KR100758611B1 - Multi-Chamber Reaction Device for Nucleic acid Amplification and Assay - Google Patents

Abstract

The present invention relates to a multi-chamber reaction apparatus for nucleic acid amplification and nucleic acid analysis reaction of trace samples of 10 μl or less, and specifically, a plurality of reaction chambers separated for containing biological sample solutions and an isolation formed between the reaction chambers. A biological sample comprising a reaction block consisting of a groove, a transparent sealing cover for sealing the reaction chamber on the reaction block, and a dried nucleic acid amplification reagent attached to the bottom of the reaction chamber and a dried nucleic acid analysis reagent. A multi-chambered reaction apparatus for nucleic acid amplification and nucleic acid assays of solutions. The reactor of the present invention is suitable for reacting a small amount of sample, and because the thickness of the reactor is very thin and the heat capacity is small, the heating and cooling rate of the reactor is very fast. The light to be measured can be easily measured.

Reactor, reaction chamber, PCR, sealing cover, nucleic acid amplification

Description

Multi-Chamber Reaction Device for Nucleic acid Amplification and Assay

1 is an overall perspective view of the present invention.

2 is a plan view and a cross-sectional view taken along the A and A 'line of the reaction block of the present invention.

3 is a plan view and a right side view of the sealing cover of the present invention.

Figure 4 is a diagram showing the results of polymerase chain reaction according to the present invention.

5 is a diagram showing the results of polymerase chain reaction according to the present invention.

<Brief description of the main parts of the drawing>

10: reaction block 11: reaction chamber

12: Containment Home 13: Border

21: sealing cover 30: dried nucleic acid amplification reagent and nucleic acid analysis reagent

The present invention relates to a multi-chamber reactor for use in performing nucleic acid amplification and nucleic acid analysis reactions of trace biological samples.

Recently, a real-time PCR method has been developed that can be monitored in real time while performing polymerase chain reaction. This method does not require electrophoresis on the gel, can identify amplification products during the reaction cycle, and has the advantage of obtaining quantitative results. The apparatus used for the real-time PCR is a device incorporating a thermal cycler (PCR) for the PCR reaction and a fluorometer for the real-time detection of the reactants. In general, fluorescence detection using a fluorescent reagent is used for real-time PCR monitoring. This allows quantitative analysis of the product by simultaneously measuring the fluorescence generated from the PCR product while performing PCR. Recently, when the real-time PCR is performed, the amount of reaction sample is generally used in 10 to 50 µl. If the reaction sample can be used in a small amount, the amount of reagents required for the reaction is reduced, which is a great economic benefit. In order to detect the fluorescence generated at the same time while PCR reaction of such a small amount of sample, the reaction vessel should be small to fit the sample amount. In order to react such a microsample, U.S. Patent No. 5948673 proposes a reactor called a 'film reactor' or a 'DNA card', which is a very thin film of three layers. The lower layer forms the bottom of the reactor, the middle layer forms the side of the reactor, and the upper layer forms the sample inlet. However, after injecting a small sample solution through the pipette into the DNA card reactor, the reaction inlet should be completely sealed. Otherwise, all reaction solution will evaporate during PCR reaction. However, it is very difficult to seal the reaction inlet of the film reactor. When the adhesive tape is attached, the reaction solution flows out due to the property of the film and the pressing force during the adhesion, which makes it difficult to completely seal the adhesive tape because it is not attached to the reactor. In this case, when bubbles enter, fluorescence measurement becomes difficult due to light scattering. In addition, due to the nature of the thin film, the sample solution is easily mixed with the solution in the other chamber, and the liquid is removed from the reactor due to the liquid when reacting at a high temperature. Therefore, the sealing of the reactor during the PCR reaction is easy and a sealing cover with good sealing effect is required. In addition, a reactor in which a plurality of reaction chambers are formed in a concave shape on a flat block is presented. In such a reactor, bubbles are generated at a high temperature during a PCR reaction, and light is scattered during the actual fluorescence measurement, making it difficult to measure, and bubbles overflow the solution. Transfer to another reaction chamber will result in contamination. Therefore, there is a need for a transparent reaction vessel that must be isolated so as not to be contaminated between the reaction chambers and has a good sealing effect and can measure light generated from the reactants in real time.

Therefore, the reaction and analysis are performed simultaneously as in the real-time PCR apparatus, using a trace or a very small amount of sample, suitable for measuring the light generated during the reaction in real time, easy to seal, and very little risk of contamination There is a demand for a reaction apparatus for a sample.

It is therefore an object of the present invention to provide a multichamber reactor for trace samples suitable for reacting trace samples of 10 μl or less.

Another object of the present invention is to provide a multi-chambered reactor with low heat capacity and good heat transfer rate capable of performing cooling and heating very quickly.

Still another object is a multi-chambered reactor equipped with a sealing cover that can accurately measure the fluorescence generated from the reaction product due to the perfect sealing of the reaction chamber during or after the reaction to eliminate scattering and disturbance of light caused by bubbles during fluorescence measurement. To provide.

Another object of the present invention is to provide a multi-chamber reactor that can be suitably used for various biological reactions, ie, nucleic acid amplification reactions, ligand chain reactions (LCRs), hybridization reactions, and hybridization probe reactions.

In order to achieve the above object, the present invention seals the reaction block and the reaction chamber made of a separation groove formed between a plurality of separate reaction chamber and a plurality of reaction chamber for containing the reaction sample solution, To provide a multi-chamber reactor for performing nucleic acid amplification and nucleic acid analysis of a biological sample solution consisting of a transparent sealing cover.

The present invention further comprises a dried nucleic acid amplification reagent and a dried nucleic acid analysis reagent attached to the bottom of the reaction chamber.

In the present invention, the reaction chamber is suitable for reacting 1 to 20 μl of sample.

In the present invention, the sealing cover is transparent and is made of a material having elasticity and elasticity having heat resistance of 100 ° C. or higher.

In addition, the reaction block of the present invention is made of aluminum coated with plastic or polymer having a thickness of 1 to 8 mm.

The reaction block of the present invention is formed with a rim of a certain height to prevent leakage of the sample solution.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Figure 1 shows an overall perspective view of the present invention, the reactor of the present invention consists of a reaction block 10 and the sealing cover (21). A plurality of reaction chambers 11 are formed in the thin block 10. The reaction chamber 11 is formed in a constant arrangement. The reaction chamber may be cylindrical, square or conical. In the present invention, the outer diameter of the reaction chamber is about 2 to 4 mm, preferably about 2 to 2.5 mm, and the height from the bottom of the chamber to the top of the chamber is about 0.5 to 5 mm, preferably about 1 to 3 mm (see FIGS. 2 and 3). . Thus, the reaction chamber of the present invention is suitable for reacting trace samples. In the present invention, 24 reaction chambers are provided on the reaction block. The number of reaction chambers can be adjusted.

An isolation groove 12 is formed between each reaction chamber 11. The chamber and the chamber are separated from each other by forming a recessed recess between the chamber and the chamber. In the present invention, since the isolation groove is formed between the chambers, even if the sample overflows during the reaction, the sample does not enter the other reaction chamber, so there is no risk of contamination.

Edge 13 is formed at the edge of the reaction block 10 of the present invention. The height of the rim was 1 to 5mm, preferably 2 to 2.5mm from the top of the reaction block. The edge is formed at the edge of the block so that when the sample overflows from the reaction chamber, the sample solution does not flow out of the reaction block. Even when the reactor of the present invention is mounted and used in a device such as a PCR device, there is no risk of leakage of a sample solution out of the reactor, and thus there is no danger of damaging the PCR device.

At the edge of the reaction block of the present invention, any display means for indicating the position may be formed. In the present invention, the hole 14 is formed in the diagonal corner so that the direction of the block can be known.

As shown in FIG. 3, the present invention includes a sealing cover 21 for sealing a reaction chamber formed on the reaction block. The sealing cover is made of transparent material. In the case of using a transparent material, the injected reaction sample can be checked visually, and the light generated from the reaction chamber covered with the sealing cover during the reaction can be measured in real time. When carrying out the PCR reaction by mounting the reactor of the present invention to the real-time PCR device, the fluorescence generated during the reaction can be easily detected by the fluorescent detector of the PCR device through the sealing cover. The material of the sealing cover is preferably made of transparent silicone, transparent, urethane, transparent PVC, or a mixture thereof, etc., in order to facilitate the sealing of the reaction chamber. It is good. In addition, the portion in which the sealing cover and the reaction chamber are in contact may form a protrusion 22 to fit the reaction chamber in order to increase the sealing effect. When sealing the reaction chamber with the projection cover formed projections are inserted into the reaction chamber is a complete sealing. The sealing cover of the present invention seals the reaction chamber so that no bubbles or sample solution leak out during the reaction. When the reactor of the present invention is mounted on a PCR device, a conventional PCR device is provided with a means for pressing the upper part after mounting the reactor. Therefore, the reactor of the present invention has an excellent sealing effect due to the sealing cover when used in a PCR device. The size of the sealing cover is preferably fitted into the rim 13 of the reaction block (10). In the embodiment, the thickness of the sealing cover was 0.5 to 2 mm, preferably about 1 to 1.5 mm, and the thickness of the protrusions was about 0.4 to 0.6 mm from the cover surface.

As the material of the reaction block 10 of the present invention, aluminum, plastic, or the like may be used. In the case of aluminum, they may be used by anodizing them or by coating a thin film such as a polymer. As the polymer used for coating, teflon, polycarbonate, polyethylene terephthalate, polybutylene terephthalate, polypropylene, polyethylene, polyamide, polyester resins and modified resins thereof may be used. The reaction block 10 is preferably in a color that can prevent diffuse reflection of light during the reaction, it is preferable that the opaque color, such as a black system. In the present invention, the reaction chamber 11, the isolation groove 12 and the rim 13 are preferably formed in a block made of aluminum, and then anodized and coated with a black Teflon membrane to be used as a reaction block. It was. In this case, light or fluorescence generated from the reaction sample is reflected only to the upper portion of the reaction chamber and is not transmitted to other parts of the reaction chamber.

The reactor of the present invention may further include reaction reagents required for a desired analysis in the reaction chamber, including amplified nucleic acid amplification and nucleic acid analysis reagents. Nucleic acid amplification reagents and nucleic acid analysis reagents (30) preferably used in the present invention are various polymerase chain reaction (PCR), ligand chain reaction (LCR), oligonucleotide ligation assay, hybridization assay (hybridization) As an assay, it may mean a reaction reagent commonly used in various probe assays. Specifically, as a nucleic acid amplification reagent, a reagent necessary for nucleic acid amplification is a solution consisting of a buffer solution, four types of dNTPs, and a nucleic acid polymerase. A nucleic acid analysis reagent is a reagent that can monitor the reaction process during a nucleic acid amplification reaction. Fluorescent reagents have been used as reagents for generating light that can be detected. When using a fluorescent reagent it is possible to observe the reaction state through the fluorescence generated during the reaction. Nucleic acid assay reagents include, for example, SybrGreen (10X, Molecular probe, USA Oregon) embedded in double-stranded DNA. SybrGreen is a reagent that fluoresces at a particular wavelength by inserting double-stranded DNA that is amplified during nucleic acid amplification. These nucleic acid amplification reagents and nucleic acid assay reagent solutions are placed in a reaction chamber, dried, and stored until reaction. In the reaction, put the sample solution to be analyzed by using a pipette into a reaction chamber containing the dried reaction reagent, seal it with a sealing cover, and then perform the reaction.

The size of the entire reaction block of the present invention was about 68mm in width, about 34mm in length, and the height from the bottom of the block to the top of the reaction chamber was 1 to 8mm, preferably 2 to 4mm. The size of the reaction block, the thickness of the reaction block, the number of reaction chambers, the shape and capacity of the chamber, the thickness of the sealing cover is adjustable according to the user's intention.

The multi-chambered reactor of the present invention is suitable for reacting a small amount of sample, is suitable as a multi-chambered reactor for detecting in real time the fluorescence generated from the sample during the reaction, and is used for mounting on a real-time PCR apparatus It is convenient to

Experimental Example PCR Reaction Using the Present Invention

PCR reaction was carried out using the reactor of the present invention. PCR reaction and detection were carried out as follows.

The polymerase used in this experiment was a Taq DNA polymerase expressed by Thermus aquaticus (strain YT1) polymerase gene. One unit of Taq DNA polymerase used at this time is the amount of enzyme required to convert 10 nmole of dNTPs into an insoluble form in acid solution for 30 minutes at 72 ° C. Additional reagents used were dilution SybrGreen (10X, Molecular probe, USA Oregon), reaction buffer [(10X), (100 mM Tris-HCl, 400 mM KCl, 15 mM MgCl ₂ , pH 9.0)], dilution Buffer [20 mM Tris-HCl, 100 mM KCl, 0.5 mM EDTA, 1 mM DTT, 0.5% Tween 20, 0.5% IGEPAL CA-630 (pH 8.0)], dNTPs (2.5 mM each) and storage buffer [20 mM Tris -HCl, 100 mM KCl, 0.5 mM EDTA, 1 mM DTT, 0.5% Tween 20, 0.5% IGEPAL CA-630 (pH 8.0)] was used Taq DNA polymerase containing 5000 units / mL. In addition, the DNA used in the real-time PCR experiments includes E. coli genomic DNA (unknown concentration), E. coli genome specific forward primer (10 pmoles / uL), E. coli genome specific backward primer (10 pmoles / uL). Was used. At this time, the reagent storage temperature was kept constant at -20 ℃.

Using the reagents mentioned above, 2 uL of 10x reaction buffer, 2 uL of 10 mM dNTP (2.5 mM / each), 0.2 uL of Taq DNA polymerase 1 unit (5 unit / uL), and 1 uL of the fluorescent reagent SybrGreen (10X) were added to the reaction chamber. And then lyophilized. After lyophilization, it was stored at room temperature or -4 ° C until reaction. In the reaction, 10 ul of genomic DNA (unknown concentration), 2 uL of forward primer (10 pmoles / uL), 2 uL of backward primer (10 pmoles / uL) and distilled water were added to the lyophilized composition. uL to mix.

PCR circulating conditions used for real-time PCR proceeded with initial denaturation at 94 ° C for 30 seconds, and denaturation, annealing and extension at 94 ° C, 57 ° C, and 72 ° C for 10 seconds each. A total of 35 cycles were performed. After all cycles were completed, all PCR reactions were completed by standing at 72 ° C. for 30 seconds.

The reaction was carried out under the PCR cycling conditions mentioned above by covering the sealing cover with 8 uL of the PCR reaction product prepared in the reactor (6 x 4 well, 24 well) of the present invention in a real-time PCR device of Bioneer. 10 μL of distilled water was added instead of 10 μL of E. coli genomic DNA as a control.

As shown in Figs. 4 and 5, the amount of PCR reaction products amplified using E. coli specific primers increases with increasing cycle number, whereas the amount of PCR reaction products in the control group is delayed. You can see the increase. In FIGS. 4 and 5, brown and purple color represent amplification products amplified by PCR using E. coli genomic DNA, and green and sky colors are the same reaction using distilled water (DW) instead of E. coli genomic DNA as a control. The results obtained are shown.

Since the multi-chamber reactor of the present invention has a thin thickness and uses a small amount of sample, the heat capacity is low, and thus the cooling and heating of the reactor can be quickly performed. Its low cost allows for mass production, making it suitable for single use. In addition, since the reactor is suitable for measuring the light generated during the reaction in real time, it is suitable for use in a real-time PCR device.

Claims (8)

The reaction consists of a plurality of separate reaction chambers for containing biological sample solution and isolation grooves provided between the reaction chambers to prevent the sample solution from mixing between the reaction chambers, and made of aluminum coated with plastic or polymer. A block; A multi-chamber reactor for performing nucleic acid amplification and nucleic acid analysis of a biological sample solution, characterized in that it comprises a transparent sealing cover for sealing the reaction chambers on the reaction block. The multi-chambered reactor of claim 1, wherein the reaction chamber has a capacity of 1 μl to 20 μl. The multi-chambered reactor of claim 1, wherein the sealing cover is made of an elastic material having a heat resistance temperature of 100 ° C or higher. The multi-chambered reactor according to claim 1, wherein an edge of a predetermined height is formed at an edge of the reaction block. delete According to claim 1, wherein the reaction block is a multi-chamber reactor, characterized in that having a thickness of 1 to 8mm. The multi-chambered reactor according to claim 1, further comprising a dry nucleic acid amplification reagent and a dry nucleic acid analysis reagent attached to the bottom surface of the reaction chamber. The reactor according to claim 7, wherein the nucleic acid assay reagent is a fluorescence assay reagent for nucleic acid analysis.

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