EP0259453A1 - Detection d'acides nucleiques par la determination de l'agglutination de particules - Google Patents

Detection d'acides nucleiques par la determination de l'agglutination de particules

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Publication number
EP0259453A1
EP0259453A1 EP19870901958 EP87901958A EP0259453A1 EP 0259453 A1 EP0259453 A1 EP 0259453A1 EP 19870901958 EP19870901958 EP 19870901958 EP 87901958 A EP87901958 A EP 87901958A EP 0259453 A1 EP0259453 A1 EP 0259453A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
particles
acid sequences
sample
bound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19870901958
Other languages
German (de)
English (en)
Inventor
Malcolm L. Gefter
Christie A. Holland
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cambridge Biotech Corp
Original Assignee
Angenics Inc
Cambridge Biotech Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Angenics Inc, Cambridge Biotech Corp filed Critical Angenics Inc
Publication of EP0259453A1 publication Critical patent/EP0259453A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • 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
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • This invention is in the field of ligand assays and in particular relates to the detection and quantification of nucleic acid sequences through nucleic acid hybridization.
  • Nucleic acid hybridization is the basis for many methods used for the detection and identifica ⁇ tion of nucleic acids in a sample.
  • Hybridization is the process by which a single stranded nucleic acid (i.e., DMA or RNA) recognizes its complementary strand and hydrogen bonds to it, forming a double stranded molecule. That is, when single stranded nucleic acids are combined under appropriate condi ⁇ tions, complementary base sequences pair and. double- stranded hybrid molecules are formed.
  • DMA or RNA single stranded nucleic acid
  • sample DNA or RNA is attached to a solid support (e.g., a cellulose nitrate filter) by simply allowing it to adhere to the support.
  • a labelled probe DNA or RNA is then added under conditions appropriate for hybridization of complementary sequences to occur. The presence of sequences complementary to the probe sequence is determined by detecting binding of the labelled probe to bound (sample) DNA or RNA.
  • Attachment of DNA to a solid support can be accomplished by non-specific physical adsorption of single stranded nucleic acid (e.g., to nitrocellu ⁇ lose papers) and by chemical bonding (e.g., to agarose/Sepharosef a inoethyl-Sepharose Sephadexes cellulose) .
  • nucleic acid hybridization provides a very sensitive and specific approach to detecting and identifying nucleic acids in samples.
  • methods presently available require enzyme - or radioactive tracer - labelled nucleic acid probes, time-consuming ' procedures and/or sophisticated equipment.
  • nucleic acid hybrids are detected by observing a change in the absorbance of a DNA solution; by physically isolating hybridized DNA from nonhybridized DNA using chromatography or hydroxy patite and quantitating the hybridized DNA; or capturing the hybridized DNA on nitrocellulose.
  • nucleic acid sequences are often radio- actively labelled using phosphorous 32 ( 32P) , which can be introduced into DNA molecules as phosphate groups while they are being synthesized by host bacteria or by an _ir ⁇ vitro reaction. Radioactively labelled nucleic acid sequences are widely used, but radioactive material can pose a risk to the user.
  • 32P phosphorous 32
  • EPO European Patent Office
  • Dattagupta and Crothers describe a solid support for nucleic acids and an immobilized nucleic acid probe capable of hybridizing with complementary nucleic acids.
  • the solid support to which a nucleic acid can be bound by irradiation, is des ⁇ cribed as comprising a solid substrate which has reactive groups; a photochemically reactive inter- calator compound or other ligand capable of binding nucleic acids; and a divalent radical chemically linking the solid and the nucleicacid binding ligand.
  • the ligands chemi- cally link with nucleic acids.
  • the solid substrate is nitrocellulose paper having hydroxyl groups and linked by a bifunctional reagent to an amino-substituted compound, which in turn is photochemically linked to a nucleic acid.
  • the resulting immobilized nucleic acid is described as being useful in hybridization assays in which the support with coupled DNA is mixed with an unknown (possibly containing sequences complementary to that on the support) and a detection (labelled) probe. Testing the solid support for presence of a label (e.g., radioactivity) shows whether hybridization has occurred or not (and thus whether complementary DNA is present) .
  • a label e.g., radioactivity
  • Dattagupta et a_ describe a method for detecting the presence in a sample of a particular nucleic acid sequence which involves dual nucleic acid hybridization.
  • a sample containing unknown DNA is mixed with two nucleic acid probes which are complementary to two nonover- lapping portions of the nucleic acid sequence to be detected.
  • One probe is labelled and soluble in the sample and the other probe is fixed to a solid support (e.g., nitrocellulose).
  • the mixture is allowed to stand under hybridizing conditions; hybridization to both probes by DNA in the sample occurs only if it contains sequences complementary to both probes.
  • Kohne describes a method for detecting and quantifying bacteria and viruses containing RNA. After the nucleic acids in a sample and a marked probe (radioactively labelled nucleic acid sequences complementary to the RNA of the organism to be detected) have been incubated under hybridization conditions, the degree of hybridization with the marked probe is measured. The method is described as being useful for in solution hybridization or hybridization with an immobilized nucleic acid probe.
  • a marked probe radioactively labelled nucleic acid sequences complementary to the RNA of the organism to be detected
  • the method of this invention has very broad application, both in terms of the types of samples for which it is useful and the types of organisms which can be detected in such samples.
  • the nucleic acid content of any type of biological sample e.g., blood and other tissues; urine; and foodstuffs such as milk
  • the presence in biological samples of bac ⁇ teria and viruses can be detected using particle agglutination.
  • bacteria have common nucleic acid sequences, as well as sequences specific to a strain or class within the species, it is possible to detect all bacteria in a sample by using a shared nucleic acid sequence or to detect specific bacteria by using a nucleic acid sequence unique to that strain or class.
  • the present invention is based on the discovery that nucleic acid segments attached to a suspendable solid support, such as latex particles, and comple ⁇ mentary nucleic acid segments in solution will hybridize and cause particle agglutination.
  • the nucleic acid segments which can be either DNA or RNA, thus initiate particle agglutination (i.e., cause particles to agglutinate) .
  • the invention described herein is a method of detecting, identifying and/of quantitating nucleic acids in a biological sample, as well as particles having nucleic acids bound thereto.
  • the nucleic acids can be either bound directly to the particle surfaces or are attached through a spacer molecule which can, in turn, be either covalently bound or adsorbed to the particle surfaces.
  • nucleic acid sequences are used either to produce agglutination of inert particles having bound thereto nucleic acid sequences comple ⁇ mentary to nucleic acid sequences to be detected in the sample or to interfere with agglutination of such particles.
  • a sample contains nucleic acid sequences complementary to those attached to the solid support, hybridization will occur and cause particle agglutination.
  • inhibition of agglutination can be used to detect the presence of nucleic acid sequences of interest in a sample.
  • two different nucleic acid sequences e.g., + and -
  • the sample contains nucleic acids complementary to either of the attached sequences, agglutination of the solid support is inhibited.
  • detection of the degree of agglutination can be carried out visually or by another method known in the art.
  • the degree of agglutination is indicative of the extent of hybridization of complementary nucleic acid sequences, which is, in turn, indica ⁇ tive of the presence of nucleic acid sequences in the sample.
  • nucleic acids of interest in biological samples (e.g., body fluids, tissues, foodstuffs) and other samples using techniques and equipment which do not require highly skilled personnel for successful operation.
  • An important characteristic of the use of in solution or in suspension hybridization as described herein is that the reactants are not immobilized as, for example on large particles or filter membranes, and, as a result, hybridization occurs more rapidly because the reactive (hybridizable) sites can diffuse together more readily.
  • the resulting speed and the specificity with which nucleic acid se- quences of interest can be detected are important advantages of the present invention.
  • nucleic acid hybridiza ⁇ tion is highly specific - that is, a nucleic acid sequence will hybridize only with a complementary sequence - the particle agglutination method of the present invention is a very reliable means of
  • nucleic acid sequences of interest from among the numerous sequences found in biological samples.
  • the nucleic acid sequences to be detected can be characteristic of or shared by all members of a bacterial or viral species; as a result, all bacteria (or viruses) in a sample can be detected. This is particularly useful, for example, in detect ⁇ ing all bacteria in a foodstuff (e.g., a complete plate count for bacteria present in a milk sample) .
  • the nucleic acid sequences to be detected in the foodstuff can be specific to members of a given strain or class.
  • Figure 1 is a block diagram of one embodiment of the method of detecting nucleic acid sequences in a sample in which nucleic acid sequences are bound to particles.
  • Figure 2 is a block diagram of one embodiment of the method of detecting nucleic acid sequences in a sample in which probe nucleic acid sequences are bound to particulate support material and complementary nucleic sequences are bound to other particles.
  • Figure 3 is a block diagram of one embodiment of the method of detecting nucleic acid sequences in a sample in which nucleic acid sequences comple ⁇ mentary to those to be detected in the sample are bound to particles.
  • Figure 4 is a block diagram of one embodiment of the method of detecting nucleic acid sequences in a sample in which probe nucleic acid sequences are bound to inert support material and complementary nucleic acid sequences are bound to inert support material of relatively smaller size.
  • Figure 5 is a block diagram of one embodiment of the method of detecting nucleic acid sequences in a sample in which sample nucleic acid sequences are bound to particles.
  • detection of nucleic acids in a sample using agglutination of particles to which nucleic acid sequences are attached can be carried out directly or indirectly.
  • nucleic acid sequences complementary to those of interest are attached to latex particles and contacted with an appropriately treated sample (i.e., one which has been treated by methods known to those skilled in the art to make the nucleic acid sequences available for hybridiza ⁇ tion) .
  • an appropriately treated sample i.e., one which has been treated by methods known to those skilled in the art to make the nucleic acid sequences available for hybridiza ⁇ tion
  • Hybridization of the bound nucleic acids with those in the sample causes particle agglutina ⁇ tion; agglutination does not occur if the sequences of interest are not present in the sample.
  • a solution containing one set of latex particles having nucleic acid sequences ABC and a second set of latex particles having nucleic acid sequences DEF is contacted with a sample containing the nucleic acid sequence complementary
  • the presence in a sample of the nucleic acid egments of interest inhibits agglutination of particles bearing complementary nucleic acid sequences.
  • Two sets of nucleic-acid- bearing latex particles are used. For example, latex particles bearing strands of DNA (designated +) and other latex particles bearing DNA strands complementary to (+) strands (designated -) are contacted with a sample to be analyzed for the presence of a particular DNA sequence. The DNA strands attached to some of the latex particles are complementary to the DNA to be detected in the sample.
  • sample tested e.g., blood
  • the DNA sequence of interest will hybridize to its complement (which is bound to latex particles) and prevent hybridization of the particlebound (+) and (-) DNA segments. Agglutination of the particles will thus be inhibited or prevented and its absence will indicate that the DNA sequence sought is present in the sample.
  • Deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) to be used as the probe can be attached to a solid support or included in a solution which is contacted with the sample DNA-particle complex. It can be any gene or nucleic acid sequence (DNA or RNA) of interest. For example, it can " be sequences complementary to a ribosomal RNA sequence that is present in either all bacteria (inclusive) or sequences complementary to a ribosomal RNA sequence that is characteristic of a single type of bacteria (exclusive) . If an inclusive ribosomal nucleic acid is used as the probe, contact with a sample contain ⁇ ing RNA from any bacteria will result in hybridi ⁇ zation and agglutination of the particles.
  • RNA from a specific group of bacteria is present. Because RNA is rapidly degraded when an organism dies, only viable cells will be detected. In the first case, all viable bacteria in a sample will be detected; in the second, only the one type of viable bacteria having RNA complementary to the probe selected will be detected. This could be used, for example, in detecting viable bacteria in a milk sample pretreated to release bacterial RNA; it provides a simple, rapid and specific alternative to the standard plate count presently used in the dairy industry. As described in greater detail in Example 1, any single strand of DNA and its complement can be attached to the solid support.
  • one clone provides one DNA strand (designated +) and a second clone provides the second (or complementary) DNA strand (designated -) .
  • the src gene and the beta globin gene can be attached to the latex particles.
  • the nucleic acid sequences attached to the solid support can be of almost any length. It has been demonstrated that a stable bond or hybrid is formed when the nucleic acid has five or more base pairs. Generally, therefore, the nucleic acid sequences bound to the solid support will be five or more bases in length.
  • Nucleic acid, sequences to be attached to the solid supports for use as probes can be obtained by cloning of isolated DNA or RNA segments according to methods well known in the art. See, for example, Maniatis, T. et al. , Molecular Cloning - A Labora ⁇ tory Manual, Cold Spring Harbor Laboratory (1982) .
  • any restriction enzyme such as EcoRI, can be used to produce a desired DNA fragment for use as a probe.
  • Bacterial DNA can be cleaved at selected sites on either side of the DNA fragment of interest; the resulting fragments of interest can be isolated from other fragments (and thus purified) electrophoretically.
  • the isolated DNA fragments can then be amplified by inserting them into a plasmid or a bacterial virus (bacteriophage) , which is in turn inserted into an appropriate bacterial host cell.
  • a plasmid or a bacterial virus bacteria
  • the plasmid also replicates, producing many copies of the DNA fragment to be used as a probe.
  • the hybrid plasmids are isolated and purified, resulting in the isolation of many copies of the DNA fragment.
  • Nucleic acid sequences to be attached to the solid supports could also be obtained synthetically or, if they occur in nature in sufficient quanti ⁇ ties, simply by isolation and purification.
  • nucleic acid sequences from an appropriately treated biological sample to the solid supports.
  • the nucleic acid sequences in solution would be those complementary to the sequences of interest in the sample; that is, the probe sequences would be in solution.
  • the solid support to which the DNA or RNA is attached can be essentially any insoluble material to which the DNA or RNA can be covalently attached or irreversibly adsorbed; that is, the material must be reactive with the DNA or RNA or must adsorb a substance which can be covalently linked to the DNA or RNA.
  • the solid support can be, for example, latex, charcoal, colloidal gold, bentonite or glass. in addition, silica gel, controlled pore glass, red blood cells and liposomes can be used. In fact, any such particles to which nucleic acids can be attached can be employed in the application of the present invention. Nucleic acids have reactive amino functional groups, as well as terminal phos ⁇ phate groups and reactive hydroxyl groups in the sugar "backbone".
  • ribose in RNA can be partially cleaved oxidatively (e.g., with periodate, as in Fischer, U.S. Patent 4,264,766) to produce a reactive aldehyde function.
  • re ⁇ active groups on the nucleic acid can be reacted with reactive groups on latex or other particles.
  • Glass or other particles can be derivatized to form reactive functional groups (see, for example, Weetall, U.S. Patent 3,652,761; Koster, et al. , Tetrahedron Letters 2_ ⁇ 747 (1983)) capable of reacting with nucleic acids and proteins. Nucleic acids can also be linked directly to polystyrene (e.g., Ito, et al. ; Nucleic Acid Research 10; 755 (1982)).
  • the solid support need not have a particu ⁇ lar shape (configuration) but will often be spheri- cal. It must be small enough to remain in suspen ⁇ sion and will generally have a large particle size relative to the molecular weight of the DNA or RNA probe (e.g. , less than 500 microns) .
  • Nucleic acid sequences are attached to a solid support.
  • they can be covalently bound; the binding can occur randomly t along the length of the DNA or RNA, at the 5 end or at the 3 end.
  • the sequences can also be bound to the solid support through a protein or other spacer material.
  • a carbodiimide coupling reaction has been used by those in the field for the purpose of covalently linking DNA to solid supports, such as agarose (Sepharose ®) , ammoethyl-Sepharose Sephadexes and cellulose. Allfrey, V.G. and A.
  • nucleic acid sequences bound to solid supports are linked to latex particles along the length of the fragments.
  • Example 1 One method which can be used for covalent attachment is described in detail in Example 1. Nucleic acid sequences bound to latex particles using this method appear to be covalently attached randomly along their length. The method in Example l'is based on the method for forming an amide bond between latex and protein described by Dor an in U.S. Patent 4,045,384. There are three steps to the procedure.
  • carboxylated latex particles are activated; that is, an active ester is formed at the latex surface through reaction with a water-soluble N-hydroxy compound (e.g., N-hydroxybenzotriazole or N-hydroxysuccinimide) and a water-soluble carbodi- i ide (e.g., l-cyclohexyl-3-[2-morpholinoethyl]- carbodiimide methyl-p-toluene sulfonate (CMC) ) .
  • CMC methyl-p-toluene sulfonate
  • Single stranded DNA can be covalently bonded to the activated latex particle by combining the latex-hydroxybenzotriazole complex with single stranded DNA (or RNA) and agitating the combination (e.g., by rocking) at room temperature.
  • the product of this procedure has been shown to be single stranded DNA covalently attached to the latex particle at random along the length of the DNA. Similarly, this can also be done using RNA.
  • nucleic acids can also be at ⁇ tached to particles by being attached to other "bridging" molecules which can be adsorbed or covalently attached to latex.
  • Nucleic ' acids can be covalently linked to polysaccharides through one of many known reactions (Allfrey, V.G. and A. Inoue, In: Methods in Cell Biology 18: 253-270 (1978)). Polysaccharides can be partially oxidized to yield reactive aldehyde groups which can, in turn, be reacted with particles having suitably reactive groups such as amino groups (e.g., amino functional latex) .
  • DNA can be covalently attached to proteins by numerous methods (Nucleic Acid Research, 12: 3435
  • test or probe DNA or RNA can be bound to support material, such as latex particles, and DNA (or RNA) complementary to the probe DNA (designated
  • I t I A B C (-) can be bound to other latex particles.
  • unknown nucleic acid e.g., ribosomal RNA
  • I t t sample nucleic acid is complementary (e.g., A B C ) to that of the probe DNA, it will interfere with agglutination of the particles bearing the comple ⁇ mentary nucleic acid sequences. Particle agglutin ⁇ ation will be inhibited.
  • the extent of interference with particle agglutination is directly related to the amount of complementary nucleic acids in the sample. If there are only a few complementary sequences in the sample, particle agglutination will be interefered with to a lesser extent than if the sample contains a large number of sequences comple ⁇ mentary to * sequences on the latex particles.
  • probe nucleic acid is covalently bound to all latex particles.
  • the presence of complementary nucleic acid sequences in a sample will be detected when the latex parti ⁇ cles agglutinate.
  • one group of latex particles can have the nucleic acid sequences represented by ABC bound to them and another group sequences represented by DEF bound to them. If the sample nucleic acid contains the complementary
  • probe DNA can be attached to the larger particles and its complement to the smaller particles.
  • small particles would not be agglutinated to large particles and would pass through a filter of selected pore size. If the complementary sequences are not present in the sample, the probe DNA (bound to the larger particles) and its complement which is bound to the smaller particles will hybridize, preventing the small particles from passing through the filter.
  • Determination of agglutination or aggregation of particles having covalently-bound DNA or RNA can be carried out by any method capable of detecting the degree of agglutination present after sample and probe have been contacted under conditions appropri ⁇ ate for hybridization to occur. For example, it can be carried out visually using the unaided eye (e.g., visualization against a black or other dark back- ground) ; microscopically; or by turbidimetric measurements. In addition, a particle counter having a size threshhold can be used to detect aggregated/unaggregated particles.
  • Selective counting techniques which are well known in the art, make it possible to count the number of part ⁇ icles in a given size range and thus allow quanti ⁇ tative assays to be carried out. See, for example, U.S. Patent 4,184,849 to CL. Cambiaso et al. , in which such techniques are described. It is also possible to use a filter having a defined pore size; the pore size is selected so as to allow nonaggre- gated particles to pass through but to prevent aggregated particles from doing so. See, for example, U.S. Patent 4,459,361 to M.L. Gefter.
  • the particles can be enzyme labeled in such a way that the enzymes attached to the particle surfaces catalyze a color-producing change (thus aiding particle detection) .
  • amplification techniques are particularly useful when the nucleic acid sequences of interest are present in a sample in low concentrations.
  • A. Preparation of single stranded complementary DNA To prepare single stranded complementary DNA, the following method was used. A DNA sequence (i.e., a portion of the src gene) was cloned into an M13 bacteriophage using protocols described previ ⁇ ously. Hu, N. and J. Messing, The making of strand- -specific Ml3 probes. Gene, 1/7:271-277 (1982);
  • (+) and (-) strands of DNA can hybridize by hydrogen bonding.
  • Another method is to isolate the complementary DNA strands by preparative electrophoresis as described by Maniatis and co- workers. Maniatis, T. et al. , Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory (1982) .
  • Carboxylated latex used as the solid support in covalent binding with DNA was prepared according to the following technique, which is based on the method described by Dorman in U.S. Patent 4,045,384.
  • N hydroxybenzo ⁇ triazole solution 0.2 ml. of an N hydroxybenzo ⁇ triazole solution was added to 1 ml. of carboxylate latex (1 micron in diameter, 2.5% solids).
  • the N hydroxybenzotriazole solution was made by dissolving 93 mg. of Aldrich N-hydroxybenzotriazole in 1.6 ml. dimethylformamide; this was diluted to 4 ml. with water.
  • the combination was placed in an ice water bath and 0.1 ml. of CMC (100 mg. CMC solution in 2 ml. water at 0°C) was added dropwise. The mixture was stirred in the cold for four hours ' or overnight.
  • the mixture was placed in Eppendorf tubes (1 ml. each) and sonicated for 10 seconds. They were spun and resuspended three times in water and then resuspended in 50 mM phosphate buffer at pH 8.0.
  • DNA to be bonded to the latex particles was added to the dialyzed particles described above.
  • the DNA prepared as in part A was added while the particles were being stirred in an ice water bath or at 5°. Also present at that time was 1 ml. of pH 8.0 phosphate buffer and 0.1 M NaCl solution. About 0.06 ml. of the N-hydroxybenzotriazole solution (prepared as described above) was also added to aid the forward reaction.
  • the pH of the mixture (6.8) was raised to 7.2 by the addition of 0.5M dibasic sodium phosphate. The mixture was stirred for 5 days at about 4°C and washed with distilled water containing azide.
  • Latex particles prepared according to the method described above were spun down in Eppen- dorf tubes and washed three times with 0.1% SDS; each time they were resuspended in 1 ml. of 0.1% SDS. Microscopic assessment showed that most of the particles were monomers or dimers.
  • Hybridization of (-) strand DNA to (+) strand DNA covalently bound to latex particles was carried out. About 0.1 ml of latex (2% solids) to which (+) strand DNA was covalently bound was placed in each of three tubes. The solid latex particles were pelleted by centrifugation and resuspended in 50 microliters of 0.4M sodium phosphate buffer (pH 7.8) and 0.1% SDS containing one of the following:
  • the three tubes were incubated at 60°C for 10 to 12 hours. After the incubation the latex was pelleted again and resuspended in 0.1% SDS. Approximately 10 microliters were placed on a glass microscope slide and examined microscopically under oil immersion at 100 X magnification under a cover slip. The examination showed the following: (1)
  • Example 2 The procedure described in Example 1 was used except that the latex preparation was resuspended in 50 microliters of 0.4 M sodium phosphate buffer, 0.1% SDS containing
  • RNA can be extracted from a sample of milk and used to cause the agglutination of a latex that has a probe specific for ribsomal RNA (vRNA) attached to it.
  • vRNA ribsomal RNA
  • Such a probe is DNA or RNA complementary to the sequences of vRNA of organisms which occur in milk. This can be carried out as follows: (1) To 10 ml of skim milk add SDS to 0.1%, Proteinase K to 200 ug/ l
  • Nucleic acid probes and hybridization assays according to this invention have a variety of possible applications in which the ability to detect, quantify and/or identify complementary nucleic acid sequences of interest in biological samples of all kinds is of great value. For ex ⁇ ample, they are useful in a research context as tools for studying gene structure and inheritance. In addition, they are useful in clinical settings for the detection and identification of infectious agents and for prenatal diagnosis of genetic dis ⁇ orders. Finally, DNA probes have utility in the diagnosis of cancer (by providing information on the structure of oncogenes) ; in tissue typing; in veterinary and plant diagnostics; and in food testing (by providing a quicker, more convenient means of testing for the presence of pathogens) .

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Abstract

Procédé de détection, identification et/ou quantification d'acides nucléiques dans un échantillon par la détermination de l'agglutination ou de l'inhibition de l'agglutination de particules en suspension auxquelles sont liés les acides nucléiques. Ces dernierspeuvent être fixés directement à la surface des particules ou être fixés par l'intermédiaire d'une molécule d'écartement pouvant, à son tour, être liée de manière covalente ou adsorbée sur les surfaces des particules. Les particules en suspension sont suffisamment petites pour rester en suspension et présentent généralement une taille particulaire relativement grande par rapport au poids moléculaire de l'ADN ou de l'ARN fixé sur les surfaces. La présence ou l'absence de séquences d'acides nucléiques dans un échantillon est déterminée en détectant l'agglutination ou l'inhibition de l'agglutination des particules sur lesquelles sont fixées des séquences d'acides nucléiques complémentaires à celles à l'étude dans l'échantillon.
EP19870901958 1986-03-04 1987-03-03 Detection d'acides nucleiques par la determination de l'agglutination de particules Withdrawn EP0259453A1 (fr)

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US83610086A 1986-03-04 1986-03-04
US836100 2001-04-17

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EP0259453A1 true EP0259453A1 (fr) 1988-03-16

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EP19870901958 Withdrawn EP0259453A1 (fr) 1986-03-04 1987-03-03 Detection d'acides nucleiques par la determination de l'agglutination de particules

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EP (1) EP0259453A1 (fr)
JP (1) JPS63502875A (fr)
CA (1) CA1313485C (fr)
WO (1) WO1987005334A1 (fr)

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US5124246A (en) * 1987-10-15 1992-06-23 Chiron Corporation Nucleic acid multimers and amplified nucleic acid hybridization assays using same
US5359100A (en) * 1987-10-15 1994-10-25 Chiron Corporation Bifunctional blocked phosphoramidites useful in making nucleic acid mutimers
US5104791A (en) * 1988-02-09 1992-04-14 E. I. Du Pont De Nemours And Company Particle counting nucleic acid hybridization assays
CA2032203C (fr) * 1989-12-29 2009-05-19 Christine L. Brakel Essai de capture par amplification
US5147777A (en) * 1990-06-18 1992-09-15 Eastman Kodak Company Biologically active reagents prepared from carboxy-containing polymer, analytical element and methods of use
DE69112309T2 (de) * 1990-06-28 1996-01-25 Wakunaga Seiyaku Kk Verfahren zum Nukleinsäurenachweis.
GB9019512D0 (en) * 1990-09-06 1990-10-24 Ici Plc Assay method
US5578498A (en) 1991-05-22 1996-11-26 Behringwerke Ag Metal chelate containing compositions for use in chemiluminescent assays
US6251581B1 (en) 1991-05-22 2001-06-26 Dade Behring Marburg Gmbh Assay method utilizing induced luminescence
JPH0538300A (ja) * 1991-08-07 1993-02-19 Hitachi Ltd 核酸の測定方法及び核酸測定用自動分析装置
FR2720508B1 (fr) * 1994-05-25 1996-08-09 Rech Developp Activ Commun Procédé de mise en évidence d'un analyte dans un échantillon et dispositif pour mettre en Óoeuvre le procédé.
US5747256A (en) * 1995-12-19 1998-05-05 Beckman Instruments, Inc. Homogeneous DNA probe titration assay
US5914230A (en) * 1995-12-22 1999-06-22 Dade Behring Inc. Homogeneous amplification and detection of nucleic acids
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WO2015095929A1 (fr) * 2013-12-23 2015-07-02 The University Of Queensland Procédé et kit de détection d'acide nucléique

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US4605686A (en) * 1984-03-13 1986-08-12 Sekisui Kagaku Kogyo Kabushiki Kaisha Latex for immunoserological tests and a method for the production of the same
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CA1313485C (fr) 1993-02-09
JPS63502875A (ja) 1988-10-27
WO1987005334A1 (fr) 1987-09-11

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