KR20190058439A - Method for Preparation of Nucleic Acids Probe-Coated, Porous Solid Supporter Strip - Google Patents

Abstract

The present invention relates to a method for manufacturing a nucleic acid probe-coated porous solid support strip. A method for manufacturing the same 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.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a nucleic acid probe-coated porous solid support strip,

The present invention relates to a method of preparing a porous solid support strip coated with a nucleic acid probe, and more particularly, to a method of preparing a conjugate by binding a nucleic acid probe to a carrier, and fixing the nucleic acid probe to a porous solid support by a vacuum transfer method. The present invention also relates to a method for producing a porous solid support strip coated with a nucleic acid probe.

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

In general, techniques for analyzing the sequence of a specific nucleic acid from a nucleic acid sample separated from a bacterium or a cell or a result obtained by amplifying the nucleic acid sample by a nucleic acid polymerization reaction include those having a complementary base sequence that specifically binds to a part of a nucleic acid to be analyzed And nucleic acid fragments, that is, nucleic acid probes, are used to analyze the presence or absence of nucleic acids to determine the presence or absence of nucleic acid.

At present, there is a method in which a specific nucleic acid probe is attached to the surface of a microwell plate to perform a hybridization reaction with a nucleic acid to be analyzed, a method in which a specific nucleic acid probe is immobilized on a membrane and a nucleic acid hybridization reaction is performed by injecting the amplified nucleic acid thereto / line blot hybridization), and a method in which a plurality of probes are fixed on a plate made of a material such as glass, and nucleic acid hybridization reaction is performed by injecting amplified nucleic acid (DNA chip).

To prepare the diagnostic strip used in the nucleic acid hybridization reaction as described above, a step of fixing the nucleic acid probe to the solid support is required.

Specifically, the method of attaching the nucleic acid includes a chemical bonding process including the most commonly used physical adsorption process and ultraviolet (UV) irradiation or covalent bonding (R Lutgarde et al. , Applied Environmental Microbiology, 62, 300-303, 1996).

Physical adsorption is the process of physically adsorbing biomolecules onto solid surfaces and is of great interest in in vitro diagnostics.

The obvious mechanism by which the biomolecules are adsorbed to the solid phase is unclear, but it should be accompanied by virtue of interaction with the surface, which is probably due to 1) molecular transport to the surface, 2) adsorption to the surface, 3) , 4) potential desorption of adsorbed molecules, and 5) migration of desorbed molecules away from the surface (see W Norde, Advances in Colloid and Interface Science, 25, 267-340, 1986 ).

This summary suggests that the desorption is potentially inherent, but the binding is irreversible if the molecule is sufficiently large in nature (see AW Adamson, " The Solid-liquid Interface: Adsorption From Solution ", in Physical Chemistry surface, 5th ed. (Chichester, UK: Wiley, 1990), 421-459).

This is because, as the molecular weight increases, the number of binding sites increases, so that even if one binding site dissociates on the surface, many of the remaining binding sites remain in the binding state.

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

However, a major problem with the physical adsorption method is that the nucleic acids immobilized on the surface are rearranged and may be in an inappropriate conformation to interact with any incoming base sequence.

In the case of a nucleic acid having a long base sequence, this may not be a problem. However, as the base sequence of the nucleic acid becomes shorter, the potential decrease in activity may become a serious problem. If there are some bases immobilized on the surface that do not remain free to interact with the solution, the degenerated structure will not allow the hybridization reaction to occur normally.

In addition, nucleic acid probes are relatively difficult to immobilize because of their relatively small molecular size and relatively weak physical forces (electrostatic forces, hydrophobic interactions, etc.) that can be adsorbed on porous solid supports for direct application of the vacuum transfer method .

There is a risk that the nucleic acid probe will not be uniformly coated, and if there is no additional fixing step, there is a high possibility of desorption during the assay reaction. In order to use it for a hybridization assay requiring high reproducibility and high sensitivity, There is a problem of falling. Considering these problems, a method using UV is widely used.

UV cross-linking is one of the simplest methods of covalently bonding nucleic acid probes to nylon membranes. Linkage is primarily made up of the thymine residues (other nucleotides are less frequent), which react with the amine group of the nylon membrane when the thymine residue is activated (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 hybridization reaction, so if the membrane is overexposed to UV, the UV linking eventually interferes with the hybridization reaction. Therefore, it is important to determine the UV dose correctly.

That is, exposing the membrane to a small amount of UV does not result in efficient cross-linking, and overexposure can reduce the efficiency of the cross-reaction.

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

On the other hand, the technique in which photoactivatable compounds are used in membranes or probes is an advanced cross-linking method (JK Veilleux and LW Duran, IVD Technology 2, no. 2, 26-31, 1996). The use of photoactive compounds (eg, photobiotin) can provide the probe with the correct directionality and freedom, but there are few applications because exposure to UV irradiation often results in uncontrolled reactions.

Covalent linkage is the most popular technique for immobilizing nucleic acid probes on solid support surfaces using conventional chemistry. Among the various linking chemistries used, the most common are amines, carbonyls, carboxyls, and thiols.

It is possible to control the orientation of the probe by covalent bonding and to control and attach the nucleic acid through a terminal residue with a controlled-chemistry linkage. By introducing a spacer of the correct size, It is possible to freely perform hybridization reaction with the added probe.

The covalent bond between the probe and the solid support is commonly applied in the form of a microarray system, that is, a glass slide or a bead, but is expected to be applicable 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. In the case of using 1-Ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC or EDAC) reagent, which is mainly carbodiimide, But was limited to the covalent bond between activated membranes.

Also, the technique of covalently immobilizing nucleic acid probes in a controlled attachment fashion on the membrane is inherently difficult to switch to mass production and automation processes in a batch process. It also takes a lot of time and is costly.

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

Therefore, there is a high demand for development of membrane strips in which manufacturability is ensured in order to solve the above-mentioned problems and to apply membrane-based nucleic acid tests throughout the diagnostic field.

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

Specifically, it is an object of the present invention to provide a nucleic acid probe-coated porous solid support strip in which a nucleic acid probe can be bound to a carrier to secure the directionality of the nucleic acid probe, And a part of the hybridization base sequence region of the probe which is to be complementarily bound to the target base sequence is not fixed, so that an abnormal hybridization reaction due to a decrease in activity does not occur.

It is a further object of the present invention to provide a method and apparatus for mass spectrometry that does not require the adjustment of the optimal UV dose during UV irradiation and does not utilize the batch process that is essentially required in the prior art for fixing the nucleic acid probe by chemical bonding, And a method for producing the same.

It is still another object of the present invention to provide a method for producing a nucleic acid probe which is simple, rapid, inexpensive and reproducible by mass production, and has a stable membrane surface without interfering with the interaction so as to facilitate hybridization reaction between the target base sequence and the nucleic acid probe. And a method for producing a nucleic acid-probe assay system capable of exhibiting high sensitivity.

Accordingly, a method of preparing a porous solid support strip coated with a nucleic acid probe according to the present invention comprises: preparing a conjugate by binding a nucleic acid probe to a carrier; To a porous solid support.

In-vitro Diagnostics Techniques for immobilizing nucleic acid probes on porous solid supports such as membranes for hybridization reactions for use in nucleic acid testing vary, but there are a number of factors to consider. As a result, there are advantages and disadvantages in terms of binding effectiveness and ease of manufacturing process associated with assay performance. The key is how easy it is to manufacture in large quantities at competitive prices.

In this regard, the method of preparing a porous solid support strip coated with a nucleic acid probe according to the present invention can ensure the directionality of the nucleic acid probe by binding the nucleic acid probe to the carrier, There is an effect that an abnormal hybridization reaction due to a decrease in activity is not caused since a part of the hybridization reaction base sequence region of the probe which is not arranged and complementarily binding with the target base sequence is not fixed.

In addition, as in the prior art, there is no need to adjust the optimum amount of UV irradiation, and the batch process essentially required in the chemical bonding technique is not used, so mass production and automation processes are effective.

In the present invention, the nucleic acid probe may be composed of sense (forward) and antisense (backward) nucleic acids recognizing a target gene sequence such as DNA, RNA, PNA (peptide nucleic acid)

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

The methods of immobilizing these different molecules are similar, but are actually different due to differences in the structure and size of the molecules. Since the method of immobilizing a large fragment of DNA on a solid phase is no longer used for diagnostic testing, a method of immobilizing DNA shorter than this on a solid phase is mainly used.

In the present invention, the binding of the nucleic acid probe to the carrier may be preferably a chemical bond, and the chemical bond may include covalent bonds, noncovalent bonds such as ionic bonds, hydrogen bonds, and the like. Of these, covalent bonding is more preferable.

However, a binding method using an affinity binding, which is not a covalent bond but has a strong bonding force at a covalent bonding level, is also applicable.

In connection with the affinity binding, a biotin-avidin / streptavidin immobilization technique capable of forming strong bonds with a binding constant of ~ 10 ^-15 M is most commonly used. Glutathione-GST (Glutathione S-transferase), histidine-metal ion (Co, Ni, Cu), maltose-MBP (maltose binding protein) and lectin (eg, concanavalin) -mannose / glucose binding protein Coupling technique can be utilized between the probe and the solid support.

It is therefore desirable to introduce a functional group into the nucleic acid probe for covalent binding with the carrier or to introduce a ligand to the nucleic acid probe for affinity binding with the carrier so that the base sequence of the entire nucleic acid probe can participate in the hybridization reaction .

Surface modification for covalent coupling between a nucleic acid probe and a solid support can be carried out using a carboxyl group (-COOH), a phosphate group, an amino group (-RNH ₂ , -ArNH ₂ where R is H or Various functional groups such as a C ₁ -C ₆ alkyl group), methyl chloride (-ArCH ₂ Cl), amide (-CONH ₂ ), aldehyde (-CHO), hydroxyl group (-OH), epoxy, (References: 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.

On the other hand, techniques for affinity coupling between nucleic acid probes and solid supports may be implemented by various systems such as biotin-avidin / streptavidin, Glutathione-GST, histidine-metal ion, lectin-glycoprotein, .

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

Also, the immobilization technique of nucleic acid probes is most efficient when the reaction is through the end group, so that the linking group is introduced into the 5 ' or 3 ' -active terminal group of the nucleic acid .

Thus, the 5'-terminal or 3'-terminal of the nucleic acid probe may preferably be modified to a functional group capable of covalently binding to the carrier. Thus, the step of modifying the nucleic acid probe can be performed in solution prior to nucleic acid probe production or prior to application to the membrane.

Further, in the present invention, the nucleic acid probe may be composed of, for example, 16 to 22 nucleotides as a synthetic oligonucleotide.

Specifically, in the case of a nucleic acid probe capable of detecting the tubercle bacilli and determining the resistance to rifampin and the ion drug, a nucleic acid probe targeting the Mycobacterium tuberculosis group-specific ITS (Inter Transcriptional Spacer) gene region is used for identifying tuberculosis bacteria .

To determine the susceptibility and tolerance of the anti-tuberculosis drug rifampin, a wild-type or mutant-type nucleotide sequence encoding the RNA polymerase-subunit was inserted May be used.

In order to discriminate the susceptibility and tolerance of a child, a gene coding region of katG (catalase peroxidase) and a phenotype or mutant base sequence of promoter region of inhA (enoyl acyl carrier protein (ACP) reductase) and ahpC (alkyl hydroperoxide reductase) May be used.

Meanwhile, a nucleic acid probe capable of discriminating species of Mycobacterium tuberculosis (TB) and non-tuberculous mycobacteria (NTM) can be used as a nucleic acid probe targeting the microbial genus-specific ITS (Inter Transcription Spacer) gene site.

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

Such labels include, for example, enzymes (e.g., horseradish peroxidase, alkaline phosphatase), radioactive isotopes (e.g., ³² P), fluorescent molecules, chemical groups , And biotin).

The spacer is preferably a nonspecific poly T tail and may be added after the amino group modification site. The length of the poly T tail used as a spacer may be comprised of 5 to 30 thymine bases.

For example, a nucleotide sequence of a hybridization site capable of complementarily binding to a nucleic acid sequence of a target gene by linking a poly T tail consisting of 10 thymine bases with an amino-C6 linker at the 5'- A nucleic acid probe 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 preferable, and dextran or a derivative thereof, which binds covalently or affinity to a nucleic acid probe, is more preferable.

In the case of affinity binding, avidin / streptavidin, maltose binding protein, glycoprotein, metal ion (Co, Ni, Cu etc) are ligated to the carrier and biotin, maltose, lectin, histidine Or a derivative thereof may be labeled with a nucleic acid probe. Among them, the biotin-avidin / streptavidin linking technique is more preferable.

Since dextran has multiple activation groups per molecule, dextran can immobilize several DNA probes per molecule, as well as multiple 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). Also, since dextran is a long and flexible polymer, the immobilized DNA molecules can be easily cross-reacted, and the steric hindrance due to the solid support surface can also be avoided (Reference: Goris J. et al., Can 15, p.13, 1987. Penzol G., et al., Nucleic Acids Res., 25, 1155-1161, 1999, Gingeras T. R et al. et al., Biotechnol Bioeng. 60, 518-523, 1998).

As a representative example, in the case of aldehyde-aspartic-dextran, an extremely high molecular weight substance having an anion and an aldehyde group can form a very strong covalent bond with an aminated DNA probe, Binding is very stable to the extent that it is durable to extreme pH, high temperature or high concentrations of salts, solvents, nucleophiles of the hybridization reaction mixture (see Fuentes M. et al., Biomacromolecules, 5, 883-888, 2004).

In one preferred example, the dextran may be composed of a polysaccharide which can be oxidized to form an aldehyde functional group.

For example, polydehyde dextran can be prepared by oxidizing dextran at 43 kDa, which is highly conjugated with a nucleic acid probe, compared to dextran at 500 kDa.

The polyaldehyde dextran is prepared by mixing dextran and KIO ₄ in an amount corresponding to 62.5 mmol of glucose, rotating the mixture at a temperature of 40 rpm at room temperature for 6 hours or more, adding 2 ml of sterile distilled water Dialyzed three times per hour.

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 covalently bonding with the amino group or the thiol group of the protein or the synthetic polymer, May be introduced into the nucleic acid probe.

A nucleic acid probe labeled with a polyaldehyde dextran transporter and an amine group is reacted at a pH of 11 at room temperature to form an immune intermediate of Schiff's base and reacted with a reducing agent, Stable covalent bonding can be achieved by reducing the Schiff base with sodium borohydrate, which is a gentle reducing agent, to induce irreversible bonding.

Nucleic acid is essentially non-reactive, but if sufficient energy is given, it reacts with other groups. If the nucleic acid is exposed to UV light in the presence of a photosensitizer such as methylene blue without visible light or photosensitizer, (R Lalwani et al., J Photochemistry and Photobiology 7, 57-73, 1990; JW Hockensmith et al., Method in Enzymology, 208, 211-236, 1991).

There are two major problems with producing an activated solid porous support, e.g., an activated membrane, to directly cross-link the nucleic acid.

The first problem is the loss of activity, the chemically active surface of the membrane reacting with atmospheric gases to lose activity.

The greater the chemical activity, that is, the faster the cross-linking with the target, the more rapidly the activity is lost. To overcome this problem, recently, manufacturers use a less reactive group that specifically reacts with the target probe itself. Typical examples of reactions between aldehydes and amines are both relatively stable.

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

Membranes that provide surface chemistry that can specifically bind to the probe usually provide less reactivity and are less active over time and less active when stored, with amine or aldehyde groups on the membrane surface It is dominant.

The advantage of the aldehyde-activated surface is that the binding takes place in significant amounts, primarily through the formation of Schiff's bases, without any additional reagents.

Since the Schiff base is relatively stable but can be reverted under physiological conditions, it should be reduced to a irreversible bond by reducing the Schiff base with a mild reducing agent, sodium borohydride (MB "The latex Course in Covalent Binding in Diagnostic Test Design", 2000).

The second problem is non-specific binding, and the activated membrane must be blocked before use. Otherwise, the sample is additionally bonded to the membrane. The blocking step should be simple, rapid, and very efficient, ideally blocking the formation of hydrophilic, non-volatile residues on the membrane surface.

Passivation of the membrane surface by blocking may introduce a new chemical group and thus may not be reactive with the nucleic acid. However, charge binding or detection by binding of the sample or detection system The hydrophobic attraction is increased and the nonspecific signal can be increased.

Most covalent bonds, except for disulfide bonds, are essentially batch processes, which take about 30 minutes to several hours to couple the target to the membrane followed by blocking the membrane.

These deviations cause difficulties in automation and mass production. The introduction of photoactive linking chemistry simplifies mass production, but is relatively inexpensive 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 specifically reacting with polyaldehyde dextran which is activated (oxidized) as a carrier, dextran, (EDC or EDAC) as well as other covalently bonded reagents as shown in the covalent bond between amine groups and carboxyl groups, the use of 1-Ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride The cost of using 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, but are not limited to, membranes (nitrocellulose or nylon membrane), controlled-pore glass beads, acrylamide gels, polystyrene matrices, Activated dixtran, avidin-coated polystyrene beads, and glass, and a variety of such solid supports have been applied to DNA diagnosis (see, for example, Saiki RK et al., PNAS, Acids Res., 15, 5353-5372, 1987; Rasmussen SR et al., ≪ RTI ID = 0.0 > Lund V. et al., Nucl. Acids Res., 16, 10861-10880, 1988). .

The present invention focuses on how porous target supports can be immobilized on porous solid supports among these various solid supports, especially membranes.

Membranes include a wide range of substrates that can be used for nucleic acid immobilization. Specifically, a new membrane such as a conventional molded membrane such as nitrocellulose, nylon, a ceramic or a ceramic or track-etched membrane, and a microarray And the type of substrate used.

Many membranes recommended for nucleic acid fixation should be first determined for optimal binding conditions, and in some linkage techniques nucleic acids may be added to the membrane in an uncontrolled manner.

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

The main advantage of nitrocellulose is its quick availability, but it is fragile 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 greater physical strength and binding capacity, and a wider surface chemistry range that can be optimized for attachment of nucleic acids.

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

As mentioned, the present invention solves the problems of conventional physical adsorption processes. However, from a production manufacturing standpoint, physical adsorption is very simple and robust technique (TC Tisone, IVD Technology 6, no. 3, 43-60, 2000).

The fabrication of a simple membrane-based system is easiest when the material is applied so that it can be combined upon drying in a continuous process. Fixable materials can be applied to a continuous process, where the linkage is made up of a simple, well-controlled process, namely a drying process.

Physical adsorption is, of course, useful as a technique for the mass production of nucleic acid diagnostic tests, provided that a clear problem of physical adsorption can be solved, i. E., Enhanced to allow stronger and directional bonding.

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

Most intensified physical adsorption techniques not only increase the coupling efficiency for particularly short probes, but are also arranged so that the important base sequences of the probes protrude in a direction away from the solid surface. As a result, the incoming sample and the important base sequence can freely interact. Because selectivity and sensitivity are improved, it is very effective, especially when short probes are used.

In this aspect, according to the present invention, the covalent bond has a direction in which the important base 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 is preferably a method in which a conjugate of a nucleic acid probe and a carrier is dispensed to a nucleic acid slit line of an automated line blotting apparatus, a vacuum pump is operated, May be adsorbed on a porous solid support.

The present invention also provides a porous solid support strip coated with a nucleic acid probe, which is 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 that is different from conventional ones by a special manufacturing process as described above.

As described above, the present invention provides a probe capable of securing the directionality of a nucleic acid probe by binding a nucleic acid probe to a carrier, preventing the nucleic acid from being rearranged on the surface of the solid support and complementarily binding with the target base sequence Since a part of the sequence of the hybridization reaction sequence is not fixed, there is an effect that the abnormal hybridization reaction due to the decrease of the activity is not caused.

In addition, there is no need to adjust the optimal UV irradiation amount in UV irradiation, and mass production and automation processes can be effected since the batch process, which is essentially required in the conventional technology for fixing the nucleic acid probe by covalent bonding, is not used .

In other words, mass production enables simple, rapid, and inexpensive reproducibility, and that the membrane surface can exhibit stability and high sensitivity without interfering with the interaction so as to facilitate hybridization between the target base sequence and the nucleic acid probe There is an effect that a nucleic acid-probe assay system can be produced.

FIG. 1 is a view showing a whole process of preparing a strip by conjugating a nucleic acid oligonucleotide probe to a carrier dextran in a covalent bond manner and fixing the oligonucleotide probe to a membrane by a vacuum transfer method
FIG. 2 is a photograph of a nucleic acid probe coated with a patent blue V dye, which is a blue staining solution, on a membrane strip, wherein the top is a strip capable of discriminating the susceptibility / immunity of rifampin and Iina of the example, Is a case of the class of mycobacterial genus and detectable strips in the examples
FIG. 3 is a diagram showing a resultant pattern when reverse-hybridization line blotting is performed after performing PCR on rifampin and aniso / susceptibility / resistance sample
FIG. 4 is a diagram showing a resultant pattern when PCR was performed on a mycobacterial specimen and a control (negative, positive), followed by reverse-hybridization line blotting.

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

The following experiment relates to a process of fabricating a strip by fixing a nucleic acid probe to a membrane by a vacuum transfer method. In two specific examples, the membrane strip is used to remove sputum, bronchial washing solution, cerebrospinal fluid, urine, (Ii) detection and classification of mycobacteria, ie, Mycobacterium tuberculosis (TB) and non-tuberculous mycobacteria (NTM), and (iii) ) Were tested by a reverse hybridization line blot assay. The test was carried out to verify the validity of the test.

Example 1: Preparation of polyaldehyde dextran (PAD)

(1) Preparation of required reagents and equipment

(Cut off 12000: Spectra / Por, MWCO 12-14000), hydroxyamine hydrochloride (hydroxylamine hydrochloride), dextrane (Sigma, D-4133, Mr ~ 43 kDa), KIO ₄ (Aldrich, 32242-3, FW 230.00) hydrochloride (NH ₂ OH-HCl = 69.49), methyl orange powder, 10N hydrochloric acid, pH meter, 0.1N sodium hydroxide (NaOH), freeze dryer or speed bag were used.

(2) Manufacture of oxidized dextran

0.5 g of dextran (Sigma, D-4133, corresponding to Mr ~ 43 kDa / 62.5 mmol of glucose) was completely dissolved in autoclaved DW and adjusted to a final volume of 10 ml. The KIO ₄ (corresponding to 62.5 mmol same eq) of 0.72g was added (reference:.. Azzam T et al, J Med Chem, 45, 1817-1824, 2002; Azzam T et al, Macromolecules, 35, 9947- Hosseinkhani H. et al, Gene Therapy, 11, 194-203, 2004).

And allowed to stand at room temperature for 6 to 12 hours while being rotated at a speed of 40 rpm. The oxidized dextran was placed in a cellulose dialysis membrane, dialyzed once in 2 L of sterilized distilled water, and dialyzed three times in total for 2 hours, replacing the sterile distilled water. After dialysis, the exact volume of the polyaldehyde dextran was measured.

(3) Measurement of amount of aldehyde group in oxidized dextran

The amount of aldehyde group of oxidized dextran was measured according to the method described in Huiru Zhao et al. (Pharmaceutical Research, 8, 400-402, 1991). First, a reagent of 0.25 N 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.

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

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

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

The calculation of the oxidation-reduction reaction and the degree of substitution of aldehyde in polyaldehyde dextran is shown in the following equations and the total mole number of aldehyde X contained in the total volume of polyaldehyde dextran (PAD) Respectively.

(4) Freeze-drying and stock solution preparation of polyaldehyde dextran

A small amount of polyaldehyde dextran was dispensed in 10.0 μL into the screw cap tube, and then lyophilized in O / N with SpeedVac or freeze-dryer. Stock solution was prepared by completely dissolving lyophilized polyaldehyde dextran by adding sterilized distilled water to a final concentration of 100 nmol / μl.

In the case of the following Table 2, since the total aldehyde mole of the lyophilized polyaldehyde dextran was 468.3 μmol, 468.3 μl of sterilized distilled water was added to show that 1.0 μ mol / μl of stock was completely dissolved.

(5) Preparation of 1 pmol / l of polyaldehyde dextran

100 nmol / 의 of polyaldehyde stock solution was diluted 10-fold in order and adjusted to 1 pmol / 인, which is a necessary concentration for covalent bond reaction with the nucleic acid probe.

Example 2: Preparation of nucleic acid probe

The probes used in the present experiment include (i) probes capable of detecting resistance to rifampin and a drug or resistance to drug resistance, (ii) detection and classification of mycobacteria, i.e., Mycobacterium tuberculosis (TB) (NTM) species.

First, with respect to (i) above, in the case of a probe capable of detecting resistance to rifampin and an agent for resistance to mycobacteria, a probe targeting the Mycobacterial group specific ITS (Inter Transcriptional Spacer) gene site is identified as Mycobacterium tuberculosis .

As probes for determining the susceptibility and tolerance of rifampin, an anti-TB drug, rpoB or an oligonucleotide containing a wild type or mutant type nucleotide sequence encoding a gene encoding the RNA polymerase β-subunit Respectively.

In addition, probes for discriminating isoniazid sensitivity and resistance include a gene encoding region of katG (catalase peroxidase) and a promoter region of inhA (enoyl acyl carrier protein (ACP) reductase) and ahpC (alkyl hydroperoxide reductase) A probe containing a phenotype or mutated base sequence was used.

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

Meanwhile, with respect to (ii), probes capable of distinguishing between the mycobacterial genus and the species of Mycobacterium tuberculosis (TB) and non-tuberculous mycobacteria (NTM) As in the case of the probe (i), the structure of these probes is composed of a poly-T tail spacer consisting of an amino-C6-linker and 10 thymine bases at the 5'- It is designed to be modified at the 5'-side of the nucleotide sequence.

In the following examples, probes prepared according to the purpose of the probe were not specifically shown but the dextran derivative (polyaldehyde dextran) as a carrier was subjected to a 5'-amino linker and a poly T tail spacer Lt; RTI ID = 0.0 > a < / RTI > 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 of probes for discriminating susceptibility / immunity against rifampin or iin) Probe brief information).

Example 3: Preparation of conjugate of nucleic acid probe and polyaldehyde dextran

(1) Preparation of necessary reagent

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

(2) Preparation of master mixture for conjugation reaction

Master mixture was composed of polyaldehyde dextran (1 pmol / l), 0.5M sodium borate buffer (pH 11.0) and sterilized distilled water.

(3) Conjugation reaction

To prepare 40 membranes having a standard 16.7 cm x 5.5 cm line width and 0.8 mm line width per line, 48.00 l of a 100 lm probe solution and 4752.00 l of sterilized distilled water were mixed to give a final volume of 4800.00 l. 11200.00 [mu] l of the mastering mixture for conjugation in Table 5 was added and mixed, and O / N reaction was carried out while rotating at 40 rpm at room temperature.

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

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 of polyaldehyde dextran-probe imine conjugate total volume was added in total for 2 hours for 1 hour every 1 hour. After the addition of the reducing agent, the reaction was carried out by rotating at 40 rpm at room temperature.

Example 4: Fabrication of Nucleic acid probe-coated membrane strip

(1) Automatic line blotter setting

A line blotting machine with a slit of approximately 0.8 mm for one membrane (16.7 cm x 5.5 cm) was self-manufactured and the top and bottom plates were sonicated by a washing process before use.

The line blotting equipment was mounted so that the top plate and the bottom plate were in good contact with each other, washed several times with sterilized distilled water several times and 1X PBS, and the conjugate was prepared for automatic dispensing.

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

Patent blue V dye (5000X = 3g / 500ml), a blue staining solution, was mixed with 1X PBS at an appropriate concentration. The dye was added to the membrane to ensure that the line was evenly drawn. For each line (each probe), 400 μl of PAD-nucleic acid probe conjugate was mixed with 600 μl of 1 × PBS (containing patent blue V dye) to prepare a total of 1 ml.

The membrane used in this example was a Pall Biodyne B membrane, and 3MM paper was cut to meet the specifications of the slit blotting equipment. One 3MM paper was laid on the bottom plate of the slit blotting machine, the membrane was covered, and the top plate was attached to the membrane. The solution dispensed in the slit was washed with 1X PBS solution containing patent blue V dye Were tested by dividing them first.

In this method, 3MM paper was placed on the lower plate of the line brothting equipment, and the Biodyne B membrane prewetted in 1X PBS was covered thereon. Then, the plate was tightly bonded to the upper plate to prepare the 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 was adsorbed onto the Biodyne B membrane by vacuuming the vacuum pump at a speed of 150 for 5 seconds and then air-dried to enhance the physical adsorption. The desiccator was then pre-cross- lt; RTI ID = 0.0 > prehybridization < / RTI > buffer.

(3) Pretreatment of membrane with pre-hybridization reaction buffer solution

In this example, the membrane-stripping pretreatment of the pre-hybridization reaction buffer to the membrane was performed to secure the rapidity of the nucleic acid hybridization assay and to increase the signal-to-noise ratio (S / N ratio).

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

(4) Making a strip attached to a housing in a membrane

The dried membrane was attached to an adhesive backbone (size: 6 cm length, transparency: 0.5 cm) on the opposite side of the coated surface, and cut at a width of 3.4 mm. The backbone is made of an adhesive film on both sides and is attached to a housing that can store the strips of the membrane using the opposite side of the backbone side to which the membrane is attached.

When attaching the membrane strip to the housing, pressing with a roller of rubber may cause the coated probe to fall off, so it is pressed with soft 3MM paper or a sponge so that the membrane strip adheres well to the housing.

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

Experimental Example 1: Reverse-hybridization line blot assay for detection of rifampin / iNA drug susceptibility / resistant Mycobacterium tuberculosis

A concrete principle of this experimental example will be described 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 promoter region of rpoB (RNA polymerase ㅯ -subunit), katG (catalase peroxidase) gene coding region and inhA (enoyl acyl carrier protein (ACP) reductase) and ahpC (alkyl hydroperoxide reductase) (Biotin-labeled, single-stranded target DNA) by amplification with each target gene-specific biotin-adherent primer.

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

During the reverse-hybridization reaction, each biotinylated label single-stranded target DNA was hybridized with a nylon membrane-adsorbed probe (rpoB, katG, inhA, ahpC wild-type or mutant probe).

After the hybridization reaction was completed, the drug-resistant Mycobacterium tuberculosis detection strip was subjected to a stringent wash process and then subjected to enzyme conjugation reaction to obtain a streptavidin-alkaline phosphatase (AP) conjugate Conjugated biotin-labeled target with a membrane-adsorbed oligonucleotide probe and a control ampullicon.

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

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

In this case, since NBT also produces dark blue dye as an oxidizing agent, the blue color becomes thicker and the detection sensitivity can be increased.

The pattern of the blue colored band was compared with the test reference guide / reading card and the result was interpreted. Also, using AdvanSure ™ GenoLine Scan, an automatic analysis equipment of LG Life Sciences, Can distinguish.

(1) Preparation of necessary solution

Sample DNA Extraction, PCR Reaction, Reverse Dissociation Line Blot Assay In order to carry out the experiment, the reagents of Table 6 below were prepared.

(2) Preparation of sample and DNA extraction

Sputum of patients with tuberculosis or 1-2 ml of standard culture medium and 1N NaOH in the same volume of 1N NaOH were added to a 15-ml tube and thoroughly stirred and centrifuged at 4,000 rpm for 20 minutes.

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

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

(3) PCR reaction

For the PCR reaction, 12.5 ㎕ of the 2X PCR mixture in Table 6, 5 프 of the primer mixture and 7.5 시 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 culture, Mycobacterium tuberculosis)

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

(4) Reverse hybridization line blot assay

A reverse cross line blot assay was performed using the reagents of Table 6 above. 20 μl of the PCR reaction product and 250 μl of a pre-warmed hybridization buffer were added to prepare a hybridization reaction mixture. The mixture was dispensed into the membrane probe adsorption strip and hybridized with a hybridization chamber / oven ) And subjected to hybridization with stirring for 30 minutes (9 rpm for vertical agitation and 80 rpm for horizontal agitation).

After the hybridization reaction, the reaction solution of the probe adsorption strip was removed by vacuum suction (aspiration), and 250 쨉 l of pre-warmed stringent wash solution was dispensed into the strip, and the mixture was vertically stirred at 55 rpm for 15 minutes at 9 rpm The reaction was carried out and washed. After removing the reaction solution from the washed strips, 250 μl of a rinse solution was dispensed into the strips, and the strips were washed by performing a vertical stirring reaction at 9 rpm for 1 minute at room temperature.

The reaction solution of the washed strip was removed. The enzyme conjugate solution was used after being left at room temperature for 10 minutes or more. 250 μl of the enzyme conjugation solution was dispensed into the strip, and the reaction mixture was subjected to a vertical stirring reaction at 9 rpm for 30 minutes at room temperature. Respectively.

To rinse again, 250 l of a rinse solution was dispensed into the strip and washed by a vertical agitation reaction at 9 rpm for 1 minute at room temperature. After removing the reaction solution from the washed strips, 250 μl of sterilized distilled water was dispensed into the strips and subjected to a vertical stirring reaction at 9 rpm for 1 minute at room temperature.

After removing the reaction solution from the washing strip, the color development reaction was carried out. The coloring solution was used after being left at room temperature for 10 minutes or more. 250 μl of a coloring solution was dispensed into strips, and then subjected to a vertical stirring reaction at 9 rpm for 10 minutes at room temperature under light-shielding conditions .

After the reaction solution of the color-developed strip was removed, it was rinsed with 250 占 퐇 of sterilized distilled water to completely remove the remaining coloring solution and the precipitate. Membrane strips can be dried quickly using a hair dryer or air dried in the air and then analyzed using the AdvanSure ™ GenoLine Scan program from LG Life Sciences or a test reference guide (Reading Card) or Data Interpretation sheet The results were read visually.

Based on the probe information in Table 3, the specific results of Experimental Example 1 were able to be interpreted in FIG. 3 as to whether susceptibility / tolerance of rifampin or Iina to Mycobacterium tuberculosis. The results were evaluated by comparing the susceptibility / resistance to rifampin (rpoB), aina (katG, inhA, ahpC) against wild type and mutant band intensities of each locus. If the intensity of the wild type band is stronger than the mutated type band intensity, it is susceptible, and if it is weak, it can be judged to be resistant.

Experimental Example 2: Reverse Dissociation Line Blot Assay for 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 method using a one-tube nested multiplex asymmetric PCR method was applied.

During the PCR reaction, the ITS region of M. tuberculosis (M. TB) and non-tuberculous antimicrobial bacteria (NTM) was amplified using an asymmetric PCR method using a biotin-adhered mycobacterial primer to obtain a final biotin-labeled single stranded target DNA biotin-labeled, single-stranded target DNA, and at the same time a PCR amplification control was also generated.

Then, as in Experimental Example 1, the membrane strips thus prepared were subjected to hybridization, washing, and coloring reaction steps to detect and differentiate mycobacteria (M. tuberculosis (M.TB) or non-tuberculous mycobacteria (NTM) Respectively.

(1) Preparation of necessary solution

In order to perform sample DNA extraction, PCR reaction, and reverse-hybridization line blot assay, the reagents of Table 8 below were prepared.

(2) Preparation of sample and DNA extraction

Preparation of the sample and DNA extraction are the same as in Experimental Example 1, and thus are omitted.

(3) PCR reaction

The PCR reaction was carried out by adding 12.5 2 of the 2X PCR mixture in Table 8, 5 프 of the primer mixture and 7.5 시 of the sample DNA.

(4) reverse crossing line blot assay

The reverse-hybridization line blot assay is the same as in Experimental Example 2 and is 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 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 (18)

A nucleic acid probe and a conjugate wherein the polyaldehyde dextran is coupled as a carrier,
Moving in the conjugate vacuum transfer mode and adsorbing to the porous solid support, and
A method for preparing a porous solid support strip having a nucleic acid probe immobilized thereon via polyaldehyde dextran, comprising drying and fixing the porous solid support on which the conjugate is adsorbed,
The step of preparing the conjugate comprises reacting a nucleic acid probe labeled with an amine group with a polyaldehyde dextran as a carrier to form an imine intermediate of the Schiff base, reducing the Schiff base with a reducing agent to form irreversible bonds, An amine conjugate of polyaldehyde dextran was prepared and performed,
The adsorption step is performed by sucking the conjugate from a solution to a porous solid support in a suction manner by dispensing the conjugate solution and operating a vacuum pump,
Wherein the securing step is performed by fixing the carrier of the conjugate between the apertures of the porous solid support.
Wherein the nucleic acid probe is immobilized via polyaldehyde dextran as a carrier. The method according to claim 1, wherein the nucleic acid probe is at least one selected from DNA, RNA, and PNA. The method according to claim 1, wherein the binding of the nucleic acid probe to the carrier is an affinity binding having a covalent bond or an equivalent level of binding force. The method according to claim 1, wherein the amine group is attached to the 5'-terminal or 3'-terminal of the nucleic acid probe. The nucleic acid probe according to claim 1, wherein the nucleic acid probe is modified to a ligand capable of covalent binding or affinity binding with a carrier at a 5'-terminal or 3'-terminal,
Wherein the ligand is at least one selected from the group consisting of biotin, glutathione, maltose, histidine, lectin or derivatives thereof. 6. The method of claim 5, wherein the ligand is attached directly to the nucleic acid probe. 2. The method of claim 1, wherein the nucleic acid probe comprises a label that is detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunological or chemical means. 2. The method of claim 1, wherein the nucleic acid probe is a synthetic oligonucleotide. 9. The method of claim 8, wherein the synthetic oligonucleotide is comprised of 16 to 22 nucleotides. 5. The method of claim 4, wherein a spacer is added to the internal region of the nucleic acid probe to increase the efficiency of the hybridization reaction. 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. The method of claim 1, further comprising the step of physically adsorbing the conjugate between the carrier and the orifice of the porous solid support by drying. The vacuum transfer method according to claim 1, wherein the vacuum transfer method comprises: dividing a nucleic acid probe and a carrier conjugate into a nucleic acid slit line of an automated line blotting apparatus; and operating the vacuum pump to suction the conjugate into a porous solid Wherein the adsorbent is adsorbed on a support. 2. The method of claim 1, comprising pre-treating the solid support with a pre-crossbased reaction buffer. 17. The method of claim 16, wherein the pre-hybridization reaction buffer is a composition comprising denhardt's solution, SSC, SDS and denatured salmon sperm DNA. 17. A porous solid support strip coated with a nucleic acid probe, which is produced by a process according to any one of claims 1 to 17.

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