or kits that can be used to implement these methods (reagents, probes, primers, antibodies, chips, cells, etc.), their preparation and their uses. The invention is useful for detecting the presence of an infection in mammals, including in the early phase.

  Prion diseases, also known as transmissible spongiform encephalopathies or unconventional transmissible diseases (NCTA), are diseases of the central nervous system found in some mammals, including man.

  The best-known forms of these diseases are sheep scrapie in sheep, Bovine Spongiform Encephalopathy (BSE) in cattle, Creutzfeld-Jakob disease (CJD), kuru and fatal familial insomnia in dogs. man.

  The infectious agent is not yet definitively determined, but the most accepted hypothesis is that these diseases are associated with the accumulation in the brain of a prion protein (PrP) of abnormal conformation with respect to the conformation observed in healthy individuals.

  The discovery of a new variant of MJC (vMJC) after the BSE outbreak in England confirms that these diseases are transmissible and are likely to cross the species barrier through food. Their slow and fatal evolution is associated with lesions that exclusively affect the central nervous system.

  With the discovery of vCJD, emergency measures were put in place to assess the extent of the BSE epidemic and to protect public health. The European Union is now imposing a systematic test of all meat from slaughtered cattle over 30 months old. As the incubation time of BSE is around five years, during which time the infection can spread laterally and vertically, the development of a diagnostic test on live animals is of vital importance. An early test would provide a safe way to exclude infected animals from the food chain. The test used today detects only post-mortem animals infected at a late stage of the disease.

  There is an urgent need for a diagnostic test capable of detecting encephalopathies at an early stage in live animals in a rapid manner. Such a test would track all the animals at risk by testing them multiple times in their lifetime.

  Application WO02 / 074986, belonging to the applicant, describes several genetic markers of encephalopathies. Through extensive research using an innovative approach, several additional markers of BSE have been identified and validated in hybridization experiments, making it possible to establish a pre-symptomatic test that can be used from the blood of a living mammal.

  The identified markers were isolated by the DATAS technique (patent application WO99 / 46403). DATAS identifies qualitative differences in gene expression and provides a systematic analysis of spliced RNA between two conditions: healthy / infected. DATAS leads to the identification of functionally distinct RNA variants. The DATAS technique consists of three distinct steps: tissue collection, RNA isolation, and the construction of a directory containing qualitative differences and identifying new gene fragments, which can not be isolated by other genetic techniques.

  By comparing the qualitative expression of genes in the blood cells of healthy mammals infected naturally or experimentally with BSE, different signatures of genetic markers have been isolated. Naturally infected mammals were at the final stage of the disease, while mammals infected orally with 1 g of BSE-infected brains represented the early stage of the disease.

  The implementation of the DATAS methodology on cow blood cells has enabled the identification and isolation of several thousand genetic markers, divided into two repertoires representative of the qualitative expression of genes between healthy cows and infected cows. naturally on the one hand, and between healthy and experimentally infected cows on the other.

  The different clones of the two DATAS experiments were deposited on glass slides. The slides were hybridized with probes produced from biological material from naturally or experimentally infected cows and healthy cows used as controls. Through two types of statistical analysis, SAM (Significance Analysis of Microarray) and PAM (Prediction Analysis of Microarray) comparing healthy animals versus infected animals, 15 clones were observed to show deregulation in healthy versus infected conditions. The nucleic acid sequences are described below as SEQ ID NO: 1-15.

  The present application thus provides a set of biological markers that can be used, alone or in combination (s), to detect, characterize or track a transmissible spongiform encephalopathy in a mammal. The invention is useful in particular for detecting the presence of prion diseases in mammalian subjects, in particular sheep, cattle or humans. It is particularly advantageous insofar as it can be carried out on living mammals, from biological fluids such as blood, plasma, platelets, etc. An object of the present application relates to a method for detecting the presence or risk of developing encephalopathy in a mammal, comprising determining the presence (or absence) of a target molecule in a biological sample of the mammal selected from: a) a nucleic acid comprising a sequence selected from SEQ ID NOs: 1 to 30 or a fragment thereof having at least 5, preferably 6, 7, 8, 9 or 10 consecutive bases, b) a nucleic acid having a sequence complementary to a sequence according to a), c) a functional analogue of a nucleic acid according to a) or b), or d) a polypeptide encoded by a nucleic acid according to a) to c), the presence (or absence) of such a target molecule in the sample being an indication of the presence or risk of developing encephalopathy in that mammal.

  In a particular variant of implementation, the method comprises the (simultaneous) determination of the presence or absence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the target molecules such as as defined above.

  The target molecule may be the complete sequence of the gene or of the RNA or of the protein corresponding to the sequences SEQ ID NOs: 1-15, or a fragment thereof, for example having a domain of variability (splicing, deletion, polymorphism, etc.). A functional analogue refers more particularly to an analogue from another species (eg human, sheep, etc.), or a natural variant resulting for example from polymorphism, splicing, etc. In a particular embodiment, the method comprises determining the presence of at least one nucleic acid according to a) to c). Different techniques that can be used to detect a species of nucleic acid in a sample can be used in the present invention, such as, for example, Northern Blot, selective hybridization, the use of probed oligonucleotide-coated supports, and the amplification of acid. nucleic acid as for example by RT-PCR, quantitative PCR and ligation-PCR, etc. These methods may include the use of a nucleic probe (eg an oligonucleotide) capable of selectively or specifically detecting the target nucleic acid in the sample. The amplification may be carried out according to various methods known per se to those skilled in the art, such as PCR, CSF, transcription-mediated amplification (TMA), strand displacement amplification (SDA), NASBA, the use of allele-specific oligonucleotides (ASO), allele-specific amplification, Southern blotting, SSCA conformational analysis, in situ hybridization (eg, FISH), gel migration, heteroduplex analysis, etc. According to a preferred mode of implementation, the method comprises the detection of the presence or absence of a nucleic acid according to a) to c) by selective hybridization or selective amplification.

  Selective hybridization is typically performed using nucleic probes, preferably immobilized on a support, such as a solid or semi-solid support having at least one surface, flat or not, allowing the immobilization of nucleic probes. Such supports are for example a blade, ball, membrane, filter, column, plate, etc. They can be made of any compatible material, such as glass, silica, plastic, fiber, metal, polymer, etc. The nucleic probes may be any nucleic acid (DNA, RNA, PNA, etc.), preferably single-stranded, comprising a sequence specific for a target molecule as defined in a) to c) above. The probes typically comprise from 5 to 400 bases, preferably from 8 to 200, more preferably less than 100. The probes may be synthetic oligonucleotides, produced on the basis of the target molecule sequences of the invention according to conventional synthesis techniques. . The probes can also be synthesized directly in situ, on the support, according to methods known per se to those skilled in the art. The probes can also be manufactured by genetic techniques, for example by amplification, recombination, ligation, etc. Such a probe is another object of the present application, as well as its use (essentially in vitro) for the detection of encephalopathy in a subject. In a particularly preferred manner, a set of nucleic probes comprising all or a fragment of at least 5 consecutive bases of each of the sequences SEQ ID NO: 1-15, or of a complementary strand thereof, advantageously immobilized on a support.

  Hybridization can be carried out under standard conditions, known to those skilled in the art and adjustable by it (Sambrook et al). In particular, the hybridization can be carried out under conditions of high, medium or low stringency, depending on the desired level of sensitivity, the quantity of available material, etc. For example, suitable hybridization conditions include a temperature of 62 to 67 C for 2 to 18 hours. After hybridization, different washes can be performed to remove unhybridized molecules, typically in SSC buffers comprising SDS, such as a buffer comprising 0.1 to 10 X SSC and 0.1% SDS.

  In a typical embodiment, the nucleic acids (or the chips or supports) are prehybridized in hybridization buffer (Rapid Hybrid Buffer, Amersham) typically containing 100 g / ml of salmon sperm DNA. 65 C for 30 min. The nucleic acids of the sample are then placed in contact with the probes (typically applied on the support or the chip) at 65 ° C. for 2 to 18 hours. Preferably, the nucleic acids of the sample are marked beforehand with any known labeling (radioactive, enzymatic, fluorescent, luminescent, etc.). The supports are then washed in a 5 × SSC buffer, 0.1% SDS at 65 ° C. for 30 min, then in a 0.2 × SSC buffer, 0.1% SDS. The hybridization profile is analyzed according to conventional techniques, for example by measuring the marking on the support by means of a suitable instrument (for example Instantlmager, Packard Instruments). Hybridization conditions can naturally be adjusted by those skilled in the art, for example by modifying the hybridization temperature and / or the salt concentration of the buffer.

  The selective amplification is preferably performed using a primer or pair of primers for amplifying all or part of one of the target nucleic acids in the sample, when present therein. The primer may be specific for a target sequence according to SEQ ID NO: 1-15, or a region flanking the target sequence in a nucleic acid of the sample. The primer typically comprises a single-stranded nucleic acid, preferably between 5 and 50 bases long, preferably between 5 and 30. Such a primer constitutes another object of the present application, as well as its use (essentially in in vitro) for the detection of encephalopathy in a subject.

  In another embodiment, the method comprises determining the presence of a polypeptide according to d). The detection of a polypeptide in a sample can be carried out by any technique known per se, such as in particular by means of a specific ligand, for example an antibody or an antibody fragment or derivative. Preferably, the ligand is an antibody specific for the polypeptide, or a fragment of such an antibody (for example an Fab, Fab ', CDR, etc.), or a derivative of such an antibody (for example a single antibody). chain, ScFv). The ligand is typically immobilized on a support, such as a slide, ball, column, plate, etc. The presence of the target polypeptide in. the sample can be detected by demonstrating a complex between the target and the ligand, for example using a labeled ligand, using a second labeled revealing ligand, etc. Immunological techniques that can be used and well known are ELISA, RIA, etc. Antibodies specific for the target polypeptides may be produced by conventional techniques, including immunizing a non-human animal with an immunogen comprising the polypeptide (or an immunogenic fragment thereof), and recovering antibodies (polyclonal) or producing cells (to produce monoclonals). Techniques for producing poly- or monoclonal antibodies, ScFv fragments, human or humanized antibodies are described for example in Harlow et al., Antibodies: A laboratory Manual, CSH Press, 1988; Ward et al., Nature 341 (1989) 544; Bird et al., Science 242 (1988) 423; WO94 / 02602; US5,223,409; US5,877,293; WO93 / 01288. The immunogen may be synthetically manufactured, or by expression, in a suitable host, of a target nucleic acid as defined above. Such an antibody, monoclonal or polyclonal, as well as its derivatives having the same antigenic specificity, are also an object of the present application, as well as their use for detecting encephalopathy.

  The method of the invention is applicable to any biological sample of the mammal under test, in particular any sample comprising nucleic acids or polypeptides. A sample of blood, plasma, platelet, saliva, urine, stool, etc., may be advantageously mentioned, more generally any tissue, organ or, advantageously, biological fluid comprising nucleic acids or polypeptides. In a preferred embodiment, the sample is a sample of blood or plasma. The sample may also be pre-treated to facilitate the accessibility of the target molecules, for example by lysis (mechanical, chemical, enzymatic, etc.), purification, centrifugation, separation, etc. The sample can also be labeled, to facilitate the determination of the presence of the target molecules (fluorescent, radioactive, luminescent, chemical, enzymatic labeling, etc.).

  The invention is applicable to any mammal, preferably selected from cattle, sheep and humans. The method of the invention is particularly useful for the detection of sheep scrapie in sheep, Bovine Spongiform Encephalopathy (BSE) in cattle, and Creutzfeld-Jakob disease (CJD), kuru and lamb familial fatal insomnia in men.

  A particular object of the present application relates to a method for detecting the presence or risk of developing BSE in cattle, including determining the presence (or absence) in a biological sample of cattle of one or more a plurality of target molecules selected from: a) a nucleic acid comprising a sequence selected from SEQ ID NO: 1 to 15 or a fragment thereof having at least 5, preferably 6, 7, 8, 9 or 10 consecutive bases, b) a nucleic acid having a sequence complementary to a sequence according to a), c) a polypeptide encoded by a nucleic acid according to a) or b).

  Another particular object of the present application relates to a method for detecting the presence or risk of developing BSE in cattle or sheep, comprising contacting a biological sample of the bovine or ovine containing nucleic acids with a product. comprising a support on which is immobilized at least one nucleic acid comprising a sequence selected from SEQ ID NO: 1 to 15, a fragment thereof having at least 5 consecutive bases, or a nucleic acid having a sequence complementary thereto , and the determination of the hybridization profile, the profile indicating the presence or risk of developing BSE in cattle or sheep. Preferably, the product comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 different nucleic acids selected from the nucleic acids mentioned above. In a particular embodiment, the product each comprises nucleic acids of sequence SEQ ID NO: 1 to 15, a fragment thereof having at least 5 consecutive bases, or a nucleic acid having a sequence complementary to those this.

  Another subject of the present application relates to a product comprising a support on which at least one nucleic acid comprising a sequence chosen from SEQ ID NO: 1 to 15, a fragment thereof having at least 5 consecutive bases, an acid, is immobilized. nucleic acid having a sequence complementary thereto or a functional analogue thereof. Preferably, the product comprises at least 2, 3, 4, 5, 6, 7, 8, 9, different nucleic acids selected from the nucleic acids mentioned above.

  In a particular embodiment, the product each comprises nucleic acids of sequence SEQ ID NO: 1 to 15, a fragment thereof having at least 5 consecutive bases, or a nucleic acid having a sequence complementary to those Another object of the present application relates to a product comprising a support on which is immobilized at least one polypeptide encoded by a nucleic acid comprising a sequence selected from SEQ ID NO: 1 to 15, a fragment thereof having at least 15 consecutive bases, a nucleic acid having a sequence complementary thereto or a functional analogue thereof. Preferably, the product comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 different polypeptides selected from the polypeptides mentioned above.

  The support can be any solid or semi-solid support having at least one surface, flat or not, allowing the immobilization of nucleic acids or polypeptides. Such supports are for example a blade, ball, membrane, filter, column, plate, etc. They can be made of any compatible material, such as glass, silica, plastic, fiber, metal, polymer, polystyrene, teflon, etc. The reagents can be immobilized on the surface of the support by known techniques, or, in the case of nucleic acids, synthesized directly in situ on the support. Immobilization techniques include passive adsorption (Inouye et al., J. Clin Microbiol 28 (1990) 1469), the covalent bond. Techniques are described for example in WO90 / 03382, WO99 / 46403). The reagents immobilized on the support can be ordered according to a pre-established scheme, to facilitate the detection and identification of the complexes formed, and in a variable and adaptable density.

  In one embodiment, the product of the invention comprises a plurality of synthetic oligonucleotides, of a length of between 5 and 100 bases, specific for one or more target nucleic acids defined in a) to c).

  The products of the invention typically comprise control molecules for calibrating and / or normalizing the results.

  Another object of the present application relates to a kit comprising a compartment or container comprising less a nucleic acid comprising a sequence chosen from SEQ ID NO: 1 to 15, a fragment thereof having at least 5 consecutive bases, a nucleic acid having a sequence complementary thereto or a functional analogue thereof. Preferably, the product comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 different nucleic acids selected from the nucleic acids mentioned above. In a particular embodiment, the product each comprises nucleic acids of sequence SEQ ID NO: 1 to 15, a fragment thereof having at least 5 consecutive bases, or a nucleic acid having a sequence complementary to those this. The kit may furthermore comprise reagents for a hybridization or immunological reaction, as well as, if appropriate, controls and / or instructions.

  Another subject of the invention relates to the use of a product or kit as defined above for the detection of encephalopathy in a mammalian subject.

  Another subject of the invention relates to a nucleic acid comprising a sequence chosen from SEQ ID NO: 1-15, or a fragment thereof comprising at least 5 consecutive bases, or a nucleic acid having a sequence complementary thereto. , or a functional analogue thereof, in particular an analogue from another species. The invention also relates to a cloning vector or expression comprising these nucleic acids, as well as any recombinant cell comprising such a vector or nucleic acid.

  Another subject of the invention relates to the use of a nucleic acid comprising a sequence chosen from SEQ ID NO: 1-15, or a fragment thereof comprising at least 5 consecutive bases, or a nucleic acid having a sequence complementary thereto, or a functional analogue thereof, in particular an analogue from another species, for the detection (essentially in vitro) of encephalopathy in a mammalian subject.

  According to a particular embodiment of the invention, a blood sample is taken from a mammal to be tested. The blood sample is processed to make the nucleic acids more accessible, and these are labeled. The nucleic aids are then applied to a product as defined above and the hybridization profile is determined, to diagnose the presence or absence of encephalopathy in the subject. The method of the invention is simple, practiced ex vivo, on live animals, and allows the early detection of prion disease.

SEQUENCE LIST

  SEQ ID NO: 1 EXB-NROA0576: Homology with NM_003576: Homo sapiens serine / threonine kinase 24 (STE20 homolog, yeast) (STK24), mRNA. 4/2003 January 5 GGTGTGGAGG TGTTCAAAGG CATTGACAAT CGGACTCAGA AAGTAGTCGC 51 CATAAAAATC ATTGACCTGG AGGAGGCAGA AGATGAGATC GAGGACATTC 10,101 AGCAGGAAAT CACAGTGCTG AGTCAGTGTG ACAGTCCCTA CGTAACCAAA 151 TATTACGGAT CCTACCTGAA GGACACTAAA TTGTGGATAA T SEQ ID NO: 2 - Cluster NROA_c584_1: Homology with CB424646: 598 918 MARC 6BOV Bos taurus cDNA 3 ' , mRNA sequence. 3/2003 1 TATCTGCAGA ATTTCCCCTT GAGAAGCGTT ATGGGGTGCA GGTAAGTTAT 51 TACACAAGAG AAAGAAGTTT TCTTACTAAC AGCAAGATTA ATGGCACAAT 101 TCAACCAAAA CTCATATACA TTTTACTGCT TAATTTACAT ATTATTTTGG 151 TGGAAAAAAT AGTATTCTTT ATTCTTTCAG TTTCTTTATG CAAAAATACA 201 CTTCTACAGG GACATCACTT AGATGTTATG CAAACCTCCC CCCC SEQ ID NO: 3 EXB NROA1108: Homology with NM_138402: Homo sapiens hypothetical protein BC004921 (LOC93349), mRNA. 4/2003 1 GAGACATTTG GCCAAAAGAG GAATTTCCAG GACACCAACA ACATCCATTA 51 TTCCATTATT CATTTGTTTC CTGAAGAGCA AACACTTCCT TGAAATTCTT 101 CTCAAATTCT GCCTCCAGTC TAAGCCCCAT TTGGCCAAAA TCATTGAACT 151 TGAAAGATGC CCTGTGGTTC TGAAAGATGA GACGCATGTC CCACACAAAC 201 CCTTCCACAT TGGAGTAGCC CTGCTCATTC AGCCTCTTCT TGATCTTGTC 251 CAGCCACATG GGCTCCTTGA GGTTTTTAGA AGCCTCTTTC ATATAATAAT 301 AATAGGGAAT CCTCACTATA ACGCT SEQ ID NO: 4 - Cluster NROA_c450_1: No homology 1 AAGCGTTATG CAGGTAGGCC GACAAGGCGA AGTGGGATGC CGGAGAGCGG 51 CCGAGTTATT GCTCCGAGGA GACCACGTTC ACCGGTTACT ATGGCGACCG 101 CCCCATCCCG GATCACTATC AGCCGTTCAC CGCCGATGAG GCGACGTGGT 55,151 TCCAGCTCTG GGAGACGGTG AGCGAGGGCA CTCCTACGTC GCCGCCCTTC 201 GCGACGATTG AGGAACTGGC AGCCTACCTC GCCGAGTGGG GCGACTTCTG 251 TGATCACAGG CGCGCCGTCG AGTCCATGGA CGCGCGCGAG ATTGAGCGCC 301 TCCTGACGCT GAATGACCGG CACTAGTTCA AGGTGCGGCT GGGGGCAGCA 351 GCGCGCCTAA GCTTTCTGCA AGACTGGCTG GGCGCCCAGC ATGATGGTCC 401 GCGGCGGCGA GATCCTGACC AACCCTGGGG ACATGGTGTC GTCGTGACCC 451 TCGCCTAGCT CTCTCACACA C CTAGGAGGA AGAGATGACC ACCCCCAACA 501 TTCGCGGCCA CGAGACCGAA GCCAAGGCCC GCAAGGCGGC GATGAAGTGG 551 TTCACCTTCA CGGACGGCAC CAAGCCTGTC GAGGGCGTCC ACTTCCACAT 601 CAAGCAGAAC CACTTCGGGC TCTGGACCTT CCGGGAGGGC CCGGCTCCGA 651 AGTCCGCCGG ACCCCGCATC ACTCATAACG CTTCTCAA SEQ ID NO: 5 EXB-NROB1323: Homology with NM_000982 Homo sapiens ribosomal protein L21 (RPL21), mRNA. 5/2003 1 AGAAGCGTTA TTGCTGATAC CCGCTACATG TTCTCCAGGC CTTTCAGAAA 51 ACATGGAGTT GTTCCTTTGG CCACATACAT GCGAATCTAC AGGAAGGGTG 101 ATATTGTAGA TATCAAGGGA ATGGGTACTG TTCAAAAAGG AATGCCCCAC 151 AAATGTTACC ATGGCAAAAC TTGAAAAGTC TATAATGTCA CCCAGCATGC 201 TGTTGGCATC ATTGTAAACA AACAAGTTAA GGGCAAGATT CTTGCCAAGA 251 GAATTAATGT GCGTATCGAG CATATTAAGC ACTCTAAGAG CCGAGATAGC 301 TTCCTGAAAC GTGTGAAGGA AAATGATCAG AAAAAGAGGG AAGCCAAAGA 351 GAAAGGGACT TGGGGTTAAC ACC SEQ ID NO: 6 EXB-NROA0588: Homology with AW325879: 17199 MARC 1BOV Bos taurus cDNA 5 ', mRNA sequence. 4/2001 1 GGGCGGAGGT CACCCTGGGG ATCCTCCAGG GCCAGGCCCT GGCACAACTC 51 GTCTCCATCA CACAGATGGG CCGTCGCCTG GTCGTGGCTC TCAGGAGTCA 101 GACCGGAAAA AGCCAGCCCT GGGGCAACCA GGAGCACCGA GGTGATGAGC 151 AGGACAGCCC AGGAGGTCAT GTTGAGGCAG CTGAAAGGTC TGTGCAAGTC 201 AATCATGAAG AAATTTCTCC GTACCATCAC CTCCC SEQ ID NO: 7 EXB-NROB1540: Homology with D37952: Bovine NB10 mRNA for MHC class II (BoLA-DQB), partial cds. 50 2/1999 1 CTTGTGTAGG CAGAGGTTCC AGGGTCAGTG GAGGAAGCAG CATCACAGCC 51 AGATCCATGG TTGGGGGATG GCCACGGGAA ATGACTTGGT GACTGACTCT 101 GATCTCAGAG TGGGACAGGC TGACAGGCAT CTGGGAATTC CGGGCAAGGT 151 CAGGCACGTA TTATAGAAGA GCAAACACCA ATCCCAAAAT ATCCTCAGGA 201 ATCAGCGCAT GAGCCCCTTC TGGCTCCTGT GATGGATGAT GAGGCCCAGC 251 CCAAGGAAGA TCAGCCCCAG CACAAAGCCT CCAAC SEQ ID NO: 8 Cluster NROB_cl_- 64: Homology with D76416: Bovine mRNA for MHC class II alpha-chain DM (BoLA-DMA), complete cds. 2/1999 1 TCTGCAGAAT TCGCCTCTGA GAAGCGTTAT CCGTTGGACC CAANNNNNNN 20 51 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNCAAGA GAAAAGATCA 101 GAGGGTGCTG GTGTGACGTT TAAGTAGGAA AAGGCCTGGA AGGTGAGTCC 151 ATCAACCGCG GAGACAAAAG TGGGCCCGGC TCCTTCCACA GGTGCCGACT 201 GATGCTGCCA GTTCACGGTC AGTGTGGGTC AACAC SEQ ID NO: 9 EXB-NROB1371: Homology with NM_006098 Homo sapiens guanine 30 nucleotide binding protein (G protein), beta 2-like polypeptide 1 (GNB2L1), mRNA. 4/2003 1 AAGCGTTATT TAGATAANNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 35 51 NNNNNNNTAC ACCCAGAGTA TTCCATAGTT TGATGGTTTT GTCTCGGGAG 101 CCAGAGACAA TTTGCCGGTT GTCAGAAGAG AAGGCCACAC TCAGCACATC 151 TTTGGTATGG CCTACAAATC GGCGAGTGGT GGTGCCCGTT GTGAGATCCC 201 AAAGGCGAAG GGTTCCATCC CAGGAGCCTG AGAGGGCAAATTGGCCATCT 251 GAGGAAATGA CCACATCACT AACAAAGTGG GAGTGACCCC GAAGAGCACG 301 CTGTGGGATA CCATAGTTGG TTTCATCTCT GGTCAGCTTC CACATAATGA 351 TGGTCTTATC TCGAGAGGCG GACGATATCA TGTCCGGGAA CTGGGGAGAG SEQ ID NO: 10 - NROB_c579_1: Homology with D87076: Homo sapiens mRNA for KIAA0239 gene, partial cds. 1/2003 1 GGGCCAGGGG ATGATATGAA TGTCACAGGA GGAGACACCT TCTGTCTTTG 55 51 TTTCAAAGAA AGTTGATGTG CCATTTGTTA ATATACAAGA GAAATATTGA 101 AAATATATTG AAAAGAGCAA TTTTAAATTA TTTTTGGCTT ATGTTGCAAT 151 ATTTATTTTC TTGTATTAGG AAAGATTCCT TTGTAGAAAA AAAATGTATT 201 TTTCATTAAC GCAAAAACCT ATTTCTCCTT TTTGTACATT GTCCATGTTC 251 GCTACCCTTA ACGAGCAATA GAATGTATGG CTGCCTCGGG GTGGCCGGTG 301 CCCGCGTGCC CTGCATGATT CTGTGGTCCC ACCACCATGT AGCTCCCAGT 351 CCCATCCTGT CCTGCTCACT CATGGGGGTT TCCAGAGCCT AGCCCCT SEQ ID NO: 11 Cluster NROA_c125_1: Homology with D45359: Bos taurus mRNA 15 for MHC Class II DRB3, partial cds. 1/2003 1 TGGATTGCAG GTGACTGAGA AAACCATCGA GGACAGTTTT TAAGGGGTCA 51 CTGAGCCAGG AGCAAATGAG ATCCTGAGAA AGTACTTCAT TGTGGAAGAG 101 TTAGCACTAA GCAGGAAACC TTTCCATGCT GTGAAGAAGC TGGGACAGAA 151 GGTTCTTCCT TGAGTGTGAC CATCTTCACT TCAGCTCAGG AGCCCTGTTG 201 GCTGAAGTGT AGGGCGTCCT TTCTGATTCC TGAAGTATAT TTATTAGCCC 251 CACGGCAAGG AAGAACAGAC TCAGAACGAA GCCCCCGACT CCACTCATCA 301 TCTTGCTCTG AGCAGAGTCA GACCGTGCCC TCCATTCTAC TGTGATAGGG 351 CTTGTCTGGC TGGGGTGCTC CACTTGGCAA GTGTAGACCT GGCACCA SEQ ID NO: 12 Cluster NROB_c0_1: Homology with J01394: Bos taurus 35 mitochondrion, complete genome. 4/2001 1 GCTGTCCAAA AAGGCCTCCG TTATGGAATA ATTCTTTTTA TTATCTCCGA 51 AGTACTATTC TTTACCGGAT TTTTCTGAGC TTTCTACCAC TCAAGCCTCG 101 CCCCCACCCC TGGGGGAGGC GGCTGCTGAC CCCCAACAGG CATTCACCCA 151 CTAAACCCCC TAGAAGTCCC ACTGCTCAAC ACCTCTGTCC TATTGGCTTC 201 CGGAGTTTCT ATTACCTGAG CCCATCATAG TTTAATAGAA GGGGACCGAA 251 AGCATATATT ACAAGCCCTA TTTATCACCA TCACATTAGG AGTCTACTTC 301 ACACTACTAC AAGCCTCAGA ATACTATGAA GCACCTTTTA CTATCTCCGA 351 CGGAGTTTAC GGCTCAACTT TTTTTGTAGC CACAGGCTTC CACGGCCTCC 401 ACGTCATCAT TGGGTCCAAC AAATAACGCT TCTCNNNNNN NNNNNNTGCA 55 451 GATA SEQ ID NO: 13 - Cluster NROA c1045 -1: Homology with BM363411: BS320054B20G01 Subtracted Lewin Cattle Spleen Bos taurus cDNA clone BS320054B20G01 5 ', mRNA sequence. 1/2002 1 GGGGAGGTAT CTGTCACCCA CGCAGAAATG CTTCTGACAG GCGGCANNNN 51 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 101 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 151 CTTGGCCGAA AAGCCTGAGG TAGTCTCGGC GGCAGAGCTT CCGGCCCAGC TTGTAGTAGA 201 GGCGCCGGCC CACCTACCC SEQ ID NO: 14 Cluster NROA_c69_1: Homology with NM_003127 Homo sapiens spectrin, alpha, non erythrocytic 1 (alpha-fodrin) (SPTAN1), mRNA. 4/2003 1 GAAGCGTTAT TGGAGGAGGC TAACCTAGGA GCAGAGGATC AGTTCACGAA 51 GAGCGAGCGG GTGAACTCGA CGTAGTCAAA AGCAGTGGGG AGTTCGCGGC 101 CCTTGCTGTC CACGTAGGGC TTCATGTGGG AGACGCAGTA GTCGGCTTGT 151 TCCCGAGTCA AGTTCTGGTA CAGCTCCTCC TTGGTCACAT AAGGCTTCCC 201 TTCAGAGCTC ACGGCCCGGA AGGCGCTCTC AATCTCCTCG CTGGACTTGA 251 CGTTCTCCGT CTCACGGCTG ATCATAAAGG ACATGTACTC TTGCATGGAG 301 ACGTGGACGT CCCTGTTAGG ATCCACAGTG TCCAAGATGG ACTCGAACTC 351 AAGGTCGGGC TCCCCTTCCT CCAACATGGG CAGGTC SEQ ID NO: 15 Cluster NROBcl_12: Homology with BM431390: lDuol5D10 Bos taurus Duodenum # 1 library Bos tails cDNA, mRNA sequence. 1/2002 1 TCTGCAGAAT TCGCCTTTGA GAAGCGTTAT GGGGGCGAGG TGGTAAAGGA 51 AGCTTACAAA ACAACTATTC TTTAAAAAAA AACAAAAAAA CAAAAAAGCA 101 AAAAACAGCA AAAGCCAACC GGCCCAATTT TGTCTCCAGT TTTCAACGTG 151 TGCTTTCGAG CATTTCAGCT GTTTCCAGTT ACTTTAGTTT CCAGATATTA 201 GTCTTCCATT TAGTTTTAAG ACTAAATCTC ACTTTTGGAT AAACACAAGG 251 AAATATTTTA CTTGCTGAAA AATCACTTTA CTGGATAAAG TTACCTCTTA 301 TGCCTTTCAG TTTTCTAATC CAACTTTCTG ACAACCAGTG GTAATTAGGA 351 AGTTCTAAGT TGCAGTTGTC CCTATGACTT TGGGCTTCCC TGGTGGCTCA 55 401 GCTGGTCAAA AATCTGCCTG CAATGCGGGA GACCTCCACC CCATAACGCT 451 TCTCAAAGGC GAATTCTGCA GA

Claims (2)

17 CLAIMS   A method for detecting the presence or risk of developing encephalopathy in a mammal, comprising determining the presence, in a biological sample of the mammal, of a target molecule selected from: a) a nucleic acid comprising a sequence selected from SEQ ID NO: 1 to 15 or a fragment thereof having at least 5, preferably 6, 7, 8, 9 or 10 consecutive bases, b) a nucleic acid having a sequence complementary to a sequence according to a), c) an analogue of a nucleic acid according to a) or b) from another species or a natural variant of such a nucleic acid, or d) a polypeptide encoded by a nucleic acid according to a) to c), the the presence of such a target molecule in the sample being an indication of the presence or risk of developing encephalopathy in that mammal.   The method of claim 1 comprising (simultaneous) determination of the presence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the target molecules.   3. Method according to claim 1 or 2, comprising detecting the presence or absence of a nucleic acid according to a) to c) by selective hybridization or selective amplification.   The method of claim 1 or 2 comprising detecting the presence or absence of a polypeptide according to d) using a specific antibody or a fragment or derivative thereof.   The method of claim 1 for detecting the presence or risk of developing BSE in cattle, comprising determining the presence, in a biological sample of the bovine, of one or more target molecules selected from: a) a nucleic acid comprising a sequence selected from SEQ ID NO: 1 to 15 or a fragment thereof having at least 5, preferably 6, 7, 8, 9 or 10 consecutive bases, 2867485 18 b) a nucleic acid having a sequence complementary to a sequence according to a), c) a polypeptide encoded by a nucleic acid according to a) or b).   A method for detecting the presence or risk of developing BSE in cattle or sheep, comprising contacting a biological sample of the bovine or ovine containing nucleic acids with a product comprising a carrier on which is immobilized at at least one nucleic acid comprising a sequence selected from SEQ ID NO: 1 to 15, a fragment thereof having at least 5 consecutive bases, or a nucleic acid having a sequence complementary thereto, and determining the profile of hybridization, the pattern indicating the presence or risk of developing BSE in cattle or sheep.   7. Use of a nucleic acid probe specific for a target nucleic acid as defined in claim 1, said probe comprising from 5 to 400 bases, for the in vitro detection of encephalopathy in a subject.   8. Use of a nucleic primer allowing the amplification of all or part of a target nucleic acid as defined in claim 1, said primer being single-strand, of a length of between 5 and 50 bases, for the in vitro detection of encephalopathy in a subject.   9. A product comprising a support on which is immobilized at least one nucleic acid comprising a sequence chosen from SEQ ID NO: 1 to 15, a fragment thereof having at least 5 consecutive bases, or a nucleic acid having a sequence complementary to them.   A product comprising a support on which is immobilized at least one polypeptide encoded by a nucleic acid comprising a sequence selected from SEQ ID NO: 1 to 15, a fragment thereof having at least 15 consecutive bases, or a nucleic acid having a complementary sequence of these.   A kit comprising a compartment or container comprising at least one nucleic acid comprising a sequence selected from SEQ ID NO: 1 to 15, a fragment thereof having at least 5 consecutive bases, or a nucleic acid having a complementary sequence of these.   12. Use of a nucleic acid comprising a sequence selected from SEQ ID NO: 1-15, or a fragment thereof comprising at least 5 consecutive bases, or a nucleic acid having a complementary sequence thereof, for the in vitro detection of encephalopathy in a mammalian subject.