Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

• the present invention relates to the field of techniques for detecting and / or identifying bacteria of the genus Mycobacterium and more particularly to those using a genetic marker.
• the ribosomal RNA of bacteria is used as a target, because firstly it is found in all cells of all living organisms, in quantity, and secondly it has a particular nucleotide sequence made up of a succession of regions characterized by a speed of variable evolution.
• Patent application WO-88/03957 describes probes specific for detecting mycobacteria, including the sequences nucleotides are capable of hybridizing to the 16S and 23S subunits of the ribosomal RNA of said bacteria.
• the probes described are 7 probes of species or groups of species and 4 probes of genus, and are collated in Table 1 below which are respectively listed the targeted ribosomal RNA subunit, the targeted nucleotide sequence taking as references the nucleotide sequence of the ribosomal RNA of E. Coli and the specificity of the probes with regard to the species, group of species or genus Mycobacterium.
• the regions variable from one species to another or from one species to a group of species, used to develop probes are located on the sub- 16S unit of ribosomal RNA, and that on the other hand, the 23S subunit comprises homologous regions within the genus Mycobacterium making it possible to detect groups of species of mycobacteria.
• certain authors have sought, from the partial determination of the nucleotide sequences of the 23S subunit of the ribosomal RNA of some pathogenic mycobacteria, to develop probes of species which , by specific hybridization, would make it possible to selectively detect a species of mycobacteria.
• variable areas within the genus Mycobacterium have been determined on the ribosomal RNA of the 23S subunit, which makes it possible to meet the current need to diagnose a pathogenicity in particular in immunocompromised subjects, by providing species-specific detection probes and species groups of mycobacteria.
• the starting point of the invention is the complete determination of the nucleotide sequences of the 23S subunit of the ribosomal RNA for a sufficient number of species of mycobacteria, which made it possible to highlight several variable regions allowing to discriminate several species of mycobacteria.
• a nucleotide fragment or an oligonucleotide is a chain of monomers, characterized by the informational sequence of natural nucleic acids, capable of hybridizing to a nucleotide fragment under predetermined conditions, the sequence being able to contain monomers of different structures and to be obtained from a natural nucleic acid molecule and / or by genetic recombination and / or by chemical synthesis ,
• a monomer can be a natural nucleotide of nucleic acid whose constituent elements are a sugar, a phosphate group and a nitrogenous base; in DNA the sugar is ribose, in RNA the sugar is deoxy-2-ribose; depending on whether it is DNA or RNA, the nitrogen base is selected from adenine, guanine, uracil, cytosine, thymine; or a nucleotide modified in at least one of the three constituent elements; by way of example, the modification may take place at the base level, generating modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2,6-purine , bromo-5-deoxyuridine and any other modified base promoting hybridization, at the sugar level, namely the replacement of at least one deoxyribose by a polyamide [PE Nielsen et al, Science, 254, 1497-1
• information sequence means any ordered sequence of monomers, the chemical nature and order in a reference direction constitute information of the same quality as that of natural nucleic acids
• hybridization means the process during which, under appropriate conditions, two nucleotide fragments, having sufficiently complementary sequences, pair to form a double strand
• a probe is a nucleotide fragment comprising from 5 to 100 monomers, advantageously from 8 to 50 monomers, having a specificity of hybridization under determined conditions to form a hybridization complex with a nucleotide fragment having a nucleotide sequence included in the Ribosomal RNA, DNA obtained by reverse transcription of said ribosomal RNA and DNA of which said ribosomal RNA is the transcription product;
• a probe can be used for diagnostic purposes such as capture and / or detection probes or for therapy purposes, - the capture probe can be immobilized on a solid support by any suitable means, i.e. -to say directly or indirectly, for example by covalence or passive adsorption,
• the detection probe is marked by means of a marker chosen from radioactive isotopes, enzymes notably chosen from peroxidase and alkaline phosphatase and those capable of hydrolyzing a chromogenic, fluorigenic or luminescent substrate, chromophoric chemical compounds, chromogenic, fluorigenic or luminescent compounds, analogs of nucleotide bases, and biotin,
• a marker chosen from radioactive isotopes, enzymes notably chosen from peroxidase and alkaline phosphatase and those capable of hydrolyzing a chromogenic, fluorigenic or luminescent substrate, chromophoric chemical compounds, chromogenic, fluorigenic or luminescent compounds, analogs of nucleotide bases, and biotin,
• the probes used for diagnostic purposes of the invention can be used in all known hybridization techniques and in particular the so-called “DOT-BLOT” techniques [MANIATIS et al, Molecular Cloning, Cold Spring Harbor, (1982 )], "SOUTHERN BLOT” [SOUTHERN. E.M., J. Mol. Biol., 98, 503 (1975)], "NORTHEN BLOT” which is a technique identical to the "SOUTHERN BLOT” technique but which uses RNA as target, the SANDWICH technique [DUNN A.R.,
• the SANDWICH technique is used in the present invention comprising a specific capture probe and / or a specific detection probe, it being understood that the capture probe and the detection probe must have an nucleotide sequence at least partially different,
• a therapy probe for treating infections due to mycobacteria said probe being capable of hybridizing in vivo on the 23S ribosomal RNA of said bacteria and / or on genomic DNA to block the translation and / or transcription phenomena
• a primer is a probe comprising from 5 to 30 monomers, having a specificity of hybridization under conditions determined for the initiation of an enzymatic polymerization, for example in an amplification technique such as PCR (Polymerase Chain Reaction), in an elongation process, such as sequencing, in a reverse transcription method or the like,
• the probes and primers are those whose sequences are identified below or those which have at least 70% homology and at least 5 consecutive monomers of identical information sequence with the latter.
• nucleotide sequence of the 23S ribosomal RNA was determined for 16 species of mycobacteria, sequencing from which single-stranded nucleotide fragments belonging to variable regions have been defined. therefore specific to a species of mycobacteria.
• the single-stranded nucleotide fragments of the invention have a nucleotide sequence chosen from the following sequences:
• nucleotide N ° 1409 starting at nucleotide N ° 1409 and ending at nucleotide N ° 1420, excluding the fragments of ribosomal RNA 23S of M. tuberculosis, M. marinum, M. scrofulaceum, M. avium TMC 724, M. leprae, M. kansasii and M. asiaticum, M. ulcerans ATCC 19423, M. malmoense ATCC 29571,
• nucleotide N ° 1854 starting at nucleotide N ° 1854 and ending at nucleotide N ° 1876, excluding fragments of ribosomal RNA 23S from M. tuberculosis, M. kansasii and M. fortuitum, - starting at nucleotide N ° 2126 and ending at nucleotide
• nucleotide sequences of the fragments of the invention are chosen from the sequences SEQ ID NO 1 to SEQ ID N028, SEQ ID N030 to SEQ ID N037, SEQ ID N039 to SEQ ID N041, SEQ ID N043 to SEQ ID N049, SEQ ID N051 to SEQ ID N067, SEQ ID N069 to SEQ ID N075, SEQ ID N077 to SEQ ID N093, SEQ ID N095, SEQ ID N097, SEQ ID N098, SEQ ID N0100 to SEQ ID N0136, SEQ ID N0138 to SEQ ID N0144, SEQ ID N0146 to SEQ ID N0148, SEQ ID N0150 to SEQ ID N0152, SEQ ID N0154 to SEQ ID N0158, SEQ ID N0160, SEQ ID N0161, SEQ ID N0163 to SEQ ID N0174 and SEQ ID N0178, and their complementary sequences .
• E. coli 23S rRNA sequence applied to the sequences identified in the present description does not always reflect the number of nucleotides or monomers constituting these sequences. Indeed, mycobacteria have additional genetic material compared to E. coli, which is particularly highlighted for the sequences SEQ ID NO 29 to SEQ ID NO 41.
• the fragments of the invention are RNA fragments, but they can also be single-stranded DNA fragments resulting from the reverse transcription of the above-mentioned RNA fragments, or their complementary fragments, as well as single-stranded fragments of genomic DNA whose transcripts are the abovementioned RNA fragments, or their complementary fragments.
• the preferred probes are those having a sequence chosen from the sequences:
• a particularly advantageous probe of the invention is that whose nucleotide sequence begins at nucleotide N ° 1 and ending at nucleotide N ° 21 of the variable zone corresponding to SEQ ID N ° 94, whose interest lies in its specificity vis- against the M. tuberculosis species, among other species of mycobacteria, but also within the M. tuberculosis group itself.
• Another subject of the invention is a primer for the specific reverse transcription of a 23S ribosomal RNA sequence of mycobacteria belonging to a variable region, into a complementary DNA sequence, or a primer in particular for specific amplification, by polymerase chain reaction of the sequence of DNA complementary to a 23S ribosomal RNA sequence of mycobacteria, belonging to a variable region.
• the therapy probes of the present invention comprise a nucleotide sequence belonging to the sequence of a fragment having at least one of the following nucleotide sequences of the 23S RNA:
• nucleotide N ° 2126 starting at nucleotide N ° 2126 and ending at nucleotide N ° 2161, and their complementary sequences.
• the invention also relates to a reagent for detecting and / or identifying at least one species of mycobacteria in a biological sample comprising at least one probe of the invention, and in particular a capture probe and a detection probe one and / or the other corresponding to the definition of a probe according to the invention.
• the reagent may include a mixture of probes of the invention for the purpose of detecting at least two species of mycobacteria.
• the invention provides a method for selectively detecting and / or identifying a species of mycobacteria in a biological sample according to which the 23S ribosomal RNA, extracted from mycobacteria and possibly denatured, or DNA is exposed. genomic, extracted and denatured bacteria, or DNA obtained from reverse transcription of ribosomal RNA 23S, to at least one probe of the invention and hybridization of said probe is detected.
• an amplification of this DNA is carried out in the presence of a suitable enzymatic system and at least one amplification primer of the invention.
• Figures 7, 8 and 9 are formed by the alignment of Figures 7a and 7b respectively; 8a, 8b and 8c; 9a, 9b and 9c. More specifically, Figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
• 1 1, 12 and 13 relate to the variable zones in the order of their appearance on the 23S rRNA of the mycobacteria and correspond to the sequences respectively:
• the amplification products obtained were cloned into the double-stranded forms of phage M13.
• the recombinant M13 phages obtained served as a template for the sequencing reaction using the chain termination method (Sanger et al., Proc. Natl. Acad. Sci. USA, 1977, 74: 5463-5467).
• Hybridization of the ribosomal RNA of a target bacterium was carried out according to the non-radioactive and semi-automated detection method described in French patent n ° 90 07249 and modified for the detection of ribosomal RNA by the addition destabilizing.
• a capture probe 34 nucleotides in M. tuberculosis inserted corresponding to 15
• a microtiter plate (Nunc 439454) is deposited a solution of 1 ng / ⁇ l of the capture oligonucleotide, the 5 'end of which reacted with the reagent Aminolink 2 (Applied Biosystems, Foster city, California) in PBS 3X (0.45 M NaCI 0.15 M sodium phosphate pH 7.0). The plate is incubated for 2 h at 37 ° C. and then washed 3 times with 300 ⁇ ⁇ of PBST [PBS 1 x, Tween 20: 0.5 ° / 00 (Merck 822184)].
• the Aminolink 2 reagent makes it possible to add an arm comprising a 6-aminohexyl group to the 5 ′ end of the probe.
• the probe thus coupled to a ligand having a polar group (primary amine), and passively attached to the support, provides an improved signal; see application FR 91 09057.
• the target constituted by 10 ⁇ ⁇ of total RNA extract is mixed with 40 ⁇ ⁇ of salmon PBS buffer (PBS-3X + salmon sperm DNA 10 ⁇ g / ml [Sigma D 9156)].
• the whole is added to the well at 50 ⁇ ⁇ of a solution of the oligonucleotide-peroxidase conjugate, constituting the detection probe, at the concentration of 0.1 ng / ⁇ l of oligonucleotide in a PBS-horse buffer [PBS 3X + 10% horse serum (BioMérieux 55842)].
• the plate is incubated for 1 h at 37 ° C. and then washed 3 times with 300 ⁇ ⁇ of PBST buffer.
• OPD substrate ortho-phenylenediamine Cambridge Medical Biotechnology ref / 456
• citric acid buffer 0.1 M sodium monohydrogen phosphate, pH 4.93
• 30% hydrogen peroxide diluted 1/1000 is added immediately.
• the enzymatic activity is blocked with 100 ⁇ l of 1 N sulfuric acid and the reading is carried out on an Axia Microreader device (AXIA: registered trademark) (bioMérieux) at 492 nm.
• Axia Microreader device AXIA: registered trademark
• bioMérieux bioMérieux
• M. bovis including ATCC 19210) and 2 strains of M. bovis BCG
• M. avium-intracel / ulare including ATCC 35764), M. asiaticum (ATCC 25276), M. chelonae (ATCC 14472), M. flavescens, M. fortuitum, M. gastri, M. gordonae, M. kansasii, M. marinum, M. phlei, scrofulaceum, M. simiae, M. smegmatis, M. terrae, M. vaccae, M. xenopi, Nocardia asteroides (ATCC 3308), N. brasilensis (ATCC 19296).
• the combination of probes used is, a posteriori, specific for the tuberculosis complex. It does not cross-react with nucleic acids, in particular ribosomal RNA, from other bacterial species. It has been checked that the ribosomal target RNAs of the other species are well released during the lysis step since they react with the combination of capture and detection probes directed against 16S ribosomal RNA and of eubacterial specificity.
• the adaptation of the specific combination on the VIDAS machine has been carried out.
• the wall of the microplate well is here replaced by the SPR (trademark) ("Solid Phase Receptacle") which is a conical support made from a material sold under the name K resin (butadiene-styrene copolymer) and marketed by the company bioMérieux- VITEK (USA).
• the various reagents are placed in a strip with several cuvettes and the different stages take place in the SPR which is capable of sucking and discharging the reagents and which therefore acts as a pipette.
• the sandwich hybridization reaction described in the protocol below takes place on the internal wall of the cone.
• the capture oligonucleotide is passively attached comprising at its 5 ′ end the ligand Aminolink 2 (Applied Biosystems-ref. 400808) at a concentration of 1 ng / ⁇ l in a volume of 315 ⁇ ⁇ d ' a 4x PBS solution (200 mM sodium phosphate pH 7.0, 600 mM NaCl). After overnight at room temperature or two hours at 37 ° C., the cones are washed twice with a PBS Tween solution, then dried under vacuum.
• the strip contains in cuvettes the reagents necessary for detection, that is to say: 200 ⁇ ⁇ of a solution at 0.1 ng / ⁇ l of the oligonucleotide-phosphatase detection conjugate 17
• the cone After incubating the cone for 30 minutes with the target mixture plus detection probe, the cone is washed twice with a PBS Tween solution. 250 ⁇ l of MUP substrate (4-methyl umbelliferyl phosphate) in solution in a diethanolamine buffer are aspirated into the cone, then released into a reading bowl. The device measures the fluorescent signal expressed in URF (relative fluorescence units) of the cuvette.
• MUP substrate 4-methyl umbelliferyl phosphate
• a collection of mycobacterial strains was tested by hybridization on the 23S rRNA and made it possible to observe the specificity of this probe a posteriori.
• the hybridization of the rRNAs of a target bacterium was carried out according to the method described in example 3 above for the VIDAS robot.
• a capture probe (30 nucleotides in M. xenopi corresponding to the coordinates 81-1 10 of SEQ ID N0173) and an oligonucleotide-enzyme detection conjugate, corresponding to the coordinates 59-76 of SEQ ID N0173, are used.
• the target bacteria tested were notably the following: 18
• mycobacteria various other mycobacteria (56 isolates) including 2 strains of M. bovis (including ATCC 19210), M. asiaticum (ATCC 25276), M. avium- intracellulare (including ATCC 35764), M. chelonae (ATCC 14472), M. flavescens, M. fortuitum, M. gastri, M. gordonae, M. kansasii, M. marinum, M. non chromogenicum, M. phlei, M. scrofulaceum, M. simiae, M. smegmatis, M.szulgai, M. terrae , M. vaccae, Nocardia asteroides (ATCC 3308), M. brasilensis (ATCC 19296).
• M. bovis including ATCC 19210
• M. asiaticum ATCC 25276
• M. avium- intracellulare including ATCC 35764
• the hybridization of the rRNAs of a target bacterium was carried out according to the method described in example 3 above for the VIDAS robot.
• a capture probe 24 nucleotides in M. fortuitum corresponding to the coordinates 1468-1493 of E. coli
• an oligonucleotide-enzyme detection conjugate corresponding to the coordinates 6-22 of SEQ ID NO 1 17, are used.
• the target bacteria tested were notably the following:
• mycobacteria various other mycobacteria (45 isolates) including 2 strains of M. bovis (including ATCC 19210), M. asiaticum (ATCC 25276), M. avium-intracel / u / are (including ATCC 35764), M. chelonae (ATCC 14472 ), M. flavescens, M. fortuitum, M. gastri, M. gordonae, M. kansasii, M. marinum, M. non chromogenicum, M. phlei, M. scrofulaceum,
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• IviB STRAIN CIP 140 120001 lviC INDIVIDUAL / ISOLATED: lviD STAGE OF DEVELOPMENT lviE HAPLOT ⁇ PE: lviF TYPE OF FABRIC: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANIC: lvii IMMEDIATE SOURCE: bioMérieux SA lviII BIB: THE GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSITION RELATIVE
• IViB STRAIN CIP 140310001 lviC INDIVIDUAL / ISOLATED: lviD STAGE OF DEVELOPMENT lviE HAPLOT ⁇ PE: lviF TYPE OF FABRIC: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANIC: lvii IMMEDIATE SOURCE: bioMérieux SA lviiA BIB: GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSITION RELATIVE
• IViB STRAIN CIP 140220001 lviC INDIVIDUAL / ISOLATED: lviD STAGE OF DEVELOPMENT lviE HAPLOTYPE: lviF TYPE OF FABRIC: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANIC: lvii IMMEDIATE SOURCE: bioMérieux SA lviiA BIB: GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSITION RELATIVE
• IViB STRAIN A 253 lviC INDIVIDUAL / ISOLATED: lviD DEVELOPMENT STAGE lviE HAPLOT ⁇ PE: lviF TYPE OF ⁇ SSU: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANELLE: lvii IMMEDIATE SOURCE: bioMérieux SA lviiI BIBLIOT GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSITION RELATIVE
• IViB STRAIN A 255 lviC INDIVIDUAL / ISOLATED: lviD DEVELOPMENT STAGE lviE HAPLOTYPE: lviF TYPE OF FABRIC: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANELLE: lvii IMMEDIATE SOURCE: bioMérieux SA lviiA LIBRARY GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSI ⁇ ON RELATIVE
• IviB STRAIN A 203 lviC INDIVIDUAL / ISOLATED: lviD DEVELOPMENT STAGE lviE HAPLOTYPE: lviF TYPE OF FABRIC: lviG TYPE OF CELL: lviH CELL LINE: lvii ORGANELLE: lvii IMMEDIATE SOURCE: bioMérieux SA lviiI LIBRARY GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSI ⁇ ON RELATIVE
• IViB STRAIN CIP 140030001 lviC INDIVIDUAL / ISOLATED: lviD DEVELOPMENT STAGE lviE HAPLOTYPE: lviF TYPE OF FABRIC: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANIC: lvii IMMEDIATE SOURCE: bioMérieux SA liiiviii GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSITION RELATIVE
• IViB STRAIN CIP 140120001 lviC INDIVIDUAL / ISOLATED: lviD STAGE OF DEVELOPMENT lviE HAPLOTYPE: lviF TYPE OF FABRIC: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANIC: lvii IMMEDIATE SOURCE: bioMérieux SA lviiA BIB: GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSITION RELATIVE
• IviB STRAIN CIP 140 310 001 lviC INDIVIDUAL / ISOLATED: lviD STAGE OF DEVELOPMENT lviE HAPLOTYPE: lviF TYPE OF FABRIC: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANIC: lvii IMMEDIATE SOURCE: bioMérieux SA lviiA BIB: IN THE GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSITION RELATIVE
• IViB STRAIN CIP 140220001 lviC INDIVIDUAL / ISOLATED: lviD STAGE OF DEVELOPMENT lviE HAPLOTYPE: lviF TYPE OF FABRIC: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANIC: lvii IMMEDIATE SOURCE: bioMérieux SA lviiA BIB: GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSITION RELATIVE
• IViB STRAIN A 253 lviC INDIVIDUAL / ISOLATED: lviD DEVELOPMENT STAGE lviE HAPLOTYPE: lviF TYPE OF FABRIC: lviG TYPE OF CELL: lviH CELL LINE: lvii ORGANELLE: lvii IMMEDIATE SOURCE: bioMérieux S.A. lviiA BIBii
• IviB STRAIN CIP 140 410 001 lviC INDIVIDUAL / ISOLATED: lviD STAGE OF DEVELOPMENT lviE HAPLOTYPE: lviF TYPE OF FABRIC: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANAL: lvii IMMEDIATE SOURCE: bioMérieux SA liiiviii THE GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSITION RELATIVE
• IViB STRAIN A 251 lviC INDIVIDUAL / ISOLATED: lviD DEVELOPMENT STAGE lviE HAPLOTYPE: lviF TYPE OF FABRIC: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANELLE: lvii IMMEDIATE SOURCE: bioMérieux SA lviiI BIBLIOT GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSITION RELATIVE
• IviB STRAIN A 203 lviC INDIVIDUAL / ISOLATED: lviD DEVELOPMENT STAGE lviE HAPLOTYPE: lviF TYPE OF FABRIC: lviG TYPE OF CELL: lviH CELL LINE: lvii ORGANELLE: lvii IMMEDIATE SOURCE: bioMérieux SA lviiI LIBRARY GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSITION RELATIVE
• IviB STRAIN HOST. PASTEUR lviC INDIVIDU / ISOLE: lviD DEVELOPMENT STAGE lviE HAPLOTYPE: lviF TYPE OF TISSUE: lviG TYPE OF CELL: lviH CELL LINE: lvii IMMEDIATE SOURCE: bioMérieux SA lviiA BIBLIOTHEME: lviiB / SEGMENT: lviiiB POSITION RELATIVE
• IViB STRAIN CIP 140030001 lviC INDIVIDUAL / ISOLATED: lviD DEVELOPMENT STAGE lviE HAPLOTYPE: lviF TYPE OF FABRIC: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANIC: lvii IMMEDIATE SOURCE: bioMérieux SA liiiviii GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSITION RELATIVE
• IViB STRAIN CIP 140120001 lviC INDIVIDUAL / ISOLATED: lviD STAGE OF DEVELOPMENT lviE HAPLOTYPE: lviF TYPE OF FABRIC: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANIC: lvii IMMEDIATE SOURCE: bioMérieux SA lviiA BIB: GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSITION RELATIVE
• IviB STRAIN CIP 140 310 001 lviC INDIVIDUAL / ISOLATED: lviD STAGE OF DEVELOPMENT lviE HAPLOTYPE: lviF TYPE OF FABRIC: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANIC: lvii IMMEDIATE SOURCE: bioMérieux SA lviiA BIB: IN THE GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSITION RELATIVE
• IviB STRAIN CIP 140 220 001 lviC INDIVIDUAL / ISOLATED: lviD STAGE OF DEVELOPMENT lviE HAPLOTYPE: lviF TYPE OF ⁇ SSU: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANIC: lvii IMMEDIATE SOURCE: bioMérieux SA liiiviii IN THE GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSITION RELATIVE
• IViB STRAIN A 253 lviC INDIVIDUAL / ISOLATED: lviD STAGE OF DEVELOPMENT lviE HAPLOTYPE: lviF TYPE OF FABRIC: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANELLE: lvii IMMEDIATE SOURCE: bioMérieux SA lviiI BIBLIOT GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSI ⁇ ON RELATIVE
• IViB STRAIN A 255 lviC INDIVIDUAL / ISOLATED: lviD DEVELOPMENT STAGE lviE HAPLOTYPE: lviF TYPE OF FABRIC: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANELLE: lvii IMMEDIATE SOURCE: bioMérieux SA lviiA LIBRARY GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSITION RELATIVE
• IViB STRAIN A 203 lviC INDIVIDUAL / ISOLATED: lviD DEVELOPMENT STAGE lviE HAPLOTYPE: lviF TYPE OF ⁇ SSU: lviG TYPE OF CELL: lviH CELL LINE: lvii ORGANELLE: lvii IMMEDIATE SOURCE: bioMérieux SA lviiA BIBLIOT GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSI ⁇ ON RELATIVE
• GUGUGGAGCC GUGAUGAUGA AUCACCGGUA CUAACCACCC AAAACCGAUC GAUCGACCUU 60 CCUUCGGGUG UGGAGUUCUG GGGCUGCGUG GAACUUCGCU GGUAGUAGUC AAGCGAA 117 114
• IViB STRAIN CIP 140 120001 lviC INDIVIDUAL / ISOLATED: lviD STAGE OF DEVELOPMENT lviE HAPLOTYPE: lviF TYPE OF USSU: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANAL: lvii IMMEDIATE SOURCE: bioMérieux SA lviII BIB: THE GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSI ⁇ ON RELATIVE
• IviB STRAIN CIP 140 220001 lviC INDIVIDUAL / ISOLATED: lviD STAGE OF DEVELOPMENT lviE HAPLOTYPE: lviF TYPE OF ⁇ SSU: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANIC: lvii IMMEDIATE SOURCE: bioMérieux SA liiiviii THE GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSI ⁇ ON RELATIVE
• GUGUGGCGUC CCUGAUGAAU CAGCGGUACU AACCACCCAA AACCGGAUCG ACCAUUCCUU 60 CGGGGCGUGG AGUUUCGGGG CUGCGUGGAA CCUUCGCUGG UAGUAGUCAA GCAAU 115 119
• IViB STRAIN A 253 lviC INDIVIDUAL / ISOLATED: lviD DEVELOPMENT STAGE lviE HAPLOT ⁇ PE: lviF TYPE OF ⁇ SSU: lviG TYPE OF CELL: lviH CELL LINE: lvii ORGANELLE: lvii IMMEDIATE SOURCE: bioMérieux SA lviii BIBLIOT GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSI ⁇ ON RELATIVE
• IViB STRAIN A 232 lviC INDIVIDUAL / ISOLATED: lviD DEVELOPMENT STAGE lviE HAPLOTYPE: lviF TYPE OF ⁇ SSU: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANELLE: lvii IMMEDIATE SOURCE: bioMérieux SA lviiA BIBLIOT GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSI ⁇ ON RELATIVE
• IViB STRAIN CIP 140410001 lviC INDIVIDUAL / ISOLATED: lviD STAGE OF DEVELOPMENT lviE HAPLOTYPE: lviF TYPE OF ⁇ SSU: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANIC: lvii IMMEDIATE SOURCE: bioMérieux SA liiiviii GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSIUON RELATIVE
• IViB STRAIN A 203 lviC INDIVIDUAL / ISOLATED: lviD DEVELOPMENT STAGE lviE HAPLOTYPE: lviF TYPE OF ⁇ SSU: lviG TYPE OF CELL: lviH CELL LINE: lvii ORGANELLE: lvii IMMEDIATE SOURCE: bioMérieux SA lviiA BIBLIOT GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSI ⁇ ON RELATIVE
• IviB STRAIN HOST. PASTEUR lviC INDIVIDU / ISOLE: lviD DEVELOPMENT STAGE lviE HAPLOTYPE: lviF TYPE OF ⁇ SSU: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii IMMEDIATE SOURCE: bioMérieux SA lviiA BIBLIOTHEME: lviiI / SEGMENT: lviiiB POSI ⁇ ON RELATIVE
• IViB STRAIN CIP 140030001 lviC INDIVIDUAL / ISOLATED: lviD STAGE OF DEVELOPMENT lviE HAPLOTYPE: lviF TYPE OF ⁇ SSU: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANELLE: lvii IMMEDIATE SOURCE: bioMériIB liiiviiiviii GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSI ⁇ ON RELATIVE
• IviB STRAIN CIP 140 120001 lviC INDIVIDUAL / ISOLATED: lviD STAGE OF DEVELOPMENT lviE HAPLOTYPE: lviF TYPE OF ⁇ SSU: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANIC: lvii IMMEDIATE SOURCE: bioMérieux SA liiiviii THE GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSI ⁇ ON RELATIVE
• IViB STRAIN A 232 lviC INDIVIDUAL / ISOLATED: lviD DEVELOPMENT STAGE lviE HAPLOT ⁇ PE: lviF TYPE OF USSU: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANELLE: lvii IMMEDIATE SOURCE: bioMérieux SA lviiI BIBLIOTHE GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSIUON RELATIVE
• IViB STRAIN CIP 140410001 lviC INDIVIDUAL / ISOLATED: lviD STAGE OF DEVELOPMENT lviE HAPLOTYPE: lviF TYPE OF USSU: lviG TYPE OF CELL: lviH CELLULAR LINE: lvii ORGANELLE: lvii IMMEDIATE SOURCE: bioMérieux SA lviiA BIB: GENOME: lviiiA CHROMOSOME / SEGMENT: lviiiB POSI ⁇ ON RELATIVE

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