JP2000514644A - Use of carrier materials for detection and sample preparation in genetic engineering analysis methods - Google Patents

Abstract

  (57) [Summary] Investigation of sorbent material in the form of compacts, coarse packing of sorbent material in the form of particles, sorbent material in the form of gel or as a dispersion by means of sample preparation and genetic engineering analysis methods Use for detection, wherein the sorbent material has at least one component for performing a solid phase assay and is fluid permeable; the sorbent material has an average of 0.1-100 μm The sorbent material has no or very low non-specific sorption capacity for the affinity material; the sorbent material has a specific concentration of the complementary component. When a certain amount of the diluted solution is flowed once in a homogeneous manner, the component (affinity material) for performing the solid phase assay contained in a given unit volume of the carrier material has an average loading of ± 40%. Between some statistically related fillings near the volume The binding amount is standardized by the provision that it specifically binds to its complementary component, the binding amount being the carrier material filled with components for blocking specific and non-specific adsorption sites and for performing solid phase assays. The use of a suitable liquid in the carrier material, such as washing and supply of the affinity reaction material, which remains constant within the above range during several subsequent flow steps.

Description

Detailed description of the invention Use of carrier material for detection and sample preparation in genetic engineering analysis methods The subject of the present invention is the use according to claim 1. For example, amplification methods using molecular biology methods such as the polymerase chain reaction (PCR), or "branched DNA" systems using detection amplification, e.g., by increasing the number of labeled compounds of complementary DNA. Using gene-based genetic engineering methods such as hybridization methods, such as those described in "Profiting from Gene-based Diagnostics", CTB International Publishing Inc., Maplewood 1996, ISBN 1-887566-04-x Analysis methods have become important analytical tools. Such amplification methods are known for their sensitivity. That is, for example, PCR is an analysis method with the highest sensitivity, and has an important possibility of an analysis system in the future. In this case, inconsistently, very high sensitivity is a major deficiency in that the selectivity in PCR is seen to be limited by the so-called "collateral contamination" from large amounts of foreign DNA in the sample and the environment. Become. Complex detection and interpretation of test results are often performed later. In addition, the low degree of automation has hindered the spread of PCR. Furthermore, in samples diluted at very high magnification, it is lethal that the labeling amplification does not guarantee at all that the nucleic acid to be amplified or detected is present in the sample collected and applied to the amplification. That is, for example, viral titers are very low in many diseases, especially in the treated viral diseases, and existing methods for direct detection of the virus determine whether the virus is still present in the patient. The problem cannot be pinpointed. This is due to the fact that the volume of blood sample taken during the test is usually not very large (Poisson area). Another disadvantage of currently used amplification reactions is their interference by inhibitors. That is, for example, in a whole blood analysis test, the polymerase chain reaction is impossible without removing the inhibitor. It is a technical problem underlying the present invention to avoid the disadvantages of the prior art as described above and to enable direct virus detection even with very low titers of free virus. In addition, inhibitors of the amplification reaction must be removed. Surprisingly, the above disadvantages can be avoided by the use according to the invention of the carrier materials described in PCT / EP 97/00405. PCT / EP 97/00405 describes carrier materials in the form of gels or as dispersions, sorption in the form of particles, in the form of particles, for the preparation of samples and for detection as determined by genetic engineering analysis methods. With respect to loose packings of material, the adsorbent material has at least one component for performing a solid phase assay and is fluid permeable. This carrier material has an average pore diameter of 0.1 to 100 μm and is characterized by showing no or very little specific adsorption capacity for the affinity material. In addition, the adsorbent material comprises a component (affinity material) for performing a solid-phase assay contained within a given unit volume of the carrier material, specifically binds to its complementary component, and a specific concentration of the complementary component. According to the provision that once a given amount of a dilute solution of the complementary component having the formula (1) flows once in a homogeneous manner, it binds in an amount that varies between several statistically related fillings around an average filling value of ± 40%. Some of the appropriate liquids in the carrier material, such as blocking of standardized and specific and non-specific adsorption sites, washing of the loaded carrier material for performing solid phase assays, and supply of affinity reacting materials During the subsequent flow step, the amount of binding is kept constant within the above range. Here, the use according to the invention is particularly advantageous when using a hydrophilic carrier material in combination with a DNA probe and a sensitive dye, such as those described in WO 97/19354, for example, a simple machine-readable result of the PCR results. This enables the development of a simple detection system. In doing so, the carrier material used according to the invention functions as an analytical enrichment column, for example with high specificity, selectivity and high degree of enrichment. A substance having an affinity for the required nucleic acid, for example, preferably a monoclonal antibody, but optionally with an antisense nucleic acid or an oligonucleotide that at least partially hybridizes to the required nucleic acid Use after degrading the material allows for enrichment of the required material such as a virus from a sample such as a larger sample volume or a pooled sample than is required directly for PCR, washing Steps can remove unwanted nucleic acid and DNA / RNA containing substances or other interfering substances, such as inhibitors. In addition, multiple carrier material layers with different affinity partners allow different substances to be concentrated from the sample (with a larger volume) without collecting additional samples, and if necessary, the layers can be concentrated before elution. By separating, the above substances can be separately amplified. Each device on which the carrier material according to the invention is arranged is preferably eluted directly into the respective reaction vessel by the starting buffer of the respective method, for example the polymerase chain reaction. By pre-purification, this immunoaffinity treatment results in a higher selectivity of the amplification reaction, especially in PCR tests, whereby the high sensitivity of the method is obtained in an ideal form. At the same time, this change reduces the bandwidth of the amplified DNA on a suitable electrophoresis gel, or results in a deep chromophore shift upon detection in the device of the invention, or, as indicated by that embodiment, the change. Only allows for safe detection in very low concentration ranges. If a carrier material with a high loading of affinity reactants is used, and the loading still binds the total amount of substance present from the larger sample volume, the measurement range for standardized PCA amplification is Distinctly moves to lower concentrations by at least two step sizes (standardized sample collection). In the PCR method, the device of the present invention for sample preparation (concentration and / or purification) and subsequent detection (quantitative and / or qualitative) complements the actual PCR, but this device is It is used as the only device when DNA is used, ie the required virus (or the required DNA if necessary) is concentrated in the device of the invention. The detection is then carried out directly with the branched multi-label affinity partner, or alternatively the virus is eluted and degraded and bound and detected in a corresponding form in the second device of the invention. The use of the sorption material according to the invention also removes inhibitors of the amplification reaction. The device used according to the invention, comprising the carrier material used according to the invention, can be used to quantify the corresponding nucleic acids, for example DNA, after amplification of the sample. The carrier material that binds the nucleic acids (DNA) formed during the first stage amplification reaction (PCR) is used for quantification within the meaning of an immunoassay. Thereafter, a quantitative detection, for example with a very sensitive dye bound to streptavidin, is performed with a DNA probe, preferably a biotinylated DNA probe. This procedure is particularly advantageous in that automated concentration and / or quantification can be performed using the above-described carrier materials used according to the invention. The carrier material according to the invention for carrying out the solid-phase assay may be a sorbent material in the form of a gel, in the form of a compact, in the form of a coarse packing, in the form of particles, as described in more detail in PCT / EP 97/00405. Can exist as The carrier material has at least one component for performing a solid-phase assay and is permeable to fluids. Said carrier material has an average pore diameter of 0.1-100 μm, and in suitable materials pores are understood as interparticle gaps and are not porous at the surface of the corresponding particles. The carrier material has no or very low capacity for adsorbing affinity substances. However, it has a high affinity for specific components that are complementary to the affinity substance located on the carrier material. In the carrier material of the present invention, a component (affinity material) for performing a solid-phase assay present in a given unit volume of the carrier material specifically binds to its complementary component, and a predetermined amount of this complementary component is defined. Once flowing in a homogeneous manner a predetermined amount of a predetermined solution of this complementary component having a concentration, it binds in an amount that varies between several statistically related fills around an average fill of ± 40%. Is standardized by the provision that The change is not more than ± 30%, preferably ± 20%, particularly preferably ± 10%. The amount bound also remains constant in the range mentioned above in the subsequent flow steps of the carrier material with a suitable liquid. Similarly, eluting a liquid is not appropriate. Such additional flow steps may include blocking of specific and non-specific adsorption sites, particularly affinity washing for labeling and / or amplification of the carrier material loaded with components for performing solid phase assays. And the like. The carrier material according to the invention can be produced relatively easily without significant time and equipment expenditures, and in particular components that are readily available to the user himself, ie sorption materials on the one hand, in particular sorption according to PCT / EP 97/00405 Advantageously, the material and one or more affinity substances required to perform the solid phase assay can be easily prepared from the flow vessel. Further provided is a buffer solution that can be used to fill the carrier material. The carrier material used in accordance with the present invention is prepared by applying a single flow of a specific amount of at least one solution having a specific concentration of at least one component for performing a solid phase assay to PCT / EP 97/00405. Obtained by filling with a sorption material. Furthermore, it is preferred that the carrier material has at least one component for performing a solid-phase assay, wherein free non-specific adsorption sites of the carrier material are blocked. In particular, the carrier material used according to the invention consists of molecules, groups of molecules or particles having an affinity for another substance. In particular, affinity substances are hybridized with enzymes, substrates interacting with enzymes, antibodies, antigens, such as high molecular weight substances or pollen or other allergens, haptens, biotin or streptavidin, nucleic acids of the RNA and DNA type, especially other nucleic acids. From the group consisting of those that can soy, receptors or ligands for the receptors, viruses, bacteria, cells, cell organelles, blood cells, particles, such as colloidal particles of metals, metal oxides, polymers, or combinations of the above affinity substances Selected. The carrier material of the present invention can also be produced from the carrier material of the present invention without prior modification with a non-specific substance by contacting one or more analytes with an adsorbent material. The device for use according to the invention is preferably a hollow body having in its cavity one or more carrier materials used according to the invention. If one or more carrier materials with different components for the solid-phase assay are arranged in the device, a homogeneous flow in the hollow body, in particular in the region of the carrier material, is ensured. In particular, it prevents edge effects from occurring or other areas where the flow is faster or slower than elsewhere. This property is essentially determined by the standardization of the carrier material. It is also important in the present invention that the adsorption or carrier material having the most accurate geometrical shape possible (a cylinder whose cross section corresponds to the cross section of the flow vessel) is exactly matched. The adsorbent and / or carrier material can be fixed in the form of a coarse filling or ker by means arranged in the cavity of the hollow body. Preferably such means exhibit a uniform flow behavior for the fluid. Such means are those which have little or preferably no non-specific intrinsic adsorptivity, especially for affinity substances. The process for the preparation of the carrier material used according to the invention comprises the step of combining the carrier material used according to the invention with one or more affinity materials, each in essentially a solution having a certain concentration in a certain amount of liquid. It is started by processing in. This can be done in a batch mode, but it is preferably done as a homogeneous flow in a hollow body having an inlet and an outlet arranged therein. The latter method has the advantage that in a very short time and without the use of large-scale equipment, a standardized carrier material with a high and uniform filling for the concentration used is obtained. In the batch mode, unlike the continuous flow mode, in which the material is rapidly contacted with the affinity material, including the inner surface, only the outer region of the adsorbent material is filled first, so an equivalent filling is performed in the batch mode. It takes a very long time to get in. Furthermore, according to the latter method, the carrier material is produced directly by the user in a simple manner. However, if this is not desired, the corresponding carrier material can be produced on a larger scale and then loaded into a device for performing a solid phase assay. In a preferred embodiment of the method according to the invention for the production of the carrier material according to the invention, the free adsorption sites of the carrier material are blocked by a suitable substance which is inert in the solid-phase assay to be performed. Optionally, the carrier material is washed one or more times. Further examples of carrier materials and pure intermediates can be found in PCT / EP 97/00405. According to the invention, a suitable homogeneous continuous flow to be ensured by the sorbent material and the carrier material used according to the invention is the entire surface of the carrier material, but also its entire cross-section, i.e. in the direction of flow, especially Correlates to a homogeneous pore structure for the adsorption inner surface. Such a symmetric and homogeneous pore structure is advantageous. However, it is also possible to use an asymmetrical pore structure which is initially small in the direction of flow, e.g. larger pores in order to give a larger inner surface and thereafter the flow velocity is not too low. . Further features and advantages of the adsorbent material and the support material for the production of the support material used according to the invention can be found in PCT / EP 97/00405 and WO 97/19354, which are hereby incorporated by reference. Department. Hereinafter, the present invention will be described more specifically. Example 1 Polymerase Chain Reaction Material with HBV (Hepatitis B Virus) and HDV (Hepatitis D Virus) Circular polyethylene frit, 5 mm in diameter, 5 mm in height, just fitted to the cross section of a flow vessel and obtained by passing through a screen Cut out from a filter plate manufactured by firing from polyethylene powder having a narrow particle size distribution (particle size less than 250 μm), nominal pore size 50 μm (foaming point), pore size distribution measured by Coulter Porometer: 5.492 to 138.6 μm, Average pore diameter 13.64 μm, 1.063% of pores exceeding 27.5 μm, 15.45% of pores exceeding 15.92 μm (Abion GmbH, Julich, Germany), circular or hollow with a tapered lower part in accordance with DE 41 26 436 A1 Flow vessel, 5 mm diameter, volume for sample solution through a 5 mm high frit fitted to the top of a cone: about 0.8 ml (Abion GmbH, Julich, Germany), carrier material 80 With a volume of μl, the loading of the affinity components (murine anti-HDV-IgG and HBV-IgG respectively bound to the carrier material) was 40 μg. The following describes a conventional PCR for HDV DNA and HBV RNA compared to the treatment of the carrier material used according to the invention. This is a standard method for detecting HVD genome by PCR. Separation of DNA from Serum 16 μl of denaturing buffer containing 100 mM Tris HCl, pH 8.0, 2% (weight / volume) was added to a 100 μl aliquot of serum. For the determination of molecular weight, 1.λ / Bst E1 molecular weight marker was added. Serum was analyzed. To the fragment serving as a positive control, 50 mM EDTA was added together with SDS. The obtained solution was incubated at 60 ° C. for 30 minutes, and then extracted with phenol / chloroform. Nucleic acids were precipitated with ethanol, recovered by centrifugation, washed twice with 70% ethanol and dissolved in 100 μl of water. PCR of HBV DNA A 30 μl DNA solution was amplified in a 50 μl PCR reaction mixture. The reaction mixture was mixed with 10 μl of PCR buffer (105 mM Tris HCl, pH 8.8, 81.5 mM ammonium sulfate, 30 mM magnesium dichloride, 0.5% Nonidet P40-Tween 20), 4 μl of a mixture of each dNTP, and Taq polymerase (Biomaster, Moscow). And 200 pM of each primer was added. Primers were selected using the computer program PRIMER MASTER R 1.0 and flanked by a 359 bp surface gene region. PCR was performed in 30 cycles using the following temperature profile: 93 ° C, 1 minute, 58 ° C, 1 minute, 72 ° C, 1.2 minutes. The amplified fragments were analyzed in an agarose gel and stained with ethidium bromide. Detection of HBV Genome Using Carrier Materials Used According to the Invention Isolation of DNA from Serum 100 μl of serum was diluted with 50 mM Tris HCl, pH 8.0, 150 mM sodium chloride, 0.1% sodium azide, 0.5% It was diluted with an equal volume of w-500 buffer containing Tween 20, which was diluted and loaded onto the column with the carrier material used according to the invention. After washing the column with 500 μl of w-500 buffer, virus particles were eluted with 100 μl of a denaturing solution containing 50 mM HCl, pH 8.0, 1% SDS, and 25 mM EDTA. The eluate was collected in an Eppendorf vessel and incubated at a temperature of 60 ° C. for 30 minutes. Then, it was precipitated with ethanol. The pellet was collected by centrifugation, washed twice with 70% ethanol, and dissolved in 100 μl of water. Thereafter, HBV DNA was amplified using PCR. During this step, 30 μl of the DNA solution was amplified in a 50 μl PCR reaction mixture. Standard Method of RT (Reverse Transcriptase) PCR for Detection of HBV Genome Serum RNA was obtained by guanidinium thiocyanate phenol / chloroform extraction. Thereafter, the following reagents were added to 100 μl of the serum. 1) Denaturing solution of 4M guadinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarkosyl 2) 50 μl of 2 M sodium acetate, pH 4.0 3) 3 μl of 2-mercaptoethanol 4) 10 μg of tRNA 5 ) 550 μl water-saturated phenol and 100 μl chloroform The resulting suspension was shaken well and cooled on ice for 5 minutes. Samples were centrifuged at 10,000g for 20 minutes at 4 ° C. The aqueous phase was extracted with an equal volume of chloroform, and the resulting aqueous phase was precipitated with an amount of isopropanol. After centrifugation and treatment with 70% cold ethanol, the DNA pellet was dissolved in 50 μl of water and treated with diethyl pyrocarbonate. cDNA synthesis and PCR 2 μl of the resulting serum RNA was added to 17 μl of the reverse transcription (RT) mixture. This mixture was mixed with 4 μl of 5 × RT buffer (250 mM Tris HCl, pH 8.3, 250 mM potassium chloride, 50 mM magnesium dichloride, 50 mM DTT, 2.5 mM spermidine), 2 μl each of 10 mM dNTP, 1 μl (100 ng ), 0.5 μl (20 units) of HPRI (obtained from Promega), and 9.5 ml of water. The mixture was heated at 94 ° C. for 3 minutes and then cooled on ice. After denaturation, 1 μl (10 units) of AMV reverse transcriptase (Promega) and 0.5 μl (20 units) of HPRI were added. cDNA synthesis was performed at 37 ° C. for 45 minutes. 10 μl of the cDNA was amplified in a 50 μl PCR reaction mixture. The reaction mixture was 10 ml of 5 × PCR buffer (150 mM Tris HCl, pH 8.8, 81.5 mM ammonium sulfate, 0.5% Nonidet P40-Tween 20), 4 μl of 10 mM each dNTP, 1 μl (5 units) of Taq polymerase (Biomaster). ), 1 μl (200 pM) of each backward and / or forward primer. PCR was performed for 40 cycles at the following temperature profile: 94 ° C, 1 minute, 57 ° C, 1 minute, 72 ° C, 1.2. Amplified fragments were analyzed in agarose skeletal and stained with ethidium bromide. Isolation of HDV RNA with the carrier material used according to the invention 100 μl of serum was diluted with an equal volume of 150 mM sodium chloride and applied on the column described above. After washing the column with 500 μl of 150 mM sodium chloride solution, virus particles were eluted with 200 μl of a denaturing solution containing 3.2 M guanidium thiocyanate, 20 mM sodium citrate, pH 7.0, 0.4% sarkosyl. The eluate was collected in an Eppendorf vessel with 2 mM sodium acetate, pH 3.0 and 2 μl of 2-mercaptoethanol, mixed well, and then precipitated with ethanol. The RNA pellet was centrifuged, dissolved in 20 μl of water, and 10 μl of the RNA solution was used for a reverse transcription reaction (RT reaction). RT reactions and PCR were performed as described above. The following results were clarified by the examples. DNA was extracted from 10 HBV PCR positives and 5 HBV PCR negatives by standard methods of DNA isolation. This DNA was used in a suitable column with the carrier material used according to the invention. In a total of 10 cases, amplification of these DNA samples indicated the presence of specific fragments. Amplification of DNA samples isolated by standard methods did not yield specific fragments. Carrier material used according to the invention for the analysis of low virus titer samples HBV DNA and HDV RNA extracted using the carrier material used according to the invention and purified virus particles correspond to 100 μl It can be used for small amounts of PCR testing. In this case, an increase in the synthesis efficiency is observed. Twenty-five therapeutically treated patients whose HBV DNA was not detected by standard methods were tested. 1000 μl of each serum was applied to the column containing the carrier material used according to the invention. DNA was isolated without phenol-chloroform extraction. The entire amount of the isolated DNA was transferred to a PCR reaction. Five HBV DNA positive sera were found in 25 samples. In addition, six serum samples from anti-HD positive and HDV RNA negative patients were examined. In this case, 500 μl of each serum was applied to the column containing the carrier material used according to the invention, and the RNA was recovered from the virus particles, extracted using the phenol-chloroform method and then precipitated with ethanol. The entire amount of the isolated RNA was subjected to a reverse transcription reaction, and half of the RT mixture was used for PCR. At the same time, sera were tested using a standard RT PCR assay. Specific HDV genomic fragments were observed in three samples treated with the carrier material used in accordance with the present invention packed in a column, while specific fragments were not detected in samples by standard assays. It could not be observed. Similar results were obtained by detection using a column packed with DNA probes, PCR performed in the usual manner did not show red staining, but showed color that was clearly visible by pre-concentration. This makes it possible to obtain a template for PCR in a quick and simple manner by using the column containing the carrier material used according to the present invention, and to eliminate factors of PCR and RT inhibition. It is particularly advantageous that complete template isolation can be achieved in less than one hour, and does not require the use of routine, routine laboratory equipment. The use according to the invention for selecting specific HBV and HDV of the described virus particles increases the efficiency of PCR detection.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, S D, SZ, UG, ZW), EA (AM, AZ, BY, KG) , KZ, MD, RU, TJ, TM), AL, AU, BA , BB, BG, BR, CA, CN, CU, CZ, DE, EE, GE, GH, HU, IL, IS, JP, KP, K R, LC, LK, LR, LT, LV, MG, MK, MN , MX, NO, NZ, PL, RO, SG, SI, SK, SL, TR, TT, UA, US, UZ, VN, YU, Z W (72) Inventor Erhard Christoph             Jürich D, Germany             52428, Carl-Heintz-Beckarts-             Strasse 13, Avion Betairi             Gungs-und Velwaldung             Soo Gezer Shaft Mitt Bechlen             Ktel Hachtung (72) Inventor Mies Peter             Jürich D, Germany             52428, Carl-Heintz-Beckarts-             Strasse 13, Avion Betairi             Gungs-und Velwaldung             Soo Gezer Shaft Mitt Bechlen             Ktel Hachtung

Claims (1)

[Claims] 1. Sorbent material in the form of a compact, coarse packing of sorbent material in the form of particles, Sample preparation and genetic engineering of sorbent material in gel form or as dispersion Used for detection investigated by a scientific analysis method, The sorbent material has at least one component for performing a solid phase assay; Fluid permeable The sorption material has an average pore diameter of 0.1-100 μm, The sorption material has no or very little nonspecific sorption capacity for the affinity material Low The sorption material comprises a fixed amount of a dilute solution of the complementary component having a specific concentration of the complementary component; Is contained within a given unit volume of the carrier material once it has flowed in a homogeneous manner. Components (affinity materials) for performing solid phase assays around ± 40% average loading The amount that varies between several statistically related fills in Standardized by the provision of differential binding, -The amount of binding is determined by blocking specific and non-specific adsorption sites, Such as washing the carrier material filled with the components to perform, and supplying the affinity reaction material Within the above range during several subsequent flow steps of a suitable liquid in the carrier material Use maintained constant. 2. The genetic engineering analysis method is used for amplification such as polymerase chain reaction (PCR) A method or a hybridization method, such as a "branched DNA" method. Use according to 1. 3. 3. Use according to claim 1 and / or 2 for concentrating nucleic acids to be amplified. for. 4. The nucleic acid to be tested is present at low concentrations, and the selectivity of nucleic acid amplification in the sample and Use according to at least one of claims 1 to 3 for enhancing sensitivity. 5. If the desired nucleic acid is present at low concentrations, 6. Use according to claim 5, for avoiding the son region. 6. At least one of claims 1 to 5 for removing an inhibitor of a genetic engineering analysis method. Use also as described in 1.