KR20080030997A - A method of isolating a dna from a microorgansim cell using a nonplanar solid substrate, a method of amplifying a nucleic acid using the isolated nucleic acid as a template and a device for isolating and amplifying a nucleic acid comprising the nonplanar solid substrate - Google Patents

Abstract

A method for isolating a DNA from a microorganism cell is provided to perform effectively microorganism isolation and nucleic acid isolation simultaneously, simplify the isolation process separate nucleic acids without use of additional nucleic acid isolating agent such as chaotropic agent, and perform the isolation by using small devices such as a lap-on-a-chip. A method for isolating a DNA from a microorgansim cell by using a nonplanar solid substrate comprises the steps of: contacting the nonplanar solid substrate with a sample containing microorganisms including bacteria, fungi or viruses at pH 3.0-6.0 to adhere the microorganisms to the solid substrate; removing unadhered materials by washing the solid substrate; and crushing the microorganisms adhered to the solid substrate at pH 3.0-8.0 or pH 11-14, wherein the sample is blood, urine or saliva.

Description

A method of isolating a nucleic acid from a microorganism using a non-planar solid support, a method for amplifying a nucleic acid using the separated nucleic acid as a template, and a nucleic acid separation and amplification apparatus comprising the solid support a microorgansim cell using a nonplanar solid substrate, a method of amplifying a nucleic acid using the isolated nucleic acid as a template and a device for isolating and amplifying a nucleic acid comprising the nonplanar solid substrate}

The present invention relates to a method for separating nucleic acids from a microorganism using a non-planar solid support, a method for amplifying a nucleic acid using the separated nucleic acid as a template, and a nucleic acid separation and amplification apparatus including the solid support.

There has been known a method for purifying nucleic acids using solid materials. For example, US Pat. No. 5,234,809 discloses a method for purifying nucleic acids using solid materials that bind to nucleic acids. However, the method is complicated because it takes time and is not suitable for lab-on-a-chip. In addition, the method has a problem that must be used chaotropic material. In other words, when the chaotropic material is not used, the nucleic acid does not bind to the solid material.

In addition, US Pat. No. 6,291,166 discloses a method for archiving nucleic acids using a solid matrix. Since the nucleic acid binds irreversibly to the solid matrix, the nucleic acid solid matrix complex may be stored and then analyzed later or repeated. However, according to this method, a positively charged material such as alumina must be activated with a basic material such as NaOH, and the nucleic acid cannot be separated from the alumina because it irreversibly binds to the activated alumina.

US Pat. No. 5,705,628 describes a method for mixing reversible and nonspecifically binding DNA by mixing magnetic microparticles having a surface coated with a carboxyl group by mixing a solution comprising DNA with a salt and polyethylene glycol. The method utilizes magnetic particles, salts and polyethylene glycols having a carboxyl group surface to separate DNA.

As described above, conventional nucleic acid isolation and purification methods require the addition of high concentrations of reagents separately to bind DNA, which may affect subsequent processes such as PCR and is difficult to apply on LOC. there was. In addition, the conventional nucleic acid isolation and purification method is configured as a separate step separate from the purification or enrichment of the cells or viruses, and the nucleic acid from the purified or concentrated cells or viruses to separate the nucleic acid from the purified cell or virus There is no known method for separating nucleic acids by a continuous process.

Therefore, there has been a demand for a method capable of separating or concentrating microorganisms by attaching microorganisms on a solid surface such as a substrate, and at the same time, purifying or concentrating nucleic acids derived from the microorganisms with high affinity for nucleic acids.

It is an object of the present invention to provide a method of isolating cells or viruses and separating nucleic acids therefrom using a solid support having a non-planar surface.

Another object of the present invention is to provide a method for amplifying a nucleic acid separated by the above method.

The present invention comprises the steps of attaching the microorganism to the solid support by contacting a non-planar solid support and a sample containing the microorganism in the range of pH 3.0 to 6.0,

It provides a method for separating a nucleic acid from a microorganism, comprising the step of crushing the microorganism attached to the solid support.

The method for separating nucleic acid from the microorganism of the present invention includes contacting a non-planar solid support with a sample containing the microorganism in the range of pH 3.0 to 6.0 to attach the microorganism to the solid support. The contact causes the microorganism to adhere to the solid support.

Among the liquid media in which the solid support is included, the microorganisms may be present in or attached to the solid support. The distribution between this liquid medium and the solid support is known to be determined by the difference between the surface tension of the liquid medium and the surface tension of the bacteria. That is, if the surface tension of the liquid medium is greater than the surface tension of the bacteria, the bacteria adhere better to the solid support, that is, the hydrophobic solid support, the surface tension of the bacteria is greater than the surface tension of the liquid medium, The bacteria adhere better to a solid support having a high surface tension, i.e., a hydrophilic solid support, and when the surface tension of the bacteria is equal to the surface tension of the liquid medium, the attachment of the solid support of the bacterial cells has no effect of surface tension and is electrostatic Other interactions, such as interactions, have been reported to affect (Applied and Environmental Microbiology, July 1983, p. 90-97). It is also known that through thermodynamic approach based on surface tension, bacterial cells can adhere to surfaces not only by bacterial cells but also through electrostatic attraction characteristics. However, these phenomena have a problem that their attachment speed is very slow.

In order to solve the problems of the prior art as described above, the present inventors use a non-planar solid support, and at the same time by contacting the sample containing microorganisms with the solid support in the range of pH 3.0 to 6.0, It was confirmed that the microorganisms can be separated. This is believed to increase the surface area of the solid support and at the same time, by using a liquid medium of pH 3.0 to 6.0, the cell membrane of the microorganisms is denatured, solubility in the solution is lowered, so that the rate of attachment to the solid surface is relatively high, but of the present invention The scope is not limited to this particular mechanism.

In the contacting step, the sample includes any sample containing a microorganism. Preferably, it is selected from the group consisting of biological samples, clinical samples and laboratory samples containing the microorganisms. In the present invention, "biological sample" means a sample containing or consisting of cells or tissues, such as cells or biological liquids isolated from an individual. The subject may be an animal including a human. Examples of biological samples include saliva, sputum, blood, blood cells (e.g. white blood cells, red blood cells), amniotic fluid, serum, semen, bone marrow, tissue or microneedle biopsy samples, urine, peritoneal fluid, pleural fluid and cell culture. Included. Biological samples include tissue sections, such as frozen sections taken for histological purposes. Preferably, the biological sample is a "clinical sample" which is a sample derived from a human patient, and more preferably, the sample is blood, urine, saliva, or sputum.

In the present invention, microorganisms to be isolated include bacterial cells, fungi and viruses.

In the contacting step, the sample may be diluted with a solution or a buffer that can serve as a buffer for the microorganism at low pH. Preferably, the buffer may be diluted with phosphate buffer (eg sodium phosphate, pH 3.0 to 6.0) or acetate buffer (sodium acetate, pH 3.0 to 6.0). The degree of dilution is not particularly limited, but may be preferably diluted at a magnification of 1: 1 to 1: 1,000, more preferably 1: 1 to 1:10.

In the contacting step, the sample is one having a salt concentration of 10 mM to 500 mM, preferably 50 mM to 300 mM. The sample has an ion concentration selected from the group consisting of 10 mM to 500 mM, preferably 50 mM to 300 mM acetate and phosphate.

In the contacting step, the solid support has a non-planar shape and thus has a surface whose surface area is increased compared to the plane. The solid support may be a concave-convex structure is formed on the surface. In the present invention, the "concave-convex structure" refers to a structure having a concave and convex surface without smooth surface. The uneven structure includes a surface having a columnar structure in which a plurality of pillars are formed or a surface having a network structure in which a plurality of pores is formed, but is not limited to these examples.

In the contacting step, the non-planar solid support may have any shape. For example, a solid support having a pillar structure having a plurality of pillars formed on a surface thereof, a solid support having a bead shape, and a sieve structure having a plurality of pores formed on a surface thereof. It may be selected from the group consisting of a solid support. The solid support may be used alone or as a collection of a plurality of the solid supports (eg, an aggregate packed in a tube or a container).

In the contacting step, the solid support may be in the form of an inner wall of the microchannel or microchamber in the microfluidic device. Thus, the method of the present invention may be carried out in a fluidic device or microfluidic device having one or more inlets and outlets, the inlets and outlets communicating through channels or microchannels. have.

In a first embodiment of the method of the present invention, in the contacting step in the method of the present invention, the solid support may have a columnar structure in which a plurality of pillars are formed on the surface thereof. The manufacture of pillars on solid supports is well known in the art. For example, fine pillars can be formed at high density using a photolithography process or the like used in a semiconductor manufacturing process. Preferably, the pillar has an aspect ratio of 1: 1 to 20: 1, but is not limited thereto. In the present invention, the "aspect ratio" means the ratio of the cross-sectional diameter to the height of the pillar. The pillar structure may be a ratio of the distance between the pillar height and the pillar is 1: 1 to 25: 1. The pillar structure may have a distance between the pillars in a range of 5 μm to 100 μm.

In the contacting step in the method of the present invention, the non-planar solid support may have a hydrophobicity of 70 ° to 95 ° water contact angle. The hydrophobicity with a water contact angle of 70 ° to 95 ° consists of octadecyldimethyl (3-trimethoxysilyl propyl) ammonium (OTC) and tridecafluorotetrahydrooctyltrimethoxysilane (DFS). The compound selected from the group may be coated and given. The surface having a water contact angle of 70 ° to 95 ° preferably has octadecyldimethyl (3-trimethoxysilyl propyl) ammonium (OTC) and tridecafluorotetrahydrooctyltrimethoxy in the SiO ₂ layer of the solid support. Compounds selected from the group consisting of silanes (DFS) may be self-assembled monolayer (SAM) coated.

In the method of the present invention, in the contacting step, the non-planar solid support may have one or more amine-based functional groups on its surface. Surface having at least one functional group of the amine-based may be a polyethyleneimine trimethoxysilane (PEIM) is coated on the surface of the solid support. The coated surface may preferably be a self-assembled monomolecular (SAM) coating of polyethyleneiminetrimethoxysilane (PEIM) on the SiO ₂ layer of the solid support. The amine-based functional group has a positive charge in the range of pH 3.0 to 6.0.

In the method of the present invention, in the contacting step, the solid support includes a support of any material as long as it has water contact angle characteristics as described above or one or more amine-based functional groups on its surface. For example, glass, silicon wafers, plastic materials, and the like are included, but are not limited to these examples. The surface having a water contact angle of 70 ° to 95 ° or a surface having at least one amine-based functional group on the surface thereof is considered to be attached to the microorganism when the sample containing the microorganism contacts the solid support having the surface. The present invention is not limited to a specific mechanism.

The method of the present invention may further comprise washing the material other than the target microorganism that is not attached to the solid support after the contacting step. The washing may be any solution capable of removing impurities that may adversely affect subsequent processes without leaving the target microorganisms attached to the solid support surface from the solid support. For example, acetate buffers and phosphate buffers used as binding buffers can be used. The wash solution is preferably a buffer having a pH in the range of pH 3.0 to 6.0.

In the present invention, "separation of microorganisms" includes not only pure separation of microorganisms in a sample but also concentration.

The method for separating nucleic acid from the microorganism of the present invention includes the step of crushing the microorganism attached to the solid support.

The microbial cell crushing may be a crushing method made by any crushing method known in the art. For example, it may be crushed by a crushing method selected from the group consisting of boiling lysis, laser crushing, crushing with chemicals, and electrochemical crushing (eg, electrolysis), but is not limited thereto. no.

In a second embodiment of the method of the present invention, in the method of the present invention, the microbial disruption step is performed in the range of pH 3.0 to 6.0 to attach the nucleic acid from the microorganism to the solid support. The crushing may be carried out in any liquid medium as long as it is in the range of pH 3.0 to 6.0. The crushing may be performed in phosphate buffer or acetate buffer.

In the second embodiment of the method of the present invention, the nucleic acid released from the disrupted microorganism is attached to the solid support. The nucleic acid thus attached can be separated by removing nucleic acid, cells, viruses, or fragments of the cells or viruses that are not attached to the surface of the solid support.

Thus, the second embodiment of the method of the present invention may further comprise washing the solid support by washing the solid support after the crushing step.

In the washing step, the washing solution may be any substance as long as it has a property of removing a substance such as nucleic acid not attached to the solid support without leaving the nucleic acid attached to the surface. Preferably, the wash solution is a solution having the above characteristics and capable of removing impurities that may adversely affect subsequent processes. Examples of the washing solution include acetate buffer, phosphate buffer and the like used as the binding buffer. The wash solution is preferably a buffer having a pH in the range of pH 3.0 to 6.0.

In the second embodiment of the method of the present invention, the nucleic acid attached to the solid support can be used as it is or eluted.

Therefore, the second embodiment of the method of the present invention may further comprise eluting the nucleic acid attached to the solid support.

In the eluting step, any solution known in the art may be used as long as the solution used for eluting has a property of leaving the nucleic acid attached to the solid support. For example, high pH solutions may be used. The high pH solution allows the hydroxide ions (OH-) to change the surface anionic, leaving the DNA with anionic properties. Preferably, as a solution of pH 11 or higher, typically a NaOH solution can be used.

In a third embodiment of the method of the present invention, in the method of the present invention, the microbial disruption step is performed in the range of pH 11-14. In this way, DNA elution can be easily performed by minimizing the attachment of DNA to the solid support. High pH solutions allow the hydroxide ions (OH-) to anionicize the surface of the solid support so that the anionic nature of the crushed DNA does not adhere to the solid support. That is, for the purpose of attaching the microorganisms to the solid support and eluting the DNA derived therefrom, it is preferable to perform the microbial disruption step at high pH and elute through the same high pH solution.

The third embodiment of the method of the present invention may further comprise the step of purifying the nucleic acid from the lysate obtained in the crushing step. As a method for purifying nucleic acids from microbial lysates, any method known in the art may be used.

The present invention also provides a method for amplifying a nucleic acid, comprising amplifying a target nucleic acid by using the isolated nucleic acid as a template by the method for separating nucleic acid from the microorganism of the present invention as described above.

In the method for amplifying the nucleic acid of the present invention, any amplification method known in the art may be used for the nucleic acid amplification, and is preferably performed by PCR. The method for separating nucleic acids from microorganisms is as described above.

In the method for amplifying a nucleic acid of the present invention, the "nucleic acid" includes DNA and RNA. Preferably, the nucleic acid is DNA.

In the method for amplifying a nucleic acid of the present invention, the nucleic acid separation and the nucleic acid amplification may be performed in the same container containing the solid support. The vessel may be, for example, microchannels, microchambers and tubes, but is not limited to these examples. Preferably, the microchamber formed in the microfluidic device is provided with a PCR device. The PCR device includes a heater and a cooler. Thus, one embodiment of the method of amplifying a nucleic acid of the present invention is that the nucleic acid extraction is performed in a microchamber and the nucleic acid amplification is performed in the same microchamber.

In the method for amplifying a nucleic acid of the present invention, the nucleic acid separation and the nucleic acid amplification may be performed in different containers. For example, the nucleic acid separation may be performed in a container comprising the solid support, and the nucleic acid amplification may be performed in another container in communication with the container containing the solid support or in another container that is not in communication. have. The vessel may be, for example, microchannels, microchambers and tubes, but is not limited to these examples. For example, the nucleic acid separation may be performed in a microchannel or microchamber of a microfluidic device, and the separated nucleic acid may be transferred to another microchannel or microchamber and eluted, and used for amplification.

The present invention also comprises a reaction chamber containing a non-planar solid support, a heating unit for heating the chamber, a temperature control unit for controlling the heating unit to heat the chamber, nucleic acid separation and amplification apparatus To provide.

In the nucleic acid separation and amplification apparatus of the present invention, the solid support has a non-planar shape, and has a surface whose surface area is increased compared to the plane. The solid support may be a concave-convex structure is formed on the surface. In the present invention, the "concave-convex structure" refers to a structure having a concave and convex surface without smooth surface. The uneven structure includes a surface having a columnar structure in which a plurality of pillars are formed or a surface having a network structure in which a plurality of pores is formed, but is not limited to these examples.

In the nucleic acid separation and amplification apparatus of the present invention, the non-planar solid support may have any shape. For example, a solid support having a pillar structure having a plurality of pillars formed on a surface thereof, a solid support having a bead shape, and a sieve structure having a plurality of pores formed on a surface thereof. It may be selected from the group consisting of a solid support. The solid support may be used alone or as a collection of a plurality of the solid supports (eg, an aggregate packed in a tube or a container).

In the nucleic acid separation and amplification apparatus of the present invention, the solid support may be in the form of the inner wall of the microchannel or microchamber in the microfluidic device. Accordingly, the nucleic acid separation and amplification apparatus of the present invention is provided with one or more inlets and outlets, and the inlet and outlet are fluidic devices or microfluidic devices in communication through channels or microchannels. Can be. That is, the separation and the PCR of the nucleic acid may be a fluidic device (fluidic device) or a microfluidic device (microfluidic device) can be performed in the same chamber.

In a first embodiment of the nucleic acid separation and amplification apparatus of the present invention, in the apparatus of the present invention, the solid support may have a columnar structure in which a plurality of pillars are formed on the surface thereof. The manufacture of pillars on solid supports is well known in the art. For example, fine pillars can be formed at high density using a photolithography process or the like used in a semiconductor manufacturing process. Preferably, the pillar has an aspect ratio of 1: 1 to 20: 1, but is not limited thereto. In the present invention, the "aspect ratio" means the ratio of the cross-sectional diameter to the height of the pillar. The pillar structure may be a ratio of the distance between the pillar height and the pillar is 1: 1 to 25: 1. The pillar structure may have a distance between the pillars in a range of 5 μm to 100 μm.

In the nucleic acid separation and amplification apparatus of the present invention, the non-planar solid support may have a hydrophobicity with a water contact angle of 70 ° to 95 °. The hydrophobicity with a water contact angle of 70 ° to 95 ° consists of octadecyldimethyl (3-trimethoxysilyl propyl) ammonium (OTC) and tridecafluorotetrahydrooctyltrimethoxysilane (DFS). The compound selected from the group may be coated and given. The surface having a water contact angle of 70 ° to 95 ° preferably has octadecyldimethyl (3-trimethoxysilyl propyl) ammonium (OTC) and tridecafluorotetrahydrooctyltrimethoxy in the SiO ₂ layer of the solid support. Compounds selected from the group consisting of silanes (DFS) may be self-assembled molecule (SAM) coated.

In the nucleic acid separation and amplification apparatus of the present invention, the non-planar solid support may have one or more amine-based functional groups on its surface. Surface having at least one functional group of the amine-based may be a polyethyleneimine trimethoxysilane (PEIM) is coated on the surface of the solid support. The coated surface may preferably be a self-assembled monomolecular (SAM) coating of polyethyleneiminetrimethoxysilane (PEIM) on the SiO ₂ layer of the solid support. The amine-based functional group has a positive charge in the range of pH 3.0 to 6.0.

In the nucleic acid separation and amplification apparatus of the present invention, the heating portion may be any device known in the art for heating the chamber. The heating unit includes, for example, a heater and a micro heater.

In the nucleic acid separation and amplification apparatus of the present invention, any temperature control unit known in the art may be used to control the heating unit to heat the chamber to generate a temperature cycle used for PCR. The temperature control unit may include a temperature sensor for sensing the temperature of the chamber and means for turning on / off the operation of the heating unit.

According to the method for separating nucleic acid from the microorganism according to the present invention, it is possible to efficiently perform the microbial separation and nucleic acid process at the same time. In addition, the nucleic acid can be separated without using a separate nucleic acid separating agent such as chaotropic agent, so the separation process is simple. In addition, the method of the present invention can be usefully implemented using a miniaturized device such as a lab-on-a-chip (LOC).

According to the method for amplifying DNA according to the present invention, cell separation and nucleic acid separation can be performed continuously, and nucleic acid can be efficiently amplified using a miniaturized device such as a lab-on-a-chip (LOC).

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example  1: solid support having a columnar structure DNA  Confirmation of binding properties

In this example, the binding characteristics were confirmed by flowing a DNA sample into a flow apparatus having a chamber having an inlet and an outlet and having an array of pillars formed on a substrate having a size of 10 mm x 23 mm. In the pillar array, the distance between the pillars is 12 µm, the pillar height is 100 µm, and the cross section of each pillar has a square length of 25 µm on one side.

The surface of the area where the pillar array is formed has a surface coated with PEIM having one or more amine-based functional groups or OTC (water contact angle 80 °) each having a water contact angle in a range of 70 ° to 95 °.

DNA samples using DNA in 0.01 N NaOH (pH 12) and DNA in 100 mM NaH ₂ PO ₄ (pH 7.0) were flowed at 200 μl at 200 μl / min.

DNA bound to the column array in the flow device was measured using a picogreen kit (Molecular Probes) and a spectrometer. Experiments were performed according to the manufacturer's instructions.

FIG. 1 shows the correlation between absorbance intensity and the amount of picogreen labeled DNA in NaOH solution at pH 12.0 and phosphate buffer at pH 7.0 in vitro. As shown in FIG. 1, there is a proportional relationship between DNA concentration and absorbance intensity, and it is shown that DNA quantification is possible through a proportional expression (fluorescence intensity and DNA concentration) derived from this proportional relationship.

Figure 2 is a diagram showing the degree of adhesion of the DNA according to pH after flowing the DNA sample through the flow device is formed columnar array. As shown in FIG. 2, in the case of DNA in NaOH at pH 12.0, the DNA recovery was 95% or more regardless of the nature of the surface of the pillar array region. For DNA in phosphate buffer at pH 7.0, the DNA recovery was less than about 40%. This indicates that DNA adhesion hardly occurs in the high pH range (11-14), and the efficiency of DNA attachment to the solid support is high in the relatively low pH range (3-8). By using this property, the DNA can be easily attached and eluted to the solid support having the columnar array. In other words, the DNA should be treated at low pH if the DNA is to be attached to the solid support, and at the high pH if the DNA is to be eluted.

Example 2: Cells bound, solidified and elution of DNA to a solid support having a columnar array in a flow apparatus

In this example, the cell sample was flowed into a flow apparatus provided with an inlet and an outlet, and an array of pillars was formed on a substrate having a size of 10 mm x 23 mm, the cells were disrupted, and the DNA was eluted. In the pillar array, the distance between the pillars is 12 µm, the pillar height is 100 µm, and the cross section of each pillar has a square length of 25 µm on one side.

The surface of the region where the pillar array is formed has a surface coated with OTC (water contact angle 80 °) having a water contact angle in a range of 70 ° to 95 °.

The cell sample used Escherichia coli of 0.01 OD ₆₀₀ in LB medium. The sample was given 250 μl at a flow rate of 200 μl / min.

Next, E. coli was bound to the surface of the region where the pillar array is formed. Cell disruption was performed by flowing a 50 μl solution of 0.01 N NaOH for 2 minutes at a flow rate of 25 μl / min (hereinafter also referred to as 0.01 N NaOH_2), or a 10 μl solution of 50 μl of 0.01 N NaOH for 10 minutes at a flow rate of 5 μl / min. (Now referred to as 0.01N NaOH_10), a 3.5 μl solution of 0.01N NaOH was filled into the chamber, then boiled for 2 minutes, and a 45 μl solution of 0.01N NaOH was flowed at 200 μl / min (hereinafter also referred to as 0.01N NaOH / boiling). .

40 μl of the lysate flowing out after cell crushing was recovered, and the concentration of DNA was measured using a picogreen kit (Molecular Probes) and a spectrometer. DNA elution efficiency was calculated as (sample amount of fluorescence predicted when cell disruption / 100% cell disruption) × 100. The DNA standard curve was determined by crushing a sample containing Escherichia coli having a known concentration using NaOH at the same concentration in a tube and measuring the fluorescence intensity according to the cell concentration.

The expected amount of fluorescence at 100% cell disruption can be obtained as follows. First, assuming that all the injected cells bind to the solid support, that is, assuming that the binding efficiency is 100%, the total number of cells added and bound to the solid support = 0.01 OD600 x 1.0 Cell concentration per volume OD600 x volume = 0.01x 10 ⁹ cells / ml × 0.25 ml = 2.5 × 10 ⁶ cells (1.0 OD600 is usually at a cell concentration of 10 ⁹ cells / ml). Therefore, the amount of DNA that can be eluted from the cells at 100% cell disruption = 2.5 x 10 ⁶ cells x the amount of DNA per cell, wherein the amount of DNA per E. coli cell is about 5fg DNA, so 2.5 x 10 ⁶ cells x 5x10 ^-15 g DNA = 12.5 ng DNA. Dividing this by the volume of about 50 μl of total NaOH volume used for elution, the concentration is 0.25 ng / μl. Substituting this into the standard curve obtained in Example 1 gives the expected amount of fluorescence upon 100% cell disruption.

3 is a standard curve showing the correlation between the amount of DNA present in the lysate and the concentration of the cells after cell disruption. As shown in FIG. 3, there is a proportional relationship between cell concentration and DNA concentration in cell lysate.

FIG. 4 flows E. coli samples through a flow apparatus comprising a chamber coated with octadecyldimethyl (3-trimethoxysilyl propyl) ammonium (OTC) and having a columnar array; Next, it is a figure which shows the result of measuring the efficiency of DNA collect | recovering the said crushed object. As shown in Fig. 4, in the case of using NaOH, the method used in combination with the boiling method was most efficient. For 0.01 N NaOH / boiling, 32% DNA was recovered, assuming 1.OD ₆₀₀ of 10 ⁹ cells / ml. Considering the above results, the DNA recovery after cell disruption, the error of the cell OD (generally 1.0 OD600 is 5x10 ⁸ to 10 ⁹ cells / ml), is considered to be 30-60%.

Example 3: Binding Cells to Cell Solids , Cell Fracture, DNA Purification and Amplification

In this embodiment, the cell sample is flowed into a flow apparatus having an inlet and an outlet, and an array of pillars is formed on a substrate having a size of 7.5 mm x 15 mm. Unwashed material was washed away and DNA amplified with the template bound to the substrate. In the pillar array, the distance between the pillars is 15 µm, the pillar height is 100 µm, and the cross section of each pillar has a square length of 25 µm on one side.

The surface of the region where the pillar array is formed has a surface coated with OTC (water contact angle 80 °) having a water contact angle in a range of 70 ° to 95 °.

As a cell sample, an E. coli sample of 0.01 OD ₆₀₀ in LB medium was used. The sample was adjusted to pH 4.0 using 100 mM sodium acetate buffer and flowed for 5 minutes from the inlet to the outlet through the chamber at a flow rate of 100 μl / min.

Next, E. coli was bound to the surface of the region where the pillar array is formed. Cell disruption was accomplished by filling the chamber with 100 mM phosphate buffer (pH 4.0), treating it for 2 minutes at 95 ° C. and then cooling to room temperature five times. Next, after cell crushing, foreign substances that were not bound to the surface of the column array formed region were washed by flowing 200 μl at a flow rate of 200 μl / min using 100 mM phosphate buffer (pH 4.0).

Next, PCR was performed using DNA attached to a solid support to DNA bound to the surface of the region where the pillar array was formed. PCR was performed using Taqman probe.

FIG. 5 is a diagram showing the result of E. coli samples flowing through a chamber having a surface formed with a columnar array in a flow apparatus, and cell disruption, washing and real time PCR amplification. In Figure 5, the sample is as shown in Table 1 below.

Table 1.

Sample Name process Ct (min) One dPCR1 14.14 2 dPCR2 14.13 3 Purified PCR1 13.29 4 Tablets dPCR2 14.63 5 0.01 OD cells 25.72 6 Negative control 25.23

In Table 1, purification PCR refers to samples that were crushed / washed (lanes 3 and 4 of electrophoresis below) and dPCR means samples (lanes 1 and 2 of electrophoresis below) that were not.

As shown in FIG. 5, when the E. coli sample was flowed through a chamber having a surface on which a columnar array was formed in the flow apparatus, and cell crushing, washing and real time PCR amplification, 10 times compared to a 0.01 OD cell standard sample PCR cycle differences over (DNA enrichment rate 1,000 times). There was no significant difference in Ct values between E. coli cells binding, cell disruption, washing and PCR, and E. coli cells binding and PCR without cell disruption. Since this has a large concentration and purification effect at the cellular level, it is believed that the purification effect at the DNA level is not large.

FIG. 6 shows the result of measuring the concentration of E. coli samples by flowing them through a chamber having a surface on which a columnar array is formed in a flow apparatus, cell crushing, washing and real time PCR amplification. Drawing. In Figure 6, lanes 1 and 2 show the results of PCR performed without binding to E. coli, and cell disruption, lanes 3 and 4 show the results of PCR performed after binding to E. coli and cell disruption, lane 5 is a negative control (Sample 6 of Table 1) is shown, and lane 6 is a figure which shows the result of PCR of 0.01 OD Escherichia coli sample (Sample 5 of Table 1) with a template. The final product concentrations measured are shown in Table 2 below. It can be seen that the concentration of the final product increased by more than 70% through the cell disruption process.

Table 2.

Cell Binding / Cell Breaking / Washing / PCR Cell Binding / PCR PCR using 0.01 OD E. coli sample DNA concentration (ng / μl) 24.45 17.35 735 Incremental Ratio for PCR Using 0.01 OD Escherichia Coli Sample 226% 131% One

Example 4 Binding, Cell Disruption, DNA Purification and Amplification to Solid Supports with Columnar Arrays in Flow Devices Using Clinically Simulated Samples

In this embodiment, a sample containing Escherichia coli cells in a urine is flowed into a flow apparatus having an inlet and an outlet and an array of pillars is formed on a substrate having a size of 7.5 mm x 15 mm, and the cells are crushed to disperse DNA. Bound to the phase, the unbound material was washed away, and the DNA bound to the substrate was amplified with the template. In the pillar array, the distance between the pillars is 15 µm, the pillar height is 100 µm, and the cross section of each pillar has a square length of 25 µm on one side.

The surface of the region where the pillar array is formed has a surface coated with OTC (water contact angle 80 °) having a water contact angle in a range of 70 ° to 95 °.

Cell samples were prepared using sodium acetate buffer (pH 4.0), a sample containing E. coli 0.01 OD ₆₀₀ in urine diluted with sodium acetate buffer; cells = 4: 1, and the chamber was removed from the inlet at a flow rate of 100 μl / min. Flowed through the exit for 5 minutes. As a control sample, 0.01 OD Escherichia coli sample was used (pH 4).

Next, E. coli was bound to the surface of the region where the pillar array is formed. Cell disruption was accomplished by filling the chamber with 100 mM phosphate buffer (pH 4.0), treating it for 2 minutes at 95 ° C. and then cooling to room temperature five times. Next, after cell disruption, foreign substances that were not bound to the surface of the column array formed region were washed by flowing 200 μl at a flow rate of 200 μl / min using 100 mM phosphate buffer (pH 4.0). Next, PCR was performed using DNA attached to a solid support to DNA bound to the surface of the region where the pillar array was formed. PCR was performed using Sybr Green.

FIG. 7 shows urine samples containing Escherichia coli flowed through a chamber having a surface on which pillar arrays are formed in a flow apparatus, and resulted from cell disruption, washing and real time PCR amplification. In Figure 7, the sample is shown in Table 3 below.

Table 3.

Sample Name process Ct (min) One Purified PCR1 11.43 2 Purified PCR2 11.21 3 dPCR1 11.94 4 dPCR2 13.16 5 Negative Control 1 23.76 6 Negative Control 2 23.38

In Table 3, purified PCR was crushed / washed and the rest was not processed.

As shown in FIG. 7, the urine sample containing E. coli was flowed through a chamber having a surface on which a columnar array was formed in the flow apparatus, and the urine sample was flowed in case of cell crushing, washing and real time PCR amplification. The Ct value preceded about 1 cycle compared to the case where the column array in the device was flowed through a chamber having a surface on which it was formed and real time PCR amplification without cell disruption and washing.

Example 4 Binding, binding, cell disruption, DNA elution and amplification of cells to a solid support having a columnar array in a flow apparatus using clinically simulated samples -Comparative experiment with a commercial DNA purification kit

In this embodiment, a sample containing E. coli cells in whole blood is flown into a flow apparatus having an inlet and an outlet and an array of pillars is formed on a substrate having a size of 10 mm x 23 mm, and the cells are crushed. The DNA was eluted from the substrate, and the eluted DNA was amplified with the template. This method was compared with a commercially available DNA purification kit from Qiagen.

In the pillar array, the distance between the pillars is 12 µm, the pillar height is 100 µm, and the cross section of each pillar has a square length of 25 µm on one side. The surface of the region where the pillar array is formed has a surface coated with OTC (water contact angle 80 °) having a water contact angle in a range of 70 ° to 95 °.

The cell sample was prepared by adding 10 μl of 1.0 OD ₆₀₀ E. coli to 1 ml of whole blood to prepare a clinical simulation sample having E. coli 0.01 OD ₆₀₀ . 200 μl of the sample was diluted 1: 1 with sodium acetate buffer (pH 3.0, 100 mM) and flowed from the inlet to the outlet through the chamber at 200 μl / min. Next, E. coli was bound to the surface of the region where the pillar array is formed. Cell disruption was infused with 5ul of 0.01N NaOH, boiled for 2 minutes, and further added 45µl NaOH solution to elute DNA at a flow rate of 200µl / min.

Control Sample (Qiagen Kit, Cat 13323, Blood & Cell Culture DNA Mini Kit) was prepared through a protocol provided by Qiagen, 200 μl of the clinical simulation sample.

Next, the two samples obtained above were subjected to PCR (Sybr Green). As shown in Table 4 below, the Ct value or the PCR product concentration showed similar results to commercialized kits. That is, it can be seen that the DNA prepared by the method of the present invention is of sufficient quality for PCR without additional DNA purification. The time between the two methods of DNA preparation is different. The present invention took less than 10 minutes and the Qiagen method took about 45 minutes. In addition, the present invention is possible from cell acquisition to DNA elution in one chip, it is very advantageous to automate. Therefore, it will be very useful for future LOC production. In Table 4, SAIT is the present invention, the concentration represents the electrophoresis result of FIG.

Table 4.

chip CT  Concentration (ng / μl) SAIT1 18.7 11.8 SAIT2 18.9 13.1 Qiagen1 18.4 13.0 Qiagen2 17.8 11.5 Negative Control 28.8 -

FIG. 8 is a diagram showing a result of real-time PCR amplification of a whole blood sample containing Escherichia coli through a chamber having a surface on which a columnar array is formed in a flow apparatus, and after cell disruption and DNA elution. .

9 is a result of flowing whole blood samples containing Escherichia coli through a chamber having a surface on which a columnar array is formed in a flow apparatus, and electrophoresis of a product obtained by cell disruption, DNA elution, and real time PCR amplification. It is a figure which shows.

1 is a graph showing the correlation between the amount of DNA in the NaOH solution at pH 12.0 and the phosphate buffer at pH 7.0 and the absorbance intensity.

Figure 2 is a diagram showing the degree of adhesion of the DNA according to pH after flowing the DNA sample through the flow device is formed columnar array.

3 is a standard curve showing the correlation between the amount of DNA present in the lysate and the concentration of the cells after cell disruption.

FIG. 4 flows E. coli samples through a flow apparatus comprising a chamber coated with octadecyldimethyl (3-trimethoxysilyl propyl) ammonium (OTC) and having a columnar array; Next, it is a figure which shows the result of measuring the efficiency of DNA collect | recovering the said crushed object.

FIG. 5 is a diagram showing the result of E. coli samples flowing through a chamber having a surface formed with a columnar array in a flow apparatus, and cell disruption, washing and real time PCR amplification.

FIG. 6 shows the result of measuring the concentration of E. coli samples by flowing them through a chamber having a surface on which a columnar array is formed in a flow apparatus, cell crushing, washing and real time PCR amplification. Drawing.

FIG. 7 shows urine samples containing Escherichia coli flowed through a chamber having a surface on which pillar arrays are formed in a flow apparatus, and resulted from cell disruption, washing and real time PCR amplification.

FIG. 8 is a diagram showing a result of real-time PCR amplification of a whole blood sample containing Escherichia coli through a chamber having a surface on which a columnar array is formed in a flow apparatus, and after cell disruption and DNA elution. .

9 is a result of flowing whole blood samples containing Escherichia coli through a chamber having a surface on which a columnar array is formed in a flow apparatus, and electrophoresis of a product obtained by cell disruption, DNA elution, and real time PCR amplification. It is a figure which shows.

Claims (38)

Attaching the microorganism to the solid support by contacting a non-planar solid support with a sample containing the microorganism in a range of pH 3.0 to 6.0, Washing the solid support to remove material that is not attached to the solid support, and Crushing the microorganism attached to the solid support. The method of claim 1, wherein the microorganism is a bacterium, fungus or virus. The method of claim 1, wherein the sample is a biological sample. The method of claim 3, wherein the sample is blood, urine, or saliva. The method of claim 1, wherein the sample is diluted with phosphate buffer or acetate buffer. The method of claim 5, wherein the dilution is carried out in a ratio of 1: 1 to 1:10. The method of claim 5, wherein the sample has a salt concentration of 10 mM to 500 mM. 8. The method of claim 7, wherein the sample has a salt concentration of 50 mM to 300 mM. According to claim 1, The non-planar solid support is a solid support having a columnar structure is formed with a plurality of pillars (pillar) on the surface, a solid support having a bead shape and a sieve having a plurality of voids formed on the surface and a solid support having a (sieve) structure. The method of claim 9, wherein the pillar has an earth pack ratio of 1: 1 to 20: 1. 10. The method of claim 9, wherein the columnar structure has a ratio of column height to column distance between 1: 1 and 25: 1. The method of claim 9, wherein the columnar structure has a distance between the pillars in the range of 5 μm to 100 μm. The method of claim 1, wherein the non-planar solid support has hydrophobicity with a water contact angle of 70 ° to 95 °. The method of claim 13, wherein the hydrophobicity is coated on the surface of the solid support with a compound of octadecyldimethyl (3-trimethoxysilyl propyl) ammonium (OTC) or tridecafluorotetrahydrooctyltrimethoxysilane (DFS). Method characterized in that the property is given by. The method of claim 1, wherein the non-planar solid support has at least one amine-based functional group on its surface. The method of claim 15, wherein the surface having at least one amine-based functional group is coated with polyethyleneiminetrimethoxysilane (PEIM). The method of claim 1 further comprising washing material other than the desired microorganism that is not attached to the solid support after the contacting step. The method according to claim 1, wherein the microbial crushing is crushing by crushing method selected from the group consisting of boil crushing, laser crushing, crushing with chemicals and electrochemical crushing. The method of claim 1, wherein the microbial disruption step is performed in the range of pH 3.0 to 8.0 to attach the nucleic acid derived from the microorganism to the solid support. 20. The method of claim 19, further comprising, after the shredding step, washing the solid support to wash material not attached to the solid support. 20. The method of claim 19, further comprising eluting the nucleic acid attached to said solid support. The method of claim 1, wherein the microbial disruption step is performed in the range of pH 11-14. 23. The method of claim 22, further comprising separating nucleic acids from the lysate obtained in the crushing step. 24. A method for amplifying a nucleic acid, the method comprising amplifying a target nucleic acid with a nucleic acid isolated according to any one of claims 1 to 23 as a template. The method of claim 24, wherein the nucleic acid is DNA or RNA. The method of claim 24, wherein the nucleic acid amplification is performed in the same chamber as the chamber comprising a non-planar solid support or in another chamber fluidly connected to the chamber. The method of claim 26, wherein the amplification is performed by PCR. The method of claim 27, wherein the template nucleic acid uses nucleic acid present in the microbial lysate as a template without undergoing further nucleic acid separation. And a chamber including a non-planar solid support, a heating unit for heating the chamber, and a temperature control unit for controlling the heating unit to heat the chamber. 30. The nucleic acid separation and amplification apparatus of claim 29, further comprising a nucleic acid amplification chamber fluidly connected to a chamber in which the non-planar solid support is included. The solid support of claim 29, wherein the non-planar solid support is a solid support having a columnar structure in which a plurality of pillars are formed on the surface, a solid support having a bead shape, and a sieve in which a plurality of voids are formed on the surface. and a solid support having a (sieve) structure. 32. The apparatus of claim 31, wherein the pillar has an earth pack ratio of 1: 1 to 20: 1. 32. The apparatus of claim 31 wherein the columnar structure has a ratio of column height to column distance between 1: 1 and 25: 1. 32. The apparatus of claim 31 wherein the columnar structure has a distance between the pillars in the range of 5 μm to 100 μm. 30. The apparatus of claim 29, wherein the non-planar solid support has hydrophobicity with a water contact angle of 70 degrees to 95 degrees. 36. The method of claim 35, wherein the hydrophobicity is coated on the surface of the solid support with a compound of octadecyldimethyl (3-trimethoxysilyl propyl) ammonium (OTC) or tridecafluorotetrahydrooctyltrimethoxysilane (DFS). Apparatus characterized in that the property is given by. 30. The device of claim 29, wherein the non-planar solid support has one or more amine-based functional groups on its surface. 38. The device of claim 37, wherein the surface having at least one amine-based functional group is coated with polyethyleneiminetrimethoxysilane (PEIM).

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