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  • 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 detection and / or amplification techniques, using oligonucleotide probes or primers, and their application to the search for the presence or identification of bacteria of the genus Listeria.
• Listeria are Gram + bacteria belonging to the family of Listeriaceae, which is subdivided into two genera the genus Listeria and the genus Brochothrix (Collins et al, 1991, International Journal of Systematic Bacteriology, 41, 240-246)
• the genus Listeria groups the following bacterial species Listeria monocytogenes, Listeria innocua, Listeria seeligert, Listeria welshimeri, Listeria ivanovu, Listeria murrayi and Listeria grayi
• Listeria monocytogenes has a pathogenic capacity for humans This species is particularly responsible in humans for severe pathologies such as meningoencephalitis, septicemia or abortion
• the species Listeria monocytogenes has been recognized as the agent responsible for several epidemics of food poisoning, notably in Canada in 1981, in the United States from 1983 to 1985, in Switzerland from 1983 to 1987 and very recently in France in 1992 and 1993
• the mortality rate associated with these pidripartis is very high, about a third of
• the present invention overcomes the aforementioned drawbacks for demonstrating the presence of bacteria of the genus Listeria, and more particularly of the species Listeria monocytogenes, by using a genetic marker in a detection process by nucleic acid hybridization , combining specificity, sensitivity and speed
• RNA molecules Bacterial ribosomes contain at least three distinct RNA molecules called 5S, 16S and 23S rRNAs. Historically, these names have been chosen by reference to their sedimentation rate, which is related to the size of these RNA molecules
• the ribosomal RNA (rRNA) of bacteria can be used as a target.
• rRNA ribosomal RNA
• nucleic acid extracted from bacteria means either the total nucleic acid, or the ribosomal RNA, in particular the 23S rRNA, or the genomic DNA, or even the DNA obtained from the reverse transcription of 23 S ribosomal RNA;
• nucleotide fragment or an "oligonucleotide” are two synonymous terms designating a sequence of nucleotide patterns characterized by the informational sequence of natural (or possibly modified) nucleic acids and capable of hybridizing, like natural nucleic acids, with a nucleotide fragment
• the chain may contain nucleotide motifs with a structure different from that of natural nucleic acids.
• a nucleotide fragment (or oligonucleotide) can contain, for example, up to 100 nucleotide units. It generally contains at least 10, and in particular at least 12 nucleotide units and can be obtained from a natural nucleic acid molecule and / or by genetic recombination and / or by chemical synthesis,
• a nucleotide motif is derived from a monomer which can be a natural nucleotide of nucleic acid, the constituent elements of which are a sugar, a phosphate group and a nitrogenous base; in DNA the sugar is deoxy-2-ribose, in RNA the sugar is ribose; depending on whether it is DNA or RNA, the nitrogen base is chosen from adenine, guanine, uracil, cytosine, thymine; or the monomer is a nucleotide modified in at least one of the three constituent elements; by way of example, the modification can take place either at the base level, with modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2,6- purine, bromo-5-deoxyuridine or any other modified base capable of hybridization, either at the sugar level, not for example the replacement of at least one deoxyribose by a
• hybridization means the process during which, under appropriate conditions, two nucleotide fragments having sequences sufficiently
• hybridization conditions are determined by "stringency", that is to say the rigor of the operating conditions. Hybridization is all the more specific as it is performed at higher stringency. Stringency is a function in particular of the base composition of a probe / target duplex, as well as by the degree of mismatch between two nucleic acids. The stringency can also be a function of the parameters of the hybridization reaction, such as the concentration and the type of ionic species present in the hybridization solution, the nature and the concentration of denaturing agents and / or the temperature of hybridization. The stringency of the conditions under which a hybridization reaction must be carried out depends in particular on the probes used.
• the temperature for the hybridization reaction is between about 20 and 65 ° C, in particular between 35 and 65 ° in saline at a concentration of about 0.8 to 1M .
• probe is a nucleotide fragment comprising for example from 10 to
• nucleotide motifs in particular from 12 to 35 nucleotide motifs, having a specificity of hybridization under determined conditions to form a hybridization complex with a target nucleic acid having, in the present case, a nucleotide sequence comprised either in a ribosomal RNA , either in DNA obtained by reverse transcription of said ribosomal RNA, or in DNA (here called ribosomal DNA or rDNA) in which said ribosomal RNA is the transcription product; a probe can be used for diagnostic purposes (in particular capture or detection probes) or for therapy purposes,
• a "capture probe” is immobilized or immobilizable on a solid support by any suitable means, for example by covalence, by adsorption, or by direct synthesis on a solid support (see in particular patent application WO 92 10092) - a "detection probe" can be marked by means of a marker chosen for example from radioactive isotopes, enzymes, in particular enzymes capable of acting on a chromogenic, fluorigenic or luminescent substrate (in particular a peroxidase or a phosphatase alkaline), chromophoric chemical compounds, chromogenic, fluorigenic or luminescent compounds, nucleotide base analogs, and iigands such as biotin,
• a “primer” is a probe comprising for example from 10 to 100 nucleotide units and 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 a sequencing process, in a reverse transcription method, etc.
• the probes according to the invention can be used, for diagnostic purposes, in the search for the presence or absence of a target nucleic acid in a sample, according to all the known hybridization techniques and in particular the techniques of point deposit on filter, called “dot-blot” (MANIATIS et al, Molecular Cloning, Cold Spring Harbor, 1982), DNA transfer techniques called “Southern blot” (SOUTHERN. EM, J. Mol.
• the technique is used sandwich, with a capture probe and / or a detection probe, said probes being capable of hybridizing with two different regions of the target nucleic acid, and at least one of said probes (generally the detection probe) being able to hybridize with a target region that is specific to the species or group of species sought, it being understood that the capture probe and the detection probe must have nucleotide sequences at least partially different.
• the nucleotide probes of the invention can be used in conventional hybridization methods
• the nucleic acid to be detected can be DNA (possibly obtained after amplification) or RNA.
• the target nucleic acid can be obtained by extraction, according to known methods, from the nucleic acids of a sample to be examined.
• the denaturation of a double-stranded nucleic acid can be carried out by known methods of chemical, physical or enzymatic denaturation, and in particular by heating to an appropriate temperature, above 80 ° C.
• the demonstration of the presence of a given Listeria species requires the use of probes allowing the detection of a point mutation. For this, it is advisable to operate with probes of a predetermined length (number of nucleotide units), under conditions which are themselves predetermined. Further details are given below, with particular reference to the sandwich hybridization method which constitutes one of the most practical hybridization methods currently.
• This method comprises contacting a first probe fixed on a solid support with a solution containing the nucleic acid to be analyzed, and bringing said support into contact with the detection probe, incubating the mixture obtained, rinsing of the support, to remove the constituents which are not fixed on the support by specific hybridization, and the qualitative or quantitative detection, using a revelation reaction of the marker fixed on the support, of the fixed detection probe.
• the revelation of the presence of the marker can be carried out for example by colorimetry, fluorescence or luminescence.
• the capture probe is brought into contact with the sample and with the detection probe can be carried out sequentially, possibly with intermediate rinsing of the support. It is also possible to operate by placing the capture probe fixed on the support in simultaneous or almost simultaneous contact with a solution containing the sample and the detection probe which can be added as a mixture or separately
• the incubation and subsequent washing stages, which constitute the key stages of the sandwich hybridization process, are each carried out at a constant temperature which can for example be included in the range mentioned above (see “Definitions”).
• nucleic acid hybrids have a dissociation temperature which depends on the number of bases hybridized (the temperature increases with the size of the hybrid) and which also depends on the nature of the bases hybridized and, for each hybridized base, of the nature of the adjacent bases.
• the dissociation of the hybrids occurs over a temperature interval of a few degrees and can be easily determined, for example by U V spectroscopy. It is possible to determine experimentally the half-dissociation temperature of the hybrid formed by a given probe with the target of complementary sequence, by simple routine experiments.
• the hybridization temperature used in the sandwich hybridization technique must obviously be chosen below the semi-dissociation temperature. More exactly, we operate at a temperature lower than the semi-dissociation temperature of the least stable hybrid. among the two hybrids formed by the target with a pan the capture probe and on the other hand the detection probe, so that the two hybrids are stable at the temperature at which one operates A point mutation, i.e.
• the subject of the invention is therefore a single-stranded oligonucleotic, characterized in that it is chosen from oligonucleotides, having at least 12 nucleotide units, the sequence of which is included in one of the sequences (a 1 ) to (a 10 ) from the following list
• oligonucleotides of the invention some can be used to characterize a species or a group of species of the genus Listeria. These are in particular the oligonucleotides defined using the sequences (b 1 ) to (bis) and (c 1 ) to (c 1 4 ) below.
• the invention relates in particular to an oligonucleotide as defined above, characterized in that it comprises a sequence of at least five consecutive nucleotide motifs included in a sequence included inside the square brackets appearing in the sequences (b 1 ) to (b 15 ) from the following list:
• the invention relates in particular to an oligonucleotide as defined above, characterized in that its sequence is included in one of the sequences (c 1 ) to (e 14 ) below
• oligonucleotides of the invention some can be used as a probe making it possible to detect any bacterium of the genus Listeria. These include
• oligonucleotides defined using the sequences (d 1 ) to (d 6 ) below.
• the invention therefore also relates to an oligonucleotide as defined above, characterized in that its sequence is included in one of the following sequences (d 1 ) to (d 6 ):
• the oligonucleotides of the invention can be immobilized on a solid support (this is notably the case with a capture probe) or labeled with a tracer agent (detection probe) .
• the subject of the invention is also a method for determining the presence or absence of at least one bacterium of the genus Listeria, in a sample containing or capable of containing nucleic acids of at least one such bacterium, according to a method in which said sample is brought into contact with at least one nucleic probe, then the formation or absence of formation of a hybridization complex between said probe and the nucleic acid of the sample, characterized in that the said probe is an oligonucleotide chosen from those which have been defined previously.
• nucleic probes For the determination of the presence or absence of a species or a group of species of bacteria of the genus Listeria, it is possible to use as nucleic probes on the one hand a first probe as defined above and on the other share a probe as defined by one of the sequences (b 1 ) to (b 15 ) or (c 1 ) to (e 14 ), it being understood that said first and second probes are capable of hybridizing with non-overlapping regions of rRNA 23 (or its complement) of a bacteria of the genus Listeria.
• the first probe is for example immobilized on a solid support and the second probe is labeled with a tracer agent.
• o can use as probe at least one oligonucleotide as defined by the sequences (d 1 ) to
• a specific probe of the genus Listeria (included in one of the sequences (d 1 ) to (d) can be used to determine the bacteria which have just been mentioned. 6 )) in combination with the specific sequences q have just been indicated.
• a two probe system is used for the determination of the presence or absence of Listeria monocytogenes. , in particular in a process characterized by the fact
• one of the probes has a sequence included in the sequence (b 1 ), (b 10 ), (b 14 ) or (b 15 ) and the other probe a sequence included in the sequence (b 4 ),
• oligonucleotides which have been defined above can also be used as nucleotide primers for the synthesis of a nucleic acid in the presence of a polymerase in a manner known per se, and in particular in amplification methods using such a synthesis in the presence of '' a polymerase (PCR, RT-PCR, etc.)
• the oligonucleotides of the invention can in particular be used as primers for the reverse reverse transcription specific for a 23 S ribosomal RNA sequence of at least one species or at least one group of species of the Listeria genus to obtain a sequence of corresponding complementary DNA
• a reverse transcription may constitute the first stage of an RT-PCR, the next stage being the PCR amplification of the complementary DNA obtained.
• oligonucleotides can also be used as primers, in particular for the amplification specific by polymerase chain reaction of the rDNA sequence complementary to a 23 S ribosomal RNA sequence of at least one species or at least one group of species of the genus Listeria
• the oligonucleotides defined above may also be used as therapy probes to treat infections caused by at least one species or group of species of bacteria of the genus Listeria
• These probes s of therapy capable of hybridizing to 23 S ribosomal RNA and / or to the genomic DNA of said bacteria can block the phenomena of translation and / or transcription and / or replication
• the principle of gene therapy methods is known and is based in particular on the use of a probe corresponding to an anti-sense strand the formation of a hybrid between the probe and the sense strand is capable of disturbing at least one of the steps of decrypting genetic information
• the sequencing was carried out according to the following steps: extraction of the DNA from the strains by the method of Sjöbring et al. (1990 Polymerase chain reaction for detection of Mycobacterium tuberculosis J Clin. Microbiol. 28 (10) .2200-2204), PCR amplification of 23S ribosomal DNA using eubacterial primers defined in the zones
• R means G or A
• This step allowed the amplification of 3 zones of approximately 1kb making it possible to cover the entire 23 S target and which were sequenced on each of the 2 strands by the chain termination method on an Applied Biosystems ABI 360 sequencer ( Sanger et al., Proc. Natl Acad Sci USA, 1977, 74 5463-5467)
• This probe was used as a detection probe in a sandwich hybridization test
• RNA from Gram + bacteria was extracted according to the basic protocol for the extraction of RNA from Gram + bacteria described in "Current Protocols in Molecular Biology" 1987, Ausubel FM et al., Wiley interscience, New York.
• a microtiter plate (Nunc 439454) is deposited a solution of 1 ng / ⁇ l of the capture oligonucleotide, the 5 'end of which is coupled with the reagent Aminolink 2 (registered trademark, Applied Biosystems, Foster city, California) in 3X PBS (0.45 M NaCl; 0.15 M sodium phosphate; pH 7.0). The plate is incubated for 2 h at 37 ° C.
• the Aminolink 2 reagent makes it possible to add an arm comprising a 6-aminohexyl group to the 5 ′ end of the probe.
• the target (10 ⁇ l of total RNA) mixed with 40 ⁇ l of salmon PBS buffer (PBS-3X + salmon sperm DNA 10 ⁇ g / ml (Sigma D 9156) is deposited in the well in the presence of 50 ⁇ l of a solution of the oligonucleotide-peroxidase conjugate, constituting the detection probe, at a concentration of 0.1 ng / ⁇ l of oligonucleotide in PBS-horse buffer (PBS 3X + 10% horse serum (BioMérieux 55842))
• PBS-horse buffer PBS 3X + 10% horse serum (BioMérieux 55842)
• the plate is incubated for 1 h 37 ° C. and then washed 3 times with 300 ⁇ l of PBST buffer.
• 100 ⁇ l of OPD substrate ortho-phenylenediamine Cambridge Medical Biotechnology ref / 456
• OPD substrate ortho-phenylenediamine Cambridge Medical Biotechnology ref /
• the adaptation of the specific combination of probes tested on the VIDAS machine (BIO MERIEUX-VITEK) was carried out.
• the wall of the microplate well is here replaced by the SPR ("Solid Phase Receptacle") (BIOMERIEUX- VITEK-USA) which is a conical support made from a material called K resin (registered trademark for a butadiene-styrene copolymer ).
• K resin registered trademark for a butadiene-styrene copolymer
• the various reagents are deposited in a strip with several cuvettes and the various stages take place in the SPR which is capable of aspirating and discharging the reagents.
• the sandwich hybridization reaction described in the protocol below takes place on the internal wall of the cone.
• the capture oligonucleotide comprising at its 5 ′ end the ligand Aminolink 2 (Applied Biosystems-ref. 400808) is fixed passively on the internal surface of the SPR, at a concentration of 1 ng / ⁇ l in a volume of 315 ⁇ l of 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 associated with the VIDAS automaton contains in reagents the reagents necessary for detection, that is to say:
• the contents of the first and second wells are drawn into the pretreated cone as indicated above. After incubation for 30 minutes, the cone is washed twice with a PBS Tween solution and 250 ⁇ l of MUP substrate (4-methyl umbelliferyl phosphate) are aspirated and then released into a reading cuvette The device measures the fluorescent signal expressed in URF ( relative fluorescence units) of the cuvette
• L. mnocua can be detected using the combination of the following two probes for capture and detection, respectively:
• L. ivanovn can be detected using the combination of the following two probes for capture and detection, respectively.

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