FR2701961A1 - A method of destabilizing an intracatal secondary structure of a single-stranded polynucleotide, and capturing said nucleotide. - Google Patents

Abstract

A method of destabilizing an intracatal secondary structure of a single-stranded polynucleotide, and capturing said polynucleotide, characterized in that a device comprising an oligonucleotide attached to a support is brought into contact with a liquid phase containing said polynucleotide, at a temperature below 50.degree. C., said oligonucleotide acting as a capture probe and containing a so-called destabilizing sequence capable of hybridizing with a non-self-complementary sequence participating in said secondary structure of said polynucleotide; and device for implementing this method.

Description

 The subject of the invention is a method for destabilizing an intracatal secondary structure present in a single-stranded polynucleotide, which method can be used in a solid-phase hybridization technique when a locus of interest of the nucleic acid is present. at the level of a secondary structure.

 The invention also relates to a device for implementing this method. One of the advantages of this method is that it is easily automatable.

Hybridization of nucleic acids by formation of hydrogen bonds between two complementary sequences according to the A-T, GC for DNA, and AU, G matching laws
C for RNA, is a well-known phenomenon.

 On the basis of the nucleic acid hybridization properties, tests have been developed to identify and quantify, in a sample to be analyzed, a nucleic acid. In this so-called "nucleic probe" technology, the presence of a nucleic acid in a sample is confirmed using an oligonucleotide of known sequence (the probe) which is complementary to a nucleotide sequence of the desired nucleic acid (the target ).

 A particularly interesting hybridization technique is the so-called sandwich hybridization technique which uses a first probe fixed in excess on a solid support, making it possible to capture the desired nucleic acid, and a second, labeled probe, called a detection probe, which allows to reveal the presence of the nucleic acid thus captured. This technique, described in particular by Dunn and Hassel, Cell 12: 23-36 (1977), therefore comprises the use of two probes, a capture probe, fixed on a solid support, capable of hybridizing with a part (or capture zone) of a nucleic acid to be detected in a sample, and a labeled detection probe, which is complementary to another zone of the target, and which allows the detection by means of a marker which may be radioactive, enzymatic or chemical.

 The formation or lack of formation of a hybrid between the detection probe and the target nucleic acid is an important source of information for diagnosis. For example, if the nucleic sequence of interest is specific for a genus or species of bacteria, the presence of the genus or species in a sample is confirmed when the formation of a hybrid is detected.

 Trials based on hybridization techniques can therefore be used in the diagnosis of diseases associated with the presence of pathogenic microorganisms such as bacteria, fungi or viruses. They are also used in the agrifood industry, particularly to control the absence of bacterial contamination.

 In addition, the hybridization technique can be used in the diagnosis of genetic diseases.

 In the case of the detection of organisms, in particular of infectious organisms, in a sample, the target nucleic acid (DNA or RNA) must of course comprise at least one nucleic sequence sufficiently specific to the species, of the genus or the family of the organism to be detected and the probe, complementary to said nucleic sequence, must possibly be able to recognize its target even in a medium containing thousands of other nucleic acids which, in some cases, will differ from the target only by a pair of bases. Thus, if the desired nucleic sequence is present in the sample examined, it will form with the probe, a hybrid DNA / DNA, DNA / RNA or RNA / RNA, as the case may be.

 The target may be a single-stranded DNA molecule (obtained for example by prior thermal denaturation of a double-stranded DNA or by reverse transcription of an RNA) comprising a specific nucleic sequence of the organism to be detected.

The target may also be an RNA (ribosomal RNA, transfer RNA, or
Messenger RNA).

In the case of virus detection, the target may also be a DNA or RNA molecule. In the case of prokaryotic or eukaryotic organisms, it is often preferred to use as a target ribosomal RNA (rRNA) which, together with the ribosomal proteins, constitutes the ribosome. This ensures an essential function of the cell, namely the translation of the messenger RNA into proteins. Ribosomal RNA, which is found in all cells, is a universal marker of living things that has discriminatory variations in each species. has been the subject of much research, of which a recent compilation can be found in "The Ribosome: Structure, Function and Evolution", American Soeiety for
Microbiology, Washington DC (1990).

 The small subunit of the ribosome (16-18 S) and the large subunit (23-28 S) are molecules of choice for studying the phylogenetic relationships between the different living organisms, and for their identification. Including Takahashi et al., Biochim. Biophys.

Acta 134.124-133 (1967) showed the taxonomic value of ribosomal RNA subunits by conducting reference rRNA hybridization experiments with closely related species. These experiments show a quantifiable, reproducible percentage of homology representative of the taxonomic remoteness with respect to the reference species.

Similarly, Fox et al., International Journal of Systematic Bacteriology, 27: 44-57 (1977), have shown the interest of 16S rRNAs for the taxonomy of prokaryotes.

The taxonomic value of the nucleotide sequences of the subunits of the rRNA has been highlighted in particular by: Pace and Campbell, J. of Bacteriology 107: 543-547 (1971), which discuss the ribosomal RNAs of various bacterial species and a hybridization method to quantify levels of homology between these species; and Woese et al.
Microbiological Reviews 47: 621-669 (1983), as well as Grayet et al., Nucleic Acids Research 12: 5837-5852 (1984), which show by 16S RNA sequence comparisons that highly conserved regions are intercalated with regions of medium and low homology, even in the case of closely related species.

In fact, ribosomal RNAs are present in all living organisms, from bacteria to humans. ns have a particular nucleotide sequence made of a succession of domains characterized by a variable rate of evolution. In the latter case, certain domains are conserved in all organisms, or in groups of organisms, and can therefore be used as complementarity sites for hybridization to universal oligonucleotide primers for sequencing or amplification. When, on the contrary, the rate of evolution of a certain domain is high, it can be used to develop probes specific, depending on the case, the species, gender, family or reign. RNAs are a very abundant molecule in all cells, accounting for more than 90% of all
The result is that their sequence is representative of a species, or possibly a population, rather than an individual.

The use of ribosomal RNA as a target for hybridization reactions can be carried out on a culture, for example on a bacterial culture, or on a clinical sample, after reverse transcription optionally followed by enzymatic amplification in vitro. , using known methods, PCR (Polymerase Chain Reaction):
Saiki et al., Science, 230: 1350-1355 (1985), the Transcription Amplification System (TAS):
Kwoh et al., PNAS USA, 86: 1173-1177 (1989), 3SR (Self Sustained Sequence Replication): Guatelli et al., PNAS USA, 87: 1874-1878 (1990) or any other suitable amplification method. .

 It is also possible to directly target DNA whose ribosomal RNA is the transcription product.

 However, the development of tests aimed at highlighting the presence of rRNA in a sample faces some difficulties, particularly related to the conformation of the molecules in the sample. Indeed, the rRNA molecule, although single-stranded, has so-called secondary structures. In fact, it contains self-complementary sequences which, under specific physicochemical conditions, are able to pair locally, by intramolecular hybridization, by folding back on themselves and thus forming structures called "hairpin" or "loop-stems" (the "loops" corresponding to non-self-complementary sequence portions located between two sequences each involved in intramolecular hybridization); see, for example, Figure 1. The existence of stem-loop structures has been experimentally proven; see for example Noller et al., in the Ribosome: Structure, Function and Evolution, op.cit. The result is an organized secondary structure comparable to that of Figure 1 which represents the secondary structure of the E 16RRNA. Coli (Gutell et al., Comparative Anatomy of the 16 S-like ribosomal RNA, Prog Nucleic Acid Res.

Mol. Biol., 32: 155-216 (1985)). Analogous secondary structures are, of course, present in the single-stranded form of DNA whose rRNA is the transcript.

 The secondary structures present in the nucleic acids are at the origin of hybridization difficulties of the oligonucleotide probes because they are difficult to access to said probes; see especially Shimomaye and Salvato, Gene Anal. Techn., 6: 25-28 (1989).

For this reason, PCT Patent Application WO 91/00926 discloses a method in which a target region of rRNA is selected which contains as few secondary structures as possible.

But such a choice raises difficulties because the areas of diagnostic interest, having varied in the process of evolution, are most often located within said secondary structures.

It is possible to eliminate the secondary structures by thermal denaturation or with the aid of alkaline agents such as sodium hydroxide. However, it has been shown that part of the secondary and / or tertiary structures were not denatured under the denaturing conditions usually used; see in particular EP-0318245, whose authors have further shown that the residual secondary structures can inhibit or even block the formation of a hybrid between a probe and a target nucleic sequence integrated in a secondary structure. In addition, the thermal denaturation is not conducive to achieving hybridization in an automatic device because it requires operating at various temperatures. Finally, the use of sodium hydroxide poses problems of corrosion and accuracy of the doses introduced.

 The subject of the invention is a device for destabilizing the intracanular secondary structures of a single-stranded polynucleotide, and simultaneously allowing the capture of said polynucleotide, which further facilitates subsequent operations such as detection by hybridization, amplification or sequencing.

 In addition, the device of the invention can be used without prior denaturation of the single-stranded target polynucleotide. The device can therefore be used in an automatic apparatus operating at a fixed temperature, for example at 37 ° C.

 The invention therefore relates to a device for the destabilization of an intracatal secondary structure present in a single-stranded polynucleotide, characterized in that it contains an oligonucleotide attached to a support, said oligonucleotide containing a so-called destabilizing sequence capable of hybridizing with a non-self-complementary sequence participating in said secondary structure of said polynucleotide, and in that it is useful as a capture probe for said polynucleotide.

 In other words, the oligonucleotide attached to a support plays both the role of destabilizer and the role of capture probe. As indicated above, a capture probe is an oligonucleotide attached to a support, and allowing the isolation of the target.

 The oligonucleotide may be a naturally occurring DNA or RNA fragment, or a natural or synthetic oligonucleotide, or an unmodified synthetic DNA or RNA fragment, or comprising one or more modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2,6-purine, bromo-5-deoxyuridine or any other modified base for hybridization. Suitable chemical modifications can, for example, make it possible to improve the resistance to enzymatic degradation, in particular due to the nucleases, and also to increase the hybridization yield, of course without affecting the sequence of reactions. Examples are the introduction between at least two nucleotides of a group chosen from diphosphate, alkyl- and arylphosphonate and phosphorothioate esters, or the replacement of at least one deoxyribose with a polyamide; see P.E. Nielsen et al. Science, 254, 1497-1500 (1991).

 The support may be a macromolecular support, and in particular a solid support. The supports that can be used are known per se. This may be for example the wall of a test tube, a polystyrene or nylon plate, a cone (for example butadiene-styrene copolymer), beads or latex microspheres, etc. . The destabilizing oligonucleotide must be able to compete with the sequence complementary to the sequence it recognizes in the secondary structure of the target. For this reason, the sequence recognized by the oligonucleotide should not consist solely of a part itself self-complementary to the target, because the oligonucleotide probe would then also have a self-complementary sequence and would fold back on itself, so that it could not play its role of destabilizing probe without prior denaturation.

 It should be noted that it was not obvious that a probe fixed on a support, and therefore subjected to steric constraints, could be used as a means of destabilizing an intracatal secondary structure whose accessibility is of course strongly reduced, as a simple examination of Figure 1 makes it possible to understand it, and all the more so since in reality, polynucleotides such as rRNAs have a tertiary structure which, in general, further complicates the accessibility problems of fixed oligonucleotide probes.

This result is all the more remarkable in that, as shown in the experimental part, it is not even necessary to perform a prior denaturation of the target polynucleotide so that the device of the invention operates in a manner that satisfactory.

 In the patent application EP-0318245, liquid phase hybridization assays in which one or more auxiliary oligonucleotides whose hybridization capacity with a sequence involved in a secondary structure of the target is used to promote hybridization of another specific probe recognizing another sequence also present in said secondary structure. It is stated in this European patent application that working in a heterogeneous medium, that is to say with fixing a reagent on a solid phase, does not promote hybridization, for reasons which are due to It is obviously for this reason that the authors of the cited European patent application have opted for conducting liquid phase tests. There was, therefore, in the minds of specialists a prejudice against the possibility of using an oligonucleotide fixed on a solid support and, despite this, capable of destabilizing a secondary structure, without the aid of an auxiliary oligonucleotide.

To fix the oligonucleotide on the support, it is possible to use all the known methods such as, for example, fixation by establishing a covalent bond, either directly or via a ligand which is itself attached to the support by means of minus a covalent bond or by adsorption. In this connection, mention may be made of documents WO 88/01302 and FR-9109057.
The destabilizing sequence of the oligonucleotide used in the device of the invention must have a sufficient length (i.e., a sufficient number of nucleotide units) to cause the destabilization of the secondary structure of the target at a predetermined temperature. .

 It is known that the stability of a nucleic duplex increases with the number of paired bases, which results in the fact that the Tm temperature (dissociation temperature of 50% of the duplexes) increases, for a given oligonucleotide, when undergoes an elongation by increasing the number of nucleotide units.

 The destabilizing oligonucleotide sequence must therefore have a sufficient length to be able to both break the duplex formed by the sequences capable of intracatal hybridization, by displacement of one of the strands, and effectively inhibit the reformation of the secondary structure by intracatal annealing. the temperature at which one operates. Surprisingly, it has been found that despite the foreseeable constraints arising from the use of a destabilizing oligonucleotide attached to a support, it is possible to obtain a satisfactory destabilization of the secondary structure at temperatures below 50.degree. without prior denaturation of the target polynucleotide, while using relatively short oligonucleotides, for example having from 20 to 40 nucleotides.In practice, it can generally operate between 0 and 45 ° C, and in particular at 370 C, which represents an obvious advantage.

 The subject of the invention is therefore also a process for destabilizing an intracatal secondary structure of a single-stranded polynucleotide and for capturing said polynucleotide, characterized in that a device as defined above is brought into contact with a liquid phase containing, or likely to contain, said polynucleotide, at a temperature below 50 ° C. The liquid phase is, for example, a biological sample or a culture isolate in which it is desired to search, to assay or to characterize, a sequence polynucleotide of interest, as recalled in the introductory part above.For example, if one adds to the liquid phase a second oligonucleotide capable of hybridizing with a sequence other than the sequence recognized by the destabilizing sequence, this second oligonucleotide may be used, in sufficient quantity, either as a detection probe (the second oligonucleotide is then an oli labeled gonucleotide), either as a primer for amplification or target sequencing by elongation of the second oligonucleotide.

 Of course, the second oligonucleotide should have a suitable length, and especially short enough to ensure specificity at the temperature at which one operates. The second oligonucleotide is generally less than 25 nucleotides in length, in particular from 8 to 20 nucleotides.

 The sequence recognized by the second oligonucleotide, when present, is generally located in the destabilized part of the secondary structure.

To carry out the elongation of the second oligonucleotide, it is necessary to add to the liquid phase in sufficient quantity the elementary nucleotides and a polymerase such as a
DNA polymerase or, when the target is an RNA or a reverse transcriptase (DNA polymerase
Dependent RNA).

 By using nucleotides further containing modified nucleotides, e.g. chain terminating nucleotides or labeled nucleotides, it is possible to synthesize and characterize elongation products of the second oligonucleotide. One of the applications may be sequencing, performed by the Sanger method or any other suitable method.

 The destabilization of the secondary structure can also be used to synthesize a sequence complementary to the target by elongation of the capture probe, by addition to the liquid phase of the elementary nucleotides and of a polymerase and, in this case, the probe. capture will of course be fixed by its 5 'end. Again, it is possible to perform sequencing using nucleotides mixed with modified nucleotides.

It is also known that there are polymerases capable of moving the strands of a duplex to copy one of the two strands: this is the case, for example, of E. coli DNA polymerase I; see especially J. Sambrook et al. Molecular Cloning, A Laboratory Manual,
Cold Spring Harbor Laboratory Press (1989), chap.

 When the capture oligonucleotide is attached to the support at its 3 'end, and the second oligonucleotide is able to hybridize with a sequence of the target located downstream from the sequence recognized by the capture probe (c'). that is to say with a sequence situated between the 3 'end of the target and the sequence recognized by the capture oligonucleotide), it is possible to carry out an elongation of the second oligonucleotide so as to synthesize a sequence complementary to the region of the target between the second oligonucleotide and the capture probe, and in the case where a polymerase capable of displacing the strands of a duplex is used, the elongation of the second oligonucleotide may continue beyond of the sequence recognized by the capture probe, and the elongation product of the second oligonucleotide will be salted out in solution.

 When the capture oligonucleotide is attached to the support by its 5 'end, the addition of a polymerase under conditions allowing elongation then leads to an elongation of the capture oligonucleotide. When a second oligonucleotide, capable of hybridizing with a sequence of the target located downstream with respect to the sequence recognized by the capture oligonucleotide, is also present, the addition of a polymerase under conditions allowing the elongation leads to the elongation of the two oligonucleotides, and in the case where a polymerase capable of displacing the strands of a duplex is used, the progression of elongation of the second oligonucleotide will cause the deshybridization of the target with the capture probe (and its elongation product) .The elongation product of the capture probe will then be in single-stranded form and fixed to the solid support, while the elongation product of the second oligonucleotide will be salted out in solution in the form of a duplex formed with the target.

 Thus, the method of the present application makes it possible to easily realize, while making the most of the advantages of solid phase processes, the detection, possibly quantitative, characterization and amplification of nucleotide sequences present in secondary structures, all operations. which, until now, raised important technical difficulties.

The invention also relates to the particular devices and methods described hereinafter in the experimental part, and in particular the use of the particular capture probes described, including when they are used with the second oligonucleotides referred to as "detection probes" in the experimental part. , that is to say
use of capture probes SEQ ID No. 2, 6, 8, 10, or 12;
use, respectively as a capture probe and as a second oligonucleotide, of the following pairs of probes: 2 and 1; 6 and 5; 8 and 7; 10 and 9; 12 and 13; 12 and mixed, 14, 15 and 16.

The following examples illustrate the invention. In these examples, reference is made to the appended drawings in which:
FIG. 1 represents the secondary structure of E. coli ribosomal RNA from Gutell RR, B. Weiser, CR Woese, and HF Noller, 1985. Comparative anatomy of the 16S-like ribosomal RNA. Prog. Nucleic Acid Res. Mol. Biol. 32: 155-216, and
FIG. 2 represents a detail of FIG. 1, with details of the sequence of the probes used in example 1.

Example 1
A method which functions in particular at 37 ° C. without prior denaturation and which makes it possible to hybridize the 16S rRNA in solid phase at a locus present in a secondary structure making it possible to distinguish the Escherichia coli and Shigella species from the other species is described here. bacterial.

 The sequence of the probes used is given in Table 1 below.

 In a first step, it has been found experimentally that the presence of the secondary structure prevents the hybridization of a specific detection probe and thus the obtaining of a signal. The target is the stem-loop region of the rRNA at position 435-500 of Figure 1. The numbering is that of Gutell R.R., B.Weiser, C.R. Woese, and H.F. Noller, 1985.

Comparative anatomy of the 16S-like ribosomal RNA. Prog. Nucleic acid. Res. Mol., Biol.

32: 155-216. The detection probe G1 of Table 1 is complementary to a part of this structure and is, according to current knowledge, specific to the E. coli taxonomic entity.
Shigella. The hybridization of the rRNAs of a target bacterium was carried out according to the non-radioactive and semi-automated detection method described in French Patent No. 90 07249 using an S8L capture probe of eubacterial specificity and a detection probe. G1 conjugated with peroxidase The manipulation was carried out in a microtiter plate according to the following protocol.

In a microtitration plate (Nunc 439454) is deposited a solution of 1 ng / l of the capture oligonucleotide whose 5 'end reacted with the reagent Aminolink 2 (Applied
Biosystems, Foster city, Calif.) 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. and then washed 3 times with 300 l of PBST (1x PBS, Tween 20: 0.5% (Merck 822184)).

The target rRNA is extracted from each bacterium according to the following technique. 1.5 ml of bacterial culture is centrifuged and the pellet taken up in 75 l of Tris buffer (25 mM Tris,
10 mM EDTA, 50 mM sucrose pH 8) and 25 l of lysozyme solution (1 mg / ml buffer
Tris). After incubation for 30 minutes at 37 ° C., 50 l of phenol are added and the tube is stirred vigorously for 30 seconds, 50 μl of chloroform are added and the mixture is stirred for a further 2 seconds and after centrifugation for 5 minutes, the aqueous phase is recovered.

The target consisting of 10 μl of total RNA extract is mixed with 40 μl of buffer
Salmon PBS (PBS-3X + salmon sperm DNA 10, rg / ml (Sigma D 9156)). The whole is added in the well to 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 PBScheval buffer (PBS 3X + 10% serum of horse (BioMérieux 55842)). The plate is incubated for 1 hour at 37
C then washed with 3 times 30 + 1 of PBST. In each well, 100.1 of OPD substrate (Cambridge Medical Biotechnology ortho-phenylenediamine ref / 456) are added in a buffer of 0.055 M citric acid, 0.1 M sodium monohydrogenphosphate. pH 4.93) at a concentration of 4 mg / ml, to which 30% hydrogen peroxide diluted 1/1000 is added extemporaneously.

After 20 minutes of reaction, the enzymatic activity is blocked with 100 μl of 1N sulfuric acid and the reading is carried out on Axia Microreader (BioMérieux) at 492 nm.

The target bacteria included the following:
- 50 E. coli isolates or strains (including ATCC 25290,25922,27165, 10536, 4157, 11775T);
- Various other Escherichia (18 isolates): E. fergusonii, E. hermanii, E. vuineris:
Shigella: S. boydii, S. dysenteriae (including ATCC 29027), S. flexneri, S. sonnei;
- Various Salmonella (27 isolates or strains): S. arizonae (including ATCC 13314, 13323, 12325), S. choleraesuis (ATCC 13312), S. gallinarum, S. give, S. paratyphi A, S. shomron, S.

sophia, S. typhimurium (including ATCC 14028 and 13311), S. spp (ATCC 9712, 1202, 11997, 8388, 6962,29628);
- Various Enterobacteriaceae: Citrobacter amalonaticus including ATCC 25405, Citrobacter freundii including ATCC 11102, Enterobacter cloacae including ATCC 13047, Enterobacter sakazaki with
CUETM 79104 and ATCC 29544, Enterobacter taylorae (eUETM 452384), Hafnia alvei,
Klebsiella oxytoca, K. pneumoniae including ATCC 13886 and various other Klebsiella, Kluyvera ascorbata ATCC 33433, Moel Ierella wisconsensis (including CDC 182679 and 306575), Morganella morganii, Proteus mirabilis, Proteus morganii (including ATCC 25830), Proteus penneri, Proteus rettgeri, Proteus vulgaris (including ATCC 6380), Providencia rettgeri, Pseudomonas aeruginosa
ATCC 35422, Serratia grimesii ATCC 14460, Serratia marcescens including ATCC 8195 and 8100, various other Serratia, Yersinia enterolitica including ATCC 23715, Yersinia intermedia including ATCC 29909, Yersinia kristensenii including ATCC 35669, Yersinia pseudotuberculosis including ATCC 23207 and NCTC 8487;
- Enterococcus faecalis.

Results
The specificity results show that this detection system gives no signal under the indicated conditions. The capture probe S8L is not in question: in fact, if the specific detection probe G1 is replaced by the eubacterial detection probe E220, (capture: S8L) all the strains of E. coli-Shigella, as well as other selected bacterial species give a positive result. The negative results obtained with the G1 detection probe for the E. coli-Shigelta strains thus indicate that it can not reach its target because of the presence, under the experimental conditions used, of the secondary structure described in FIG. figure 1.

 When the S8L eubacterial capture probe is replaced under the same conditions by the oligonucleotide G3 (Table 1 and FIG. 2) located in the 435-500 coordinate rod-loop, a signal is obtained with all the E. coli and Shigella strains. . The specificity is then that desired: this combination of probes does not cross-react with nucleic acids, in particular rRNA, of any other bacterial species, including species close to Escherichia, with the exception, however, of Escherichia fergusonii gives a positive result. Thus, the oligonucleotide G3 fixed on a solid support fulfills the function of capture and destabilization. In addition, these experiments clearly show that the prior denaturation of ART is not necessary.

 The adaptation of the same system captures G3 - G1 detection on the VIDAS PLC (registered trademark - marketed by BioMérieux SA - VITEK). The wall of the microplate well is here replaced by the SPR ("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 VITEK ( USA). The various reagents are arranged in a bar with several cuvettes and the different steps take place in the SPR which is capable of aspirating and discharging the reagents and which thus acts as a pipette. The sandwich hybridization reaction described in the protocol below occurs on the inner wall of the cone.

 On the inner surface of the SPR, the destabilizing capture oligonucleotide having at its 5 'end the Aminolink 2 ligand (Applied Biosystems ref.400808) at a concentration of 1 ngF in a volume of 315 μl of a solution of PBS 4x (200 mM sodium phosphate pH 7.0, 600 mM NaCl). After a night at ambient temperature or two hours at 37 ° C., the cones are washed twice with PBS Tween solution and then dried under vacuum. The bar contains in cuvettes the reagents necessary for the detection, ie: 200, ssA1 of a 0.1 ng + solution of the oligonucleotide-alkaline phosphatase detection conjugate, twice 600 I of PBS Tween wash solution. and a substrate bowl.

 In the first well of the array, 10 μl of RNA is extracted into the same buffer as for the above microplate protocol.

After incubation of the cone for 30 minutes with the target mixture plus detection probe, the cone is washed twice with a solution of PBS Tween. 250 μl of MUP substrate (methyl-4 umbelliferyl phosphate) in solution in a diethanolamine buffer are sucked into the cone and then released into a reading cuvette. The device measures the fluorescent signal expressed in
URF (relative fluorescence units) of the cuvette.

 With this system, the same results were obtained as with the microplates.

 The Aminolink 2 reagent makes it possible to add to the 5 'end of the probe an arm comprising a 6-aminohexyl group. 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.

Example 2
A method operating in particular at 37 ° C. and for hybridizing in solid phase the 16S rRNA at a locus of interest discriminating the Salmonella genera and
Vibrio, this locus being present in a secondary structure different from that of Example 1.

 The target comprises the two secondary structures of the 16S rRNA, analogous to those present at positions 770 to 880 of Figure 1. The sequence of the probes is given in Table 1.

 The hybridization of the rRNAs of bacterial strains was carried out according to the protocol described in Example 1. It uses the destabilizing probe dSAL9-2 for capture and the oligonucleotide SAL9 as detection probe (conjugated with peroxidase). The SAL9 detection probe of Table 1 is complementary to a part of the secondary structure and must, according to current knowledge, be specific to the taxonomic genus Salmonella.

The target bacteria included the following:
- 20 isolates or strains of Salmonella arizonae including ATCC 13314, 13323 and 12325;
the other Salmonella already used in Example 1;
- various other Salmonella, and in particular: S. bakou, S. derby, S. enteritidis, S.

gallinarum, S. paratyphii A, B and C, S. panama, S. pullorum, S. thomson;
various enterobacteria (34) of the genera Citrobacter, Enterobacter, Klebsiella, Proteus,
Pseudomonas, Serratia; of which ATCC 11102, 13047, 29544, 13886, 6380, 35422, 8195, already cited and 25830 (Proteus morganii);
- various E. coli including ATCC 27165 and 4157
- various Shigella including ATCC 11060
- various other Escherichia (ATCC 29907T blattae, fergusonii, hermanii, vulgaris);
- various Vibrio (anguillarum, harveyi), Aeromonas and Pleisiomonas.

 The results obtained indicate that the combination of probes used is specific for the genera Salmonella and at least one species of the genus Vibrio (this is Vibrio anguillarum).

It does not cross-react with nucleic acids, in particular rRNA, of other bacterial species, including species close to these two genera. This is controlled by the fact that hybridization with the S8L capture probe of eubacterial specificity, and the E220 detection probe of eubacterial specificity is positive with non-eubacterial strains.
Salmonella and non-Vibrio.

 It should be noted that the SAL9 probe differs from the corresponding sequence in Escherichia coli (Fig.1) only by a single nucleotide. The SAL9 probe is, however, sufficiently specific and gives a negative result for E. coli.

Adaptation of the same combination of probes on the VIDAS automaton (bioMérieux
SA, France) made it possible to obtain the same results as in microplates.

Example 3
Here, a method that operates in particular at 37 ° C. and which makes it possible to hybridize in solid phase the rRNA R 16S at a locus of interest discriminating the Listeria monocytogenes species, this locus being present in a secondary structure of RNA 16S, similar to that between positions 1235 and 1300 of Figure 1.

 The hybridization of the rRNAs of bacterial strains was carried out according to the protocol described in Example 1, with the exception of the extraction of the target RNA which is carried out as follows: 1.5 ml of bacterial culture is centrifuged and the pellet taken up in 75 μl of Tris buffer (50 mM Tris, 10 mM EDTA, 25% sucrose pH 8) and 25 μl of mutanolysin solution (Sigma) (1 mg / ml in Tris buffer) and lysozyme (1 mg / ml in Tris buffer). After incubation for 30 minutes at 37 ° C, 50 μl of phenol is added and the tube is shaken vigorously for 30 seconds.

50 ml of chloroform are added and the mixture is stirred again for 2 seconds. After centrifugation for 5 min, the aqueous phase is recovered.

This protocol uses the destabilizing probe RL1 for capture and the oligonucleotide
RL2 as a detection probe (conjugated with peroxidase). The detection probe RL2 of Table 1 is complementary to a part of this structure and should, according to current knowledge, be specific for Listeria monocytogenes.

The target bacteria were as follows:
- eleven isolates of Listeria monoeytogenes;
- three Listeria grayi including ATCC 19120, four Listeria innocua, three Listeria murrayi including CCM 5990 (CCUG 4984), three Listeria ivanovii, four Listeria seeligeri including CIP 100100 (CCUG 15530), three Listeria spp, three Listeria welschineri including CIP 8149 (CCUG 15529).

The results obtained show that this combination of probes is specific for
Listeria monocytogenes. It does not cross-react with nucleic acids, particularly rRNA, of any other bacterial species tested, including closely related species, such as
Listeria innocua.

Adaptation of the same combination of probes on the VIDAS automaton (bioMérieux
SA, France) observed the same results as microplates.

Example 4
A method which operates in particular at 37 ° C. and which makes it possible to hybridise the 16S rRNA in solid phase at a locus of interest discriminating the Chlamydia trachomatis species, this locus being in a secondary structure similar to that located between positions 585 and 655 of Figure 1.

 Hybridization of the rRNAs of bacterial strains was carried out according to the protocol described in Example 1.

Hybridization uses the destabilizing probe dCT4 for capture and the oligonucleotide
CT3 as detection probe (conjugated with peroxidase). The CT3 detection probe of Table 1 is complementary to a part of this structure and should, according to current knowledge, be specific to Chlamydia trachomatis.

The target bacteria were as follows:
- four isolates of Chlamydia trachomatis corresponding to the classification
Serovar Ba, D, K and L2; Wang SP et al., 1975, J.Clin.Microbiol. 1: 250-255.

- various other Chlamydia including Chlamydia Pneumoniae and Chlamydia psittaci;
various non-Chlamydia strains of the genera Acinetobacter, Achromobacter,
Aeromonas, Bacillus, Branhamella, Candida, Chromobacter, Enterobacter, Enteroeoecus,
Haemophilus, Klebsiella, Escherichia, Lactobacillus, Listeria, Micrococcus, Neisseria, Proteus,
Pseudomonas, Salmonella, Serratia, Staphylococcus, Streptococcus.

 The results obtained show that this combination of probes is specific for Chlamydia trachomatis. It does not cross-react with nucleic acids, in particular rRNA, of any other bacterial species tested, including the closely related species, such as Chlamydia psittaci.

Adaptation of the same combination of probes on the VIDAS automaton (bioMérieux
SA, France) showed the same results in microplate.

Example 5
A method which operates in particular at 37 ° C. and which makes it possible to hybridize the 16S rRNA in solid phase at a locus of interest that discriminates various species of the genus is described here.
Mycobacterium, this locus being present in a secondary structure similar to that located between the positions 140-230 of Figure 1.

The hybridization of the rRNAs of the bacterial strains tested was carried out according to the protocol described in Example 1, with the exception of the extraction of the target rRNA carried out according to the basic protocol for the extraction of the RNA from the Gram positive bacteria described in "Current
Protocols in Molecular Biology 1987, Ausubel FM et al., Wiley Interscience, New York.

 The hybridization assay uses the destabilizing probe dTB 1 for capture and the oligonucleotide bovis 310 as a detection probe (conjugated with peroxidase). The bovis detection probe 310 of Table 1 is complementary to a part of the secondary structure and should, according to current knowledge, be specific to the tuberculosis taxonomic group comprising M. tuberculosis, M. bovis, M. afiicanum, M. microti.

The target bacteria were as follows:
- 27 isolates or strains of Mycobacterium tuberculosis including ATCC 27294 and NCTC 7417;
- 5 strains or isolates of Mycobacterium bovis including ATCC 19210 and M. bovis BCG (Copenhagen 1077);
various strains of Mycobacterium avium and intracellulare including ATCC 35716, 29555, 35764, 35847, 35770;
various Mycobacterium (chelonae, diernoferi, flavescens, fortuitum, gastric, gordonae, kansasii, lactis, marinum, non-chromogenicum, phlei, scrofulaceum, simiae, smegmatis, szulgai, terrae, trivial, xenopi, vaccae) including ATCC 14472, 14470, 12478 , 19981, 25275, 35799, 15755 and 23292;
- Non-mycobacteria :: Actinomadura madurae, Nocardia asteroides ATCC 3308, Nocardia brasilensis ATCC 19296, Rhodococcus aichiensis and Rhodococcus sputi.

The results obtained show that this combination of probes is specific for
Mycobacterium tuberculosis. It does not cross-react with nucleic acids, in particular rRNA, other bacterial species, including other species of the genus Mycobacterium, even though the rRNA of these strains is well available for hybridization such as confirms the observed hybridization with the S8M capture probe of eubacterial specificity, and the detection probe E220 of eubacterial specificity.

Adaptation of the same combination of probes on the VIDAS automaton (bioMérieux
SA, France) allows to obtain the same results in microplate.

In the same way, it is possible to define probes specific to the complex taxonomic group Mycobacterium avium-M. intracellulare still called "MAY". The hybridization test uses the destabilizing probe dTB1 and the mixture of detection probes MAI 1,
MAY 2, MAY 3 (Table 1).

<SEP> T <SEP> A <SEP> B <SEP> L <SEP> E <SEP> A <SEP> U <SEP> 1
<tb> Name <SEP> Sequence <SEP> Origin <SEP> SEQ <SEP> ID <SEP> No.
<tb> G1 <SEP> TCA <SEP> ATG <SEP> AGC <SEP> AAA <SEP> GGT <SEP> A <SEP> 1
<tb> G3 <SEP> TTA <SEP> ACT <SEP> TTA <SEP> CTC <SEP> CCT <SEP> TCC <SEP> TCC <SEP> CCG <SEP> CTG <SEP> E. <SEP> Coli <SEP> 2
<tb> S8L <SEP> TST <SEP> ACG <SEP> CAT <SEP> TTC <SEP> ACC <SEP> GCT <SEP> ACA <SEP> C <SEP> Eubacterium <SEP> 3
<tb> E220 <SEP> TCT <SEP> AAT <SEP> CCT <SEP> GTT <SEP> TGC <SEP> TCC <SEP> CC <SEP> 4
<tb> SAL8 <SEP> CCA <SEP> AGT <SEP> AGA <SEP> CAT <SEP> CG <SEP> Salmonella <SEP> sp. <SEP> 5
<tb> dSAL9-2 <SEP> TTA <SEP> CGG <SEP> CGT <SEP> GGA <SEP> CTA <SEP> CCA <SEP> GGG <SEP> TAT <SEP> CTA <SEP> A <SEP> 6
<tb> RL2 <SEP> ATA <SEP> GTT <SEP> TTA <SEP> TGG <SEP> GAT <SEP> TAG <SEP> C <SEP> L.monocytegenes <SEP> 7
<tb> RL1 <SEP> CTT <SEP> TGT <SEP> ACT <SEP> ATC <SEP> CAT <SEP> TGT <SEP> A <SEP> 8
<tb> CT3 <SEP> CGT <SEP> TAC <SEP> TCG <SEP> GAT <SEP> GCC <SEP> CAA <SEP> ATA <SEP> Chl. <SEP> trachomates <SEP> 9
<tb> dCT4 <SEP> TCG <SEP> CCA <SEP> CAT <SEP> TCG <SEP> GTA <SEP> TTA <SEP> GCG <SEP> GCC <SEP> GTT <SEP> T <SEP> 10
<tb> bovis <SEP> 310 <SEP> ACC <SEP> ACA <SEP> AGA <SEP> CAT <SEP> GCA <SEP> TCC <SEP> CGT <SEP> G <SEP> M.tuberculosis <SEP> 13
<tb> dTB1 <SEP> GGT <SEP> CCT <SEP> ATC <SEP> CGG <SEP> TAT <SEP> TAG <SEP> ACC <SEP> AGC <SEP> TTT <SEP> CC <SEP> Mycobacterium <SEP > sp. <SEP> 12
<tb> S8M <SEP> TCT <SEP> GCG <SEP> CAT <SEP> TCC <SEP> ACC <SEP> GCT <SEP> ACAC <SEP> Eubacterium <SEP> 11
<tb> MAY <SEP> 1 <SEP> ACC <SEP> AGA <SEP> AGA <SEP> CAT <SEP> GCG <SEP> TCC <SEP> TG <SEP> 14
<tb> MAY <SEP> 2 <SEP> ACC <SEP> TAA <SEP> AGA <SEP> CAT <SEP> GCG <SEP> CCT <SEP> AA <SEP> M.avium <SEP> intracellular <SEP> 15
<tb> MAY <SEP> 3 <SEP> ACC <SEP> AAA <SEP> AGA <SEP> CAT <SEP> GCG <SEP> TCT <SEA> AA <SEP> 16
<Tb>

Claims (18)

 1. Device for the destabilization of an intracatal secondary structure present in a single-stranded polynucleotide, characterized in that it contains an oligonucleotide attached to a support, said oligonucleotide containing a so-called destabilizing sequence capable of hybridizing with a non-homogenous sequence self-complementary participating in said secondary structure of said polynucleotide, and in that it is usable as capture probe for said polynucleotide.  2. Device according to claim 1, characterized in that said support is a macromolecular support.  3. Device according to claim 1, characterized in that said polynucleotide is an RNA.  4. Device according to any one of the preceding claims, characterized in that said destabilizing sequence has a length sufficient to cause the destabilization of said secondary structure at a predetermined temperature, less than 50 ° C, selected in particular from 0 to 45 "C.  5. Device according to the preceding claim, characterized in that said destabilizing sequence contains from 20 to 40 nucleotides.  6. Device according to any one of the preceding claims, characterized in that said oligonucleotide is chosen from those having the sequences SEQ ID No. 2, 6, 8, 10 and 12.  7. Process for destabilizing an intracatal secondary structure of a single-stranded polynucleotide, and for capturing said polynucleotide, characterized in that a device as defined in any one of the preceding claims is brought into contact with a liquid phase containing said polynucleotide, at a temperature below 50 ° C, said oligonucleotide acting as a capture probe.  8. Process according to claim 7, characterized in that said temperature is 37 ° C.  9. The method of claim 7 or 8, characterized in that one operates without prior denaturation of said polynucleotide.  10. Method according to any one of claims 7 to 9, characterized in that said liquid phase further contains a second oligonucleotide capable of hybridizing with a sequence of said polynucleotide other than the sequence recognized by said destabilizing sequence.  11. Method according to the preceding claim, characterized in that said second oligonucleotide is capable of hybridizing with a sequence participating in said secondary structure of said polynucleotide.  12. The method of claim 10 or 11, characterized in that said second oligonucleotide is labeled.  13. Method according to any one of claims 10 to 12, characterized in that said liquid phase further contains a polymerase and nucleotides, and that one elongation of said second oligonucleotide.  14. Method according to any one of claims 7 to 12, characterized in that said liquid phase further contains a polymerase and nucleotides, and that one elongates the capture probe, or an elongation of the capture probe and the second oligonucleotide.  15. The method of claim 13 or 14, characterized in that the liquid phase further contains modified nucleotides.  16. Method according to the preceding claim, characterized in that said modified nucleotides are chain terminating nucleotides or labeled nucleotides.  17. Method according to any one of claims 7 to 16, characterized in that said capture probe is chosen from those having the sequences SEQ ID No. 2, 6, 8, 10 and 12.  18. Method according to the preceding claim, characterized in that the oligonucleotides chosen from those having the sequences SEQ ID No. 1 are used as capture probe and as second oligonucleotide, respectively.  - 12 and mixed, 14, 15 and 16.  - 12 and 13  - 10 and 9  -8et7  -6et5  -2etl