KR20080017207A - A method of amplifying a nucleic acid from a microorgansim using a nonplanar solid substrate - Google Patents

Abstract

A method for amplifying a nucleic acid from a microorganism is provided to amplify conveniently, rapidly and highly sensitively a nucleic acid by performing separation of the microorganism, cell lysis and amplification of a nucleic acid in one container and use a large amount of an initial sample due to contact the microorganism with high fluid speed with a solid substrate. A method for amplifying a nucleic acid from a microorganism such as bacteria, fungi or virus comprises the steps of: (a) contacting a non-planar solid substrate with a biological sample including the microorganism under pH of 3.0-6.0 to attach the microorganism to the solid substrate; (b) washing materials not attached to the solid substrate to remove the materials; and (c) subjecting the microorganism attached to the solid substrate to PCR, wherein all the steps are performed in a same container and the solid substrate is selected from the group consisting of a solid substrate having a pillar structure where a plurality of pillar is formed, a bead-shaped solid substrate and a sieve-structured solid substrate, has a water contact angle of 70-95° and includes at least one amine-based functional group on the surface thereof.

Description

A method of amplifying a nucleic acid from a microorgansim using a nonplanar solid substrate}

Fig. 1 is a diagram showing the result of PCR by attaching E. coli to a solid support and using the DNA in the attached E. coli as a template.

2A and 2B are diagrams showing the results of PCR after attaching a blood or serum sample containing E. coli to a solid support, crushing cells by heat.

Figure 3 is a diagram showing the results of the PCR after separating the DNA using the solid support and the PCR after separating the DNA using the QIAGEN kit as a Ct value.

Figure 4 is a diagram showing the results of electrophoresis of the PCR product in the case of PCR after separating the DNA using a solid support and the PCR after separating the DNA using the QIAGEN kit.

5 is a graph showing the results of cell disruption and nucleic acid amplification after separating E. coli by contacting a support having an OTC coated surface with a blood or urine sample containing E. coli.

The present invention relates to a method for amplifying nucleic acids from microorganisms using a non-planar 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 surface to separate DNA.

As described above, existing nucleic acid isolation and purification methods require the addition of high concentrations of reagents separately to bind the nucleic acids, which may affect subsequent processes such as PCR, and are also applied on lab-on-a-chip (LOC). There was a problem that was difficult to do. In addition, the conventional nucleic acid separation and purification method is configured as a separate step separate from the purification or concentration of the microorganism, wherein the step of separating the microorganism and amplifying the nucleic acid from the microorganism in a single container Is unknown.

Therefore, microorganisms can be separated or concentrated by attaching microorganisms on a solid surface such as a substrate, and have a high affinity for nucleic acids, and using a solid support capable of purifying or concentrating nucleic acids derived from the microorganisms. There is a need for a method for amplifying nucleic acids.

It is an object of the present invention to provide a method for amplifying nucleic acids from microorganisms using a solid support having a non-planar surface.

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

Washing and removing material not attached to the solid support, and

A method for amplifying a nucleic acid from a microorganism, comprising performing a PCR on the microorganism attached to the solid support,

The contacting step, washing step, and PCR step are provided in a same vessel.

The method for amplifying nucleic acid from a 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.

As used herein, "nucleic acid" includes DNA, RNA and PNA, preferably DNA.

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 approaches based on the surface tension, bacterial cells can adhere to the surface not only by the bacterial cells but also through the 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 a biological sample and a laboratory sample containing the microorganism. 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, "aspect ratio" means the ratio of the fragment diameter to the height of the pillar. The pillar structure may have a ratio of the height between the pillar height and the pillar of 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 includes washing and removing material that is not attached to the solid support after the contacting step. In the washing, any solution capable of removing impurities and the like which may adversely affect subsequent processes may be used without causing the target microorganism attached to the surface of the solid support to detach 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.

The method also includes amplifying the nucleic acid by performing PCR on the microorganism attached to the solid support.

In the amplification step, any nucleic acid amplification method known in the art may be used for the nucleic acid amplification, and is preferably performed by PCR. "PCR" refers to a polymerase chain reaction, and is a method of amplifying a target nucleic acid from a primer pair that specifically binds to the target nucleic acid using the polymerase. PCR typically includes four types of dNTPs (dATP, dGTP, dCTP, dTTP) and buffers as templates, primers, DNA polymerases, and DNA monomers. In the amplification step of the present invention, the template DNA is a nucleic acid exposed from the microorganisms attached to the solid support by thermal cycling during PCR. Therefore, the PCR can amplify a target nucleic acid by, for example, adding a PCR reaction component other than the template DNA to a container containing the solid support to which the microorganism is attached, and performing PCR. The step of crushing cells in the thermal cycle for the PCR is considered to be a pre-denatured and / or denatured step, but the present invention is not limited to a specific mechanism. Therefore, in the amplification step, the template DNA is used in the PCR reaction as contained in the microorganism attached to the solid support without undergoing a separate separation process.

In the method of the present invention, the contacting step, the washing step, and the amplifying step are performed in the same vessel. 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, for example, a heater, a cooler, and a temperature controller. Thus, the method of the present invention may be such that the nucleic acid extraction is performed in a microchamber and the nucleic acid amplification is performed in the same microchamber.

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: Isolation, disruption and nucleic acid amplification of E. coli cells in blood using a solid support having a columnar structure

In this example, a sample containing cells was flowed into a flow apparatus having a chamber including a solid support having an inlet and an outlet and an array of pillars formed on a chip having a size of 10 mm x 23 mm. Next, PCR was performed using DNA in the cells bound to the solid support as a 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 in which the pillar array is formed has a SiO ₂ layer.

As the sample containing the cells, a sample (pH 4.7) obtained by mixing blood containing 0.1 OD ₆₀₀ E. coli (about 10 ⁸ E. coli / ml) with an equal amount of acetate buffer (pH 3.0) was used.

The sample was injected at the inlet to flow through the chamber to the outlet. The flow rate was 200 μl / min and 500 μl flowed. Then, the flow apparatus was centrifuged to remove the solution remaining in the chamber, and 3.0 µl of the PCR reaction solution was injected.

The PCR reaction solution was prepared with 1 × PCR buffer, 200 μM dNTP, 200 nM of each primer, 2.5 mM of MgCl ₂ , BSA 1 mg, 5% PEG, 0.5x Syber green, and 0.3 unit of Taq polymerase in a total volume of 3.0 μl. A PCR reaction solution containing was added to the chamber. The primers have nucleotide sequences of SEQ ID NOs: 1 and 2, respectively.

The flow apparatus including the chamber was mounted on the TMC-1000 (Samsung Tech One, Korea), and the E. coli cells were crushed by heat by repeating 10 seconds at 95 ° C and 10 seconds at 65 ° C three times. The flow apparatus used in the present embodiment is mounted on the TMC-1000 in the form of a module so that a PCR reaction can be performed.

Next, thermal cycling for PCR was performed. The thermocycling conditions are as follows. The total denaturation was performed for 10 seconds at 94 ° C., 10 seconds at denaturation 94 ° C., 10 seconds at annealing 62 ° C., and 10 seconds at extension 72 ° C. was repeated 40 times.

The concentration of amplified nucleic acid during PCR was confirmed by detecting Syber green. Fig. 1 is a diagram showing the result of E. coli attached to a solid support and PCR of the DNA in the cells as a template. As shown in FIG. 1, when E. coli cells are concentrated using a solid support, E. coli is disrupted by heat during PCR, and PCR is performed using DNA exposed therefrom as a template. The sample without concentration reduced the Ct value by the same procedure as compared to PCR. Thus, it can be seen that cell concentration occurred by the solid support of the present invention. In FIG. 1, the positive control sample was PCR under the same conditions using a sample containing DNA, and PCR of a sample without concentration was a sample before the sample containing the E. coli was flowed into the chamber. In the same amount as the sample flowed into the chamber remaining in the chamber was used, otherwise the PCR was carried out under the same conditions.

Example  2: Isolation of Escherichia Coli Cells in Blood and Serum Using a Solid Support Having a Columnar Structure, and Nucleic Acid Amplification: Effect of Washing

In this embodiment, blood and serum samples containing cells are flowed into a flow apparatus having a chamber including a solid support having an inlet and an outlet and an array of pillars formed on a chip having a size of 10 mm x 23 mm. Cells were attached to the solid support. Next, PCR was performed by washing the solid support to remove the material not attached to the solid support, or DNA in the cells attached to the solid support without the washing step.

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 in which the pillar array is formed has a SiO ₂ layer.

The blood and serum samples containing the cells were samples (pH 4.7) obtained by mixing blood and serum containing 0.1 OD ₆₀₀ E. coli (about 10 ⁸ E. coli / ml), respectively, with an equal amount of acetate buffer (pH 3.0). The serum was used isolated from the blood.

The blood and serum samples were injected into the inlet to flow through the chamber to the outlet. The flow rate was 200 μl / min and 500 μl flowed. When washing, acetate buffer (pH 4.0) was injected at the inlet to flow out through the chamber to the outlet. The flow rate was 200 μl / min and 500 μl flowed. Then, the flow apparatus was centrifuged to remove the solution remaining in the chamber, and 3.0 µl of the PCR reaction solution was injected.

The PCR reaction solution contained 1 × PCR, 200 μM dNTP, 200 nM of each primer, 2.5 mM of MgCl ₂ , 1 mg of BSA, 5% PEG, 0.5x Syber green, and 0.3 unit of Taq polymerase in 3 μl total volume. PCR reaction solution was added to the chamber. The primers have nucleotide sequences of SEQ ID NOs: 1 and 2, respectively.

The flow apparatus including the chamber was mounted on the TMC-1000 (Samsung Tech One, Korea), and the E. coli cells were crushed by heat by repeating 10 seconds at 95 ° C and 10 seconds at 65 ° C three times. The flow apparatus used in the present embodiment is mounted on the TMC-1000 in the form of a module so that a PCR reaction can be performed.

Next, thermal cycling for PCR was performed. The thermocycling conditions are as follows. The total denaturation was performed for 10 seconds at 94 ° C., 10 seconds at denaturation 94 ° C., 10 seconds at annealing 62 ° C., and 10 seconds at extension 72 ° C. was repeated 40 times.

The concentration of amplified nucleic acid during PCR was confirmed by detecting Syber green. 2A and 2B are diagrams showing results obtained by attaching a blood or serum sample containing Escherichia coli to a solid support, crushing cells by heat, and PCR using the DNA in the lysate as a template. As shown in Figs. 2A and 2B, when E. coli cells were concentrated and crushed using a solid support, and PCR was performed using DNA in the lysate as a template, a sample without cell enrichment was compared by PCR in the same procedure. Ct value decreased. As shown in FIG. 2A, for blood samples, the Ct value decreased when the solid support was washed to remove material that was not attached to the solid support. However, as shown in FIG. 2B, the serum sample had no effect upon washing. This indicates that a PCR inhibitor is present in the blood and the inhibitor is removed by the wash.

In FIG. 2, the control group is a DNA isolated from blood using a QiAamp DNA mini kit, and the DNA is PCR under the same conditions, and the "binding" is a sample without undergoing a washing step after the binding step, and "binding-washing" Is a sample that has undergone a washing step after the binding step. In FIG. 2A, the arrow indicates the Ct value decreased with washing.

Example  3: Isolation, disruption and nucleic acid amplification of E. coli cells in blood using a solid support having a columnar structure: Effect of E. coli concentration

In this embodiment, a blood sample including cells is flowed into a flow apparatus having a chamber including a solid support having an inlet and an outlet and an array of pillars formed on a chip having a size of 10 mm x 23 mm. Was attached to the solid support. Next, the solid support was washed to remove material that did not adhere to the solid support. Next, the cells adhered to the solid support were disrupted by heat to obtain cell lysates, and PCR was performed using DNA in the lysate as a 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 in which the pillar array is formed has a SiO ₂ layer.

The blood sample containing the cells was prepared by mixing blood containing 0.01 OD ₆₀₀ E. coli (about 10 ⁷ E. coli / ml) and 0.001 E. coli OD ₆₀₀ (about 10 ⁶ E. coli / ml) with an equal amount of acetate buffer (pH 3.0). It was a sample obtained (pH 4.7).

The sample was injected at the inlet to flow through the chamber to the outlet. The flow rate was 200 μl / min and 500 μl flowed. Next, acetate buffer (pH 4.0) was injected into the inlet to flow out through the chamber to the outlet. The flow rate was 200 μl / min and 500 μl flowed. Then, the flow apparatus was centrifuged to remove the solution remaining in the chamber, and 3.5 μl of the PCR reaction solution was injected (experimental group). The PCR reaction solution consisted of 200 μM dNTP, 900 nM of each primer, 2.5 mM of MgCl ₂ , 1 mg of BSA, 5% PEG, Taqman probe (SEQ ID NO: 3), 400 nM, and 0.1 units in a total volume of 3.5 μl 1 × PCR buffer. Taq polymerase. The primers have nucleotide sequences of SEQ ID NOs: 1 and 2, respectively. As a comparative experiment, DNA was isolated from the blood sample using a QiAamp DNA mini kit (QIAGEN), and PCR was performed under the same conditions using the separated sample DNA as a template (comparative experimental group).

The flow apparatus including the chamber was mounted on the TMC-1000 (Samsung Tech One, Korea), and the E. coli cells were crushed by heat by repeating 10 seconds at 95 ° C and 10 seconds at 65 ° C three times. The flow device used in the present embodiment is mounted on the TMC-1000 in the form of a module so that a PCR reaction can be performed.

After the cell disruption step, thermal cycling for PCR was performed. The thermocycling conditions are as follows. The total denaturation was performed at 94 ° C. for 10 seconds, followed by 40 times of 5 seconds at denaturation 94 ° C., 20 seconds at annealing 45 ° C. and 20 seconds at 72 ° C. extension.

The concentration of amplified nucleic acid during PCR was confirmed by detecting Syber green. 3 is a diagram showing the results of Ct values when the DNA was separated using a solid support and subjected to PCR (experimental group) and when the DNA was separated after PCR using the QIAGEN kit (comparative experimental group). The control group was experimented by putting the cells in the chamber by concentration without concentration. As shown in Figure 3, regardless of the E. coli concentration, the cell enrichment effect, and when using the QIAGEN kit, it was confirmed that the enrichment effect is greater. In Figure 3, the sample containing 10 ⁶ E. coli / ml E. coli was concentrated 80 times, the sample containing 10 ⁷ E. coli / ml E. coli was concentrated 100 times.

Figure 4 is a diagram showing the results of electrophoresis of the PCR product in the case of PCR after separating the DNA using the solid support and the PCR after separating the DNA using the QIAGEN kit. As shown in FIG. 4, in the case of using the QIAGEN kit, non-specific products were present in the PCR product, but when the PCR was performed after separating the DNA using the solid support, the non-specific product was not present. Therefore, when using the method of the present invention, the specificity of nucleic acid amplification is high.

Example  4: using a solid support having a columnar structure DNA Amplification of: Influence of Solid Support Surface

In this embodiment, PCR is performed on the solid support by incorporating DNA into a flow apparatus having a chamber including a solid support having an inlet and an outlet and an array of pillars formed on a chip having a size of 10 mm x 23 mm. It was.

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 was formed was coated with OTC and PEIM on the SiO ₂ layer, respectively.

3.5 μl of the PCR reaction solution was injected into the chamber of each support. The PCR reaction solution was prepared in a total volume of 3.5 μl 1 × PCR buffer, 200 μM dNTP, each primer 900 nM, MgCl ₂ 2.5 mM, BSA 1 mg, 5% PEG, Taqman probe 400 nM, bacterial genome DNA 1 ng, and 0.1 unit Taq polymerase. The primers have nucleotide sequences of SEQ ID NOs: 1 and 2, respectively.

The flow apparatus including the chamber was mounted on the TMC-1000 (Samsung Tech One, Korea) and thermal cycling for PCR was performed. The thermocycling conditions are as follows. The total denaturation was performed at 94 ° C. for 10 seconds, followed by 40 times of 5 seconds at denaturation 94 ° C., 20 seconds at annealing 45 ° C. and 20 seconds at 72 ° C. extension.

The concentration of amplified nucleic acid during PCR was confirmed by detecting Taqman probe. The concentration and Ct value of the amplification products for each sample are shown in Table 1 below.

Table 1.

Solid support type Ct value SiO2_1 17.75 SiO2_2 16.68 OTC-1 16.79 OTC-2 16.22 PEIM_1 30.19 PEIM_2 33

As shown in Table 1, the PCR was not affected in the solid support coated with OTC, but was not PCR in the solid support coated with PEIM. It is believed that this is because the surface coated with PEIM is positively charged, but the scope of the present invention is not limited to a specific mechanism.

Example  5: Isolation, disruption and nucleic acid amplification of E. coli cells in blood or urine using a solid support having a columnar structure: effect of sample form

In this embodiment, a blood or urine sample containing cells is flowed into a flow apparatus having a chamber including a solid support having an inlet and an outlet and an array of pillars formed on a chip having a size of 10 mm x 23 mm. Cells were attached to the solid support. Next, the solid support was washed to remove material that did not adhere to the solid support. Next, the cells attached to the solid support were disrupted by heat to obtain cell lysates, and PCR was performed using DNA in the lysates as a 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 was formed was OTC coated on a Si0 ₂ layer.

The blood and urine samples containing the cells were samples (pH 4.8) obtained by mixing blood and urine, each containing 0.01 OD ₆₀₀ (about 10 ⁷ Escherichia coli / ml), with an equal amount of acetate buffer (pH 3.0), respectively.

The sample was injected at the inlet to flow through the chamber to the outlet. The flow rate was 200 μl / min and 500 μl flowed. Next, acetate buffer (pH 4.0) was injected into the inlet to flow out through the chamber to the outlet. The flow rate was 200 μl / min and 500 μl flowed. Then, the flow apparatus was centrifuged to remove the solution remaining in the chamber, and 3.5 μl of the PCR reaction solution was injected. The PCR reaction solution contained 1 × PCR buffer, 200 μM dNTP, each primer 900 nM, MgCl ₂ 2.5 mM, BSA 1 mg, 5% PEG, Taqman probe 400 nM, and 0.1 unit of Taq polymerase in a total volume of 3.5 μl. do. The primers have nucleotide sequences of SEQ ID NOs: 1 and 2, respectively.

The flow apparatus including the chamber was mounted on the TMC-1000 (Samsung Tech One, Korea), and the E. coli cells were crushed by heat by repeating 10 seconds at 95 ° C and 10 seconds at 65 ° C three times. The flow apparatus used in the present embodiment is mounted on the TMC-1000 in the form of a module so that a PCR reaction can be performed.

After the cell disruption step, thermal cycling for PCR was performed. The thermocycling conditions are as follows. Total denaturation at 94 ° C. for 10 seconds was repeated 40 times with 5 seconds at denaturation 94 ° C., 20 seconds at annealing 45 ° C. and 20 seconds at 72 ° C. extension.

The concentration of amplified nucleic acid during PCR was confirmed by detecting Taqman probe. 5 is a graph showing the results of cell disruption and amplification after the E. coli was isolated by contacting a support having an OTC-coated surface with a blood or urine sample containing E. coli. In FIG. 5, the control group was PCR under the same conditions as described above except that PCR was performed using DNA isolated using a QIAGEN DNA mini kit (QIAGEN). As shown in FIG. 5, the cells in blood and urine were concentrated due to the decrease in Ct values compared to the control group, and the cells in blood and urine were concentrated by 117 and 20.9 times, respectively.

According to the method for amplifying a nucleic acid from a microorganism according to the present invention, by separating microorganisms, cell disruption and nucleic acid amplification in one container, the nucleic acid can be amplified conveniently, quickly and with high folklore. In addition, automation can be easily performed because the steps from the microorganism to the nucleic acid amplification are performed in a single container. In addition, a large amount of initial sample can be used because the microorganisms can be contacted with the solid support at a high flow rate to adhere to the solid support.

<110> Samsung Electronics Co. Ltd. <120> A method of amplifying a nucleic acid from a microorgansim using          a nonplanar solid substrate <130> PN068864 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer 1 <400> 1 tgtatgaaga aggcttcg 18 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer 2 <400> 2 aaaggtatta actttactc 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Taqman probe <400> 3 gtactttcag cggggaggaa 20  

Claims (15)

Attaching the microorganism to the solid support by contacting a non-planar solid support with a biological sample including the microorganism in the range of pH 3.0 to 6.0, Washing and removing material not attached to the solid support, and A method for amplifying a nucleic acid from a microorganism, comprising amplifying a nucleic acid by performing PCR on the microorganism attached to the solid support, Wherein said contacting step, washing step, and PCR step are performed in the same vessel. The method of claim 1, wherein the microorganism is a bacterium, fungus or virus. The method of claim 1, wherein the biological 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 4, wherein the dilution is performed at a ratio of 1: 1 to 1:10. The method of claim 4, wherein the sample has a salt concentration of 10 mM to 500 mM. The method of claim 6, 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 8, wherein the pillar has an earth pack ratio of 1: 1 to 20: 1. The method of claim 8, wherein the columnar structure has a ratio of column height to column distance between 1: 1 and 25: 1. The method of claim 8, 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 12, 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 14, wherein the surface having at least one amine-based functional group is coated with polyethyleneiminetrimethoxysilane (PEIM).

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