KR20180002949A - A method to prepare nucleic acid sample using a stationary liquid phase lab-on-a-chip - Google Patents

Abstract

The present invention relates to a method of preparing a nucleic acid sample by using a stationary liquid-phase lab-on-a-chip, wherein the stationary liquid-phase lab-on-a-chip comprises a sample chamber, a lysis chamber, an elution chamber, and a channel connecting the sample chamber, the lysis chamber, and the elution chamber. Specifically, the method comprises the steps of: adding a sample containing a cell and a cell capture particles (CCP) to the sample chamber to form a cell-CCP complex; transporting the cell-CCP complex to the lysis chamber, and dissolving the cell to release nucleic acids from the cell; capturing the generated nucleic acids by using a nucleic acid capture particle (NCP) to form a nucleic acid-NCP complex; and transporting the nucleic acid-NCP complex to the lysis chamber to dissolve the nucleic acids.

Description

Technical Field [0001] The present invention relates to a method for preparing a nucleic acid sample using a lab-on-a-chip and a stationary liquid phase lab-on-a-

The present invention relates to a method for preparing a nucleic acid sample using a stationary liquid phase wrap-on-a-chip, and more particularly to a method for producing a nucleic acid sample in a stationary liquid phase wrap-on-a-chip.

The process of nucleic acid sample preparation, which extracts and purifies nucleic acid from samples such as blood, tissues, cultured cells, and food, includes genotyping, transcriptional analysis, epigenetic analysis, virus / virus / bacterial detection). Although the method of analyzing nucleic acid has been rapidly developed, the method of producing a nucleic acid sample is the biggest obstacle to the analysis process because there is no great development.

Generally, a nucleic acid sample preparation method uses solid phase extraction (SPE). In the solid-phase extraction method, the nucleic acid-containing cells are first dissolved in a sample to bind the nucleic acid to a silica membrane or a silica-coated magnetic particle, washed several times, To elute the nucleic acid from the culture medium.

However, the above-described method is still complicated, takes a lot of time, requires a variety of devices, and is difficult to apply to on-site inspection. In addition, there is an automatic apparatus that makes the nucleic acid sample manufacturing process easy, but it is expensive and difficult to use in the field.

Sur et al. Developed a cartridge composed of a lysis well, in which dissolution and nucleic acid capture takes place, and an elution well, in which nucleic acid is eluted as shown in Fig. 1 for the preparation of nucleic acid samples (Journal of Molecular Diagnostics, Vol. 12, No. 5, -628).

In the above-described method, the magnetic particles and the sample to be captured with the nucleic acid are added to the dissolution well together with the dissolution / capture buffer solution, the buffer solution for elution is added to the dissolution well, and the two wells are covered with the liquid wax. The liquid wax serves to connect the two wells so that the magnetic particles can pass, and is also cleaned when the magnetic particles pass through the liquid wax. Therefore, the nucleic acid sample can be prepared by moving the magnetic particles from the dissolution well to the eluting well by the movement of the magnet.

However, the above-described methods for producing nucleic acid samples have some problems.

More specifically, first, when a sample having a large amount of residue such as food or feces is directly put into a dissolution well, there is a high possibility that the magnetic particles are contaminated by other substances and contaminated. It may not be possible. In this case, it is preferable to capture only the cells and separate them from the impurities before use.

Second, since the user must complete the cartridge by filling the sample with the liquid wax and covering the stomach with the liquid wax, it is inconvenient to use and it is difficult to expect a certain result and it is difficult to automate. If possible, the user should load the sample and start the rest of the process automatically.

Third, the movement of the magnet is complicated and costly due to the two directions of vertical and horizontal directions. In order to move the magnet, an actuator is required. The actuator for moving in two directions in comparison with the movement in one direction is complicated in structure and increases in cost.

Recently, a technique of performing immunoassay by moving solid phase particles in a stationary liquid phase wrap-on-a-chip (Patent No. 10-1398764) has been proposed. On the other hand, the stationary liquid phase wrap-on-a-chip has the advantages that the spaces in which the different processes are performed and the channels connecting the spaces are arranged horizontally, so that the particles can be moved by the horizontal movement of the magnets.

However, a method for producing a nucleic acid sample in the above-mentioned stationary liquid phase wrap-on-a-chip has not been developed.

1. KR registration No. 10-1398764

1. Journal of Molecular Diagnostics, Vol. 12, No. 5, pp620-628

The present invention provides a method for producing a nucleic acid sample using a stationary liquid phase wrap-on-a-chip.

The present invention relates to a method and apparatus for analyzing a sample using a stationary liquid phase wrap-on-a-chip composed of a sample chamber, a lysis chamber, an elution chamber and channels connecting the sample space, Forming a cell-CCP complex by adding a sample containing cells and a cell capture particle (CCP) to the space; Transferring the cell-CCP complex to a dissolution space, and dissolving the cell to release nucleic acid from the cell; Capturing the resulting nucleic acid with a nucleic acid capture particle (NCP) to form a nucleic acid-NCP complex; And transferring the nucleic acid-NCP complex to an elution space to elute the nucleic acid; The present invention provides a method for producing a nucleic acid sample using a stationary liquid phase wrap-on-a-chip.

The method for producing a nucleic acid sample using the lab-on-a-chip according to the present invention can produce a nucleic acid sample easily and rapidly from a sample containing a large amount of impurities such as food or feces.

The method for preparing a nucleic acid sample using the lab-on-a-chip according to the present invention is capable of quickly and easily separating a nucleic acid from a cell without using special equipment from a small amount of sample.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a view showing a cartridge for manufacturing a nucleic acid sample of the prior art.
FIG. 2 is a diagram illustrating a procedure of a nucleic acid sample preparation method in a stationary liquid phase wrap-on-a-chip according to an embodiment of the present invention.
FIG. 3 further illustrates a PCR mixture storage space containing a dried PCR mixture in accordance with an embodiment of the present invention. The PCR mixture storage space is separated from the elution space through a rotatable valve Lt; / RTI > shows a still liquid phase wrap-on-a-chip.
4 is a view showing the shape and size of the stationary liquid phase wrap-on-a-chip used in the embodiment of the present invention.
FIG. 5 is a graph showing the results of performing PCR with the bacterial DNA samples prepared in the examples of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. Prior to this, terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms, and the inventor should appropriately interpret the concepts of the terms appropriately It should be interpreted in accordance with the meaning and concept consistent with the technical idea of the present invention based on the principle that it can be defined.

Therefore, the embodiments described in this specification and the configurations shown in the drawings are merely the most preferred embodiments of the present invention and do not represent all the technical ideas of the present invention. Therefore, It is to be understood that equivalents and modifications are possible.

FIG. 2 is a flowchart illustrating a method of preparing a nucleic acid sample in a stationary liquid-phase wrap-on-a-chip according to an embodiment of the present invention. FIG. 3 is a flow chart of a PCR Wherein the PCR mixture storage space is further separated from the elution space through a rotatable valve, Figure 4 is a schematic diagram of a stationary liquid phase wrap-on-a- FIG. 5 is a graph showing a result of performing PCR with the bacterial DNA sample prepared in the example of the present invention. FIG.

Hereinafter, a method for producing a nucleic acid sample using the stationary liquid phase wrap-on-a-chip according to the present invention will be described in detail with reference to FIGS. 2 to 5 and Examples.

The present invention relates to a method for producing a nucleic acid sample using a stationary liquid phase wrap-on-a-chip.

The term "lab-on-a-chip" used in the present invention refers to a chip manufactured to implement various operations such as mixing, reaction, separation, and analysis performed in a laboratory. Means that the analysis can be performed by moving the solid phase instead of moving the liquid phase in the main analysis step, unlike the conventional method of moving the liquid phase.

As used herein, the term "nucleic acid" refers to a polymeric material composed of a purine base and a pyrimidine base, sugar, and phosphoric acid.

Hereinafter, a method of preparing a nucleic acid sample using the lab-on-a-chip according to the present invention will be described in detail with reference to FIG.

The stationary liquid-phase wrap-on-a-chip according to an embodiment of the present invention comprises a sample chamber, a lysis chamber, an elution chamber, and a channel connecting the sample space, the dissolution space and the elution space do.

As used herein, the term "sample space" refers to a space containing a sample containing cells, and "dissolution space" refers to a space in which cells are dissolved to release nucleic acid from cells, refers to a space containing a lysis buffer solution that can help bind nucleic acid capture particles (NCPs).

Particularly, the dissolution space may contain at least one selected from the group consisting of guanidine thiocyanate, guanidine hydrochloride, sodium dodecyl sulfate, sodium hydroxide, ethylenediaminetetraacetic acid (EDTA), triton Triton X-100 and polyoxyethylene nonylphenyl ether (NP-40). For example, the dissolution space may be filled with guanidine thiocyanate.

In addition, nucleic acid capture particles (NCP) can be placed in the sample space together with the sample. However, when the sample contains a large amount of impurities, it is preferable to place the nucleic acid capture particle in the dissolution space.

As used herein, the term "elution space" refers to a space filled with a buffer solution at a low concentration such as distilled water or 10 mM Tris, pH 8.5 buffer solution. The nucleic acid bound to silica has a property of dissociating from silica at a low ionic concentration have.

In addition, distilled water or 10 mM Tris (tris- [hydroxymethyl] -aminomethane), pH8.5 buffer solution has the advantage that it does not affect subsequent steps such as PCR.

More specifically, the method of preparing a nucleic acid sample using the stationary liquid phase wrap-on-a-chip according to the present invention includes a sample chamber, a lysis chamber, an elution chamber, Forming a cell-CCP complex by adding a sample containing cells and a cell capture particle (CCP) to the sample space using a stationary liquid phase wrap-on-a-chip composed of a channel connecting a space ); Transferring the cell-CCP complex into a dissolution space, and dissolving the cell to release nucleic acid from the cell (step 2); Capturing the resultant nucleic acid with a nucleic acid capture particle (NCP) to form a nucleic acid-NCP complex (step 2); And (3) eluting the nucleic acid by moving the nucleic acid-NCP complex to an elution space. .

On the other hand, in the stationary liquid phase wrap-on-a-chip of the present invention, the channel is filled with a hydrophobic liquid. Here, hydrophobic refers to a property opposite to hydrophilicity and having no affinity for water, and may mean a property not mixed with water.

For example, the channel may be filled with a liquid that is immiscible with water, such as olive oil, mineral oil, liquid wax, and the like.

The liquid not only prevents the solutions in different spaces from mixing with each other, but also cleanses and removes impurities when the cell-CCP complex or nucleic acid-NCP complex passes through the channel. On the other hand, it is preferable that the hydrophobic liquid has a predetermined viscosity in order not to move easily to the neighboring space.

As a specific example, when a material such as a liquid wax which exists in a solid state in a refrigerated condition and exists in a liquid state at a room temperature is filled in the channel and stored in a refrigerator, the effect of reliably separating different spaces of the lab- .

For example, the non-polar liquid may be a solid at a temperature of 10 to 20 ° C or less, and may be a liquid at a temperature of 10 to 20 ° C or more.

First, in a method of preparing a nucleic acid sample according to the present invention, a sample and a cell capture particle (CCP) are put into a sample space to form a cell-CCP complex. At this time, the cell trapping particles can be prepared by immobilizing an antibody against the cells to magnetic particles.

In addition, the surface of the cell may contain an antigen specific to the antibody. On the other hand, the method for producing the cell trapping particles may be a known technique known in the art.

Next, the cell-CCP complex can be moved to the dissolution space using a magnet under the lab-on-a-chip. At this time, the impurities in the sample space are removed when the complex passes through the channel, and only the cell-CCP complex is transferred to the dissolution space.

When the cell membrane is degraded by the action of the dissolution buffer solution in the dissolution space, the nucleic acid in the cell membrane is released, and the released nucleic acid binds to nucleic acid capture particles (NCP) to form a nucleic acid-NCP complex have.

On the other hand, the dissolution buffer solution may contain, as an example, guanidine thiocyanate as a main component, and the compound may act to promote the formation of a nucleic acid-NCP complex.

In a particular embodiment, the surface of the nucleic acid capture particle can be coated with a substance that binds to the nucleic acid, and such a nucleic acid capture particle production method can be a known technique known in the art.

In addition, using the magnet again, the nucleic acid-NCP complex in the dissolution space can be transferred to the elution space. Then, the ions contained in the lysis buffer are removed when they pass through the channel, and only the nucleic acid-NCP complex migrates to the elution space.

Accordingly, when the nucleic acid bound to the NCP is eluted due to the low ion concentration in the elution space and the elution space takes up the solution in the state where the magnet has collected the CCP and the NCP, A nucleic acid sample can be obtained.

Next, a method of manufacturing a nucleic acid sample using the stationary liquid phase wrap-on-a-chip according to another embodiment of the present invention will be described with reference to FIG.

The stationary liquid phase wrap-on-a-chip according to the present invention may comprise a PCR mixture storage space.

More specifically, the PCR mixture storage space may include a dried PCR mixture, and the PCR mixture storage space may be configured such that after eluting the nucleic acid, the solution in the elution space is moved to the PCR mixture storage space And then performing PCR.

In addition to performing the PCR, the PCR mixture storage space may perform another analysis process. In order to improve the stability of the storage, the mixture is preferably stored in a dried state.

Here, the PCR mixture may mean a mixture in which all the enzymes and compounds necessary for the PCR (polymerization chain reaction) reaction are mixed together to facilitate the use thereof.

In a particular embodiment, the elution space and the PCR mixture storage space may be separated by additional valves. More specifically, instead of moving the nucleic acid sample solution in the elution space to the PCR mixture storage space, the PCR mixture storage space and the elution space can be separated into rotatable valves. By rotating the valve, PCR can be performed.

The nucleic acid sample preparation method using the stationary liquid phase wrap-on-a-chip according to the present invention can easily prepare a nucleic acid sample, and the DNA extraction can be confirmed by carrying out PCR.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example >

Example  One. Stationary liquid phase  Using a lab-on-a-chip Vibrio parahaemolyticus  DNA sample preparation

In this example, a DNA sample of Vibrio parahaemolyticus was prepared using a stationary liquid phase wrap-on-a-chip, and real-time PCR was performed to confirm DNA extraction.

As shown in FIG. 4, the lab-on-a-chip used in this embodiment has a total size of 60 × 15 × 5 mm ³ and is composed of two channels connecting the sample space, the dissolution space, the elution space, and three spaces.

The dissolution space was filled with 50 μl of dissolution buffer solution (5.5 M guanidine thiocyanate, 20 mM Tris-HCl, pH 7.6, 20 mM EDTA), and 10 μl of 2 mg / ml NCP was added. The elution space was filled with 20 μl of distilled water. The remaining two channels (size 15 × 1 × 1 mm ³ ) were filled with liquid wax (Bio Rad, USA). NCP used magnetic particles coated with silica.

100 μl of sample containing 0-10 ⁵ cfu / ml of Vibrio parahaemolyticus and 10 μl of 1 mg / ml CCP were added to the sample space and vibration was applied to the lab-on-a-chip for 5 minutes to promote cell-CCP complex formation. CCP was prepared by immobilizing antibody specific to Vibrio parahaemolyticus on the surface of magnetic particles.

After that, the magnet was placed in the lower portion of the sample space for 30 seconds while the vibration was applied, and the cell-CCP complex was collected, and the magnet was moved to the lower space of the dissolution space to move the cell-CCP complex to the dissolution space. The magnet was placed under the dissolution space and vibrated for 5 minutes, so that the cells were lysed and the nucleic acid-NCP complex was formed.

Finally, the magnet was moved to the bottom of the elution space under vibration, and the nucleic acid-NCP complex was moved to the elution space, followed by vibra- tion for 5 minutes to elute the nucleic acid from the NCP. By taking the solution in the elution space, nucleic acid samples of Vibrio parahaemolyticus were completed.

Real - time PCR was performed to determine if Vibrio parahaemolyticus DNA was extracted from the nucleic acid samples. 6 μl of the prepared nucleic acid sample solution was used for PCR, and the remaining conditions were the same as those of the PCR kit.

As a result, as shown in FIG. 5, the amplification rate was faster as the number of Vibrio parahaemolyticus was increased, and DNA was quantitatively extracted. In addition, even at the lowest concentration of 10 ¹ cfu / ml, the amplification rate was faster than that of the negative control, and the DNA extraction efficiency was very high.

Through the above-mentioned examples, it was confirmed that the Vibrio parahaemolyticus in the sample space was captured by CCP and transferred to the dissolution space and dissolved. As a result, the released nucleic acid was captured by NCP and transferred to the elution space and eluted. I could.

While the present invention has been particularly shown and described with reference to the specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible, will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (11)

Using a stationary liquid phase wrap-on-a-chip consisting of a sample chamber, a lysis chamber, an elution chamber, and channels connecting the sample space, the dissolution space and the elution space,
Adding a sample containing cells and a cell capture particle (CCP) to the sample space to form a cell-CCP complex;
Transferring the cell-CCP complex to a dissolution space, and dissolving the cell to release nucleic acid from the cell;
Capturing the resulting nucleic acid with a nucleic acid capture particle (NCP) to form a nucleic acid-NCP complex; And
Transferring the nucleic acid-NCP complex to an elution space to elute the nucleic acid; The method comprising the steps of:
The method according to claim 1,
Wherein the channel is filled with a hydrophobic liquid.
3. The method of claim 2,
Wherein the hydrophobic liquid is a solid state at a temperature of 10 to 20 占 폚 or lower and a wax present in a liquid state at a temperature of 10 to 20 占 폚 or higher.
The method according to claim 1,
The dissolution space
Guanidine thiocyanate, guanidine hydrochloride, sodium dodecyl sulfate, sodium hydroxide, ethylenediaminetetraacetic acid (EDTA), Triton X-100 and &lt; RTI ID = 0.0 & Polyoxyethylene nonylphenyl ether (NP-40), and a solution buffer solution containing at least one selected from the group consisting of polyoxyethylene nonylphenyl ether (NP-40).
The method according to claim 1,
The dissolution space
Wherein the nucleic acid capture particle comprises Nucleic Acid Capture Particles (NCP).
The method according to claim 1,
The eluting space
Wherein the sample is filled with one of distilled water or 10 mM Tris (tris- [hydroxymethyl] -aminomethane), pH 8.5 buffer solution.
The method according to claim 1,
The cell capture particles (CCP)
Wherein an antibody that specifically reacts with the cell is immobilized on the magnetic particle.
The method according to claim 1,
The nucleic acid capture particles (NCP)
Wherein the magnetic particles are magnetic particles coated with silica.
The method according to claim 1,
The elution space of the stationary liquid phase wrap-on-a-chip is further connected to a PCR mixture storage space,
Wherein the step of eluting the nucleic acid is followed by transferring the solution in the elution space to the PCR mixture storage space to perform the PCR.
The method according to claim 1,
The elution space of the stationary liquid phase wrap-on-a-chip is further connected to a PCR mixture storage space,
Wherein the elution space and the PCR mixture storage space are separated by a rotatable valve and the eluted nucleic acid is subjected to PCR by combining the PCR mixture storage space and the elution space by rotation of the valve. A method for producing a sample;
11. The method according to claim 9 or 10,
Wherein the PCR mixture is stored in a dried state.

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