KR102089058B1 - Method for Preparation of Nucleic Acids Probe-Coated, Porous Solid Supporter Strip - Google Patents

Abstract

The present invention is a method of manufacturing a porous solid support strip coated with a nucleic acid probe, wherein a nucleic acid probe is coupled to a carrier to prepare a conjugate, and the conjugate is subjected to a vacuum transfer method to form a porous solid. It relates to a manufacturing method characterized in that it comprises a process of fixing to the support.

Description

Method of Preparation of Nucleic Acids Probe-Coated, Porous Solid Supporter Strip coated with nucleic acid probe

The present invention relates to a method for manufacturing a porous solid support strip coated with a nucleic acid probe, and more specifically, to prepare a conjugate by binding the nucleic acid probe to a carrier, 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 the step of fixing to a porous solid support by a vacuum transfer method.

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

In general, techniques for analyzing the sequence of a specific nucleic acid from a nucleic acid sample isolated from a bacterium or cell or a product amplified by a nucleic acid polymerization reaction have complementary base sequences that specifically bind to a portion of the nucleic acid to be analyzed. Nucleic acid fragments, that is, techniques for analyzing the presence or absence of nucleic acids by analyzing whether or not they are bound by using a nucleic acid probe.

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

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

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

Physical adsorption (physical adsorption) is a process that has a lot of interest in in-vitro diagnosis by physically adsorbing biomolecules on a solid surface.

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

This summary suggests that desorption is potentially inherent, but binding actually occurs irreversibly if the molecule is 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 the larger the molecular weight, the greater the number of binding sites, because even if one binding site is dissociated from the surface, the remaining majority remain in the binding state, and like proteins, the same effect is reported in nucleic acids.

In nucleic acids, the binding is not as strong as proteins, but in the case of large-sized nucleic acids, it has the property of binding well to the membrane.

However, a big problem of the physical adsorption method is that the nucleic acid immobilized on the surface is rearranged, so that it may be in a form that is not suitable for interacting with any incoming nucleotide sequence.

In the case of long-sequence nucleic acids, it may not be a problem, but the shorter the nucleotide sequence of a nucleic acid, the lower the potential activity may be a serious problem. If there are some bases immobilized on the surface that do not remain in a free form to interact with the solution, the hybridization reaction cannot normally occur due to the deformed structure.

In addition, the nucleic acid probe is very difficult to fix due to the small size of the molecule and the physical force (electrostatic force, hydrophobic interaction, etc.) that can be adsorbed on the porous solid support is relatively weak to directly apply the vacuum transfer method. .

Due to this, there is a risk that the nucleic acid probe will not be uniformly coated, and there is a high possibility of desorption during an assay reaction without an additional fixation process, and it is efficient for use in a molecular diagnostic assay requiring reproducibility and high sensitivity. There is this falling issue. In view of these problems, a method using UV is widely used.

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

Since some of these bases are covalently linked to the membrane surface and do not participate in the reaction during a hybridization reaction, when the membrane is excessively exposed to UV, the UV linkage method, as a result, interferes with the hybridization reaction. Therefore, it is important to correctly determine the UV dose.

That is, if the membrane is exposed to a small amount of UV, efficient crosslinking is not achieved, and if it is excessively exposed, the efficiency of the hybridization reaction is reduced.

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

Meanwhile, a technique in which photoactivatable compounds are used in a membrane or probe is an advanced cross-linking method (Ref. 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 very few applications since exposure to UV radiation often leads to uncontrolled reactions.

Covalent linkage is the most popular technology for immobilizing nucleic acid probes on the surface of a solid support using traditional chemistry. The most common of the various linking chemistry methods used are amine, carbonyl, carboxyl, and thiol.

The coordination of the probe is controlled by covalent bonding, and it is possible to control and attach the nucleic acid through terminal residues by a controlled-chemistry linkage.Any fixed nucleic acid is introduced by introducing a spacer of the correct size. A hybridization reaction can be made freely with the added probe.

Covalent bonding between the probe and the solid support is mainly applied to a microarray system, that is, a form such as a glass slide or a bead, but is expected to be applied to a porous solid support such as a membrane.

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

In addition, the technology of covalently immobilizing a nucleic acid probe in a controlled attachment to a membrane is essentially difficult to convert to a mass production and automated process in a batch process. Also, it is time consuming and expensive in terms of cost.

As described above, in preparing a nucleic acid probe-coated membrane strip for use in a nucleic acid hybridization assay, due to the advantages and disadvantages of physical and chemical processes, the hybridization reaction is normally performed, and at the same time in a simple and rapid method. There are limits to the mass production of membrane strips.

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

The present invention aims to solve the problems of the prior art as described above and the technical problems requested from the past.

Specifically, the object of the present invention, in manufacturing a porous solid support strip coated with a nucleic acid probe, by binding the nucleic acid probe to the carrier, it is possible to secure the orientation of the nucleic acid probe, and the nucleic acid fixed on the surface of the solid support is rearranged This is to provide a manufacturing method in which an abnormal hybridization reaction due to a decrease in activity does not occur because a portion of a hybridization reaction sequence region of a probe that must complementarily bind to a target base sequence is not fixed.

Another object of the present invention is that there is no hassle of adjusting the optimal UV irradiation amount during UV irradiation, and does not use the batch process essentially required in the prior art for fixing nucleic acid probes by chemical bonding, so mass production and automation processes It is to provide a possible manufacturing method.

Another object of the present invention is that it is simple, fast, inexpensive as well as reproducible by mass production, and the membrane surface is stable without interfering with the interaction so as to facilitate hybridization reaction between the target sequence and the nucleic acid probe. And to provide a method for producing a nucleic acid-probe assay system that can exhibit high sensitivity.

Therefore, in the method of manufacturing a porous solid support strip coated with a nucleic acid probe according to the present invention, a nucleic acid probe is coupled to a carrier to prepare a conjugate, 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 purposes for use in in-vitro diagnostic nucleic acid testing vary, but several factors must be considered. As a result, there are advantages and disadvantages in terms of binding effectiveness related to the performance of the assay and convenience in the manufacturing process. The key is how easy it is to manufacture in large quantities at a competitive price.

In this regard, in the method for preparing a porous solid support strip coated with a nucleic acid probe according to the present invention, the orientation of the nucleic acid probe can be secured by binding the nucleic acid probe to a carrier, and the nucleic acid fixed on the surface of the porous solid support is resuspended. It is not aligned, and since a part of the hybridization reaction sequence region of the probe to be complementary to the target base sequence is not fixed, an abnormal hybridization reaction due to a decrease in activity does not occur.

In addition, since there is no hassle of adjusting the optimal UV irradiation amount as in the prior art, and the batch process that is essentially required in the chemical bonding technique is not used, mass production and automation processes are possible.

In the present invention, the nucleic acid probe may be composed of sense (forward) and antisense (reverse) nucleic acids in which DNA, RNA, peptide nucleic acid (PNA) and the like recognize the 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 have similarities, but they are virtually different due to differences in the structure and size of the molecules. The method of fixing large pieces of DNA to the solid phase is no longer used for diagnostic tests, and thus a method of fixing a shorter DNA to the solid phase is mainly used.

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

However, it is not a covalent bond, but a binding method using affinity binding, which has a strong binding strength at the covalent level, is also applicable.

In connection with the affinity binding, a biotin-avidin / streptavidin fixation technique capable of forming a strong bond with a binding constant of ~ 10 ^-15 M is most often used. In addition, Glutathione S-transferase (Glutathione-GST), histidin-metal ion (Co, Ni, Cu), maltose-MBP (maltose binding protein), lectin (eg., Concanavalin) -mannose / glucose binding protein The coupling technology of can be utilized between the probe and the solid support.

Therefore, in order to allow the nucleotide sequence of the entire nucleic acid probe to participate in the hybridization reaction, preferably, a functional group is introduced into the nucleic acid probe for covalent binding with the carrier, or a ligand is introduced into the nucleic acid probe for affinity binding with the carrier. You can.

Surface modification for covalent coupling between the nucleic acid probe and the solid support is 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) and various functional groups (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. can be implemented by various systems. You can.

In one preferred example, in the case of amino group modification of the nucleic acid probe, it may be composed 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 technique of immobilizing nucleic acid probes is most efficient when the reaction is through an end group, so a linking group is introduced into the 5 'or 3'-active end group of the nucleic acid. It is preferred.

Therefore, the 5'-terminal or 3'-terminal of the nucleic acid probe may be preferably modified with a functional group capable of covalently bonding with a carrier. The step of modifying the nucleic acid probe may be performed in solution during production of the nucleic acid probe 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 a nucleic acid probe capable of detecting tuberculosis bacteria and determining whether rifampin and eye or drug resistance are present, a nucleic acid probe targeting the specific transcriptal spacer (ITS) gene region of the tuberculosis bacteria can be used for identification of tuberculosis bacteria You can.

To determine whether the anti-tuberculosis drug rifampin is susceptible and resistant, it contains a wild-type or mutant-type base sequence of a gene encoding an RNA polymerase beta subunit. A nucleic acid probe can also be used.

In addition, in order to determine whether the child is sensitive or resistant, the phenotype or mutant sequence of the gene coding region of katG (catalase peroxidase) and the promoter region of inhA (enoyl acyl carrier protein (ACP) reductase) and ahpC (alkyl hydroperoxide reductase) It is also possible to use a nucleic acid probe containing.

On the other hand, as a nucleic acid probe capable of distinguishing species of tuberculosis bacteria (TB) and non-tuberculosis antibacterial bacteria (NTM), a nucleic acid probe targeting a specific transcript spacer (ITS) gene site in the microbacteria may be used.

In addition, the nucleic acid probe, preferably, may include a label that can be detected directly or indirectly by spectroscopic, photochemical, biochemical, immunological or chemical means, and an internal region comprising a target sequencing. A spacer may be added to increase the efficiency of the hybridization reaction between the carrier and the carrier of the hybridization site that can complementarily bind.

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

The spacer is preferably a non-specific poly T tail, which can be added after the amino group modification. The length of the poly T tail used as a 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 made of 10 thymine bases is connected with a spacer, and a nucleotide sequence of a hybridization reaction site capable of complementarily binding to a nucleic acid sequence of a 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, polysaccharides, proteins, synthetic polymers, and the like. Among them, polysaccharides are preferable, and dextran or a derivative thereof that covalently or affinity-couples to the nucleic acid probe is more preferable.

In the case of affinity binding, for example, avidin / streptavidin, maltose binding protein, glycoprotein, metal ion (Co, Ni, Cu, etc.) are linked to the carrier, and biotin, maltose, lectin, and histidin ligands capable of affinity binding to the carrier Alternatively, a derivative thereof can be labeled with a nucleic acid probe. Among them, biotin-avidin / streptavidin linking technology is more preferable.

Since dextran has several activation groups per molecule, it can not only fix several DNA probes per molecule of dextran, but it can also fix several ligands such as aspartic acid and oligonucleotide probes (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, it is very easy for immobilized DNA molecules to cross-react, and also has 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, in the case of aldehyde-aspartic-dextran, a substance having a very large molecular weight, which has an anion and an aldehyde group, can form a very strong covalent bond with the amination DNA probe, and Binding is very stable to the extreme pH of the hybridization reaction mixture, high temperature or high concentrations of salts, solvents, and nucleophiles (Ref. Fuentes M. et al., Biomacromolecules, 5, 883-888, 2004).

In one preferred example, dextran can be oxidized to form a polysaccharide that can form an aldehyde (aldehyde) functional group.

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

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

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

The polyaldehyde dextran transporter and the amine group-labeled nucleic acid probe are reacted, for example, at room temperature at pH 11 to form an immine intermediate of Schiff's base, and as a reducing agent, for example, Sodium borohydrate, a mild reducing agent, reduces the CiF base to induce irreversible bonds, thereby achieving stable covalent bonds.

Nucleic acids are essentially non-reactive, but are reactive with other groups when sufficient energy is given. When nucleic acids are exposed to UV light without visible or photosensitizers in the presence of photosensitizers such as methylene blue, they cross-link directly with proteins. (direct cross-linking) (Ref. 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, where the chemically active surface of the membrane reacts with gases in the atmosphere and loses activity.

The greater the chemical activity, ie, the faster the cross-link with the target, the faster the activity is lost. To overcome this problem, recent manufacturers use less reactive groups that specifically react with the target probe itself. The reaction between the aldehyde and the amine is a typical example, both of which are relatively stable.

Production of a reactive solid phase Due to the inherent problems of manufacturing, storage and handling, it is no longer possible to provide a reactive membrane for the purpose of covalent bonding with a probe.

Usually, a membrane having a surface chemistry capable of specifically binding to the probe is provided, but a membrane having less reactivity and less activity with time and storage is used, which has an amine or aldehyde group on the membrane surface. What is dominant.

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

Since the CiF base is relatively stable, and can be reversed under physiological conditions, the CiF base should 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 non-specific binding, so activated membranes must be blocked prior to 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.

When a passivation is performed on the membrane surface by blocking, a new chemical functional group is introduced, so it may not be reactive with nucleic acids, but charge attraction or charge attraction by binding of a sample or a detection system Hydrophobic attraction may increase and non-specific signals may increase.

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

This variation causes difficulties in automation and mass production. Although the mass production is simplified by the introduction of photoactive linking chemistry, it is relatively rare because it is expensive in terms of cost.

On the other hand, in the present invention, for example, by covalent bonding of an amino group-modified nucleic acid probe that specifically reacts with a polyaldehyde dextran that activates (oxidizes) a carrier, dextran, the above problem is solved and mass Not only is it possible to produce, but also, as shown in the covalent bonding method between amine and carboxyl groups using 1-Ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC or EDAC), no other toxic additional reagents are required, so organic solvents are not required. 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 bead, acrylamide gel, polystyrene matrices, and activated dex. Tran (activated dextran), avidin-coated polystyrene beads (avidin-coated polystyrene bead), glass (glass), etc., such as a variety of various solid support has been applied to DNA diagnostics (Ref .: 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) .

Among the various solid supports, the present invention focuses on how various target materials can be fixed to porous solid supports, and in particular to a membrane.

Membrane contains a wide range of substrates that can be used to immobilize nucleic acids. Specifically, in traditional mold membranes such as nitrocellulose, nylon, new membranes such as ceramic or track-etched membranes, and microarrays. And the type of substrate used.

Many membranes recommended for nucleic acid immobilization must first determine the optimal binding conditions, among other linkage techniques nucleic acids may be added to the membrane in an uncontrolled manner.

In that case, the orientation of the nucleic acid probe may not be aligned in the preferred direction, potentially limiting the reaction. Therefore, directed adsorption, where the fixed nucleic acid is end-linked, is preferred in most cases.

The main advantage of nitrocellulose is its availability, which is quickly available, but is softer if not coated with polyester baking and has a lower binding capacity than other membranes.

In recent years, nylon has been promoted as a substrate for nucleic acid binding because it has a greater physical strength and binding capacity, and a wider range of available surface chemistries can be optimized to attach nucleic acids.

The method commonly used to bind nucleic acids to nitrocellulose is a physical adsorption method, but fixation to a nylon membrane consists of physical adsorption, UV cross-linking or chemical activation.

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

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

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

In this regard, the present invention may further include a process of physically adsorbing the conjugate between the carrier and the hole of the porous solid support by drying.

Most enhanced physical adsorption techniques not only increase the binding efficiency, especially for very short probes, but also ensure that the important sequences of the probes are arranged to project in a direction away from the solid surface. As a result, it is possible to freely interact with the incoming sample and the important base sequence. It improves selectivity and sensitivity, so it is very effective even if a short probe is used.

In this aspect, according to the present invention, covalent bonds have an orientation in which important sequences of nucleic acid probes are arranged in a direction away from the surface of the porous solid support.

In the present invention, the vacuum movement method is preferably by dispensing the conjugate of the nucleic acid probe and the carrier to the nucleic acid slit line of the automated line blotting equipment, and operating the vacuum pump to inhale the conjugate. It may include a process of adsorbing to a porous solid support.

The present invention also provides a porous solid support strip coated with a nucleic acid probe, characterized in that produced by the above method.

The porous solid support strip coated with the nucleic acid probe according to the present invention has a specific structure differentiated from the prior art by itself by a special manufacturing process as described above.

As described above, the present invention can secure the directionality of the nucleic acid probe by binding the nucleic acid probe to the carrier, and the nucleic acid immobilized on the surface of the solid support is not rearranged. Since a part of the hybridization reaction sequencing site is not fixed, an abnormal hybridization reaction due to a decrease in activity does not occur.

In addition, there is no hassle of adjusting the optimal UV irradiation amount during UV irradiation, and since a batch process that is essentially required in the technique of fixing a nucleic acid probe by conventional covalent bonding is not used, mass production and automation processes are possible. .

In other words, in mass production, it is simple, fast, inexpensive, as well as reproducible, and the membrane surface can exhibit stability and high sensitivity without interfering with the interaction so as to facilitate hybridization reaction between the target sequence and the nucleic acid probe. It has the effect of producing a nucleic acid-probe assay system.

1 is a view showing the entire process of manufacturing a strip by fixing a nucleic acid oligonucleotide probe to a carrier in a covalent bond manner to a dextran, and then fixing it to a membrane using a vacuum transfer method.
FIG. 2 is a diagram showing a result photo coated with a nucleic acid probe coated with a blue dye solution, a blue dye solution, and a membrane strip, wherein the upper part is a case of a rifampin of the embodiment and a strip capable of determining whether the child is sensitive or resistant, and the lower part Is the case of the mycobacteria of the example and the detectable strip.
FIG. 3 is a diagram showing a result pattern when a reverse cross-blot assay was performed after PCR was performed on rifampin and Aina sensitive / resistant specimens.
FIG. 4 is a diagram showing a result pattern when a reverse cross-blot assay was performed after PCR was performed on a mycobacterial sample and a control group (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 the process of producing a strip by fixing a nucleic acid probe to a membrane in a vacuum movement manner, and in two specific examples, using the prepared membrane strip, sputum, bronchial washing solution, cerebrospinal fluid, urine, tissue, whole blood , In a culture strain (i) a test that can detect the presence of rifampin and eye or drug resistance as well as detecting tuberculosis bacteria, and (ii) detection and division of mycobacteria, that is, tuberculosis bacteria (TB) and non-tuberculous bacteria (NTM) ) A test capable of classifying species was performed to verify its effectiveness by a reverse hybridization line blot assay.

Example 1: Preparation of polyaldehye dextran (PAD)

(1) Required reagents and equipment preparation

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 (hydroxylamine Hydrochloride (NH ₂ OH-HCl = 69.49), methyl orange powder, 10N hydrochloric acid, pH meter, 0.1N sodium hydroxide (NaOH), freeze dryer or SpeedVac was used.

(2) Preparation of oxidized dextran

0.5 g of dextran (Sigma, D-4133, equivalent to 62.5 mmol of Mr-43 kDa / glucose) was completely dissolved in autoclaved DW and adjusted to a final 10 ml. 0.72 g of KIO ₄ (corresponding to 62.5 mmol equivalent) was added (reference: 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 or more while rotating at a speed of 40 rpm at room temperature. The oxidized dextran was placed in a cellulose dialysis membrane, and dialyzed once by O / N in 2L of tertiary sterile distilled water, and dialysis was performed three times in total for an additional 2 hours while replacing sterile distilled water. After dialysis, the exact volume of polyaldehyde dextran was measured.

(3) Determination of the amount of aldehyde groups in oxidized dextran

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

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

Redox titration of polyaldehyde dextran was performed with 0.1N NaOH standard solution. After dialysis, 1.25 ml of 0.25N hydroxyamine hydrochloride-methyl orange (pH 4.0) solution was added to 100 µl of the obtained polyaldehyde dextran, and reacted while rotating at room temperature for 2 hours (40 rpm).

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

The oxidation-reduction reaction and the aldehyde substitution degree calculation of the polyaldehyde dextran are expressed in the following formulas, and Table 1 below is calculated by calculating the total mole number of aldehydes X contained in the total volume of polyaldehyde dextran (PAD) measured after dialysis. Shown.

(4) lyophilization of polyaldehyde dextran and preparation of stock solution

After dispensing a small amount of polyaldehyde dextran in a small amount of 10.0 uL into a screw cap tube, O / N was lyophilized using SpeedVac or freeze-dryer. Stock solution was prepared by completely dissolving the 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 was 468.3 μmol, 468.3 μl of sterile distilled water was added, and when completely dissolved, it was shown to be 1.0 μmol / µl stock.

(5) Preparation of 1 pmol / µl polyaldehyde dextran

A 100 nmol / µl polyaldehyde stock solution was sequentially diluted 10-fold to set the concentration required for covalent reaction with the nucleic acid probe to 1 pmol / µl.

Example 2: Construction of nucleic acid probe

The probe used in this experiment includes (i) a probe capable of detecting resistance to rifampin and eye or drug as well as detecting tuberculosis bacteria, (ii) detection and division of mycobacteria, namely, tuberculosis bacteria (TB) and non-tuberculous bacteria It is designed to distinguish the species of (NTM).

First, in relation to (i), in the case of a probe capable of detecting whether tuberculosis bacteria are resistant to rifampin and eye or drug, a probe targeting a specific transcriptal spacer (ITS) gene region of the tuberculosis bacteria is identified. It was used as a dragon.

The anti-tuberculosis drug, rifampin, is a probe for determining susceptibility and resistance, and includes oligonucleotides containing a wild-type or mutant-type nucleotide sequence of a gene encoding rpoB, that is, RNA polymerase β-subunit. Used.

In addition, as a probe for determining the sensitivity and resistance of iniazid, the gene coding region of katGase (catalase peroxidase) and the promoter region of inhA (enoyl acyl carrier protein (ACP) reductase) and ahpC (alkyl hydroperoxide reductase) A probe containing a phenotype or mutant base sequence was used.

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

On the other hand, in relation to the above (ii), the detection and classification of the genus Mycobacterium, that is, the specific ITS gene of each Mycobacteria genus as a probe capable of distinguishing the species of Mycobacterium tuberculosis (TB) and non-TB antibacterial (NTM) Probes targeting the sites were used, and the structure of these probes is a poly T tail spacer consisting of amino-C6-linker and 10 thymine bases at the 5'-end, as in the case of probe (i), respectively. It is designed to be modified on the 5'-side of the base sequence.

In the following examples, the 5'-amino linker and poly T tail spacer are used for the dextran derivative (polyaldehyde dextran), which is a carrier for probes designed for the purpose without specifically showing the nucleotide sequence of each probe used. The method of conjugating the nucleic acid probe modified with will be described in detail. However, in Tables 3 and 4, brief information on the probes used in (i) and (ii) is to be provided (Table 3: Brief information on probes for differentiation of rifampin or eye or sensitivity Table 4: For classification and detection of mycobacteria) Probe brief information).

Example 3: Preparation of conjugates of nucleic acid probes and polyaldehyde dextran

(1) Required reagent preparation

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

(2) Preparation of master mixed solution for conjugation reaction

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

(3) Conjugation reaction

For the production of 40 membranes with a specification 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 μМ probe solution and 4752.00 μl of sterile distilled water were mixed to achieve a final volume of 4800.00 μl. After adding 11200.00 µl of the master mixture solution for conjugation in Table 5 and mixing, the mixture was reacted O / N while rotating at 40 rpm at room temperature.

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

A stable amine conjugate was 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 as a reducing agent per 100 µl total volume of polyaldehyde dextran-probe imine conjugate was added twice in total for 2 hours per hour. After adding the reducing agent, the reaction was performed by rotating at room temperature at 40 rpm.

Example 4: Preparation of nucleic acid probe-coated membrane strip

(1) Automatic line blotter setting

Self-produced line blotting equipment with approximately 0.8 mm slits conforming to one piece of membrane specification (16.7 cm x 5.5 cm), and the upper and lower plates were ultrasonically cleaned by a washing process before use.

The line blotting equipment was mounted so that the upper and lower plates were well interlocked, washed several times with sterile distilled water and 1X PBS, and the conjugate was prepared for automatic dispensing.

(2) Nucleic acid probe coating on membrane with automated line blotting equipment

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

The membrane used in this example is Pall's Biodyne B membrane, and 3MM paper was cut to meet the specifications of the slit blotting equipment. 1x PBS solution containing Patent blue V dye is used to place one piece of 3MM paper on the lower plate of the slit blotting equipment, cover the membrane thereon, and adhere the top plate to the membrane to ensure that the solution dispensed on the slit does not leak or smear. It was confirmed by first dispensing and testing.

In this way, 3MM paper was placed on the lower plate of the line blotting equipment, and the Biodyne B membrane, which had been pre-soaked in 1X PBS, was covered thereon, followed by close contact with the upper plate, thereby preparing a nucleic acid probe to be coated on the membrane.

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

(3) Membrane pretreatment with pre-hybridization buffer solution

In this example, the pre-hybridization reaction buffer was pre-treated on the membrane to prepare a membrane strip to secure the rapidity of the nucleic acid hybridization assay and increase the signal-to-noise ratio (S / N ratio).

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

(4) Strip production with membrane attached to housing

After attaching the dried membrane to the adhesive backbone (standard: 6 cm long, transparent part: 0.5 cm) on the opposite side of the surface coated with the probe, it was cut at a width of 3.4 mm. Since the backbone is made of an adhesive film on both sides, it was attached to a housing capable of storing a strip of the membrane using the opposite side of the backbone side to which the membrane was attached.

When attaching the membrane strip to the housing, there is a risk of the coated probe falling off when compressed with a rubber roller, so that the membrane strip is well attached to the housing by pressing with a soft 3MM paper or sponge.

The membrane strip produced in this example is shown in FIG. 2. In FIG. 2, the specification of the nucleic acid-probe coated membrane strip and the state of housing attachment are presented in detail.

Experimental Example 1: Reverse cross-link blot assay for detecting rifampin / ina drug susceptibility / resistant tuberculosis

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

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

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

During the backcrossing reaction, each biotin-labeled single-stranded target DNA was hybridized with a nylon membrane-adsorbing probe (a wild type or variant probe of rpoB, katG, inhA, ahpC).

After the hybridization reaction is completed, the multi-drug resistant tuberculosis detection strip undergoes a stringent wash process, and then undergoes an enzymatic conjugation reaction to obtain a streptavidin-alkaline phosphatase (AP) conjugate. The biotin-label target hybridized with the membrane-adsorbed oligonucleotide probe and the control amplicon were bound.

After washing again, unbound products are 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 to develop color. Did.

A blue precipitate is formed at the position of the probe where the hybridization reaction occurs through the redox reaction. That is, the combined alkaline phosphatase catalyzes the dephosphorylation of BCIP to form a dark blue indigo-dye, an oxide.

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

Compare the pattern of the band colored in blue with a test card (Test Reference Guide / Reading Card) or interpret the results or use the AdvanSure ™ GenoLine Scan, an automated analysis equipment from LG Life Sciences, to determine whether the tuberculosis bacteria are resistant to rifampin and eye or drug Can be distinguished.

(1) Preparation of required solution

Sample DNA extraction, PCR reaction, backcross line blot assay Reagents in Table 6 were prepared for the following experiment.

(2) Sample preparation and DNA extraction

1 to 2 ml of tuberculosis patient's sputum or standard strain culture solution and the same amount of sample pretreatment solution 1, 1N NaOH, were placed in a 15 ml tube, stirred sufficiently, and centrifuged at 4,000 rpm for 20 minutes.

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

Again, remove the supernatant and add 50-100 μl of 5% (w / v) Chelex 100 resin (Bio-Rad), an extraction buffer solution to the precipitate, and heat it at 100 ° C. for 20 minutes, then 3 minutes at 13,000 rpm. The DNA supernatant separated by centrifugation was used as template DNA for PCR.

(3) PCR reaction

For 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 sample DNA were added, and the PCR reaction was performed under the conditions shown in Table 7.

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

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

(4) Reverse hybridization line blot assay

A backcross line blot assay was performed using the reagents in Table 6 above. A hybrid reaction mixture was prepared by adding 20 µl of PCR reaction product and 250 µl of pre-warming hybridization buffer, and then dispersing the mixed solution on a membrane probe adsorption strip to obtain a hybridization chamber at 55 ° C (hybridization chamber / oven) ) And allowed to cross-react while stirring for 30 minutes (9 rpm for vertical stirring and 80 rpm for horizontal stirring).

After removal of the reaction solution of the probe adsorption strip after the hybridization reaction is completed, the pre-warming 250 µl of a stringent wash solution is dispensed onto the strip and vertically stirred at 55 ° C. for 15 minutes at 9 rpm. The reaction was carried out to wash. After removing the reaction solution of the strip after the washing reaction, 250 µl of a rinse solution was dispensed into 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 standing at room temperature for 10 minutes or more, and 250 µl of the enzyme conjugation solution was dispensed onto the strip to remove the reaction solution of the strip after the enzymatic conjugation reaction, which was subjected to a vertical stirring reaction at 9 rpm for 30 minutes at room temperature. Did.

Again, a washing reaction was performed, and 250 µl of a rinse solution was dispensed onto the strip, followed by washing with a vertical stirring reaction at 9 rpm for 1 minute at room temperature. After removing the reaction solution of the strip after the washing reaction, 250 µl of sterile distilled water was dispensed into the strip, and the mixture was washed once again by performing a vertical stirring reaction at 9 rpm for 1 minute at room temperature.

After removing the reaction solution of the strip after the washing reaction, a color reaction was performed. The coloring solution was used after standing at room temperature for 10 minutes or more, and after dispensing 250 µl of Substrate solution on the strip, a 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 development reaction, rinsing with 250 µl of sterile distilled water completely removed the remaining color solution and precipitate. After drying the membrane strips quickly with a hair dryer or air-drying in air, perform numerical analysis using the LG Life Sciences AdvanSure ™ GenoLine Scan program, or use a test reference guide (Reading Card) or Data Interpretation sheet. Results were read with the naked eye.

Based on the probe information in Table 3, the specific result of Experimental Example 1 was able to read whether rifampin or eye or susceptibility / resistance of TB bacteria in FIG. The determination of the results was determined by comparing susceptibility / resistance of rifampin (rpoB) and aina (katG, inhA, ahpC) by comparing wild-type and variable band strengths of each locus. If the strength of the wild-type band is stronger than that of the mutant-type band, it is sensitive, and if it is weak, it can be judged as resistant.

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

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

During the PCR reaction, the ITS sites of Mycobacterium tuberculosis (M. TB) and non-TB Mycobacterium tuberculosis (NTM) are amplified using a biotin-attached mycobacterial primer by an asymmetric PCR method, resulting in 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.

Subsequently, as in Experimental Example 1, a hybrid membrane, a washing, and a color reaction step are used to detect and classify mycobacteria (M.TB) or non-TB antibacterial bacteria (NTM) in a sample, and the prepared membrane strip is applied. Did.

(1) Preparation of required solution

Reagents in Table 8 were prepared to perform sample DNA extraction, PCR reaction, and backcross line assay.

(2) Sample preparation and DNA extraction

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

(3) PCR reaction

For 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 sample DNA were added, and PCR reaction was performed under the conditions shown in Table 9 below.

(4) Reverse hybrid line blot assay

The inverse hybridization line blot assay is the same as in Experimental Example 2, and is therefore omitted. The specific results of Experimental Example 2 were able to detect and distinguish each mycobacteria in the example of FIG. 4 based on the probe information of Table 4.

Those skilled in the art to which the present invention pertains will be able to make various applications and modifications within the scope of the present invention based on the above.

Claims (18)

Preparing a polyaldehyde dextran-conjugated conjugate as a nucleic acid probe and carrier,
Moving the conjugate by a vacuum transfer method to adsorb on a porous solid support, and
A method of manufacturing a porous solid support strip in which a nucleic acid probe is fixed through polyaldehyde dextran, comprising drying and fixing the porous solid support to which the conjugate is adsorbed,
In the step of preparing the conjugate, an amine group-labeled nucleic acid probe is reacted with a polyaldehyde dextran as a carrier to form an imine intermediate of the Cipro base, and a reciprocating agent reduces the Cipro base to form an irreversible bond to form a nucleic acid probe. It is performed by preparing an amine conjugate of polyaldehyde dextran,
The adsorption step is performed by adsorbing the conjugate from the solution to the porous solid support by dispensing the conjugate solution and operating the vacuum pump to inhale.
In the fixing step, the conjugate-adsorbed solid phase support is pretreated with a pre-hybridization buffer containing denhardt's solution, SSC, SDS, and denatured salmon sperm DNA, and the porous solid support adsorbed with the conjugate is dried. Thus, it is carried out by fixing the carrier of the conjugate between the holes of the porous solid support,
A method for producing a porous solid support strip in which a nucleic acid probe is immobilized through a polyaldehyde dextran as a carrier. 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 between the nucleic acid probe and the carrier is a covalent bond or affinity binding having an equivalent level of binding force. The method of claim 1, wherein the amine group is attached to the 5'-end or 3'-end of the nucleic acid probe. The nucleic acid probe is modified with a ligand capable of covalent or affinity binding to a carrier at the 5'-end or 3'-end,
The ligand is biotin (biotin), Glutathione, maltose, histidin, lectin or a method of manufacturing, characterized in that at least one selected from the group consisting of derivatives thereof. The method according to claim 5, wherein the ligand is directly attached to a nucleic acid probe. 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. 9. The method according to claim 8, wherein the synthetic oligonucleotide is composed of 16 to 22 nucleotides. 5. The method according to claim 4, wherein a spacer for increasing the efficiency of hybridization reaction is added to the internal region of the nucleic acid probe. 11. The method of claim 10, wherein the spacer is a non-specific poly T tail. The method of claim 1, wherein the porous solid support is a membrane. 13. The method of claim 12, wherein the membrane is nitrocellulose or nylon. delete The method of claim 1, wherein the vacuum movement method is to disperse the conjugate of the nucleic acid probe and the carrier to the nucleic acid slit line of the automated line blotting equipment, and operate the vacuum pump to inject the conjugate into a porous solid. Manufacturing method comprising the step of adsorbing to the support. delete delete delete

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