KR20120067401A - Method for preparation of nucleic acids probe-coated, porous solid supporter strip - Google Patents

Abstract

PURPOSE: A method for manufacturing a nucleic acid probe-coated porous solid support strip is provided to prevent abnormal hybridization. CONSTITUTION: A method for manufacturing a nucleic acid probe-coated porous solid support strip comprises: a step of conjugating a nucleic acid probe with a carrier to prepare a conjugate; and a step of fixing the conjugate to a porous solid support by a vacuum transfer method. The nucleic acid probe is DNA, RNA, or PNA. The nucleic acid probe contains a marker which is directly or indirectly detectable by spectroscopic, photochemical, biochemical, immunological, or chemical device. The nucleic acid probe additionally contains a spacer for enhancing efficiency of hybridization. The space is a non-specific poly T tail.

Description

Method for preparing porous solid support strip coated with nucleic acid probe {Method for Preparation of Nucleic Acids Probe-Coated, Porous Solid Supporter Strip}

The present invention relates to a method for producing a porous solid support strip coated with a nucleic acid probe, and more particularly, to conjugate a nucleic acid probe to a carrier to prepare a conjugate (conjugate), and to move the conjugate in vacuum ( It relates to a method for producing a porous solid support strip coated with a nucleic acid probe, characterized in that it comprises a step of fixing to a porous solid support by a vacuum transfer method.

Nucleic acid diagnostics is used for disease predisposition, adequacy of treatment, and organism identification.

In general, techniques for analyzing the sequence of a particular nucleic acid from a nucleic acid sample isolated from a bacterium or a cell or a result of amplification by a nucleic acid polymerization reaction have a complementary base sequence that specifically binds to a portion of the nucleic acid to be analyzed. Nucleic acid fragments, ie, nucleic acid probes (probe) using the analysis of the binding between them to determine the presence of nucleic acids.

Currently, a method of attaching a specific nucleic acid probe to a surface of a microwell plate to hybridize with a nucleic acid to be analyzed, and a method of fixing a specific nucleic acid probe to a membrane and injecting the amplified nucleic acid into a nucleic acid hybridization reaction (reverse dot) / line blot hybridization), and a method of fixing a plurality of probes to a plate made of a material such as glass and injecting amplified nucleic acid to perform a nucleic acid hybridization reaction (DNA chip).

In order to prepare a diagnostic strip used in the nucleic acid hybridization reaction as described above, it is necessary to fix the nucleic acid probe to the solid support.

Specifically, nucleic acid attachment methods include the most commonly used physical adsorption processes and chemical linkage processes, including ultraviolet (UV) irradiation or covalent bonding (see R Lutgarde et al. , Applied) . Environmental Microbiology, 62, 300-303, 1996).

Physical adsorption is the physical adsorption of biomolecules onto a solid surface, a process of great interest in in-vitro diagnostics.

The obvious mechanism by which biomolecules are adsorbed on the solid phase is not clear, but in fact they must involve interactions with the surface, which interactions probably include 1) molecular migration to the surface, 2) adsorption on the surface, and 3) rearrangement of the adsorption molecules. 4) potential desorption of adsorption molecules, 5) desorption molecules will occur in five steps, moving away from the surface (W Norde, Advances in Colloid and Interface Science, 25, 267-340, 1986). ).

This overview suggests that desorption is potentially inherent, but bonds are in fact irreversible if the molecules are large enough (see AW Adamson, "The Solid-liquid Interface: Adsorption From Solution", in Physical chemistry of surface, 5th ed. (Chichester, UK: Wiley, 1990), 421-459).

This is because, as the molecular weight increases, the number of binding sites increases, and even though one binding site is dissociated from the surface, many remain in the bonded state. Similar effects are reported in nucleic acids as in proteins.

Although nucleic acids do not bind as strongly as proteins, large nucleic acids bind well to membranes.

However, a major problem with physical adsorption methods is that nucleic acids immobilized on the surface may be rearranged, resulting in an unsuitable conformation for any incoming nucleotide sequence.

In the case of a nucleic acid of a long sequence may not be a problem, the shorter the nucleotide sequence of the nucleic acid, the potential decrease in activity can be a serious problem. If there are some bases immobilized on the surface that do not remain free to interact with the solution, the deformed structure prevents the hybridization from occurring normally.

In addition, nucleic acid probes are difficult to fix due to their small size and relatively weak physical forces (electrostatic forces, hydrophobic interactions, etc.) that can be adsorbed on a porous solid support to directly apply a vacuum transfer method. .

This poses a risk of uncoated nucleic acid probes, and is likely to desorb during assay reactions without additional immobilization, making it efficient for use in reproducible and highly sensitive hybridization assays. There is a falling problem. In consideration of these problems, a method using UV is widely used.

UV crosslinking is one of the simplest ways of covalently binding a nucleic acid probe to a nylon membrane. Linkage consists mainly of thymine residues (other nucleotides are less frequent), and when thymine residues are activated, they react with amine groups on the nylon membrane (GM Church and W Gilbert, PNAS, 81, 1991-1995). , 1984; I Saito et al., Tetrahedron Letter, 22, 3265-3268, 1981).

Some of these bases are covalently linked to the membrane surface and do not participate in the reaction during the hybridization reaction, so if the membrane is excessively exposed to UV, UV conjugation will eventually interfere with the hybridization reaction. Therefore, it is important to correctly determine the UV irradiation amount.

In other words, exposing the membrane to a small amount of UV does not result in efficient crosslinking, and overexposure reduces the efficiency of the hybridization reaction.

On the other hand, one important problem associated with the UV irradiation of the membrane is the moisture content of the membrane. Water absorbs UV, which results in higher or lower levels of cross-linking than expected due to variations in the drying process. Here, the crosslinking through the thymine base has a problem that is very difficult to control the degree.

On the other hand, the technique of using photoactivatable compounds in the membrane or probe is an advanced crosslinking method (JK Veilleux and LW Duran, IVD Technology 2, no. 2, 26-31, 1996). When photoactive compounds (eg photobiotin) are used, they can provide the probe with the correct orientation and freedom, but there are few applications because of uncontrolled reactions often when exposed to UV radiation.

Covalent linkage is the most popular technique for fixing nucleic acid probes to solid support surfaces using conventional chemistry. Of the various linking chemistries in use, the most common are amines, carbonyls, carboxyl and thiols.

Covalent bonds control the orientation of the probe, controlled-chemistry linkage allows for controlled attachment of nucleic acids via terminal residues, and by introducing spacers of the correct size, Hybridization can also be done freely with the added probe.

Covalent bonds between probes and solid supports are commonly applied in the form of microarray systems, ie, glass slides or beads, but are expected to be applicable to porous solid supports such as membranes.

However, the prior art uses a 1-Ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC or EDAC) reagent, which is primarily a carbodiimide, in a direct covalent bond between the probe and the membrane, with an amine-labeled probe and a carboxyl group. It was limited to the covalent bond between the activated membranes.

In addition, the technique of covalently immobilizing nucleic acid probes with controlled attachment to the membrane is inherently difficult to turn into a mass production and automated process in a batch process. It is also time consuming and costly.

As described above, in the preparation of nucleic acid probe coated membrane strips for use in nucleic acid hybridization assays, due to the advantages and disadvantages of physical and chemical processes, the hybridization can be performed normally and at the same time in a simple and rapid manner. There is a limit to mass production of membrane strips.

Accordingly, there is a high need for developing a membrane strip having manufacturability in order to solve the above problems and apply the membrane-based nucleic acid test to the diagnostic field.

SUMMARY OF THE INVENTION It is an object of the present invention to solve the above-described problems of the prior art and the technical problems required from the past.

Specifically, an object of the present invention, in the preparation of a porous solid support strip coated with a nucleic acid probe, it is possible to ensure the orientation of the nucleic acid probe by binding the nucleic acid probe to the carrier, the nucleic acid immobilized on the surface of the solid support rearranged If not, a part of the hybridization nucleotide sequence region of the probe to be complementary to the target nucleotide sequence is not fixed, thereby providing a manufacturing method that does not occur abnormal hybridization reaction due to reduced activity.

Another object of the present invention is that there is no hassle to adjust the optimal amount of UV radiation when irradiating UV, and does not use the batch process that was inherently required in the prior art to fix nucleic acid probes by chemical bonding, so that mass production and automated processes It is to provide a possible manufacturing method.

Still another object of the present invention is mass production, which is simple, fast, inexpensive, reproducible, and stable without interfering with the interaction of the membrane surface to facilitate hybridization reactions between target sequences and nucleic acid probes. To provide a method for producing a nucleic acid-probe assay system that can exhibit high sensitivity with.

Therefore, in the method of preparing a porous solid support strip coated with a nucleic acid probe according to the present invention, a conjugate is prepared by binding a nucleic acid probe to a carrier, and the conjugate is vacuum transferred. It characterized in that it comprises a process of fixing to the porous solid support.

Techniques for immobilizing nucleic acid probes on porous solid supports, such as membranes, for hybridization reactions for use in in-vitro diagnostic nucleic acid testing are diverse, but several factors must be considered. As a result, there are advantages and disadvantages in terms of binding effectiveness and convenience in the manufacturing process, which are related to the performance of the assay. The key is how easy it is and how to mass-produce at competitive prices.

In this regard, the method for producing a porous solid support strip coated with a nucleic acid probe according to the present invention can secure the orientation of the nucleic acid probe by binding the nucleic acid probe to a carrier, and the nucleic acid immobilized on the surface of the porous solid support Not aligned, and since a part of the hybridization nucleotide sequence portion of the probe that is complementarily bound to the target nucleotide sequence is not fixed, there is an effect that an abnormal hybridization reaction does not occur due to a decrease in activity.

In addition, there is no hassle to adjust the optimum amount of UV irradiation as in the prior art, and does not use the batch process that was inherently required in chemical bonding technology, mass production and automated processes are possible.

In the present invention, the nucleic acid probe may be composed of sense (forward) and antisense (forward) nucleic acids in which DNA, RNA, peptide nucleic acid (PNA), etc. recognize a target gene sequence.

Four types of nucleic acid probes, such as large fragments of DNA, short DNA (including cDNA), RNA, and peptide nucleic acid (PNA), can be immobilized in the solid phase for rapid assay purposes. have.

The methods for immobilizing these different molecules also have similarities, but they are virtually different due to differences in the structure and size of the molecules. The method of immobilizing large fragments of DNA on a solid phase is no longer used for diagnostic tests, so shorter DNA is immobilized on a solid phase.

In the present invention, the binding of the nucleic acid probe and the carrier may preferably be a chemical bond, and such chemical bonds include covalent bonds and non-covalent bonds such as ionic bonds and hydrogen bonds. Among them, covalent bonds are more preferable.

However, although not covalent bonds, a binding method using affinity binding having a strong cohesion level of covalent bonds is also applicable.

In connection with the affinity binding, biotin-avidin / streptavidin fixation techniques that can form strong bonds with binding constants of ^˜10-15 M are most commonly used. In addition, Glutathione-GST (Glutathione S-transferase), histidin-metal ion (Co, Ni, Cu), maltose-MBP (maltose binding protein), lectin (eg., Concanavalin) -mannose / glucose binding protein Can be utilized between the probe and the solid support.

Thus, in order for the nucleotide sequence of the entire nucleic acid probe to participate in the hybridization reaction, a functional group is preferably introduced into the nucleic acid probe for covalent bonding with the carrier, or a ligand is introduced into the nucleic acid probe for affinity binding to the carrier. Can be.

Surface modification for covalent coupling between the nucleic acid probe and the solid support may include a carboxyl group (-COOH), a phosphate group, an amino group (-RNH ₂ , -ArNH _2, where R is H or C ₁ -C ₆ alkyl group), methyl chloride (-ArCH ₂ Cl), amide (-CONH ₂ ), aldehyde (-CHO), hydroxyl group (-OH), epoxy (epoxy), thiol (thiol) (Ref. 2005 & 2010 Integrated DNA Technologies, Strategies for Attaching Oligonucleotides to Solid Supports). These functional groups may be one kind or a combination of two or more kinds.

Meanwhile, as a technique for affinity coupling between a nucleic acid probe and a solid support, biotin-avidin / streptavidin, Glutathione-GST, histidin-metal ion, lectin-glycoprotein, antibody-antigen, etc. may be implemented. Can be.

In one preferred example, in the case of amino group modification of a nucleic acid probe, it may consist of an amino C6 linker or an amino C12 linker to which an amino group is directly attached or a short carbon chain is attached.

In addition, the immobilization technique is most efficient when the reaction is through an end group, so that the linking group is introduced into the 5 'or 3'-active end group of the nucleic acid. It is preferable.

Thus, the 5'-end or 3'-end of the nucleic acid probe may be modified with a functional group which is preferably covalently bonded to the carrier. As such, the step of modifying the nucleic acid probe can be carried out in solution during nucleic acid probe production or prior to application to the membrane.

In addition, in the present invention, the nucleic acid probe may be composed of 16 to 22 nucleotides, for example, synthetic oligonucleotides.

Specifically, in the case of nucleic acid probes capable of detecting tuberculosis, and whether rifampin and children or drug resistance can be determined, nucleic acid probes targeting the Mycobacterium tuberculosis-specific Inter Transcriptional Spacer (ITS) gene region may be used for the identification of Mycobacterium tuberculosis. Can be.

Containing a wild type or mutant type sequence of a gene encoding an RNA polymerase-subunit to determine the anti-tuberculosis drug rifampin susceptibility and resistance A nucleic acid probe may be used.

In addition, the phenotypic or mutant sequences of the gene coding region of katG (catalase peroxidase) and the promoter regions of innoA (enoyl acyl carrier protein (ACP) reductase) and aHpC (alkyl hydroperoxide reductase) for the purpose of discriminating children or sensitivity and resistance Nucleic acid probes containing these may also be used.

On the other hand, as a nucleic acid probe capable of distinguishing the species of tuberculosis bacteria (TB) and non-tuberculosis antibacterial bacteria (NTM), nucleic acid probes targeting specific ITS (Inter Transcription Spacer) gene sites in microbacteria can also be used.

In addition, the nucleic acid probe, preferably, may include a label detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunological or chemical means, the internal region and the target sequence A spacer may be added between the hybridization site sequence capable of complementarily binding and the carrier to increase the efficiency of the hybridization reaction.

Such labels include, for example, enzymes (eg horseradish peroxidase, alkaline phosphatase), radioisotopes (eg ³² P), fluorescent molecules, chemical groups (eg , Biotin).

The spacer is preferably a non-specific poly T tail, which may be added after the amino group modification. The length of the poly T tail used as the spacer may consist of 5 to 30 thymine bases.

For example, an amino C6 linker is labeled at the 5'-end, a poly T tail consisting of 10 thymine bases is linked with a spacer, and a base sequence of a hybridization site capable of complementarily binding to the nucleic acid sequence of the target gene is 16. Nucleic acid probes can be designed to consist of ˜22 nucleotides.

In the present invention, the carrier may be, for example, a polysaccharide, a protein, a synthetic polymer, or the like. Among them, polysaccharides are preferred, and in particular, dextran or a derivative thereof that covalently or affinity-bonds nucleic acid probes is more preferable.

For affinity binding, for example, avidin / streptavidin, maltose binding protein, glycoprotein, and metal ions (Co, Ni, Cu etc) are connected to the carriers and biotin, maltose, lectin, histidin, which are ligands capable of affinity binding. Or a derivative thereof can be labeled on the nucleic acid probe. Among them, biotin-avidin / streptavidin connection technology is more preferable.

Since dextran has several activation groups per molecule, dextran can not only fix several DNA probes per molecule, but also can fix several ligands such as aspartic acid and oligonucleotide probes (Ref. Goss, TA et al. ., J. Chromatogr. 508, 279-287, 1990; Schacht EH et al., Modification of Dextran and application in prodrug design.In Industrial polysaccharides Engineering: Genetic Engineering, Structure / Property Relations and Applications Yalpani, M., Ed. Elservier: Amsterdam, 1987). In addition, since dextran is a long and flexible polymer, immobilized DNA molecules are very easy to hybridize and also have the advantage of avoiding steric hindrance due to the surface of a solid support (Ref. Goris J. et al., Can J Microbiol, 44, 1148-1153, 1998; Shepinov M. S et al., Nucleic Acids Res., 25, 1155-1161, 1999; Gingeras T. R et al., 15, p.13, 1987; Penzol G et al., Biotechnol Bioeng. 60, 518-523, 1998).

As a representative example, aldehyde-aspartic-dextran is a very large molecular weight material, which has an anion and an aldehyde group, thus forming a very strong covalent bond with an aminated DNA probe. Binding is very stable enough to withstand extreme pH, high temperature or high concentrations of salts, solvents and nucleophiles in the hybridization mixture (Fuentes M. et al., Biomacromolecules, 5, 883-888, 2004).

In one preferred example, the dextran may be composed of polysaccharides that can be oxidized to form aldehyde functional groups.

For example, polyaldehyde dextran can be prepared by oxidizing 43 kDa dextran, which has higher conjugation efficiency with nucleic acid probes than 500 kDa dextran.

Such polyaldehyde dextran is, for example, mixed with dextran and KIO ₄ to 62.5 mmol equivalent amount of glucose, the mixture is rotated at a rate of 40 rpm at room temperature, left for at least 6 hours, and then sterile distilled water 2 It may be prepared by dialysis three times by time.

As described above, the carrier may be composed of a protein such as albumin or a synthetic polymer. In this case, a ligand capable of affinity binding or a functional group capable of covalently binding to an amino group or a thiol group of the protein or synthetic polymer, etc. May be introduced into the nucleic acid probe.

A polyaldehyde dextran carrier and a nucleic acid probe labeled with an amine group are reacted, for example, at a pH of 11 at room temperature to form an imine intermediate of Schiff's base and, for example, as a reducing agent. Sodium borohydrate, which is a mild reducing agent, reduces the sieve base and induces irreversible bonding, thereby achieving stable covalent bonding.

Nucleic acids are inherently unreactive, but given sufficient energy they are reactive with other groups, so if the nucleic acid is exposed to UV in the presence of a photosensitizer, such as methylene blue, without visible or photosensitizers, it crosslinks directly with the protein. direct cross-linking (R Lalwani et al., J Photochemistry and Photobiology 7, 57-73, 1990; JW Hockensmith et al., Method in Enzymology, 208, 211-236, 1991).

The method of directly crosslinking nucleic acids by producing an activated solid porous support, for example, an activated membrane, has two major problems.

The first problem is the loss of activity: the chemically active surface of the membrane loses its activity by reacting with gases in the atmosphere.

The higher the chemical activity, that is, the faster crosslinking with the target, the faster the activity is lost. To overcome this problem, manufacturers recently use less reactive groups that specifically react with the target probe itself. Reactions between aldehydes and amines are both relatively stable, with typical examples.

Due to the inherent problems in production, storage and handling of reactive solid phases, it is no longer possible to provide a reactive membrane for the purpose of covalently bonding with the probe.

Usually, membranes are provided that have a surface chemistry capable of specifically binding to the probe, but which are less reactive and less active over time and during storage, with amine or aldehyde groups on the membrane surface. It is dominant.

An advantage of the aldehyde-activated surface is that significant amounts of binding occur mainly through the formation of Schiff's base without any additional reagents.

Since the base is relatively stable but can be inverted under physiological conditions, the base must be reduced with a mild reducing agent, sodium borohydride, to form an irreversible bond (MB). Meza, "Covalent Binding in Diagnostic Test Design" in The latex Course, 2000).

The second problem is nonspecific binding, which means that the activated membrane must be blocked before use. Otherwise, the sample will additionally bind to the membrane. The blocking step should be simple, fast and very efficient, ideally blocking results in the formation of hydrophilic, non-charged residues on the membrane surface.

Blocking the membrane surface with blocking introduces a new chemical group, which may not be reactive with the nucleic acid, but may be due to charge attraction or charge attraction due to the binding of the sample or the coupling of the detection system. Increased hydrophobic attraction can lead to an increase in nonspecific signals.

Most of the covalent bonds except disulfide bonds are essentially a batch process where the reaction of binding the target to the membrane and then blocking the membrane takes approximately 30 minutes to several hours.

Such deviations create difficulties in automation and mass production. The introduction of photoactive linking chemistry simplifies mass production, but it is relatively rare because it is expensive in terms of cost.

On the contrary, in the present invention, the above-mentioned problem is solved by covalent bonding of an amino group-modified nucleic acid probe that specifically reacts with a polyaldehyde dextran that activates (oxidizes) a dextran as a carrier. In addition to the production of organic solvents, no additional toxic reagents are required, as shown in the covalent bonding between amine and carboxyl groups using 1-Ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC or EDAC). The cost of use can be reduced.

In one preferred example, the porous solid support can be a membrane and its substrate can be cellulose or nylon.

Solid supports used for nucleic acid immobilization include membranes (nitrocellulose or nylon membrane), controlled-pore glass beads, acrylamide gels, polystyrene matrices, and activated dexes. Activated dextran, avidin-coated polystyrene beads, glass, and the like, and various solid supports have been applied for DNA diagnosis (see Saiki RK et al, PNAS, 86, 6230-6234, 1989; Meinkoth J et al, Anal. Biochem, 138, 267-284, 1984; Ghosh SS et al, Nucl.Acids Res., 15, 5353-5372, 1987; Rasmussen SR et al., Anal.Biochem, 198, 138-142, 1991; Gingeras TR, Nucl.Acids Res., 15, 5373-5390, 1987; Lund V. et al., Nucl.Acids Res., 16, 10861-10880, 1988) .

The present invention focuses on how various target materials can be fixed to porous solid supports, among other things the membrane, among these various solid supports.

Membrane includes a wide range of substrates that can be used for nucleic acid immobilization. Specifically, new membranes such as nitrocellulose, traditional cast membranes such as nylon, ceramic or track-etched membranes, and microarrays. The type of substrate used.

Many membranes that are recommended for nucleic acid immobilization must first determine the optimal binding conditions, among which many nucleic acid techniques are added to the membrane in an uncontrolled manner.

In such cases, the orientation of the nucleic acid probe may not be arranged in the preferred direction, potentially limiting the reaction. Thus, directed adsorption, where the immobilized nucleic acid is in end-linked form, is preferred in most cases.

The main advantage of nitrocellulose is its usability, which is readily available, but it is fragile unless coated with polyester baking and has a lower binding capacity than other membranes.

Recently, nylon has been promoted as a substrate for nucleic acid binding because of its greater physical strength and binding capacity, and the wider range of available surface chemistries that can be optimized for attaching nucleic acids.

Commonly used methods for binding nucleic acids to nitrocellulose are physical adsorption, but immobilization to nylon membranes consists of physical adsorption, UV cross-linking or chemical activation.

As mentioned, the present invention can solve the problem of the conventional physical adsorption process. However, from a production manufacturing point of view, physical adsorption is very simple and useful as a robust technique (cf. TC Tisone, IVD Technology 6, no. 3, 43-60, 2000).

The manufacture of a simple membrane-based system is most straightforward when applied so that the material can bond upon drying in a continuous process. Immobilizable materials can be applied to a continuous process, where linkage is a simple and well controlled process, ie drying.

If the obvious problem of physical adsorption can be solved, that is, enhanced to enable stronger and directional binding, physical adsorption is naturally useful as a technique for mass production of nucleic acid diagnostic tests.

In this regard, the present invention may further comprise the step of physically adsorbing the conjugate between the carrier and the pores of the porous solid support by drying.

Most enhanced physical adsorption techniques not only increase the binding efficiency, especially for very short probes, but are also arranged so that important sequences of the probes protrude away from the solid surface. As a result, the incoming sample and important sequences can be freely interacted with. Selectivity and sensitivity are enhanced, which is particularly effective even with short probes.

In this aspect, according to the present invention, the covalent bond has a direction that the important nucleotide sequence of the nucleic acid probe is arranged in a direction away from the surface of the porous solid support.

In the present invention, the vacuum transfer method preferably dispenses the conjugate of the nucleic acid probe and the carrier to the nucleic acid slit line of the automated line blotting equipment and operates the vacuum pump to inhale the conjugate. It may include the step of adsorbing to a porous solid support.

The present invention also provides a porous solid support strip coated with a nucleic acid probe, which is prepared by the above method.

The porous solid support strip coated with the nucleic acid probe according to the present invention has a specific structure which is itself different from the conventional one by a special manufacturing process as described above.

As described above, the present invention provides the orientation of the nucleic acid probe by binding the nucleic acid probe to the carrier, the nucleic acid immobilized on the surface of the solid support is not rearranged, and must be complementary to the target sequence. Since a part of the hybridization sequence region is not fixed, there is an effect that an abnormal hybridization reaction does not occur due to a decrease in activity.

In addition, there is no hassle to adjust the optimal amount of UV irradiation during UV irradiation, and does not use the batch process that was inherently required in the technology of fixing the nucleic acid probe by covalent bonds, there is also an effect that can be mass production and automated processes .

That is, due to mass production, it is simple, fast, inexpensive, and reproducible, and the membrane surface can show stability and high sensitivity without interfering with the interaction to facilitate the hybridization reaction between the target sequence and the nucleic acid probe. There is an effect to prepare a nucleic acid-probe assay system.

FIG. 1 is a diagram illustrating the entire process of preparing a strip by covalently conjugating a nucleic acid oligonucleotide probe to a carrier, dextran, and then fixing the membrane to a membrane by a vacuum transfer method.
Figure 2 is a view showing the result of coating the nucleic acid probe is coated on the membrane strip and the patent blue V dye blue dye, wherein the top is the case of the rifampin and the eye or the strip that can determine whether the sensitivity or resistance, the bottom Is the case of the detectable strip of the genus Mycobacteria of Example
FIG. 3 is a diagram showing a result pattern when a reverse hybridization line blot assay is performed after PCR is performed on rifampin and eyena-sensitive / resistant specimens. FIG.
Figure 4 is a diagram showing the resultant pattern when performing a reverse hybridization line blot assay after performing PCR on the mycobacterial specimen and the control (negative, positive).

Hereinafter, the present invention will be described in more detail based on examples. The following examples are intended to illustrate the invention, not to limit the invention.

The following experiment relates to a process of fabricating a strip by fixing a nucleic acid probe to a membrane in a vacuum transfer method, and in two specific examples, using a membrane strip produced by the sputum, bronchial lavage fluid, cerebrospinal fluid, urine, tissue, and whole blood. (I) a test to detect tuberculosis, and to determine whether rifampin and children or drugs are resistant, and (ii) to detect and classify mycobacteria, that is, TB and non-TB bacteria (NTM). Experiments were performed to verify the effectiveness of the test using a reverse hybridization line blot assay.

Example 1 Preparation of Polyaldehyde Dextran (PAD)

(1) preparation of necessary reagents and equipment

Dextran (Sigma, D-4133, Mr ~ 43kDa), KIO ₄ (Aldrich, 32242-3, FW 230.00), cellulose dialysis membrane (cutoff 12000: Spectra / Por, MWCO 12-14000), hydroxyamine hydrochloride hydrochloride (NH ₂ OH-HCl = 69.49), methyl orange powder, 10N hydrochloric acid, pH meter, 0.1N sodium hydroxide (NaOH), lyophilizer or SpeedVac were used.

(2) oxidation Dextran ( oxidized dextran ) Produce

0.5 g of dextran (Sigma, D-4133, equivalent to Mr ~ 43kDa / glucose 62.5 mmol) was completely dissolved in autoclaved DW and adjusted to the final 10 ml. 0.72 g KIO ₄ (Equivalent to 62.5 mmol equivalents) (see Azzam T et al., J Med Chem, 45, 1817-1824, 2002; Azzam T et al., Macromolecules, 35, 9947-9953, 2002; Azzam T et al., J Controlled Release, 96, 309-323, 2004; Hosseinkhani H. et al., Gene Therapy, 11, 194-203, 2004).

It was left for 6 to 12 hours while rotating at 40 rpm at room temperature. The oxidized dextran was placed in a cellulose dialysis membrane, and dialyzed once in 2 L of tertiary sterile distilled water, and dialysis was performed three times for a total of two hours while replacing the sterile distilled water. After dialysis, the exact volume of polyaldehyde dextran was measured.

(3) oxidation Of oxidized dextran Aldehyde  Volume measurement

The amount of the aldehyde group of dextran oxide was measured according to the method described in the journal, published by Huiru Zhao et al. (Pharmaceutical Research, 8, 400-402, 1991). A reagent of 0.25N hydroxyamine hydrochloride-methyl orange (pH 4.0) was prepared first, and 1.75 g of dried hydroxyamine hydrochloride was dissolved in 15 ml of sterile distilled water. Separately 0.6 ml of the final 0.05% methyl orange indicator solution was prepared and added to the hydroxyamine hydrochloride solution.

10 N HCl was slowly adjusted to pH 4.0, followed by dilution with sterile distilled water to prepare a final 100 ml of 0.25 N hydroxyamine hydrochloride-methyl orange (pH 4.0) reagent.

Redox titration of polyaldehyde dextran was performed with 0.1 N NaOH standard solution. To 25 μl of polyaldehyde dextran obtained after dialysis, 1.25 ml of 0.25 N hydroxyamine hydrochloride-methyl orange (pH 4.0) solution was added, followed by reaction at room temperature for 2 hours (40 rpm).

Thereafter, the solution was titrated with 0.1 N NaOH standard, and the exact volume of NaOH added in the redox titration reaction was measured in μl, and the end point was identified by color change (color change from red to yellow).

The oxidation-reduction reaction and the degree of aldehyde substitution of the polyaldehyde dextran are expressed in the following formulas, and the total number of moles of aldehyde X contained in the total volume of polyaldehyde dextran (PAD) measured after dialysis was calculated. Indicated.

(4) Polyaldehyde Dextran  Freeze drying and Stock  Solution preparation

A small amount of polyaldehyde dextran was dispensed in a screw cap tube by 10.0 uL and then lyophilized by O / N using SpeedVac or freeze-dryer. The stock solution was prepared by completely dissolving lyophilized polyaldehyde dextran by adding sterile distilled water to a final concentration of 100 nmol / μl.

In the case of Table 2 below, since the total aldehyde mole number of the lyophilized polyaldehyde dextran is 468.3 μmol, 468.3 μl of sterile distilled water is added to show that 1.0 μmol / μl of stock is completely dissolved.

(5) 1 pmol / Μl Polyaldehyde Dextran  Produce

100 nmol / μl of polyaldehyde stock solution was sequentially diluted 10-fold and adjusted to 1 pmol / μl, which is the concentration required when covalently reacting with the nucleic acid probe.

Example 2: Construction of Nucleic Acid Probes

The probe used in this experiment can be used to detect (i) tuberculosis bacteria and to determine whether rifampin and children or drug resistance, (ii) detection and classification of mycobacteria, that is, TB and non-TB bacteria It is designed to distinguish species of (NTM).

First, in relation to the above (i), in the case of a probe capable of detecting tuberculosis bacteria and whether rifampin and children or drug resistance are detected, a probe targeting a tuberculosis group-specific Inter Transcriptional Spacer (ITS) gene region is identified. Used for.

As a probe for determining the anti-tuberculosis drug, rifampin susceptibility and resistance, oligonucleotides containing rpoB, a wild type or mutant type sequence of a gene encoding RNA polymerase β-subunit, may be used. Used.

In addition, probes for determining the sensitivity and resistance of the inaison (isoniazid) include gene coding sites of catGase (catalase peroxidase) and promoter sites of inhA (enoyl acyl carrier protein (ACP) reductase) and ahpC (alkyl hydroperoxide reductase). Probes containing phenotype or mutant sequences were used.

The structure of these probes was designed such that the poly T tail spacer consisting of amino-C6-linker at the 5'-end and 10 thymine bases was modified at the 5'-end of each corresponding gene sequence.

On the other hand, in relation to the above (ii), the detection and classification of the genus Mycobacteria, that is, to distinguish the species of Mycobacterium tuberculosis (TB) and nonmycobacterium tuberculosis (NTM), the probe is a specific ITS gene of each mycobacteria genus Site-targeted probes were used, and the structure of these probes was the same as that of the probe (i), and the poly T-tail spacer composed of amino-C6-linker and 10 thymine bases at the 5'-end of each gene It is designed to be modified on the 5 'side of the sequence.

In the following examples, the 5'-amino linker and poly T tail spacers are used on a dextran derivative (polyaldehyde dextran), which is a carrier, for a probe manufactured according to the purpose, without specifically presenting the nucleotide sequence of each probe used. It will be described in detail a method for conjugating the modified nucleic acid probe. However, in Tables 3 and 4, brief information on the probes used in (i) and (ii) is presented (Table 3: Brief information on rifampin or eye or susceptibility / resistant) Table 4: Identification and detection of mycobacteria Probe brief information).

Example 3 Preparation of Conjugates of Nucleic Acid Probes with Polyaldehyde Dextran

(1) preparation of necessary reagents

A buffer for the conjugation reaction was prepared by using 0.5M sodium borate buffer (pH 11.0), and a Schiff's base formed in the reaction between the aldehyde of the polyaldehyde dextran carrier and the 5'-terminal amino group of the nucleic acid probe. 0.1 mM NaBH ₄ solution was prepared as a mild reducing agent for converting to a stable covalent bond.

(2) Conjugation  For reaction master  Mixed solution manufacturing

The composition of the master mixture is polyaldehyde dextran (1 pmol / μl), 0.5M sodium borate buffer (pH 11.0) and sterile distilled water, it is mixed in the composition ratio of Table 5 below.

(3) Conjugation ( conjugation ) reaction

In order to produce 40 membranes having a size of 16.7 cm x 5.5 cm and a line thickness of 0.8 mm for each strip line, 48.00 μl of 100 μM probe solution and 4752.00 μl of sterile distilled water were mixed to achieve a final volume of 4800.00 μl. 11200.00 μl of the master mixture solution for the conjugation of Table 5 was added and mixed, followed by O / N reaction while rotating at 40 rpm at room temperature.

Specifically, if the slit thickness of the automated line blotting equipment is approximately 0.8 mm based on one membrane (16.7 cm x 5.5 cm), the amine-probe at the final concentration of 1 μМ diluted with sterile distilled water for each line coating Mix 280.00 μl of master mix per 120.00 μl of solution volume to 400.00 μl of solution at final conjugation.

Stable amine conjugates were prepared by reducing the polyaldehyde dextran-probe imine conjugate with a reducing agent. Specifically, 1.0 μl of a 0.1 mM NaBH ₄ solution of reducing agent per 100 μl total volume of polyaldehyde dextran-probe imine conjugate was added twice a day for 2 hours. After the addition of the reducing agent, the reaction was carried out by rotating at 40 rpm at room temperature.

Example 4 Preparation of Nucleic Acid Probe-coated Membrane Strip

(1) automation line Blotting  equipment( automatic line blotter ) setting

Line blotting equipment with approximately 0.8 mm slit conforming to one piece of membrane specification (16.7 cm x 5.5 cm) was self-fabricated and the top and bottom plates were ultrasonically cleaned by a washing process prior to use.

The line blotting equipment was fitted to engage the top and bottom plates well and washed several times with sterile distilled water and several times with 1X PBS to prepare for automatic dispensing of the conjugates.

(2) automation line Blotting  With equipment On the membrane  Nucleic acid Probe  coating

Patent blue V dye (5000X = 3g / 500ml), which is a blue dye, was mixed with 1X PBS at an appropriate concentration. Dye was added to check that the lines were evenly drawn on the membrane. A total of 1 ml was prepared by mixing 600 μl of 1 × PBS (containing patent blue V dye) to 400 μl of PAD-nucleic acid probe conjugate for each line (for each probe).

The membrane used in this example is Pall's Biodyne B membrane, and cut 3MM paper to the specifications of the slit blotting equipment. A sheet of 3MM paper is laid on the lower plate of the slit blotting machine, the membrane is covered thereon, and the upper plate is adhered to the membrane so that the solution dispensed on the slit is free of leakage and bleeding. Was first identified by dispensing and testing.

In this manner, by placing a 3MM paper on the bottom plate of the line blotting equipment, and covered the Biodyne B membrane previously wetted with 1X PBS on it, and then adhered to the top plate, the nucleic acid probe was prepared to coat the membrane.

1 ml of the PAD-nucleic acid probe conjugate mixed solution was automatically dispensed into the slit of the corresponding line. The PAD-nucleic acid probe conjugate was adsorbed onto the Biodyne B membrane by applying a vacuum at 150 rpm for 5 seconds, and then air dried to enhance physical adsorption, followed by pre-hybridization buffer solution in a desiccator. It was dried until pretreatment with (prehybridization buffer).

(3) free With hybridization buffer solution Membrane  Pretreatment

In this embodiment, pre-synthesis reaction buffer was pre-treated on the membrane to prepare a membrane strip, thereby securing the rapidity of the nucleic acid hybridization assay experiment and increasing the signal-to-noise ratio (S / N ratio).

The pre-hybridization buffer consists of 5X denhardt's solution, 5X SSC, 0.01-1% SDS, 0.01-1 mg / ml denatured salmon sperm DNA. The membrane was pretreated with a pre-synthesis buffer solution at 42 ° C. for 30 minutes and then dried in a drier for one day.

(4) Membrane In the housing  Attached strip production

The dried membrane was attached to an adhesive backbone (size: 6 cm long, transparent: 0.5 cm) on the opposite side of the probe-coated side, and then cut at 3.4 mm intervals. The backbone is a self-adhesive film on both sides, attached to a housing that can hold a strip of membrane using the opposite side of the backbone to which the membrane is attached.

When the membrane strip is attached to the housing, the coated probe may fall off when pressed with a rubber roller, so that the membrane strip is attached to the housing by pressing with a soft 3MM paper or sponge.

The membrane strip fabricated in this example is shown in FIG. In FIG. 2, the specification of the membrane strip coated with the nucleic acid-probe and the appearance of the housing are shown in detail.

Experimental Example 1 Reverse Hybridization Line Blot Assay for Detection of Rifampin / Ena Drug Susceptibility / Resistant Mycobacterium Tuberculosis

The specific principle of this experimental example is as follows. A reverse-hybridization line blot assay using a one-tube nested multiplex asymmetric PCR method was applied.

During the PCR reaction, the asymmetric PCR of the gene coding sites of rpoB (the sub-unit of RNA polymerase) and katG (catalase peroxidase) and the promoter sites of inhA (enoyl acyl carrier protein (ACP) reductase) and ahpC (alkyl hydroperoxide reductase) The method was amplified by each target gene specific biotin attachment primer to produce the final biotin-labeled, single-stranded target DNA.

At the same time, PCR amplification control (amplification control) and Mycobacterium tuberculosis-derived ITS (Inter-Transcriptional Spacer) amplicons were also formed.

During the reverse hybridization reaction, each biotin-labeled single stranded target DNA was hybridized with nylon membrane-adsorbed probes (wild type or variant probes of rpoB, katG, inhA, ahpC).

After completion of the hybridization reaction, the multi-drug resistant Mycobacterium tuberculosis detection strip was subjected to a stringent wash, followed by an enzymatic conjugation reaction to obtain a streptavidin-alkaline phosphatase (AP) conjugate. Biotin-labeled targets and control amplicons were hybridized with membrane-adsorbed oligonucleotide probes.

After washing again, the unbound product is removed, and a coloring solution containing 4-nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) is added to the strip. It was.

The red precipitate produces blue precipitates at the probe sites where the hybridization takes place. That is, the bound alkaline phosphatase catalyzes dephosphorylation of BCIP to form an oxide dark-blue indigo-dye.

Here, NBT also produces a dark blue dye as an oxidizing agent, so that the blue becomes darker, thereby increasing the detection sensitivity.

The pattern of the band colored in blue is compared with a test reference guide / reading card to interpret the results, or by using the AdvanSure ™ GenoLine Scan, an automated analysis equipment from LG Life Sciences, to determine whether the tuberculosis bacteria are resistant to rifampin and children or drugs. It can be distinguished.

(1) preparation of required solution

Sample DNA Extraction, PCR Reaction, Reverse Hybridization Line Blot Assay The reagents of Table 6 were prepared for the following experiments.

(2) preparation of the specimen and DNA  extraction

Sputum or tuberculosis of tuberculosis patients 1-2 ml and the same amount of sample pretreatment solution 1, 1N NaOH in a 15 ml tube was stirred thoroughly and centrifuged for 20 minutes at 4,000 rpm.

The supernatant was removed, and 10 ml of PBS buffer (0.15 M NaCl, 0.01 M NaH ₂ PO ₄ ), which was added to the precipitate, was stirred well and centrifuged at 4,000 rpm for 20 minutes. The supernatant was again removed, the precipitate was transferred to a 1.5 ml tube, 1 ml of PBS buffer was added thereto, stirred, and centrifuged at 13,000 rpm for 5 minutes.

The supernatant was removed again, and 50-100 μl of 5% (w / v) Chelex 100 resin (Bio-Rad), an extraction buffer, was added to the precipitate and heated at 100 ° C. for 20 minutes, followed by 3 minutes at 13,000 rpm. DNA supernatant isolated by centrifugation was used as template DNA for PCR.

(3) PCR  reaction

In the PCR reaction, 12.5 μl of the 2X PCR mixture solution of Table 6, 5 μl of the primer mixture solution, and 7.5 μl of the sample DNA were added, and the PCR reaction was performed under the conditions shown in Table 7 below.

* Culture samples (solid culture, liquid cultured tuberculosis strain)

** Smear-positive direct patient material (sputum, bronchial lavage fluid, cerebrospinal fluid, urine, tissue, whole blood, etc.)

(4) Backcross  line Blot  Assay Reverse hybridization line blot assay )

Reverse cross-link blot assays were performed using the reagents in Table 6 above. Hybridization reaction mixture was prepared by adding 20 µl of PCR reaction product and 250 µl of pre-warming hybridization buffer, and then dispensing the mixture on a membrane probe adsorption strip, and then using a 55 ° C hybridization chamber / oven. ) And hybridization reaction while stirring for 30 minutes (9 rpm for vertical stirring, 80 rpm for horizontal stirring).

After vacuum suction (aspiration) of the reaction solution of the hybridization probe adsorption strip, remove 250 µl of pre-warming Stringent wash solution onto the strip and stir vertically at 9 rpm at 55 ° C for 15 minutes. The reaction was carried out and washed. After removing the reaction solution of the strip after the washing reaction, 250 μl of rinse solution was dispensed onto the strip and washed by performing a vertical stirring reaction at 9 rpm for 1 minute at room temperature.

The reaction solution of the strip after the washing reaction was removed. The enzyme conjugation solution was used after being left at room temperature for at least 10 minutes, and 250 μl of the enzyme conjugation solution was dispensed on the strip to remove the reaction solution of the end of the enzymatic conjugation strip, which was vertically stirred at 9 rpm for 30 minutes at room temperature. It was.

Rinse again, 250 μl of rinse solution was dispensed onto the strip and washed by performing a vertical stirring reaction at 9 rpm for 1 minute at room temperature. After removing the reaction solution of the washed reaction strip, 250 μl of sterile distilled water was dispensed into the strip and subjected to vertical stirring at 9 rpm for 1 minute at room temperature to wash once more.

After the reaction solution of the strip was washed, the color reaction was carried out. The color developing solution was used after standing at room temperature for 10 minutes or more, 250 μl of coloring solution (Substrate solution) was dispensed on the strip, and the vertical stirring reaction was performed at 9 rpm for 10 minutes at room temperature under light blocking conditions. .

After removing the reaction solution of the strip after the color reaction, rinse with 250 μl of sterile distilled water to completely remove the remaining color solution and precipitate. Quickly dry the membrane strips with a hair dryer or air-dry them, then quantify them using the LG Life Sciences AdvanSure ™ GenoLine Scan program, or use a test reference guide (Reading Card) or Data Interpretation sheet. The results were read visually.

Specific results of Experimental Example 1 was based on the probe information of Table 3, it was possible to read whether the rifampin or eye or susceptibility / resistance of Mycobacterium tuberculosis in FIG. The results were determined by comparing the wild type and mutant band intensities of each locus with respect to susceptibility / tolerance to rifampin (rpoB) and aina (katG, inhA, ahpC). If the strength of the wild-type band is stronger than that of the mutant band, it is susceptible, and if it is weak, it can be judged to be resistant.

Experimental Example 2: Reverse Cross-Blot Assay for the Classification and Detection of Mycobacteria

The principle of Experimental Example 2 is the same as that of Experimental Example 1. A reverse-hybridization line blot assay using a one-tube nested multiplex asymmetric PCR method was applied.

During the PCR reaction, the ITS sites of Mycobacterium tuberculosis (M. TB) and non-tuberculosis mycobacteria (NTM) were amplified using biotin-attached mycobacterial primers by asymmetric PCR, resulting in the final biotin-labeled single stranded target DNA ( Biotin-labeled, single-stranded target DNA) was generated, and at the same time PCR amplification control was formed.

Thereafter, as in Experiment 1, the membrane strip was applied to detect and classify mycobacteria (M.TB or non-TB bacterium (NTM)) in the sample through hybridization, washing and color reaction. It was.

(1) preparation of required solution

The reagents of Table 8 below were prepared for sample DNA extraction, PCR reactions, and reverse hybridization line blot assay experiments.

(2) preparation of the specimen and DNA  extraction

Sample preparation and DNA extraction are the same as in Experimental Example 1 and are omitted.

(3) PCR  reaction

In the PCR reaction, 12.5 μl of the 2X PCR mixture solution of Table 8, 5 μl of the primer mixture solution, and 7.5 μl of the sample DNA were added, and the PCR reaction was performed under the conditions shown in Table 9 below.

(4) Backcross  line Blot Assay

The reverse hybridization line blot assay is the same as in Experimental Example 2 and thus is omitted. Specific results of Experimental Example 2 was able to detect and distinguish each mycobacteria in the embodiment of Figure 4 based on the probe information of Table 4.

Those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims (26)

A method for preparing a porous solid support strip coated with a nucleic acid probe, wherein the nucleic acid probe is bonded to a carrier to prepare a conjugate, and the conjugate is fixed to the porous solid support by a vacuum transfer method. A manufacturing method comprising the process of making. The method of claim 1, wherein the nucleic acid probe is at least one selected from DNA, RNA, and PNA. The method of claim 1, wherein the binding of the nucleic acid probe and the carrier is covalent or affinity binding having an equivalent level of affinity. The method of claim 1, wherein the 5′-end or 3′-end of the nucleic acid probe is modified with a functional group or a ligand capable of covalent or affinity binding with a carrier. . The method of claim 4, wherein the functional group is a carboxyl group (-COOH), a phosphate group (phosphate), an amino group (-RNH ₂ , -ArNH _2, wherein R is H or a C ₁ -C ₆ alkyl group), methyl chloride (-ArCH ₂ Cl ), Amide (-CONH ₂ ), aldehyde (-CHO), hydroxyl group (-OH), epoxy (epoxy) and thiol (thiol) is a manufacturing method characterized in that at least one selected from the group consisting of. The method of claim 4, wherein the ligand is at least one selected from the group consisting of biotin, Glutathione, maltose, histidin, lectin or derivatives thereof. 6. A method according to claim 5, wherein said amino group is attached directly to a nucleic acid probe. 7. The method of claim 6, wherein said ligand is attached directly to a nucleic acid probe. 6. The method of claim 5, wherein the amino group is an amino C6 linker or amino C12 linker with a short carbon chain attached thereto. The method of claim 1, wherein the nucleic acid probe comprises a label that can be detected directly or indirectly by spectroscopic, photochemical, biochemical, immunological or chemical means. The method of claim 1, wherein the nucleic acid probe is a synthetic oligonucleotide. The method of claim 11, wherein the synthetic oligonucleotide consists of 16 to 22 nucleotides. The method of claim 4, wherein a spacer is added to an internal region of the nucleic acid probe to increase the efficiency of the hybridization reaction. The method of claim 13, wherein the spacer is a nonspecific poly T tail. The method of claim 1, wherein the carrier is at least one selected from the group consisting of polysaccharides, proteins, and synthetic polymers. 16. The method of claim 15, wherein said carrier is dextran or dextran derivative. The method of claim 16, wherein the dextran derivative is dextran oxide. 18. The method of claim 17, wherein the dextran oxide is polyaldehyde dextran. The method of claim 1, wherein the conjugate is prepared by reacting an amine group-labeled nucleic acid probe with a polyaldehyde dex as a carrier to form an imine intermediate of the sieve base, and reducing the sieve base with a reducing agent to induce irreversible binding. Characterized in the manufacturing method. The method of claim 1 wherein the porous solid support is a membrane. The method of claim 20, wherein the membrane is nitrocellulose or nylon. The method of claim 1, further comprising physically adsorbing the conjugate between the carrier and the pores of the porous solid support. The method of claim 1, wherein the vacuum transfer method comprises dispensing the conjugate of the nucleic acid probe and the carrier in the nucleic acid slit line of the automated line blotting equipment and operating the vacuum pump to inhale the conjugate into a porous solid. Method for producing a method comprising the step of adsorbing on a support. The method of claim 1, comprising pretreating the solid phase support with a pre-crosslinking buffer. 25. The method of claim 24, wherein said pre-hybridization buffer is a composition comprising denhardt's solution, SSC, SDS and denatured salmon sperm DNA. A porous solid support strip coated with a nucleic acid probe, which is prepared by the method according to any one of claims 1 to 25.

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