EP1441825A2 - Procede pour detecter des acides nucleiques au moyen d'un test rapide a sec - Google Patents

Procede pour detecter des acides nucleiques au moyen d'un test rapide a sec

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Publication number
EP1441825A2
EP1441825A2 EP02795061A EP02795061A EP1441825A2 EP 1441825 A2 EP1441825 A2 EP 1441825A2 EP 02795061 A EP02795061 A EP 02795061A EP 02795061 A EP02795061 A EP 02795061A EP 1441825 A2 EP1441825 A2 EP 1441825A2
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EP
European Patent Office
Prior art keywords
nucleic acid
sequence
detected
zone
specific nucleic
Prior art date
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EP02795061A
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German (de)
English (en)
Inventor
Michael Weizenegger
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Hain Lifescience GmbH
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Hain Lifescience GmbH
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Publication of EP1441825A2 publication Critical patent/EP1441825A2/fr
Withdrawn legal-status Critical Current

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3204—Inorganic carriers, supports or substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
    • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/328—Polymers on the carrier being further modified
    • B01J20/3282—Crosslinked polymers
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography

Definitions

  • the present invention relates to the field of diagnosis of nucleic acids.
  • the present invention relates in particular to a highly sensitive method for the detection, differentiation and characterization of nucleic acids in the form of a rapid dry test; the rapid dry test comprising a chromatographic material
  • the sequence-specific nucleic acid probes are immobilized via a polymer linker and further characterized in that the method comprises the following steps: i) in the case of double-stranded nucleic acids, the nucleic acid to be detected is denatured and then neutralized , ii) the nucleic acid to be detected is applied to the sample receiving zone in a running buffer which contains mildly denaturing agents, iii) the nucleic acid to be detected moves from the sample receiving zone towards the liquid-absorbing zone, iv) the nucleic acid to be detected is in the binding area of the separation zone with the sequence-specific nucleic acid probe brought into contact and hybridized to the sequence-specific nucleic acid probe v) the nucleic acid to be detected or the hybridization of the nucleic acid to be detected a n
  • the sequence-specific nucleic acid probe is detected via a label which is attached to the nucleic acid to be detected or via a
  • Detection with an antibody conjugate against a DNA double strand or a DNA / RNA double strand is therefore also considered as a marker.
  • the present invention furthermore relates to a device to carry out the method according to the invention.
  • nucleic acids with probes The detection of nucleic acids with probes is described in the prior art. Methods for quick and easy detection of nucleic acids are becoming increasingly important in the fields of medicine, the environment, food and forensics. Due to the high specificity and sensitivity of nucleic acid-based methods, these tests are of great importance for the identification and differentiation of pathogens, contaminating organisms or for the subtyping of bacteria or viruses and the investigation of genetic polymorphisms.
  • the nucleic acid to be detected is amplified.
  • Various reactions can be used as the nucleic acid amplification reaction.
  • the polymerase chain reaction (PCR) is preferably used.
  • the various configurations of the PCR technique are known to the person skilled in the art, see e.g. Mullis (1990) Target amplification for DNA analysis by the polymerase chain reaction. Ann Biol Chem (Paris) 48 (8), 579-582.
  • NASBA nucleic acid strand based amplification
  • TMA transcriptase mediated amplification
  • RT-PCR reverse transcriptase polymerase chain reaction
  • ß-Q replicase Q-beta replicase amplification
  • SDA single strand displacement amplification
  • the amplify is e.g. cut specifically by digestion with a restriction enzyme and the resulting ethidium bromide-stained fragments were analyzed on an agarose gel.
  • Hybridization systems are also widespread. The hybridization usually takes place in such a way that either the composition containing the amplification product or a part thereof or the probe is immobilized on a solid phase and brought into contact with the other hybridization partner in each case.
  • solid phases for example nylon, nitrocellulose, polystyrene, silicate materials, etc. It is also conceivable that a microtiter plate is used as the solid phase.
  • the target sequence can also hybridize with a capture probe beforehand in solution and then the capture probe is bound to a solid phase.
  • At least one probe or at least one primer is labeled during the amplification of the nucleic acid to be detected.
  • Various labels are conceivable, such as fluorescent dyes, biotin or digoxigenin.
  • Known fluorescent labels are fluorescein, FITC (fluoroisothiocyanate), cyanine dyes, etc.
  • the labels are usually covalently linked to the oligonucleotides. While a fluorescent label can be detected directly, biotin and digoxigenin labels can be detected after incubation with suitable binding molecules.
  • a biotin-labeled oligonucleotide can be detected by contacting it with a solution that contains streptavidin coupled to an enzyme, the enzyme, for example peroxidase or alkaline phosphatase, converting a substrate that produces a dye or leads to chemical luminescence ,
  • the enzyme for example peroxidase or alkaline phosphatase, converting a substrate that produces a dye or leads to chemical luminescence
  • This object was achieved according to the invention by a highly sensitive method for the detection, differentiation and characterization of nucleic acids in the form of a rapid dry test; the dry rapid test containing a chromatographic material comprising a sample receiving zone,
  • the method comprises the following steps: i) the nucleic acid to be detected is denatured in the case of double-stranded nucleic acids and then neutralized, ii) the nucleic acid to be detected is applied to the sample receiving zone in a Running buffer containing mildly denaturing agents is applied, iii) the nucleic acid to be detected moves from the sample receiving zone in the direction of the liquid-absorbing zone, iv) the nucleic acid to be detected is brought into contact with the sequence-specific nucleic acid probe in the binding region of the separation zone and hybridizes to the sequence-specific one Nucleic acid probe v) the nucleic acid to be detected or the hybridization of the nucleic acid to be detected to the sequence-specific nucleic acid probe is detected by means of a label on the nucleic acid to be detected is attached or via
  • nucleic acid and oligonucleotide refers to primers, samples, probes and oligomer fragments which are detected.
  • nucleic acid and oligonucleotide is also generic to polydesoxyribonucleotides (containing 2-deoxy-D-ribose) and to polyribonucleotides (containing D-ribose) or to any other type of polynucleotide that is an N-glycoside of a purine base or a pyrimidine base , or a modified purine base or a modified pyrimidine base.
  • PNAs are thus also included according to the invention, i. H. Polyamides with purine / pyrimidine bases.
  • nucleic acid and oligonucleotide are not considered to be different within the meaning of the present invention; in particular, the use of the terms is not intended to mean any differentiation in terms of length. These terms include double-stranded or single-stranded DNA as well as double-stranded or single-stranded RNA.
  • a rapid dry test in the sense of the present invention is to be understood as a device which chromatographically separates the product to be analyzed. made possible.
  • a chromatographic material with a pore size of 4 ⁇ m and larger, most preferably larger than 8 ⁇ m.
  • the analyte is brought to the reaction zones such as the separation zone by capillary forces of the chromatographic material.
  • the chromatographic material may comprise inorganic powders such as silicate materials, magnesium sulfate and aluminum, may further comprise synthetic or modified naturally occurring polymers such as nitrocellulose, cellulose sulfate, cellulose, polyvinyl chloride or acetate, polyacrylamide, nylon, cross-linked dextran, agarose, polyacrylate etc. may also include coated materials such as ceramic materials and glass. Most preferred is the use of nitrocellulose as the chromatographic material.
  • the introduction of positively charged ion groups in e.g. Nitrocellulose or nylon membranes improve the hydrophilic properties of the chromatographic material.
  • the rapid dry test comprises a sample receiving zone, a separation zone with a binding region in which one or more sequence-specific nucleic acid probes are immobilized and a liquid-absorbing zone lying behind the separation zone with a binding region.
  • the quick dry test can have several separation zones.
  • the rapid dry test can additionally comprise zones which contain marking substances which bind to the analytes as they pass this zone. For example, a zone containing a gold conjugate for labeling the passing analyte is customary.
  • the quick dry test can in particular have the form of a test strip.
  • the chromatographic material can be mounted in a housing or the like.
  • This housing is usually water-insoluble, rigid and can consist of a variety of organic and inorganic materials. It is important that the housing does not interfere with the capillary properties of the chromatographic material, that the housing does not bind test components non-specifically, and that the housing does not interfere with the detection system. It is preferred according to the invention that the length of the polymer linker or the polymer linker with anchoring molecule is more than 30 nm.
  • the length of the linker is crucial to achieve a high sensitivity of the immobilized probe.
  • the linker acts as a spacer between the probe and the membrane. In the present case, these are mostly polymers which extend the part of the probe which is complementary to the target sequence at the 5 'or 3' end, but are not themselves coding. These can be base sequences of a non-coding nucleic acid structure or other polymer units such as e.g. Polyether, polyester and others
  • the linker must be such that it does not affect the hybridization properties of the probe or only weakly negatively. This can be avoided by the fact that there are no self-complementary structures. The chemical prerequisites for the irreversible coupling of the probe to the carrier material must also be met.
  • a key prerequisite for the probe to function well beyond its properties of forming a stable hybrid with the target sequence is the chemistry of the coupling to the surface.
  • the length of the polymer linker or the polymer linker with anchoring molecule is more than 30 nm, particularly preferably more than 40 nm.
  • the linker is polythymidine. The length of synthetic linkers is often limited.
  • the yield and product quality are reduced so much after a number of synthesis steps that the length of the oligonucleotide is limited to approximately 100 monomers. Depending on the monomer, this corresponds to a length of approx. 30 - 40 nm.
  • a terminal transferase always results in a mixture of long (up to several hundred monomers) and very short oligonucleotides.
  • the sequence-specific nucleic acid probes are preferably immobilized on the membrane or the chromatographic material via a polymer linker which is connected to an anchoring molecule.
  • the anchor molecule is psoralen.
  • psoralen offers the possibility of forming very long linkers from fully synthetic oligonucleotides.
  • Psoralen is a polycyclic compound that is able to photochemically couple with pyridine residues under UV light with a wavelength of approx. 360 nm. The reaction with thymidine residues is particularly good.
  • probes which, for example, 5 'end a psoralen-containing polythymidine possessed by the effect of UV light by cross-linking of the molecules extensions of the spacers or linkers.
  • a mixture of pure polythymidine without anchoring molecule and polythymidine with psoralen modification as an anchoring molecule at the end of the polythymidine linker can be used to build network structures which prove to be advantageous for the hybridization efficiency and the sensitivity of the probes.
  • the DNA can be mixed with psoralen-labeled oligonucleotides and photocrosslinked. This improves the attachment of the probe to the surface.
  • anchoring molecules such as, for example: derivatives of psoralen, such as, for example, bis (PIP) Cn-psoralen, or other photoreactive crosslinking and labeling reagents known to the person skilled in the art, such as, for example, simple aryl-azide crosslinking agents, fluorinated aryl azide - Crosslinkers or crosslinkers based on benzophenone.
  • derivatives of psoralen such as, for example, bis (PIP) Cn-psoralen
  • PIP bis
  • crosslinking and labeling reagents known to the person skilled in the art, such as, for example, simple aryl-azide crosslinking agents, fluorinated aryl azide - Crosslinkers or crosslinkers based on benzophenone.
  • Probe oligonucleotides can be bound to the membrane surface via proteins, for example.
  • the proteins loaded with the probe can then be bound to the porous membrane using standard methods.
  • Standard methods are, for example, coupling via homobifunctional coupling reagents or heterobifunctional coupling reagents.
  • the reactive groups are the same.
  • these are amines and / or thiols.
  • Thiols can be synthetically coupled directly to oligonucleotides and react under oxidative conditions with, for example, cysteine residues to form disulfide bridges.
  • amines as homobifunctional coupling reagents can be directly synthetically coupled to oligonucleotides and bound to the surface or the protein via imido esters or succinimide esters.
  • the reactive groups are different and allow the coupling of different functional groups.
  • the formation of amino-thiol couplings is preferred.
  • Thiolated oligonucleotides can be coupled with a heterobifunctional coupling reagent which contains both a succinimide ester maleimide or iodoacetimide.
  • Another important coupling agent are the carbodiimides, which couple carbinyl residues to amines.
  • the nucleic acid probes are immobilized on the solid phase, and then this solid phase is brought into contact with the composition which contains the labeled nucleic acids to be detected or a part thereof.
  • the composition which contains the labeled nucleic acids to be detected or a part thereof Preferably at least two probes are immobilized on the solid phase, more preferably at least five probes, more preferably at least ten probes. Different probes can be immobilized in different zones.
  • the nucleic acid to be detected is marked.
  • fluorescent labels are fluorescein, FITC, cyanine dyes, rhodamines,
  • a radioactive marking is also conceivable, e.g. I, S, P, P.
  • Particle marking such as e.g. with latex. Such particles are usually dry, in the micron range and uniform.
  • the labels are usually covalently linked to the oligonucleotides.
  • a fluorescent label can be detected directly
  • biotin and digoxigenin labels can be detected after incubation with suitable binding molecules or conjugate partners.
  • Other binding partners than, for example, biotin / streptavidin are antigen / antibody systems, hapten / anti-hapten systems, biotin / avidin, folic acid / folate-binding proteins, complementary nucleic acids, proteins A, G and immunoglobulin etc. (M: N : Bobrov, et al., J. Immunol. Methods, 125, 279, (1989).
  • a biotin-labeled oligonucleotide can be detected by contacting it with a solution containing streptavidin coupled to an enzyme, the Enzyme, eg peroxidase or alkaline phosphatase, converts a substrate that produces a dye or leads to chemical luminescence.
  • the Enzyme eg peroxidase or alkaline phosphatase
  • Possible enzymes for this purpose are hydrolases, lyases, oxido reductases, transferases, isomerases and ligases.
  • Further examples are peroxidases, glucose oxidases, phosphatases, Suchases are known per se to the person skilled in the art (Wetmur JG. Grit Rev Biochem Mol Biol 1991; (3-4): 227-59; Temsamani J. et al.
  • Another preferred conjugate comprises an enzyme which is coupled to an antibody (Williams, J. Immunol. Methods, 79, 261 (1984).
  • nucleic acid to be detected with a gold streptavidin conjugate
  • a biotin-labeled Binding partners which form covalent bonds with one another, for example sulfhydryl-reactive groups such as maleimides and haloacetyl derivatives and amine-reactive groups such as isothiocyanates, succinimidyl esters and sulfonyl halides, are also conceivable.
  • the detectable conjugate can be applied to a zone of the rapid dry test which is passed through by the nucleic acid to be detected, the conjugate partner being introduced into this nucleic acid to be detected. If the nucleic acids to be detected are labeled, the probes are usually not labeled. The nucleic acids to be detected are thus essentially labeled using the methods described in the prior art (see also US Pat. No. 6,037,127).
  • the labeling can be introduced into the nucleic acid to be detected by chemical or enzymatic methods, or by direct incorporation of labeled bases into the nucleic acid to be detected.
  • sequences to be detected which have incorporated labels are produced by labeled bases or labeled primers during the PCR.
  • Labeled primers can be made by chemical synthesis e.g. by means of the phosphoramidite method by substituting bases of the primer with labeled phosphoramidite bases during the primer synthesis.
  • primers can be prepared with modified bases to which labels are chemically bound after the primer synthesis. Methods are also conceivable without the nucleic acid to be detected being amplified or provided with a modification.
  • ribosomal RNA species can hybridize specifically with a DNA probe and can be detected as an RNA / DNA hybrid with an RNA / DNA-specific antibody.
  • T4 polynucleotide kinase or a terminal transferase enzyme Another possibility is the introduction of labels using the T4 polynucleotide kinase or a terminal transferase enzyme.
  • the introduction of radioactive or fluorescent labels (Sambrook et. Al, Molecular Cloning, Cold Spring Harbor Laboratory Press, Vol. 2, 9.34-9.37 (1989); Cardullo et. Al. PNAS, 85, 8790; Morrison, Anal Biochem, 174, 101 (1988).
  • Markers can be introduced into one or both ends of the nucleic acid sequence of the nucleic acid to be detected. Markings can also be made within the nucleus acid sequence of the nucleic acid to be detected. Several markings can be introduced into a nucleic acid to be detected.
  • the denaturation according to process step i) is carried out with NaOH and the neutralization according to process step i) with a phosphate buffer or Tris buffer. Denaturation can also be achieved by other measures such as cooking at a temperature higher than 95 ° C, possibly with the addition of mildly denaturing chemicals. Denaturation of the nucleic acid to be detected is always necessary if double-stranded nucleic acids are present in the sample. It is further preferred according to the invention that the running buffer according to process step ii) is phosphate or TRIS buffer and that one or more of the following are contained in the running buffer as mildly denaturing agent: formamide, DMSO, urea. For neutralization and as a running buffer, however, all running buffers listed below can be used according to the invention. The buffer used for neutralization can be identical to the running buffer.
  • Buffers and solvents which can be used for the process according to the invention are known and examples are described in US Pat. No. 4,740,468 and US Pat. No. 6,037,127.
  • the pH for the running buffer is normally in the range 4-11, preferably in the range 5-11 and most preferably in the range 6-9.
  • the pH is chosen so that a considerable degree of binding affinity is maintained between all binding partners, including the hybridizing nucleic acids, and an optimal signal can also be obtained from the signal-producing system.
  • Typically used buffers contain borate, phosphate, carbonate, tris, barbital and the like.
  • the choice of the suitable buffer normally does not play a critical role for the method according to the invention. However, some buffers may be more suitable than others for special tests.
  • hybridization processes are carried out with heated solutions in hybridization ovens or water baths. This is typically done at 30 ° to 70 ° C, preferably at 50 ° C.
  • work must be carried out at room temperature. To achieve good sensitivities, it is therefore necessary to add the mildly denaturing agents mentioned above to the running buffer.
  • the process according to the invention can of course be carried out at all temperatures between 4 ° C. and 50 ° C. Most convenient and therefore preferred is room temperature.
  • hybridization refers to the formation of duplex structures by double-stranded nucleic acids due to complementary base pairing.
  • Hybridization can take place between complementary nucleic acid strands or between nucleic acid strands that have smaller regions of mismatch.
  • the stability of the nucleic acid duplex is measured by the melting temperature T m .
  • the melting temperature T m is the temperature (under defined ionic strength and pH) at which 50% of the base pairs are dissociated.
  • Stringent hybridization conditions are known to the person skilled in the art (for example Sambrook et al., 1085, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). In general, stringent conditions are selected so that the melting temperature is 5 ° C lower than the T m for the specific sequence at a defined ionic strength and pH. If the hybridization is performed under less stringent conditions, then sequence mismatches are tolerated. The extent of sequence mismatches can be controlled by changing the hybridization conditions.
  • Stringent in the sim e of the present invention means that the detection method allows a clear distinction between a positive reaction and a negative reaction in the reaction field of the strip. This can be achieved in particular by the following measures:
  • Structure of the probe by the length of the structure of the probe complementary to the target sequence; 15 to 20 mers are preferred.
  • Running buffer The stringency is influenced by the salinity.
  • the ionic strength is preferably between 100-400 mM, particularly preferably around 250 mM.
  • the stringency can be individually adjusted and optimized by the above-mentioned mildly denaturing substances in the running buffer (DMSO, formamide, urea).
  • the stringency is also influenced by the pH value of the running buffer.
  • all of the above-mentioned measures are measures which have an influence on hydrogen bonds.
  • the sequence-specific nucleic acid probe is a nucleic acid with a specific sequence which hybridizes to the nucleic acid to be detected under stringent hybridization conditions.
  • Specific sequence means that a defined nucleic acid structure can be distinguished from many other structures. This can be to differentiate between microorganisms and viruses, but also to differentiate nucleic acid polymorphisms in genetic or epidemiological questions.
  • the nucleic acid to be detected can be isolated from a multitude of organisms such as bacterial and viral pathogens.
  • the nucleic acid to be detected can also be the subject of diagnostic evidence for a genetic disease.
  • the nucleic acid to be detected can come from any conceivable source if the detection of the nucleic acid or parts thereof are to be detected, differentiated or characterized. In most cases, the nucleic acid to be detected is not detected directly, but has to be amplified beforehand. (For the illustration of possible amplification methods see also US 6,037,127.)
  • the nucleic acid to be detected is a fragment from the genome of a periodontitis-associated bacterium or is complementary to this.
  • the sequence-specific nucleic acid probe is a nucleic acid with a species-specific sequence which hybridizes to the nucleic acid to be detected under stringent hybridization conditions.
  • the sequence-specific nucleic acid probe is preferably selected from one of the following sequences or is complementary to these sequences: SEQ ID No .: 1-29, or is a fragment thereof (see also FIGS. 6 and 7)
  • probes which differ slightly from the probes according to the invention, but still work. It is also conceivable to use probes that have the sequences SEQ ID no. 1-29 at the 5 'and / or 3' end have extensions or truncations by at least one, two or three nucleotides. It is also conceivable that individual or a few nucleotides of a probe can be exchanged for other nucleotides as long as the specificity of the probe is not changed too much and the The melting point of the probe is not changed too much. This includes that when modified, the melting temperature of the modified probe does not deviate too much from the melting temperature of the original probe.
  • a composition which contains the nucleic acid to be detected or a part thereof is hybridized with one or more probes.
  • the present invention furthermore relates to a device for carrying out a method according to the invention in the form of a rapid dry test comprising a chromatographic material
  • a liquid-absorbing zone located behind the separation zone with the binding area, characterized in that the sequence-specific nucleic acid probes are immobilized via a polymer linker.
  • the preferred embodiments of the device essentially correspond to those of the method according to the invention.
  • the sequence-specific nucleic acid probes are preferably immobilized on the membrane via a polymer linker which is connected to an anchoring molecule.
  • the length of the polymer linker or the polymer linker with anchoring molecule is is more than 30 nm.
  • the length of the polymer linker or of the polymer linker with anchoring molecule is preferably more than 40 nm.
  • the linker is polythymidine.
  • the anchor molecule is psoralen.
  • the chromatographic material of the device according to the invention is preferably nitrocellulose. It is further preferred that the chromatographic material has a pore size of 4 ⁇ m and larger.
  • the sequence-specific nucleic acid probe is a nucleic acid which hybridizes under stringent hybridization conditions to a fragment from the genome of a periodontitis-associated bacterium or to the sequence complementary thereto.
  • the sequence-specific nucleic acid probe is selected from one of the following sequences or is complementary to these sequences: SEQ ID No .: 1-29 or contains fragments thereof.
  • the present invention furthermore relates to the use of the device according to the invention for the detection, differentiation and characterization of nucleic acids, in particular for the detection, differentiation and characterization of periodontitis-associated bacteria.
  • the present invention furthermore relates to the use of the method according to the invention and the device according to the invention for the detection of amplification products from amplification methods such as the polymerase chain reaction (PCR) and the nucleic acid strand-based amplification (NASBA).
  • amplification products may also have been created by other nucleic acid amplification techniques such as e.g. by transcriptase mediated amplification (TMA), reverse transcriptase polymerase chain reaction (RT-PCR), Q-beta replicase amplification (ß-Q-replicase) and single-strand displacement amplification (SDA).
  • TMA transcriptase mediated amplification
  • RT-PCR reverse transcriptase polymerase chain reaction
  • ß-Q-replicase Q-beta replicase amplification
  • SDA single-strand displacement amplification
  • Actinobacillus actinomycetemcomitans 5'-PsoC6-T85-SEQ ID No.:16,
  • Porphyromonas gingivalis 5'-PsoC6-T85-SEQ ID No.:21 and
  • Figure 4 Influence of the psoralen marking on the sensitivity.
  • the probe SEQ ID No.16 was immobilized with polythymidine linker from 85 thymidine residues without psoralen labeling and in (B) with psoralen labeling.
  • Actinobacillus actinomycetemcomitans (Prime ⁇ aar 5'-Biotin-SEQ ID No.:14; 5'-Biotin-SEQ ID No.4) was amplified in each case.
  • Denatured Actinobacillus actinomycetemcomitans -Amplifikat (Prime ⁇ aar 5'-Biotin-SEQ ID No.:14; 5'-Biotin-SEQ ID No.4) was with hybridization buffer (A) without DMSO, (B) 10% DMSO, 20% DMSO and 30% DMSO applied to the quick dry test. (Probe SEQ ID No. 16, psoralen-marked and with the polymidine linker with 85 polymidine residues).
  • the probes SEQ ID No. 1-29 (the probes are modified at the 5 'end with psoralen) onto a nitrocellulose membrane and bound by UV radiation.
  • the structure of the rapid test is shown in Figure 1.
  • DNA isolation For this purpose, bacterial material was removed from solid media using a sterile inoculation loop and suspended in 300 ⁇ l lOmM Tris / HCl pH 7.5. 1 ml was removed from liquid cultures, centrifuged for 5 min at 13,000 m m in a table centrifuge, the supernatant was discarded and resuspended in 300 ⁇ l lOmM Tris / HCl pH 7.5. The cell suspensions obtained in this way were incubated for 15 minutes at 95 ° C.
  • thermomixer Eppendorf, Hamburg, Germany
  • sonicated for 15 minutes in an ultrasonic bath Bandelin electronic, Berlin, Germany
  • 10 minutes at 13,000 ⁇ m in a table centrifuge Eppendorf, Hamburg, Germany centrifuged. 5 ⁇ l of the supernatant were used in the amplification reaction.
  • the oligonucleotides were applied to the membrane after they had dried completely , in a UV crosslinker (UV-Stratalinker 2400, Stratagene, La Jolla, USA) at 1200 joules / cm 2.
  • UV crosslinker UV-Stratalinker 2400, Stratagene, La Jolla, USA
  • the coated membrane was glued to a vinyl backing and pretreated with a sample pad (grade 903 paper, Schleicher & Schüll) 0.0 IM sodium tetraborate, 1% Triton X-100, pH 7.4) and a traction cushion (grade 470 paper, Schleicher & Schüll)
  • the cut strips were packed in a plastic housing.
  • Gold particles 20 nm conjugated with streptavidin (British Biocell, Cambridge, Great Britain) OD524, 4.0 were used for the detection. 3 ⁇ l of this gold particle suspension were mixed with 20 ⁇ l biotinylated amplificate and incubated for 5min. The denaturation of the amplificate was achieved by adding a NaOH solution (final concentration 200 mM). After five minutes of incubation, the solution was neutralized in 150 ⁇ l running buffer consisting of 250 mM phosphate buffer, pH 7.5, 50 mM NaCl, 0.1% Tween 20 and 20% DMSO (SIGMA, Munich, Germany) and added completely to the application zone , The developed zones can be read after approx. 5 min.
  • the detection was carried out for the subsequent experiments without conjugated particles, but rather staining with NBT / BCIP catalyzed by an alkaline phosphatase.
  • the strip was removed from the housing after the target nucleic acid had hybridized in the dry rapid test method and washed once in 1 ⁇ SSC for 1 min.
  • 5 g / l blocking reagent Röche, Mannheim, Germany
  • maleic acid buffer pH 7.5 (8.26 g NaCl and 10.06 g
  • streptavidin-alkaline phosphatase conjugate (1: 5000 dilution, Dianova, Hamburg, Germany) for 15 min.
  • the hybridized DNA could be incubated in substrate buffer (274mM Tris / Cl pH 7.5, 68.6mM Na 3 citrate, 200mM NaCl, 27.4 mM MgCl 2 x 6 H 2 O) with NBT (75 mg / ml nitro blue tetrazolium salt in 70% dimethylformamide) and BCIP (50 mg / ml 5-bromo-4-chloro-3-indonylphosphate-toluidinium salt in 100%) dimethylformamide) ,
  • the probe SEQ ID No.16 without polythymidine linker the same probe with polythymidine linker consisting of 20 thymidine residues and with 100 thymidine residues, and hybridized with NBT / BCIP were hybridized on the strips as described in Example 1.
  • Actinobacillus actinomycetemcomitans (Prime ⁇ aar 5'-Biotin-SEQ ID No.:14; 5'-Biotin-SEQ ID No.4) was used as the amplificate.
  • a clear sensitivity gradation can be seen correlating with the length of the linker (see Figure 3).
  • the probe whose linker is 100 thymidine residues long shows the highest sensitivity.
  • the probe SEQ ID No.16 was hybridized on the strip as described in Example 1 with polythymidine linker from 100 thymidine residues with and without 5 'porsoral labeling and developed with NBT / BCIP.
  • Actinobacillus actinomycetemcomitans (Prime ⁇ aar 5'-Biotin-SEQ ID No.:14; 5'-Biotin-SEQ ID No.4) was used as the amplificate.
  • a clear gradation of sensitivity can be seen correlating with the presence of the psoralen label. (Fig. 4). Psorally labeled probes are more sensitive.
  • the probe SEQ ID No.16 was hybridized on the strip as described in Example 1 with polythymidine linker from 100 thymidine residues and with 5 'p-oral labeling and developed with NBT / BCIP.
  • Denatured Actinobacillus actinomycetemcomitans -Amplifikat was with hybridization buffer (A) without DMSO, (B) 10% DMSO, 20% DMSO and 30% DMSO applied to the quick dry test. That is, the running buffer contains increasing concentrations of DMSO n 0, 10, 20 and 30%. The test becomes more sensitive at DMSO concentrations of more than%. (Fig. 5)

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Abstract

L'invention concerne le dépistage d'acides nucléiques et, en particulier, un procédé de haute sensibilité pour détecter, différencier et caractériser des acides nucléiques, au moyen d'un test rapide à sec comportant un matériau chromatographique constitué des éléments suivants : - une zone de prise d'échantillon, - une zone de séparation avec un domaine de liaison dans lequel sont immobilisées une ou plusieurs sondes d'acide nucléique de séquence spécifique, et une zone située derrière la zone de séparation avec un domaine de liaison et absorbant le liquide. L'invention est caractérisée en ce que les sondes d'acide nucléique de séquence spécifique sont immobilisées par une liaison polymère, le procédé comprenant les opérations suivantes : i) l'acide nucléique à détecter est, dans le cas d'acides nucléiques à doubles chaînons, d'abord dénaturé puis neutralisé ; ii) l'acide nucléique à détecter est placé sur la zone de prise d'échantillon, dans un tampon mobile contenant des agents légèrement dénaturés ; iii) l'acide nucléique à détecter se déplace de la zone de prise d'échantillon vers la zone absorbant le liquide ; iv) l'acide nucléique à détecter entre en contact, dans le domaine de liaison de la zone de séparation, avec la sonde d'acide nucléique de séquence spécifique, avec laquelle il entre en hybridation ; v) l'acide nucléique à détecter ou l'hybridation de l'acide nucléique à détecter sur la sonde d'acide nucléique de séquence spécifique est détecté au moyen d'un marquage placé sur l'acide nucléique à détecter ou par repérage d'un marquage du double chaînon d'acide nucléique. La présente invention porte également sur un dispositif pour réaliser ledit procédé.
EP02795061A 2001-11-05 2002-11-05 Procede pour detecter des acides nucleiques au moyen d'un test rapide a sec Withdrawn EP1441825A2 (fr)

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DE10154291A DE10154291B4 (de) 2001-11-05 2001-11-05 Verfahren zum Nachweis von Nukleinsäuren in Form eines Trockenschnelltestes
DE10154291 2001-11-05
PCT/EP2002/012333 WO2003039703A2 (fr) 2001-11-05 2002-11-05 Procede pour detecter des acides nucleiques au moyen d'un test rapide a sec

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JP2005507674A (ja) 2005-03-24
AU2002360934B2 (en) 2008-06-26
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WO2003039703A2 (fr) 2003-05-15
DE10154291B4 (de) 2005-05-19

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