Abstract:
The present invention discloses a method to detect and identify microorganisms that are present in a sample, which comprises hybridizing total DNA isolated from the sample with DNA fragments from regions that codifies for tRNA-synthases, hereinafter ‘tRNA-synthases’, said fragments being selected due to their specificity for each taxon to be detected and identified. Furthermore, an array for detection and identification of microorganisms is disclosed, which comprises at least one tRNA-synthase fragment bound to its surface that is specific for each taxon to be detected and identified and has been designed according to the method described in the present document.

Description:
FIELD OF THE INVENTION  
       [0001]     The present invention discloses a method to detect and identify microorganisms that are present in a sample, which comprises hybridizing total DNA isolated from the sample, said method using genomic regions that codify for different tRNA-synthases.  
       BACKGROUND OF THE INVENTION  
       [0002]     Microorganism detection and identification has been carried out using different methods in time. Traditionally, it has been performed using phenotypic, physiological and structural or biochemical features of the microorganisms, by means of methods like microscopy, staining and selective culture, among many others. These techniques allow detecting and identifying a large number of microorganisms suitably, but in many cases they do not differentiate between taxonomically close microorganisms.  
         [0003]     In the last years, owing to the advances of molecular biology in the microbiology field, techniques for detection and identification of microorganisms based on the comparison of total or partial sequences of their nucleic acids, either DNA or RNA, has been developed. This makes even feasible the study of species that are not able to be cultured in the laboratory but the DNA of which can be directly extracted from the medium in which they are found. Comparing the conservation and variability features shown by nucleotide sequences of genes that are present in different organisms, it is possible to detect taxonomic groups at different levels, namely kingdom, family, genus or species. It only depends on the amount of available information and the strategy to be followed, understanding by strategy the molecular biology techniques to be employed and the selection of the nucleotide sequences on which work will be carried out.  
         [0004]     A widely diffused strategy in the art is working with the gene that codifies for the 16S ribosomal ARN molecule, which will be hereinafter referred as 16S rDNA. This region is characterized by widely variable sequences flanked by highly conserved regions in its sequence. Comparison of variable regions of 16S rDNA has allowed a satisfactory differentiation of different microbiological relevant taxons. On the other hand, the presence of highly conserved regions flanking the variable region has allowed an increase in the sensitivity of identification and detection techniques. These highly conserved regions are common to a large number of different microorganisms, and many times even to entire domains, and this has allowed the use of “universal” primers for Bacteria or Archaea domains, for instance. Using these universal primers in a polymerase chain reaction (PCR), 16S rDNA regions of all microorganisms belonging to said domains that are present in a given metagenomic sample are specifically amplified. Once said 16S rDNA region has been amplified, detection and identification techniques can be applied, based on the recognition of variable regions contained in the amplicon. The use of 16S rDNA being so extended, microorganism classification has been based mainly in the comparison of these sequences until now. The large information existent for 16S rDNA sequences facilitates the design of specific sequences that allow the identification of a determined taxon.  
         [0005]     In spite of the virtues of using 16S rDNA, its intra-genus variability has the disadvantage of being low, which makes the differentiation between species of the same genus not always possible. This represents a problem, especially in the biotechnology field, where in many cases it is necessary to work with species having specific activities, the population of which must be monitored and controlled in the processes where they are applied.  
         [0006]     When facing this difficulty, it is necessary to have an alternative strategy that allows the detection of taxonomic groups at all levels.  
         [0007]     The new proposed genomic region must fulfill the same requirements fulfilled by 16S rDNA, i.e. it must be represented in a large diversity of microorganisms and have at the same time a variability degree in its nucleotide sequence that allow the differentiation of all the relevant taxonomical groups, either families, genera or species. Moreover, it is convenient to consider a collection of sequences belonging to the proposed genomic region to be considered, which has been generated from the largest possible microorganism diversity, as this will assure a better design of specific sequences for each taxonomic level.  
         [0008]     We have solved said technical problem by selecting genomic regions codifying for tRNA-synthases to obtain specific sequences for the different microorganism taxons to be detected and identified.  
         [0009]     tRNA-synthases are present in all microorganisms and they have been studied and sequenced many times because of their relevance, said sequences being deposited in public databases. This allows the comparison of sequences of genomic regions codifying for tRNA-synthases (herein referred as tRNA-synthases) of many different types of microorganisms, which allows to carry out a good design of specific sequences for detecting and identifying the different relevant taxons.  
         [0010]     The selection of the region over which the studies will be carried out is the first stage of a strategy to detect and identify microorganisms. The second stage comprises the molecular biology techniques used in said strategy. There are many molecular biology techniques useful for detecting and identifying microorganisms, which are based in sample DNA hybridization with specific known sequences for a determined taxon. Among them, we can mention fluorescent in situ hybridization (FISH), denaturing gradient gel electrophoresis or temperature gradient gel electrophoresis (DGGE, TGGE), conjugation with specific markers, such as detection probes, polymerase chain reaction (PCR), and macro or micro DNA arrays. All these techniques are able to be performed by using genomic regions codifying for tRNA-synthases as the regions selected for the design of specific sequences for different taxons, and are to be considered within the scope of the invention.  
         [0011]     Among all mentioned techniques, we have chosen to work with DNA arrays, because we think it is the most suitable technique for fast microorganism detection and identification, as it allows simultaneous and specific detection of various microorganisms at the same time. Nevertheless, this preference must not be understood as limiting the invention, but as an application example of the same.  
         [0012]     A good definition of DNA array is the one proposed by Schena et al. (Trends Biotechnol. 16, 301-306): “a microscopic ordered nucleic acid array that allows simultaneous analysis of complex DNA samples” (Schena M., Heller, R. A., Theriault, P., Konrad, K., Lachenmeier, E. and Davis, R. W. (1998). Depending on the diameter of the deposited DNA spots, there are 2 array types: macro-arrays (300 microns or more) and micro-arrays (less than 100 microns). The first ones can be manually manufactured in the laboratory and the spots can be observed without the help of special equipment. The second ones require an automated deposition process (normally a robotic deposition platform) and a specialized image acquisition and processing equipment.  
         [0013]     The selection method using DNA fragment arrays comprise the simultaneous hybridization of the set of array “spots” with a labeled DNA extract of the studied sample. Normally, the DNA from the sample, which has been labeled and fragmented as required, is subjected to a denaturation stage wherein the DNA double helix is separated, e.g. by heating. When temperature is lowered, DNA will tend to couple with its most complementary fragment according to its physicochemical characteristics. Being this DNA in contact with the array, if there is coincidence between sample DNA and the DNA fragment contained in a spot, labeled sample DNA copies will specifically attach to said spot with the largest possibility. This is due to the larger amount of complementary DNA copies contained in the array spot. DNA labeling can be done by any known labeling technique, being fluorescence and radioactive labeling the most common one. Subsequently, in the acquisition and processing stage of the hybridized array image, this label will allow the detection of the microorganisms present in the studied sample.  
         [0014]     We have not found in the present state of the art any document that anticipates the subject matter of the present invention. Since now, structural and functional aspects of tRNA-synthases have been studied due to their relevance in protein synthesis (this enzyme promotes the bonding of the different amino acids to their corresponding transference RNA, tRNA) in different microorganisms. Even when genes codifying for tRNA synthases have been sequenced in a large variety of microorganisms, there is no study analyzing their usefulness in identification and classification of microorganisms. There are some studies based on the comparison of tRNA-synthase proteins that have been sequenced from different microorganisms, in order to establish evolutionary relationships between these genes and the phylogeny of bacteria containing them (as, for example, the review of O&#39;Donoghue P. (MMBR 2003 67 (4):550-573)), where their sequences and structures are compared. Nevertheless, there is no suggestion about the analysis of tRNA-synthase genes for taxonomic classification, and not even about its systematic use in molecular biology techniques for microorganism detection and identification, as we propose in the present invention.  
       BRIEF DESCRIPTION OF THE INVENTION  
       [0015]     The present invention discloses a method and array to detect and identify microorganisms that are present in a sample by using genomic regions codifying for different tRNA synthases.  
         [0016]     Due to their relevance, tRNA-synthases are represented in substantially all known microorganisms. Moreover, the genes that codify for them are sufficiently variable in their nucleotide sequence as to allow distinguishing between different microorganism taxons. That is, it is possible to find specific sequences for a determined genus or species.  
         [0017]     The present invention discloses a method to detect and identify microorganisms that are present in a sample, comprising hybridizing total DNA isolated from the sample with DNA regions codifying for tRNA synthases selected for their specificity for each type of microorganism to be identified. The method involves designing the specific DNA regions for each relevant taxon, from a database that comprises the sequences of different described tRNA-synthases.  
         [0018]     Once specific sequences for each relevant microorganism are obtained, these can be applied in diverse molecular biology detection techniques, such as PCR, FISH, DGGE, TGGE, detection probes, DNA arrays, or any other technique based in specific hybridization of nucleic acids.  
         [0019]     As an example of the invention, DNA arrays having specific sequences of tRNA-synthases for the detection and identification of  Agrobacterium tumefaciens; Corynebacterium glutamicum; Mycobacterium tuberculosis; Pseudomonas aeruginosa  and  Xylella fastidiosa  have been developed. 
     
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0020]      FIG. 1  shows the result of the hybridization of a metagenomic sample (M1) obtained from a mixture of commercial strains of  Agrobacterium tumefaciens, Corynebacterium glutamicum  and  Mycobacterium tuberculosis,  using an array of the invention. Used tRNA-synthase fragments are described in Table 3. Each fragment was deposited by triplicate. In Table 1, the content of each position in microarray 1 is detailed.  
         [0021]     Conclusion: The results verify the presence of  Agrobacterium tumefaciens, Corynebacterium glutamicum  and  Mycobacterium tuberculosis  in M1. All positive controls showed a hybridization signal and negative controls remained unmarked.  
         [0022]      FIG. 2  shows the result of the hybridization of a second metagenomic sample (M2) obtained from a mixture of commercial strains of  Pseudomonas aeruginosa  and  Xylella fastidiosa,  using another array of the invention. Used tRNA-synthase fragments are described in Table 3. Each fragment was deposited by triplicate. In Table 2, the content of each position in microarray 2 is detailed.  
         [0023]     Conclusion: The results verify the presence of  Pseudomonas aeruginosa  and  Xylella fastidiosa  in M2. All positive controls showed a hybridization signal and negative controls remained unmarked. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0024]     tRNA-synthases are essential components of the translation machinery in protein synthesis within cells, both in prokaryotes and in eukaryotes. These enzymes are responsible for binding each tRNA with its corresponding amino acid, catalyzing a specific amino-acylation reaction. Owing to reaction specificity, there is a specific tRNA-synthase for each tRNA-amino acid pair, and therefore each cell has at least 20 different tRNA-synthases, one for each of the natural 20 amino acids. Obviously, each of these 20 tRNA-synthases is codified in the genome, representing at least 20 different genomic regions in all organisms.  
         [0025]     As has been mentioned before, the present invention relates to the use of said genomic regions codifying for the different tRNA-synthases in molecular biology techniques for microorganism detection and identification.  
         [0026]     For a genomic region to be useful as template in molecular biology techniques for detection and identification of various microorganisms, it must fulfill 2 basic requirements: first, it has to be present in all microorganisms to be detected or identified. This requirement is fully fulfilled by tRNA-synthases, as they are not only present in all microorganisms, but also in all known organisms. The second requirement is that sequences have to be sufficiently variable to have significant differences between different taxons to be studied; this requirement is also fulfilled by tRNA-synthases as a group.  
         [0027]     A third non-essential requirement that facilitates working with a particular genomic region is the existence of a great deal of information available for said region. This requirement is not essential, as it is always possible to generate the required information, but this considerably increases the operation cost. In this case, due to the relevance of tRNA-synthases, many researchers have sequenced them for different microorganisms, public information being available for more than 200 different microorganisms.  
         [0028]     If tRNA-synthase sequences are to be used in molecular biology techniques for microorganism detection and identification, a database containing tRNA-synthase sequences from taxonomically classified microorganisms must be available in the first place. An example of a database that can be used is GenBank, from NCBI (Benson D A, Boguski M S, Lipman D J, and Ostell J. (1997)  GenBank.  Nucleic Acids Res. January 1; 25(1):1-6). We have processed the information on tRNA-synthases available in said public database to work more efficiently, thus creating our own database.  
         [0029]     Once the database to be used has been selected, DNA fragments must be designed that are taxon specific for each relevant microorganism, having a size suitable for the molecular biology technique to be used. For example, if PCR is to be used, a pair of primers having 15 to 30 nucleotides must be designed, said pair of primers being separated by a distance of 200 to 3,000 nucleotides. If a microarray is to be used, a specific fragment having 50 to 70 nucleotides, in case of using synthetic oligonucleotides, or specific fragments having 200 to 3,000 nucleotides, if PCR products are to be deposited, must be designed, and likewise for each available molecular biology technique.  
         [0030]     The design of these taxon-specific tRNA-synthase fragments can be carried out by using any method available in the art, without limiting the scope of the present invention.  
         [0031]     In our particular case, the design of DNA fragments of tRNA-synthases was carried out using a proprietary method protected by patent application CL 2102-05, owned by Biosigma. Said method is applied in the oligonucleotide design software “Massive Primer Designer”. For more clarity, we will briefly explain the method.  
         [0032]     A database containing all tRNA-synthase sequences for microorganisms disclosed up to date in the literature, corresponding to 230 microorganisms, was used. In a first stage, we will refer to nucleotide sequences as words having defined length in the alphabet {A,C,T,G}. Each sequence is computationally scanned from 5′ to 3′ obtaining all existing words for the desired DNA fragments or oligonucleotides to be designed. Each found word is then considered an oligonucleotide candidate. This oligonucleotide candidate pass through the following tests in the same detailed order, wherein a rejection in one of the tests means the total elimination of the candidate. 
        1. GC composition level: This is a filter that allows discarding a priori candidates that have very high or very low values of hybridization temperature, by performing a very cheap calculus in terms of time. The candidate is rejected if its GC composition falls off the limits imposed at the moment of executing the software.     2. Hybridization temperature: Given a sequence and environmental conditions (salt concentration, nucleotide concentration, etc.), the hybridization temperature between said sequence and its complementary one is calculated. When executing the software, an oligonucleotide is rejected if its hybridization temperature falls off the established limits to be used during a hybridization assay.     3. Secondary structure: for a reference temperature, which depends on the particular molecular biology technique to be used, it is assessed whether the candidate sequence stabilizes itself by forming a stable secondary structure (three-dimensional fold of the oligonucleotide alone) or not. If a secondary structure is formed, the oligonucleotide is replaced by its reverse complementary sequence; if this reverse complementary also forms a secondary structure, then the oligonucleotide is rejected. If only the original form of the oligonucleotide forms a secondary structure, but not its reverse complementary sequence, then this latter is selected as candidate oligonucleotide for the following tests.     4. Specificity: using one or more sequence alignment algorithms, the candidate is tested for a relevant similarity with sequences pertaining to the other microorganisms.        
 
         [0037]     In this method some other optimizations are included, such as the initial selection of sequences that are relatively specific for each microorganism, in order to limit the oligonucleotide search in selected regions.  
         [0038]     By using this method, DNA fragments can be obtained from regions codifying for tRNA-synthases that fulfill all the described requirements and can be employed in a molecular biology technique for the detection and identification of any relevant microorganism.  
         [0039]     The database used by us contains tRNA-synthase sequences disclosed in the literature for microorganisms belonging to the following taxons:  
         [0040]      Acinetobacter  spp.;  Aeropyrum pernix; Agrobacterium tumefaciens; Anabaena  spp.;  Anaplasma marginale; Aquifex aeolicus; Archaeoglobus fulgidus; Azoarcus  spp.;  Bacillus anthracis; Bacillus cereus; Bacillus clausii; Bacillus halodurans; Bacillus licheniformis; Bacillus subtilis; Bacillus thuringiensis; Bacteroides fragilis; Bacteroides thetaiotaomicron; Bartonella henselae; Bartonella quintana; Bdellovibrio bacteriovorus; Bifidobacterium longum; Blochmannia floridanus; Bordetella bronchiseptica; Bordetella parapertussis; Bordetella pertussis; Borrelia burgdorferi; Borrelia garinii; Bradyrhizobium japonicum; Brucella abortus; Brucella melitensis; Brucella suis; Buchnera aphidicola; Burkholderia mallei; Burkholderia pseudomallei; Campylobacter jejuni; Caulobacter crescentus; Chlamydia muridarum  ( Chlamydia trachomatis  MoPn);  Chlamydia trachomatis; Chlamydophila abortus; Chlamydophila caviae; Chlamydophila pneumoniae; Chlorobium tepidum; Chromobacterium violaceum; Clostridium acetobutylicum; Clostridium perfringens; Clostridium tetani; Corynebacterium diphtheriae gravis; Corynebacterium efficiens; Corynebacterium glutamicum; Coxiella burnetii; Dehalococcoides ethenogenes; Deinococcus radiodurans; Desulfotalea psychrophila; Desulfovibrio vulgaris; Ehrlichia ruminantium; Enterococcus faecalis; Erwinia carotovora atroseptica; Escherichia coli; Francisella tularensis; Fusobacterium nucleatum; Geobacillus kaustophilus; Geobacter sulfurreducens; Gloeobacter violaceus; Gluconobacter oxydans; Haemophilus ducreyi; Haemophilus influenzae; Haloarcula marismortui; Halobacterium  sp.;  Helicobacter hepaticus; Helicobacter pylori; Idiomarina loihiensis; Lactobacillus acidophilus; Lactobacillus johnsonii; Lactobacillus plantarum; Lactococcus lactis  subsp.  lactis; Legionella pneumophila; Leifsonia xyli xyli; Leptospira interrogans; Leptospira interrogans; Listeria innocua; Listeria monocytogenes; Listeria monocytogenes; Mannheimia succiniciproducens; Mesoplasma florum; Mesorhizobium loti; Methanobacterium thermoautotrophicum; Methanococcus jannaschii; Methanococcus maripaludis; Methanopyrus kandleri; Methanosarcina acetivorans; Methanosarcina mazei; Methylococcus capsulatus; Mycobacterium avium paratuberculosis; Mycobacterium bovis bovis; Mycobacterium leprae; Mycobacterium tuberculosis; Mycoplasma gallisepticum; Mycoplasma genitalium; Mycoplasma hyopneumoniae; Mycoplasma mobile; Mycoplasma mycoides mycoides; Mycoplasma penetrans; Mycoplasma pneumoniae; Mycoplasma pulmonis; Nanoarchaeum equitans; Neisseria gonorrhoeae; Neisseria meningitidis; Nitrosomonas europaea; Nocardia farcinica; Oceanobacillus iheyensis; Parachlamydia  sp.;  Pasteurella multocida; Photobacterium profundum; Photorhabdus luminescens laumondii; Phytoplasma  spp.;  Picrophilus torridus; Porphyromonas gingivalis; Prochlorococcus marinus; Propionibacterium acnes; Pseudomonas aeruginosa; Pseudomonas putida; Pseudomonas syringae; Pyrobaculum aerophilum; Pyrococcus abyssi; Pyrococcus furiosus; Pyrococcus horikoshii; Ralstonia solanacearum; Rhodopirellula baltica; Rhodopseudomonas palustris; Rickettsia conorii; Rickettsia prowazekii; Rickettsia typhi; Salmonella enterica; Salmonella typhi; Salmonella typhimurium; Shewanella oneidensis; Shigella flexneri; Silicibacter pomeroyi; Sinorhizobium meliloti; Staphylococcus aureus; Staphylococcus epidermidis; Streptococcus agalactiae; Streptococcus mutans; Streptococcus pneumoniae; Streptococcus pyogenes; Streptococcus thermophilus; Streptomyces avermitilis; Streptomyces coelicolor; Sulfolobus solfataricus; Sulfolobus tokodaii; Symbiobacterium thermophilum; Synechococcus  spp.;  Synechocystis  spp.;  Thermoanaerobacter tengcongensis; Thermococcus kodakaraensis; Thermoplasma acidophilum; Thermoplasma volcanium; Thermosynechococcus elongatus; Thermotoga maritima; Thermus thermophilus; Treponema denticola; Treponema pallidum; Tropheryma whipplei; Ureaplasma urealyticum; Vibrio cholerae; Vibrio fischeri; Vibrio parahaemolyticus; Vibrio vulnificus; Vibrio vulnificus; Wigglesworthia brevipalpis; Wolbachia  spp.;  Wolinella succinogenes; Xanthomonas axonopodis; Xanthomonas campestris; Xanthomonas oryzae; Xylella fastidiosa; Yersinia pestis; Yersinia pestis; Yersinia pseudotuberculosis  and  Zymomonas mobilis.    
         [0041]     In a preferred embodiment of the present invention, DNA arrays are developed with the designed tRNA-synthase fragments. In the examples, we have included 60 tRNA-synthase sequences specific for 5 microorganisms:  Agrobacterium tumefaciens; Corynebacterium glutamicum; Mycobacterium tuberculosis; Pseudomonas aeruginosa  and  Xylella fastidiosa.  Specific arrays containing these fragments are especially protected.  
         [0042]     It should be noted that the arrays contained in the present invention are those comprising at least one of the DNA fragments included in sequences N o  1 to 60, either entirely, or in a larger region comprising it, such as a PCR product, or partially as one of the sub-fragments contained in each of the fragments herein disclosed, or as the reverse complementary sequences of any of the former options. This is vitally relevant, as the specificity of a nucleotide sequence is the same specificity of its reverse complementary sequence, and it is this feature, i.e. specificity, the more difficult goal to achieve in the design of the DNA fragments to be used in an array. It could be possible that the stability of the reverse complementary sequence should not be suitable for the sequence to be used in an array, but nevertheless the skilled person will distinguish between thermodynamically stable and unstable oligonucleotides by means of diverse tools existing in the art. All reverse complementary sequences of fragments N o 1 to 60 of the present invention, either entirely, or in a larger region comprising it, such as a PCR product, or partially as one of the sub-fragments contained in each of the fragments herein disclosed, are to be considered within the scope of the present invention.  
         [0043]     The efficiency of the arrays of the invention is given by the specificity and stability of the fragments to be deposited. These characteristics are retained by each sub-fragment contained within the designed fragments. This means that specificity is retained if nucleotide 1 to 100 or 42 to 92, or 15 to 65, or any other possible selection is used. All selections are sub-fragments and are comprised within the scope of the present invention.  
         [0044]     It is also possible to have DNA fragments that contain fragments or sub-fragments of the invention flanked by other oligonucleotides, either by synthesis or as PCR products. These larger fragments that contain the fragments of the present disclosure, the specificity of which is given by the fragments or sub-fragments designed by us, are also to be considered within the scope of the present invention.  
         [0045]     Each selected fragment or sub-fragment have to be synthesized in many hundreds of copies and deposited as a homogeneous spot on a suitable support for an array, such as glass, silicone, nylon or other support in the art. Synthesis techniques for DNA fragments and array manufacture are known, and any of them could be used to manufacture the arrays of the present invention.  
       EXAMPLES  
     Example 1  
     Design of Specific Sequences for 5 Different Microorganisms  
       [0046]     A total of 60 fragments of tRNA-synthases having 100 nucleotides that are specific for species  Agrobacterium tumefaciens; Corynebacterium glutamicum; Mycobacterium tuberculosis; Pseudomonas aeruginosa  or  Xylella fastidiosa  were designed. The sequences of all 60 designed tRNA-synthase fragments were included in the list of sequences.  
         [0047]     To design these specific fragments, a database comprising tRNA-synthase sequences for 230 microorganisms selected from NCBI&#39;s GenBank public database was first constructed.  
         [0048]     In each case, a set of 100-letter oligonucleotides present in each sequence was determined, discarding those appearing more than once in each sequence, considering up to 3 substitutions. These oligonucleotides were the “candidate oligonucleotides”, which were evaluated according to their thermodynamic stability. Subsequently, thermodynamically favorable fragments were aligned against the entire database, in order to determine their specificity. Best fragments were selected from different tRNA-synthases for detection and identification of each of the species  Agrobacterium tumefaciens; Corynebacterium glutamicum; Mycobacterium tuberculosis; Pseudomonas aeruginosa  or  Xylella fastidiosa.    
         [0049]     Of all 60 designed sequences, sequences N o  1 to 17 are specific for  Agrobacterium tumefaciens;  sequences N o 18 to 32 are specific for  Corynebacterium glutamicum;  sequences N o 33 to 36 are specific for  Mycobacterium tuberculosis;  sequences N o 37 to 43 are specific for  Pseudomonas aeruginosa;  and sequences N o 44 to 60 are specific for  Xylella fastidiosa.    
         [0050]     The designed fragments can be deposited on the array either entire, or comprised in a larger fragment that contains them, or in partial form as any of the sub-fragments comprised in the fragment, or as the reverse complementary sequences of any of the former options. Advantageously, sub-fragments having 50 or 70 nucleotides were deposited, preferably sub-fragments having 60 nucleotides.  
       Example 2  
     Microarrays to Detect and Identify the Presence of Microorganisms  
       [0051]     Two microarrays were manufactured with the tRNA-synthase fragments designed in the former example, which specifically identify:  Agrobacterium tumefaciens; Corynebacterium glutamicum; Mycobacterium tuberculosis; Pseudomonas aeruginosa  or  Xylella fastidiosa.    
         [0052]     In each microarray, one 60-nucleotide sub-fragment for each of the designed fragments for each species to be detected, one positive control and three negative controls were included.  
         [0053]     In the first microarray, specific sub-fragments for  Agrobacterium tumefaciens; Corynebacterium glutamicum  and  Mycobacterium tuberculosis  were deposited. In the following Table 1, the content of each position in microarray 1 is detailed.  
                           TABLE 1                                       Position in microarray 1           Microorganism   tRNA-synthase                             Agrobacterium tumefaciens     A1-A3             Corynebacterium glutamicum     B4-B6             Mycobacterium tuberculosis     C4-C6           Positive control   B1-B3           Negative control   C1-C3; A4-A6; A7-A9                      
 
         [0054]     Each fragment was deposited by triplicate. The manufacture of the microarray was performed by a specialized company in the field.  
         [0055]     In the second microarray, specific sub-fragments for  Pseudomonas aeruginosa  and  Xylella fastidiosa  were deposited. In the following Table 2, the content of each position in microarray 2 is detailed.  
                           TABLE 2                                       Position in microarray 2           Microorganism   tRNA-synthase                             Pseudomonas aeruginosa     C1-C3             Xylella fastidiosa     A4-A6           Positive control   B4-B6           Negative control   A1-A3; B1-B3; C4-C6                      
 
         [0056]     Each fragment was deposited by triplicate. The manufacture of the microarray was performed by a specialized company in the field.  
         [0057]     The 60-nucleotide sub-fragments deposited on the microarrays were selected from the fragments displayed in Table 3, which are defined in the list of sequences.  
                                                               TABLE 3                                       tRNA-synthase                    Microorganism   Amino-acyl   Sequence N °                              Agrobacterium tumefaciens     Alanyl   2             Corynebacterium glutamicum     Histidyl   22             Mycobacterium tuberculosis     Phenylalanyl   33             Pseudomonas aeruginosa     Methionyl   40             Xylella fastidiosa     Arginyl   47                      
 
       Example 3  
     Use of the Obtained Microarrays to Detect and Identify Microorganisms  
       [0058]     The microarrays obtained in Example 2 were used with metagenomic samples obtained from a mixture of commercial strains of each of the species contained in the microarray. Two samples were analyzed, one hybridizing on each microarray, the first one containing a mixture of  Agrobacterium tumefaciens, Corynebacterium glutamicum  and  Mycobacterium tuberculosis  (M1), and the second one comprising a mixture of  Pseudomonas aeruginosa  and  Xylella fastidiosa  (M2).  
         [0059]     Total DNA was extracted from M1 and M2 using traditional DNA extraction methods.  
         [0060]     2 μl were taken from the DNA samples, which had a concentration between 1 and 5 μg/μl, and were put in Eppendorf tubes. In each case, the following method was carried out:  
         [0061]     36 μl of ddH 2 O and 3.3 ml of 6-nucleotide random primers were added. The mix was boiled for 5 minutes and then the assay was continued on ice.  
         [0062]     2 μl of a nucleotide mix were added, where dUTP was labeled with Cy fluorophore. Cy5 was used for M1, with green fluorescence, while Cy3 was used for M2, with red fluorescence. Subsequently, 4 μl of a polymerase and 5 μl of buffer solution were added, and the mix was incubated for 4 hours at 37° C.  
         [0063]     The reaction was stopped with 5 μl 0.5 M EDTA, pH 8. Labeled DNA was recovered using an ion exchange column. The eluate containing DNA was dried under vacuum.  
         [0064]     DNA was resuspended by adding 100 μl of a buffer solution. Resuspended samples were mixed with the complementary oligonucleotide of the positive control deposited on the microarray, which in each case was labeled with the inverted Cy fluorophore of the problem sample. In this way, the positive control of sample 1 will have red fluorescence and the positive control of sample 2 will have green fluorescence.  
         [0065]     The samples thus prepared were subjected to 100° C. for one and a half minute to denature DNA. M1 was hybridized overnight on microarray 1 and M2 was hybridized overnight on microarray 2, both at 55° C.  
         [0066]     The following morning, each microarray was washed twice with 2×SSC, 0.1% SDS, at 45° C.; once with 0.2×SSC, 0.1% SDS, at 42° C., and once with 0.2×SSC, at 42° C.  
         [0067]     Each microarray was put in a case with MilliQ water for 15 minutes and subsequently dried by centrifugation in a Falcon tube for 1 minute at 1100 rpm.  
         [0068]     Finally, the results of the microarrays could be observed, which are shown in  FIG. 1  for M1 and in  FIG. 2  for M2.  
         [0069]     In Table 4, the positions of the different fragments in microarray 1 are indicated and the result of hybridizations with DNA obtained from M1, which is shown in  FIG. 1 , is summarized. It is possible to observe that in all cases the families of spots (replicates of the same tRNA-synthase fragment) had always the same result. All positive controls showed hybridization and negative controls remained unmarked.  
                                               TABLE 4                           Sample 1 (M1)                Position in the microarray                    Microorganism   tRNA-synthase 1   R                         Agrobacterium tumefaciens     A1-A3   +             Corynebacterium glutamicum     B4-B6   +             Mycobacterium tuberculosis     C4-C6   +           Positive control   B1-B3   +           Negative control   C1-C3; A4-A6; A7-A9   −                         Legend:                (R): result;                (+): positive;                (−): negative.             
 
         [0070]     The results verify the presence of  Agrobacterium tumefaciens, Corynebacterium glutamicum  and  Mycobacterium tuberculosis  in M1.  
         [0071]     In Table 5, the positions of the different fragments in microarray 2 are indicated and the result of hybridizations with DNA obtained from M2, which is shown in  FIG. 2 , is summarized. Like for M1, it is possible to observe that the families of spots had always the same result. All positive controls showed hybridization and negative controls remained unmarked.  
                                               TABLE 5                           Sample 2 (M2)                Position in the microarray                    Microorganism   tRNA-synthase 1   R                         Pseudomonas aeruginosa     C1-C3   +             Xylella fastidiosa     A4-A6   +           Positive control   B4-B6   +           Negative control   A1-A3; B1-B3; C4-C6   −                         Legend:                (R): result;                (+): positive;                (−): negative.             
 
         [0072]     The results verify the presence of  Pseudomonas aeruginosa  and  Xylella fastidiosa  in M2.  
         [0073]     List of Sequences  
         [0074]     Sequence No.: 1  
         [0075]     Length: 100  
         [0076]     Type: DNA  
         [0077]     Microorganism:  Agrobacterium tumefaciens    
         [0078]     Category: Glutamyl and Glutaminyl tRNA-synthase  
         [0079]     Sequence:  
                           CGG TTT TTC GCG CAC TTG CCT TGT CCC GAG GGG ATG                   AGG CAT AGT CCG CGA ATC AAA AAC GGC CTG TTC CGT               TTC GGG GAA GCG CAC GAT TCC GTT GCCG          
 
         [0080]     Sequence No.: 2  
         [0081]     Length: 100  
         [0082]     Type: DNA  
         [0083]     Microorganism:  Agrobacterium tumefaciens    
         [0084]     Category: Alanyl tRNA-synthase  
         [0085]     Sequence:  
                           GGT CTT GCC GAT GAG GCC AAG GCC AAT CTC GGC TCC                   GGC GTT GTG TTG CTG ATT GCG GTT TCT GAC GAT GGC               AAG GCA AGC GCC GTC GCT GCC GTG ACCG          
 
         [0086]     Sequence No.: 3  
         [0087]     Length: 100  
         [0088]     Type: DNA  
         [0089]     Microorganism:  Agrobacterium tumefaciens    
         [0090]     Category: Phenylalanyl tRNA-synthase alpha subunit  
         [0091]     Sequence:  
                           ATG ACT GAA CTT GAT ACC TTG AAA TCG CAA CTG ATG                   TCG GAG ATC GCC GCC GCT GCC GAT GAG CCG GCC ATC               GAG GCC GTG CGC GTC TCC GCG CTC GGCA          
 
         [0092]     Sequence No.: 4  
         [0093]     Length: 100  
         [0094]     Type: DNA  
         [0095]     Microorganism:  Agrobacterium tumefaciens    
         [0096]     Category: Arginyl tRNA-synthase  
         [0097]     Sequence:  
                           ACG CGC TCG AGA CCC TCG ATC TCG TGA AAG AGA ACC                   GCG AAA AGG TCG ATT TCA GTC GAA TTA CCG TCG AAT               CCC CGC GTG ATC TCA GCC ATG GCG ATGT          
 
         [0098]     Sequence No.: 5  
         [0099]     Length: 100  
         [0100]     Type: DNA  
         [0101]     Microorganism:  Agrobacterium tumefaciens    
         [0102]     Category: Isoleucyl tRNA-synthase  
         [0103]     Sequence:  
                           CAA GCC ATG GCC GCG AGG ACT TTG ATG CCT GGA CGA                   ATA GCG TCC GCA CCC TTG AAG CAC GCG GGA TCG ATA               CCA AAA TCC CGT TCC CGG TCG ATG ACGC          
 
         [0104]     Sequence No.: 6  
         [0105]     Length: 100  
         [0106]     Type: DNA  
         [0107]     Microorganism:  Agrobacterium tumefaciens    
         [0108]     Category: Phenylalanyl tRNA-synthase beta subunit  
         [0109]     Sequence:  
                           AAC TTC GAG ACC GGT CAG GCG CTT CAC TTC CGA CAG                   CGG AAA ATC CAC GAC CTT GGG CTG ATG CCC CTT GTA               ACC GAC GAT CTT GGC TTC GCC AGC GATG          
 
         [0110]     Sequence No.: 7  
         [0111]     Length: 100  
         [0112]     Type: DNA  
         [0113]     Microorganism:  Agrobacterium tumefaciens    
         [0114]     Category: Histidyl tRNA-synthase  
         [0115]     Sequence:  
                           AGG GTG CGG GTC TTG GCG ATG AAC AGA TCG AAA AAG                   TGC TGT TCT TCG TTG GTA TCA AGG ATT ATG CGG CAA               GCG CTG ATG ATC TCG CAA AAC TGG TTGC          
 
         [0116]     Sequence No.: 8  
         [0117]     Length: 100  
         [0118]     Type: DNA  
         [0119]     Microorganism:  Agrobacterium tumefaciens    
         [0120]     Category: Methionyl tRNA-synthase  
         [0121]     Sequence:  
                           AGA CCT CGC GCA ATA TCT GGA ACC TGA TGG CCG ATA                   GCG GCG ACA TCT ACA AGG ACA GCT ATG CCG GCT GGT               ATT CCG TGC GCG ACG AGG CCT ATT ACGC          
 
         [0122]     Sequence No.: 9  
         [0123]     Length: 100  
         [0124]     Type: DNA  
         [0125]     Microorganism:  Agrobacterium tumefaciens    
         [0126]     Category: Tyrosyl tRNA-synthase  
         [0127]     Sequence:  
                           TTC AAG GAG GAG GCC CGC AAG CTA ATG ACG ATC GAC                   ATG ATC GAG GAC AAT ATC ACC TCG CTC AAG CAC GTC               TTT GCG AAT TAC CTC GAT TAC GAT CGTG          
 
         [0128]     Sequence No.: 10  
         [0129]     Length: 100  
         [0130]     Type: DNA  
         [0131]     Microorganism:  Agrobacterium tumefaciens    
         [0132]     Category: Seryl tRNA-synthase  
         [0133]     Sequence:  
                           ATG GCG CAG AAG AAC ATG GAA CTG GCG GAA AAG CTG                   AAA GCC GAA GTC GCT GAT ATC AAG GAA AAC ATG CCG               CGC GCC GAG GAG GAG GAC CGT CAG GTCT          
 
         [0134]     Sequence No.: 11  
         [0135]     Length: 100  
         [0136]     Type: DNA  
         [0137]     Microorganism:  Agrobacterium tumefaciens    
         [0138]     Category: Aspartyl tRNA-synthase  
         [0139]     Sequence:  
                           CAT CCG CAT CGA CGG TCT AGT CAA GGC GCG TTC GGA                   AGA CAC CAT CAA CAA AGG CAT GGC GAC CGG CGA GAT               CGA GCT TTA CGC GCA GGA GAT CGA AGTT          
 
         [0140]     Sequence No.: 12  
         [0141]     Length: 100  
         [0142]     Type: DNA  
         [0143]     Microorganism:  Agrobacterium tumefaciens    
         [0144]     Category: Tryptophanyl tRNA-synthase  
         [0145]     Sequence:  
                           ACA TCG ATT TCG GCG GCC ATA TTC GCA ACG CCG GTC                   TGG GCG TTA ATA TCA CGG TTG GCG ATG AGC CGG TGC               ACG CTT ATT TCC CGA TGG TGG AGC CGCT          
 
         [0146]     Sequence No.: 13  
         [0147]     Length: 100  
         [0148]     Type: DNA  
         [0149]     Microorganism:  Agrobacterium tumefaciens    
         [0150]     Category: Cysteinyl tRNA-synthase  
         [0151]     Sequence:  
                           CCG GTT GCG GGA TGA GCT TTC CGA GAA GGG TAT TCA                   GCT GAA GGA TGG CAA GGA CAA GGA GAC TGG GGA ACG               GAC GAC GAC GTG GGA GTT GAA GCG GTAG          
 
         [0152]     Sequence No.: 14  
         [0153]     Length: 100  
         [0154]     Type: DNA  
         [0155]     Microorganism:  Agrobacterium tumefaciens    
         [0156]     Category: Threonyl tRNA-synthase  
         [0157]     Sequence:  
                           ACC GTC AAC ATT CGC CGT CTC GGT TCG CAG AAC CAG                   ACG TCG ATG TCG CTC GAA GAG GCG ATC ACC AGC CTC               GTG GAC GAG GCG ACG CCG CCG GAT GTCA          
 
         [0158]     Sequence No.: 15  
         [0159]     Length: 100  
         [0160]     Type: DNA  
         [0161]     Microorganism:  Agrobacterium tumefaciens    
         [0162]     Category: Prolyl tRNA-synthase  
         [0163]     Sequence:  
                           CGC TCC TTT CGA TGT CGT GAT CAT CAA CAT GAA GGC                   TGG CGA TGC GGC CTG CGA TGC GGC TTG TGA AAA GCT               GTA TTA TCA GCT CTC CAA CGC CGG CAAG          
 
         [0164]     Sequence No.: 16  
         [0165]     Length: 100  
         [0166]     Type: DNA  
         [0167]     Microorganism:  Agrobacterium tumefaciens    
         [0168]     Category: Leucyl tRNA-synthase  
         [0169]     Sequence:  
                           CCG AAG CCG GCG ACC GAA GGC GAA GGG CTG GCG GCC                   TCC AAG GCA GCC CAC AAG ACG CTG AAG GCT GTT CAG               GAA GAT CTG GAC AAG CTC GCC TTC AACA          
 
         [0170]     Sequence No.: 17  
         [0171]     Length: 100  
         [0172]     Type: DNA  
         [0173]     Microorganism:  Agrobacterium tumefaciens    
         [0174]     Category: Valyl tRNA-synthase  
         [0175]     Sequence:  
                           GAC TTC AAC GAT TTC GAA GTC GGC AAG CGT TGC GGC                   CTG CGC GCC ATC AAC GTC ATG AAC ATC GAT GGT ACG               ATC TCC ATC AAG GAA AAC GAA GAT TTCC          
 
         [0176]     Sequence No.: 18  
         [0177]     Length: 100  
         [0178]     Type: DNA  
         [0179]     Microorganism:  Corynebacterium glutamicum    
         [0180]     Category: Glutamyl and Glutaminyl tRNA-synthase  
         [0181]     Sequence:  
                           GGT ATC GCA GCG CTG GAG GCA GTG GAG GAG TGG ACC                   ACT CCA AAC ATT GAA GCA GCA TTG AAC AAG GCT CTC               ATT GAG GAT CTA GGC CTG AAG CCT CGCG          
 
         [0182]     Sequence No.: 19  
         [0183]     Length: 100  
         [0184]     Type: DNA  
         [0185]     Microorganism:  Corynebacterium glutamicum    
         [0186]     Category: Alanyl tRNA-synthase  
         [0187]     Sequence:  
                           CAC CGG CTT TGA GGA ACT GGA TTC CCA GTC CAA GGT                   CCT CGG ACT ACT TTC CGA TGG TGC CAA GAT TTC TGA               AGC CAC AGA AGG TCA AGA AGT TGA GGTC          
 
         [0188]     Sequence No.: 20  
         [0189]     Length: 100  
         [0190]     Type: DNA  
         [0191]     Microorganism:  Corynebacterium glutamicum    
         [0192]     Category: Isoleucyl tRNA-synthase  
         [0193]     Sequence:  
                           GGC TAA GGA CCT CGT TGG TTC CTA CGC CAA GGA ACT                   CGG TGC TGA GGC TGT TAT CGT TTC TGA GCA CCC AGG               CTC TGA ACT GGT CGG ACT GAC CTA CGAG          
 
         [0194]     Sequence No.: 21  
         [0195]     Length: 100  
         [0196]     Type: DNA  
         [0197]     Microorganism:  Corynebacterium glutamicum    
         [0198]     Category: Phenylalanyl tRNA-synthase beta subunit  
         [0199]     Sequence:  
                                       TCA CCA TGA AGG TCA CTC GAC CTT CCG AGC TCG CAG                           GCG TGG ATT ATT CCG CAG AAA CTG TGA TCG CTC GTC                       TGG AAG AGG TCG GAT GCA CCG TCG CTGT          
 
         [0200]     Sequence No.: 22  
         [0201]     Length: 100  
         [0202]     Type: DNA  
         [0203]     Microorganism:  Corynebacterium glutamicum    
         [0204]     Category: Histidyl tRNA-synthase  
         [0205]     Sequence:  
                                       GTG TTC CTG ATT ACG CCC CAC CAA AAT CTG CAG CGT                           TTT TAG CAG TCC GTG ATG CCT TTG TTA ATC AAG CAC                       ATA AGG CCG GGT TTG AGC ATA TTG AGCT          
 
         [0206]     Sequence No.: 23  
         [0207]     Length: 100  
         [0208]     Type: DNA  
         [0209]     Microorganism:  Corynebacterium glutamicum    
         [0210]     Category: Methionyl tRNA-synthase  
         [0211]     Sequence:  
                                       AGT ACC TGG CGC ACT GGA AGA ATC TGA CAA GAA GAT                           CCT TGA TCT TGC TAC CGC TGC CTT TGA ATC CGT TGC                       TGC GAA CCT GGA TCA GTC CAA GTT CAAG          
 
         [0212]     Sequence No.: 24  
         [0213]     Length: 100  
         [0214]     Type: DNA  
         [0215]     Microorganism:  Corynebacterium glutamicum    
         [0216]     Category: Tyrosyl tRNA-synthase  
         [0217]     Sequence:  
                                       ACT GTT CGG CCG CGC AGA ACT CCG CGA CCT AGA CGA                           AAA AAC TCT CGC TGC ATC CGT TTC TGA AAC TGC AGT                       TGC AGA AAT CAA GGC AGG GGA GCC ACGC          
 
         [0218]     Sequence No.: 25  
         [0219]     Length: 100  
         [0220]     Type: DNA  
         [0221]     Microorganism:  Corynebacterium glutamicum    
         [0222]     Category: Seryl tRNA-synthase  
         [0223]     Sequence:  
                                       ATG TCT ACC GAT CAC GGT AGA TTG TTC TTT ATG ATC                           GAT CTG AAA TTC CTC CGT GAT AAC CCG GAC GTT GTT                       CGT GCC TCC CAG ATC ACT CGC GGC GAAG          
 
         [0224]     Sequence No.: 26  
         [0225]     Length: 100  
         [0226]     Type: DNA  
         [0227]     Microorganism:  Corynebacterium glutamicum    
         [0228]     Category: Aspartyl tRNA-synthase  
         [0229]     Sequence:  
                                       CAC CCC GCA GCA GCG TAA GGA AGC TGG CAT CGA TGC                           CAA GCC AAA GCC TAA GGC GGA AGC ACA GGC AGA GGC                       ACA GGC TGA AGA GTC TGC TGA AAA GTAA          
 
         [0230]     Sequence No.: 27  
         [0231]     Length: 100  
         [0232]     Type: DNA  
         [0233]     Microorganism:  Corynebacterium glutamicum    
         [0234]     Category: Tryptophanyl tRNA-synthase  
         [0235]     Sequence:  
                                       ACG AGT CTT GGC TAT CGG TGC TGA GCG CGC CAC CGA                           AGT TGC CAA CGA AAC CTT GGC TGA TGT GTA CGA CAA                       GAT TGG TTT CTT GGC GTC TCG TCG CTAA          
 
         [0236]     Sequence No.: 28  
         [0237]     Length: 100  
         [0238]     Type: DNA  
         [0239]     Microorganism:  Corynebacterium glutamicum    
         [0240]     Category: Cysteinyl tRNA-synthase  
         [0241]     Sequence:  
                                       AGG TAG GCG AGT GGA CGC CAG GTT TTG AAG TTG CGA                           TGG ATG AGG ATA TTG CAG TTC CTA AGG CTT TGG CTG                       AAA TCC ATA ACG CTG TCC GCG AGG GCAA          
 
         [0242]     Sequence No.: 29  
         [0243]     Length: 100  
         [0244]     Type: DNA  
         [0245]     Microorganism:  Corynebacterium glutamicum    
         [0246]     Category: Threonyl tRNA-synthase  
         [0247]     Sequence:  
                                       ACG TGA GCG CAC GAA GGA GCG CAA TTT TCG TGA ACA                           CCA CAG ATA CCG CAG TAG CAA ACC TTG TTG TTT TTG                       AGG TCC CTG CAG GCA CCG CAA TTG GTGC          
 
         [0248]     Sequence No.: 30  
         [0249]     Length: 100  
         [0250]     Type: DNA  
         [0251]     Microorganism:  Corynebacterium glutamicum    
         [0252]     Category: Prolyl tRNA-synthase  
         [0253]     Sequence:  
                                       ATC GAA GGC CTG CCA GAA GCA GTC ACC TAC GAA ACT                           CCT GTT TCT GAA ACC ATC GAT GCT TTG GTT GAT TGG                       GCT AAC TCC ATC GAC GTG CAG ATC GAGG          
 
         [0254]     Sequence No.: 31  
         [0255]     Length: 100  
         [0256]     Type: DNA  
         [0257]     Microorganism:  Corynebacterium glutamicum    
         [0258]     Category: Leucyl tRNA-synthase  
         [0259]     Sequence:  
                           TCG CCA CCG TTT TGG GTC TGC CTA TCA AGG AAG TTG                   TCG CAG GTG GCA ACA TCG AAG AGG CTG CTT TCA CCG               AAT CTG GCG AAG CAG TCA ACT CTG CGAA          
 
         [0260]     Sequence No.: 32  
         [0261]     Length: 100  
         [0262]     Type: DNA  
         [0263]     Microorganism:  Corynebacterium glutamicum    
         [0264]     Category: Valyl tRNA-synthase  
         [0265]     Sequence:  
                           ACC TGG CTA ACG CAC AGA AGG AAT TGG AAA CCA CCG                   CAA AGA AGC TGG GTA ATG AGG CTT TCC TGT CCA AGG               CAC CTG ATG CAG TGG TAG ACA AGA TCCG          
 
         [0266]     Sequence No.: 33  
         [0267]     Length: 100  
         [0268]     Type: DNA  
         [0269]     Microorganism:  Mycobacterium tuberculosis    
         [0270]     Category: Phenylalanyl tRNA-synthase beta subunit  
         [0271]     Sequence:  
                           CGA GGA GCT CAC CGG CTA CAA GAA GCC GAT CCG GGC                   CTG CGC GGT AGA TAT CGG CGA TCG GCA GTA TCG CGA               GAT TAT TTG TGG TGC AAC CAA TTT CGCG          
 
         [0272]     Sequence No.: 34  
         [0273]     Length: 100  
         [0274]     Type: DNA  
         [0275]     Microorganism:  Mycobacterium tuberculosis    
         [0276]     Category: Histidyl tRNA-synthase  
         [0277]     Sequence:  
                           GAG GCC GGG ACG GTC GCA GTG AAG GAC TTG ACG ACG                   GGT GAG CAA GTT TCG GTC TCG ATG GAT TCG GTT GTG               GCC GAA GTA ATT TCG CGG CTG GCT GGGT          
 
         [0278]     Sequence No.: 35  
         [0279]     Length: 100  
         [0280]     Type: DNA  
         [0281]     Microorganism:  Mycobacterium tuberculosis    
         [0282]     Category: Cysteinyl tRNA-synthase  
         [0283]     Sequence:  
                           AAG CGA TTG CTG AAA TGG TCG AGC TCA TCG AAA AAA                   TGC TGG CGT GCG GGG CGG CCT ATG TCA TAG ACC GGG               AAA TGG GAG AGT ACC AGG ACA TCT ACTT          
 
         [0284]     Sequence No.: 36  
         [0285]     Length: 100  
         [0286]     Type: DNA  
         [0287]     Microorganism:  Mycobacterium tuberculosis    
         [0288]     Category: Leucyl tRNA-synthase  
         [0289]     Sequence:  
                           TCG TGG AAG TAA TTG CCG GCG GCA ATA TTT CGG AAT                   CCG CGT ATA CAG GCG ATG GCA TCC TGG TCA ACT CGG               ATT ACC TCA ATG GAA TGA GCG TGC CAGC          
 
         [0290]     Sequence No.: 37  
         [0291]     Length: 100  
         [0292]     Type: DNA  
         [0293]     Microorganism:  Pseudomonas aeruginosa    
         [0294]     Category: Glutamyl and Glutaminyl tRNA-synthase  
         [0295]     Sequence:  
                           TCC TGG CTG AAC GGG CAG TGG ATC CGC GAG CAG TCC                   GTG GAG GAG TTC GCC CGT GAG GTG CAG AAG TGG GCG               TTG AAT CCC GAA TAC CTG ATG AAG ATCG          
 
         [0296]     Sequence No.: 38  
         [0297]     Length: 100  
         [0298]     Type: DNA  
         [0299]     Microorganism:  Pseudomonas aeruginosa    
         [0300]     Category: Phenylalanyl tRNA-synthase beta subunit  
         [0301]     Sequence:  
                           AGC CCT GGA ACT AAC GGT GGT AGC CGA CGG GGA AGG                   GCA GTG GTC GGT CGG CGT ACC GAG CCA TCG CTT CGA               CAT TTC CCT GGA AGT CGA CCT GAT CGAG          
 
         [0302]     Sequence No.: 39  
         [0303]     Length: 100  
         [0304]     Type: DNA  
         [0305]     Microorganism:  Pseudomonas aeruginosa    
         [0306]     Category: Histidyl tRNA-synthase  
         [0307]     Sequence:  
                           TGG TTG GCG CGC CGA CCC TGC ACG ACT ATC TCG ACG                   AAG AAT CGA TCG CCC ACT TCG AGG GTC TCA AGG CCC               GCC TGG ATG CTG TCG GAC TTC GCT ACGA          
 
         [0308]     Sequence No.: 40  
         [0309]     Length: 100  
         [0310]     Type: DNA  
         [0311]     Microorganism:  Pseudomonas aeruginosa    
         [0312]     Category: Methionyl tRNA-synthase  
         [0313]     Sequence:  
                           TTC GAG CCC CTG ATG ACC CGT ATC GAG CCC GCG AAA                   GTA GAA GCC ATG ATC GAA GCC TCC AAG GAA GAC CTC               GCC GCC GCA TCC CAA CCC GCC GGC AACG          
 
         [0314]     Sequence No.: 41  
         [0315]     Length: 100  
         [0316]     Type: DNA  
         [0317]     Microorganism:  Pseudomonas aeruginosa    
         [0318]     Category: Threonyl tRNA-synthase  
         [0319]     Sequence:  
                           CGA TTT CAT CAA GCT GAC CTT GCA GGT CTA CCG CGA                   CTT CGG TTT CAC CGA CAT CGC CAT GAA GCT GTC GAC               CCG TCC GGC CAA GCG CGT CGG TTC CGAC          
 
         [0320]     Sequence No.: 42  
         [0321]     Length: 100  
         [0322]     Type: DNA  
         [0323]     Microorganism:  Pseudomonas aeruginosa    
         [0324]     Category: Prolyl tRNA-synthase  
         [0325]     Sequence:  
                           CGA AGG CGT CCT GGA ATA CAA GGG CCG CCG CGA CAG                   CGA GTC GCA GAA CCT GCC GAT CGG CGA ACT GAT GTC               GTT CAT CAC CGA AAA ACT CAG CCG CTGA          
 
         [0326]     Sequence No.: 43  
         [0327]     Length: 100  
         [0328]     Type: DNA  
         [0329]     Microorganism:  Pseudomonas aeruginosa    
         [0330]     Category: Leucyl tRNA-synthase  
         [0331]     Sequence:  
                           ATG CAC GAG CAA TAT CAG CCA CTC GAG ATC GAA ACC                   CAG GCC CAG AAC TAC TGG AAA GAA CAC CAG TCC TTC               CTC GTC AGG GAG CTA CCC GAC AAG GAGA          
 
         [0332]     Sequence No.: 44  
         [0333]     Length: 100  
         [0334]     Type: DNA  
         [0335]     Microorganism:  Xylella fastidiosa    
         [0336]     Category: Glutamyl and Glutaminyl tRNA-synthase  
         [0337]     Sequence:  
                           CCG AGC CCA GCC TGA GAA GAA AGA CTT TAT CCG GCA                   GAT CAT CAG AGA AGA CTT AGC CCA CGG CAC ACA CAC               TCA CAT CCG CAC CCG TTT CCC CCC AGAA          
 
         [0338]     Sequence No.: 45  
         [0339]     Length: 100  
         [0340]     Type: DNA  
         [0341]     Microorganism:  Xylella fastidiosa    
         [0342]     Category: Alanyl tRNA-synthase  
         [0343]     Sequence:  
                           CGT GAG GTT GGC GCG TTT ACA GTG CCA GCC TAC TGT                   TAA CTC CAT GAA CAT ACC TAC CAA ATT CAC CAC TTC               AAA GAT TCG TAG CGA TTT TCT AGA ATTT          
 
         [0344]     Sequence No.: 46  
         [0345]     Length: 100  
         [0346]     Type: DNA  
         [0347]     Microorganism:  Xylella fastidiosa    
         [0348]     Category: Phenylalanyl tRNA-synthase alpha subunit  
         [0349]     Sequence:  
                           GCA GAA CTT AGA TCA TTT GGA AGT TTT GCG CGT AGC                   GCT TCT TGG TAA AAA TGG CAG CAT CAC CTT GCA GCT               CAA GCA ACT TGG TAA ATT GCC AGT TGAG          
 
         [0350]     Sequence No.: 47  
         [0351]     Length: 100  
         [0352]     Type: DNA  
         [0353]     Microorganism:  Xylella fastidiosa    
         [0354]     Category: Arginyl tRNA-synthase  
         [0355]     Sequence:  
                           ATG CTA ACG CGC TTC TCA TAC AAA CGG TCA GAT AAA                   ATT ACG CTA TCT ATC GCC ACA CAC CCA CAC CCA CAC               GTG AAA GCC CCA CTC CGC GCC CTG ATTT          
 
         [0356]     Sequence No.: 48  
         [0357]     Length: 100  
         [0358]     Type: DNA  
         [0359]     Microorganism:  Xylella fastidiosa    
         [0360]     Category: Isoleucyl tRNA-synthase  
         [0361]     Sequence:  
                           CGG TGC CGT CCA CAC CGC CCC TGG CCA CGG CCA AGA                   AGA CCA CCA AGT CTT TCA ACA ATA CGG ATT ACT CAA               TCA CTA CAG CGC TGC CGA ACT CAA CCCC          
 
         [0362]     Sequence No.: 49  
         [0363]     Length: 100  
         [0364]     Type: DNA  
         [0365]     Microorganism:  Xylella fastidiosa    
         [0366]     Category: Phenylalanyl tRNA-synthase beta subunit  
         [0367]     Sequence:  
                           TGG AGC GAA CTG GCA ATC ACC GTG CGC GAT GTC ATT                   GGA TCG TTA CTT CGT GAC GTG AAA CTA TTT GAC CGC               TAC GTC GGT AAG GGT ATC GAA CCA GGCT          
 
         [0368]     Sequence No.: 50  
         [0369]     Length: 100  
         [0370]     Type: DNA  
         [0371]     Microorganism:  Xylella fastidiosa    
         [0372]     Category: Histidyl tRNA-synthase  
         [0373]     Sequence:  
                           TTG CCA CCC GTT CCA CCA GCC ATG CCG ATG CAT TGG                   TGC GCC TGG ATG CAC TGA TCG AAG ATT CAG GGC CAG               AGG CAC ACG AGA TGC TGC GCC AAG GTGT          
 
         [0374]     Sequence No.: 51  
         [0375]     Length: 100  
         [0376]     Type: DNA  
         [0377]     Microorganism:  Xylella fastidiosa    
         [0378]     Category: Methionyl tRNA-synthase  
         [0379]     Sequence:  
                           TCG GTG GCC ACA CCA TCC ACC CCT ACT CCC CCT TAT                   TCA CCC GCA TCG ATA AAA AAG TCA TTG AAG TGA TGA               TCA ACG CCT CCA AAG ACA CCC TCG CCCC          
 
         [0380]     Sequence No.: 52  
         [0381]     Length: 100  
         [0382]     Type: DNA  
         [0383]     Microorganism:  Xylella fastidiosa    
         [0384]     Category: Tyrosyl tRNA-synthase  
         [0385]     Sequence:  
                           TAA GCC ACT TAG TCA TGA GGA TGT ATT GGC TAA TGC                   CCG TAC CTA CGA GGA TCA GGT ATT TAA GGT TTT GGA               TCG TAC CCT TAC CGA GGT GAG GTT CAAT          
 
         [0386]     Sequence No.: 53  
         [0387]     Length: 100  
         [0388]     Type: DNA  
         [0389]     Microorganism:  Xylella fastidiosa    
         [0390]     Category: Seryl tRNA-synthase  
         [0391]     Sequence:  
                           GAA GAG GAC ATG TTC AGT ACT CAG CTC GGT GAG CAT                   CGG CGC TAT TTG ATT TCG ACC TCT GAA ATT TCG CTG               ACT AAT TTA GTA CGT AAC GAG ATT ATTG          
 
         [0392]     Sequence No.: 54  
         [0393]     Length: 100  
         [0394]     Type: DNA  
         [0395]     Microorganism:  Xylella fastidiosa    
         [0396]     Category: Aspartyl tRNA-synthase  
         [0397]     Sequence:  
                           TGA CGG TGG ATT CGC GTG CCA TCG ACC AGA ACA ACT                   CAG AAC TGT TCA AGG TTG CTA GTG GCT TGA GTT ATG               AGG ATG TAC TAC AAG TTG AAG GCG TCGT          
 
         [0398]     Sequence No.: 55  
         [0399]     Length: 100  
         [0400]     Type: DNA  
         [0401]     Microorganism:  Xylella fastidiosa    
         [0402]     Category: Tryptophanyl tRNA-synthase  
         [0403]     Sequence:  
                           GGA AGG GGA TTC TTA ATC GTG CGC ATG CTT ATA AGG                   CGG TGG TGG ATA AGA ATC GTA GTG AGG GGG GGG ATG               ATG ATG CCG GGA TCA CGG CCG GTC TGTT          
 
         [0404]     Sequence No.: 56  
         [0405]     Length: 100  
         [0406]     Type: DNA  
         [0407]     Microorganism:  Xylella fastidiosa    
         [0408]     Category: Cysteinyl tRNA-synthase  
         [0409]     Sequence:  
                           GTG CAT GGG TTG GTG GCA AAG CAT CCT CCC GAA GCA                   CTA CGT TAC GCG CTG CTG TCG GCA CAT TAT CGG AAA               CCG CTG GAC TGG TCA GAG GCA CTC ATCG          
 
         [0410]     Sequence No.: 57  
         [0411]     Length: 100  
         [0412]     Type: DNA  
         [0413]     Microorganism:  Xylella fastidiosa    
         [0414]     Category: Threonyl tRNA-synthase  
         [0415]     Sequence:  
                           AGC ATG ACG CCA TGC TGC GAA TCA TCA CTC CTG AGG                   ATC AAG AAG CTC TGG ACA TCA TAC GTC ACT CTT GTG               CTC ACT TGG TTG GCC ATG CGG TCA AGCA          
 
         [0416]     Sequence No.: 58  
         [0417]     Length: 100  
         [0418]     Type: DNA  
         [0419]     Microorganism:  Xylella fastidiosa    
         [0420]     Category: Prolyl tRNA-synthase  
         [0421]     Sequence:  
                           ACT CGT TCC ATC TGA ACG ATG CTG ATT TGG TAC GTG                   AGT ACG AAA ATA TGA GGG CGA CGT ATA CAC GTA TCT               TTA CCA GGC TGG GGT TGG AGT TCC GTGC          
 
         [0422]     Sequence No.: 59  
         [0423]     Length: 100  
         [0424]     Type: DNA  
         [0425]     Microorganism:  Xylella fastidiosa    
         [0426]     Category: Leucyl tRNA-synthase  
         [0427]     Sequence:  
                           AAA TAC GGT TTA CCC ATC CGT CAA GTG ATC GCA CTC                   AAA GAA CCT AAA AAC CAA GAC GAA TCC ACA TGG GAA               GGA GAT GTC TGG GGT GAT TGG TAC GCTG          
 
         [0428]     Sequence No.: 60  
         [0429]     Length: 100  
         [0430]     Type: DNA  
         [0431]     Microorganism:  Xylella fastidiosa    
         [0432]     Category: Valyl tRNA-synthase  
         [0433]     Sequence:  
                           GGA AGG GTT GAT CGA TGT GGA TGC CGA GCG CGT GCG                   CCT GGA TAA AGA AAT CAA ACG TGT CGA AAG CGA AAT               CGA TAA AAG CAA CGG GAA ATT GAG TAAC          
 
         [0434]