KR20160097881A - Non-Amplification Method for DNA Detection Using Gold Nanoparticle and Magnetic Bead Particle - Google Patents

Abstract

The present invention relates to a method for detecting non-amplified DNA, wherein the method comprises the steps of: (a) producing a DNA probe which is complementary to target DNA; (b) coupling gold nano-particles (GNP) with the DNA probe which is complementary to target DNA and is produced in step (a); (c) coupling magnetic bead particles (MP) with the DNA probe which is complementary to target DNA and is produced in step (a); (d) inducing a DNA binding in a sandwich formation form of target DNA and materials produced in step (b) and step (c); and (e) detecting target DNA by using silver enhancement detection. According to the present invention, a detection limit is enhanced, a specific genetic information is stably collected and analyzed in a short time from a small quantity of disease-related genes, so a possibility of diagnosing a disease in an early stage is improved. Accordingly, the method according to the present invention can be widely utilized in the medical industrial field and the molecule diagnosing industrial field. Also, a sensing platform based on nano-particles according to the present invention can be easily standardized, and a process flow can be easily established. Thus, basic technology which is foundational for studies of miniaturizing a diagnosing device can be ensured. Moreover, through a fusion of fundamental technology with respect to a high sensitivity/multi-sensing material and a sensing scheme and information communications technology, the method according to the present invention can be utilized in developing a ubiquitous sensing system. Consequently, the method according to the present invention can be utilized as technology for not only detecting disease-related materials but also detecting various materials such as environmentally harmful factors.

Description

[0001] The present invention relates to a non-amplified DNA detection method using gold nanoparticles and magnetic bead particles,

(A) preparing a DNA probe complementary to the target DNA; (b) binding a complementary DNA probe to the gold nanoparticle (GNP) with the target DNA prepared in step (a); (c) binding a DNA probe complementary to the target DNA prepared in step (a) to magnetic bead particles (MP); (d) inducing DNA binding in the form of a sandwich formation of the target DNA and the substance prepared in the steps (b) and (c); And (e) relates to a method for detecting non-amplified DNA, comprising detecting target DNA using silver enhancement.

In the era of aging and well-being, the importance of prevention through early diagnosis is emphasized rather than the treatment of diseases. For this purpose, it is required to develop a detection apparatus for diagnosis of diseases with high sensitivity and accuracy as well as portability. In addition, the market for molecular diagnostics is a clear market for advanced technology by combining various fields, and products that complement the shortcomings of real-time PCR have been developed and released.

Detection of DNA having a specific nucleotide sequence has attracted a great deal of attention because it can be applied to diagnosis of pathogenic diseases or genetic diseases. The paradigm of technological development is shifting from past medical technology, which has been in therapy for a long time, to predictive or preventive medical technology. The development of a new concept diagnosis method such as gene diagnosis by disease, The need for the development of technologies for the realization of customized treatments.

Recently, as interest in early diagnosis of diseases has increased, researches on related medical business and new diagnostic methods are being actively carried out. In particular, various diagnostic techniques using nanoparticles have been developed as the usefulness of nanoparticles has been demonstrated in various fields such as diagnosis, drug delivery, nanostructures, and disease treatment. Korean Patent Registration No. 10-1062183 discloses a method for quantifying a protein using nanoparticles. Such a method requires an antibody in the quantitative determination, and accordingly, there is a problem that the reactivity can not be maintained constantly.

Research on the point of care (POC), one of the most remarkable and developing fields in various academic and industrial foundations including the present life, along with the vigorous development of the life industry including the medical industry, A device redesigned for its intended use by combining a biomaterial such as an enzyme, protein, antibody, DNA, microorganism, animal or plant cell / organ, or nerve cell having a specific function of a specific function with a manifold composed of silicon, It was started to apply the new functions of biological organism in the 1970s, namely, multiprocessing function, infinite memory function, self-assembly function, diagnosis of disease using biomaterial specific reaction, It is showing a lot of progress. It is a future fusion convergence industry capable of converging technologies across the whole of science, including electronics, chemistry, and biology, and is expected to have high growth potential.

Unlike other analytical methods, the advantage of POC is to analyze materials quickly and accurately by reacting with samples to be directly measured in the field. In addition, it is possible to quickly and accurately detect the analyte, thereby reducing the detection limit of the disease diagnosis in the medical field, simplifying the measurement, speeding up the sensitivity, and improving the sensitivity. The recognition process for a specific substance is reversible and continuous measurement is possible, and only the analyte can be selectively detected by the biosensor.

In the POC, the selectivity, which is the ability of the bioreceptor to selectively detect the substance to be measured and show it as a detection signal, the detection limit of the concentration (density) range to which the analyte can be measured, (Range & Linear Range), Reproducibility to verify the activity of biosensors such as enzymes after a certain period of time through a standard test, Measurement results after biochemical reaction (Life Time Limits) problem that occurs as the function of the organic material deteriorates over time is required.

Recently, as a POC, biochips are being developed not only for analytical systems that diagnose diseases fast and cheaply but also for biomedical researches and for high-speed and high-speed analytical applications for customized therapies. This innovative diagnostic and analysis system has various interdisciplinary It is developing and applying various chips such as protein chips and cell chips as well as DNA chips that were studied in the early stage of biochip because they are developing at a rapid pace thanks to cooperation and development of new principles. For example, DNA / RNA chips are mainly used to check for the presence of bacterial or viral infections such as sepsis, sexually transmitted diseases, or hepatitis. Tumor-related diseases, including cancer in which a protein biomarker exists, It also uses protein chips. Recently, a cell chip using its own cells is used for the development of therapeutic agents for customized treatment in connection with a specific disease. Recently, the importance of these various biochips has been highlighted in the fields of microarray that can measure simultaneous re-concentration, nano-sensor using nanotechnology for ultra-low concentration measurement, lab-on-a-chip -on-a-chip.

The detection equipment for disease diagnosis using lab-on-a-chip is fully automated by fully automating all necessary processes from the first sample, such as blood, urine, and saliva, to be used in clinical experiments using the biochip As a biochip, it is necessary to include all components necessary for sample analysis on a unit chip, and each component must be connected through a microfluidic conduit. Research results and commercial achievements enable the use of small quantities of samples (<1 μL) in the analysis of organisms and also reduce the amount of reagents used in the analysis, thereby reducing the analytical costs required per sample.

In order to measure infectious pathogens and viruses in various living environments such as saliva, urine, blood, and the like, which can be provided by the human body, nucleic acid-based technology using nucleic acid such as DNA / RNA and specific Antibody-based technology using antibodies is mainly used for molecular diagnosis. Unlike antibody-based technologies, nucleic acid-based technologies can increase diagnostic sensitivity by the powerful sample amplification technology of PCR (Polymerase Chain Reaction), and measurement technology that mainly uses DNA is mainly used for the presence or absence of highly sensitive pathogens. The market for molecular diagnostics using these DNAs is now divided into real-time PCR devices including DNA chip arrays. These methods include Presence-Absence Method, Most-Propable-Number Method, MMOMUG (minimal medium o-nitrophenyl-β- galactosidase-4-methylumbelliferyl-β-D-glucuronide method and Membrane filter (MF) method are accurate, but the measurement time is long and the result is interpreted by the identification method depending on morphological method It was designed to overcome the difficulty of this difficulty.

Unlike antibody-based technologies, the following basic processes are required to measure bacteria and viruses using nucleic acid-based technologies: 1. Cell destruction processes; 2. nucleic acid purification / concentration process; 3. sample amplification process; And 4. sample measurement process. This continuous process requires a labor force equal to that of the conventional cell culture measurement method, requires a specialist for measurement, has a problem that the measurement error is high due to the error of the measurer, and the measurement cost is relatively high .

A method proposed and developed to solve this problem is a lab-on-a-chip (LOC) that can perform all the steps for pathogen measurement on one chip. LOC technology based on MEMS technology and microfluidic technology has been developing in recent years with the convergence of nanobiotechnology. In particular, the LOC technology, which can automate the entire process, is a concept suitable for nucleic acid-based diagnosis that frequently causes diagnosis errors due to possibility of contamination by sample exposure when moving the diagnostic process. , Which is an essential method for transitioning from desktop to small portable.

Particularly, there is a problem that a very small amount of a substance with a concentration that can not be detected, such as a viral infectious disease-related substance, is not detected without an amplification process such as PCR or is detected as an unidentified signal. A method for detecting a HBV (Hepatitis B virus) gene or protein, which is a kind of viral infectious disease-related substance, is disclosed in Korean Patent Publication No. 10-1093783 and Korean Patent Registration No. 10-1209312, There is a problem including an amplification process such as PCR.

The present inventors have conducted continuous research on molecular diagnostic technology such as DNA, and have developed a non-amplification detection method using sandwich formulations using gold nanoparticles and magnetic bead particles. By using a DNA probe complementary to a target DNA, It is possible to detect the genetic information of a substance with a concentration which is difficult to detect in a short period of time in a stable manner, without completing the further process of the present invention.

It is an object of the present invention to provide a non-amplified DNA having a high detection limit by a non-amplification method and capable of stably collecting and analyzing characteristic genetic information from a disease- And to provide a detection method.

In order to accomplish the above object, the present invention provides a DNA probe comprising: (a) preparing a DNA probe complementary to a target DNA; (b) binding a complementary DNA probe to the gold nanoparticle (GNP) with the target DNA prepared in step (a); (c) binding a DNA probe complementary to the target DNA prepared in step (a) to magnetic bead particles (MP); (d) inducing DNA binding in the form of a sandwich formation of the target DNA and the substance prepared in the steps (b) and (c); And (e) providing a non-amplified DNA detection method comprising detecting target DNA using silver enhancement (Fig. 1).

In the present invention, "nanoparticles" means particles having an average size of 100 nanometers or less, preferably 10 nanometers or less (1 nanometer is 10 ^-9 meters).

In the present invention, the target gene may be a viral infection disease gene. The viral infection disease may be hepatitis virus, influenza virus, linovirus, adenovirus, coronavirus, respiratory syncytial virus, The present invention is not limited thereto.

In one embodiment of the present invention, the target gene is HBV (hepatitis B virus DNA of SEQ ID NO: 1, and the DNA probe complementary to the target gene of step (b) is SEQ ID NO: 8, ) Is a DNA probe complementary to a target gene of SEQ ID NO: 9. However, the present invention is not limited thereto.

In the present invention, when the target DNA is in the form of a plasmid DNA, it may further include a step of pre-denaturing the target DNA before the step (a) Denaturing the DNA, but is not limited thereto.

In the present invention, the gold nanoparticles of step (b) may be gold nanoparticles (cGNP) concentrated.

In the present invention, a thiol group may be labeled at the end of a DNA probe complementary to the target DNA of step (b), and a DNA probe complementary to the target DNA of step (c) An amine group may be labeled at the end of the molecule.

In the present invention, the concentration of the DNA probe complementary to the target DNA of step (b) may be 1 to 5 μM, but is not limited thereto.

In the present invention, a DNA probe complementary to the target DNA of step (b) may be activated by using DTT (dithiothreitol) or TCEP (tris (2-carboxyethyl) phosphine) no.

In the present invention, in order to increase the reaction efficiency of the step (b) or (c), a shaking incubator may be used. The shaking incubator may preferably have a speed of 300 to 1350 rpm, Is preferably 650 to 1350 rpm, and most preferably 1350 rpm. However, the present invention is not limited thereto.

In the present invention, the step (e) is of enhanced detection is preferably capable of being carried out at a temperature of 50 ℃, the step (e) of the concentration of the target gene when the detection enhancement ^10-7 Or more, but is not limited thereto.

According to the non-amplified DNA detection method of the present invention, it is possible to detect one-step from DNA extraction to reaction dramatically (up to 20%) in comparison with real-time PCR test commonly used in molecular diagnostics.

In the present invention, "qualitative analysis" is a generic term used to identify which components constitute a sample in a chemical analysis method. The term &quot; qualitative analysis &quot; Reaction and physical properties (for example, spectral and rotational polarization intensities). It is a chemical analysis that refers to a method of detecting what constituent of a substance is, which element, group or root. Each has its own unique chemical reaction or physical properties.

In the present invention, the term "quantitative analysis" refers collectively to analytical methods that clarify the quantitative relationship constituting a substance. When classified by the analytical method, the analytical method using the physicochemical machinery / The chemical analysis is simple and easy to operate and has high precision and low individual variation. Conventionally, mass spectrometry and volumetric analysis were the main methods. Recently, however, quantitative analysis by absorption (absorption) analysis, luminescence analysis, polarography and electrochemical methods have been widely used.

In the present invention, "real-time PCR" refers to the application of a fluorescent material to a PCR technique. The amplification of a target gene existing in a specimen during the reaction and the emission of the fluorescent material And analyze quantitatively the amplification of the target gene and the manner of its amplification in a rapid and accurate manner. This real-time PCR is divided into two methods, one using SYBR Green and the other using double-labeled probes. In the SYBR green technique, the SYBR green dye binds to the amplified DNA in the real-time PCR amplification process and binds to the double-stranded DNA synthesized by the PCR reaction to generate a fluorescence signal. The fluorescence signal is detected and the presence or absence of the target gene The amount of the amplified product of the test substance can be measured. In addition, a real-time PCR using a dual labeled probe is a method using a double-labeled probe in which a fluorescent substance is labeled at the 5 'end and a quencher is labeled at the 3' end, It is possible to confirm whether or not the double-labeled probe is annealed with the PCR amplification product of the target gene through the fluorescence signal from the probe, and the presence or absence of the target gene and the amount of the amplification product from the amplification product can be measured.

In the present invention, "molecular diagnostics" is a generic term for genetic materials such as DNA and RNA, which are used in molecular biology and molecular genetic techniques. It is a diagnostic technique. It is more accurate than blood or urine tests that indirectly read pathologic changes and can avoid histopathology. It is a technology that can be divided into DNA and gene analysis, protein or metabolism analysis, and molecular imaging medicine.

In the present invention, "Point of care" is a technique that provides a great potential for detecting and monitoring infectious diseases in a resource-limited area by eliminating unnecessary steps that may occur in preparing the sample preparation and experiment It is a technology that can be used in a limited situation.

In the present invention, "complementary" means a pairing of nucleotide sequences according to the Watson / Creek pairing rules. For example, sequence 5'-GCGGTCCCA-3 'has the complementary sequence 5'-TGGGACCGC-3'. The complementary sequence may also be an RNA sequence complementary to the DNA sequence. Certain bases which are not commonly found in natural nucleic acids are not limited, but may be included in complementary nucleic acids including indocyanine, 7-diazabicyl, lock nucleic acids (LNA), and peptide nucleic acids (PNA). Complementarity need not be perfect; Stable double strands may have mismatched base pairs, degenerate, or unmatched bases. Those of skill in the art of nucleic acid arts can experimentally determine double-stranded stability, taking into account various variables including, for example, oligonucleotide length, oligonucleotide base composition and sequence, ionic strength and incoincidence base pair frequencies.

In the present invention, "hybridization" means a process in which two complementary nucleic acid strands are recovered from each other under suitable stringency conditions. Oligonucleotides or probes suitable for hybridization are 10-100 nucleotides in length (e.g., 18-50, 12-70, 10-30, 10-24, 18-36 nucleotides in length). Nucleic acid hybridization techniques are well known in the art (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N. Y., 1989).

In the present invention, "probe" means a DNA or RNA fragment of variable length (for example, 3-1000 base length) and is used to detect the presence of target nucleotide sequences complementary to the probe sequence. Typically, the probe hybridizes with a single-stranded nucleic acid (DNA or RNA) capable of probe-target base pairs due to the base sequence due to the complementarity between the probe and the target.

As described above, the non-amplified DNA detection method of the present invention enhances the possibility of early diagnosis of diseases by stably collecting and analyzing characteristic genetic information from a disease-related gene in a short time by increasing the detection limit, It can be widely used in the medical industry field, the molecular diagnosis industry field, and the like. In addition, since the sensing platform based on nanoparticles according to the present invention is easy to standardize and easy to establish a process sequence, it is possible to provide a basic technology for studying miniaturization of diagnostic devices, Through the convergence of source technology and information communication technology, it can be utilized in the development of ubiquitous sensing system. As a result, it can be applied not only to the detection of disease-related substances but also as a technology for detecting various substances such as environmental harmful factors.

1 is a schematic diagram showing a DNA detection method of the present invention.
Fig. 2 shows the sequences of HBV target DNA and probe DNA of Example 1-1.
Fig. 3 shows electrophoresis results of Example 1-2. Fig.
4 shows the activation method of the GNP probe of Example 2-1 and the result of GNP polymerization according to the buffer composition. (A) shows the result when activated using DTT, and (B) shows the result when activated using TCEP.
5 shows the polymerization efficiency according to the concentration of the GNP and GNP probes of Example 2-1.
Fig. 6 shows the results of the possibility evaluation using the synthetic target oligonucleotide of Example 5-1. Fig.
FIG. 7 shows silver detection results of the synthetic HBV gene according to the temperature of Example 5-2. FIG.
8 shows the result of detection of silver enhancement using the oligonucleotide target DNA of Example 5-3.
Fig. 9 shows the detection results of silver enhancement for each plasmid target DNA type in Example 5-4. Fig.
10 shows the results of evaluation of the usefulness of the pre-denaturation process of the plasmid target DNA of Example 5-5.
Fig. 11 shows the results of detection of silver enhancement by plasmid target DNA concentration in Examples 5-6. Fig.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

[ Example  ]

Example  1. Target DNA  And probes probe ) DNA Preparation of

1-1: Target DNA  And Probe DNA Preparation of

The HBV target DNA sequence (SEQ ID NO: 1) was selected. The concrete selection method is as follows. First, using sequencing data of six strands (SEQ ID NOS: 2 to 7), conserved regions that are common to each strand were searched. Using the BLAST (Basic Local Alignment Search Tool; NCBI home page http://blast.ncbi.nlm.nih.gov/Blast.cgi), the relationship between the target sequence and the previously reported HBV gene was confirmed.

Next, the probe DNA sequences (SEQ ID NOS: 8 and 9) to be attached to the nanoparticles were selected. To attach the nanoparticles, thiol groups and amine groups were labeled at the ends of the probes, respectively (FIG. 2). Specifically, an oligonucleotide sequence capable of binding complementarily to both ends of the target DNA was set. For polymerization with nanoparticles, literature review (Edgar D. Goluch et al., Lab on a Chip , 6: 1293-1299, 2006). The type and direction of modification of each oligonucleotide sequence were set.

To summarize, a HBV target DNA sequence of 82 bases was set, a complementary sequence was set up to form 20 bp at both ends, and a 10 base oligodeoxidase (T) sequence was added to ensure space. The terminal was modified for binding with GNP and MP (terminal modification). For visual detection, fluorescent substances were labeled on each probe sequence.

1-2: Target DNA Wow Probe DNA Of sequence complementarity

Polyacrylamide electrophoresis was performed to confirm the complementarity of the sequence of the target DNA and the probe DNA (Fig. 3). As a result, when hybridization was performed by reacting with a GNP probe or an MP probe of lanes 3, 4, 5, and 8 as compared to the 82-mer HBV target DNA detected in lane 1, band migration (band shift) occurred.

1-3: Plasmid DNA Metamorphosis

A sample derived from the human body was used in the form of plasmid DNA. Since the plasmid DNA has a double helix structure, the double helix structure was disassembled at the stage of binding with the probe to form a single helix structure. As a control group, distilled water (DW), which does not cause a sandwich type bioassay, was used, and plasmid DNA in which DNA was extracted from a sample derived from a human body and amplified by cloning was used as an experimental group (provided by SK Telecom; HBV hepatitis patient (2219, 2515, 2618, 2633) were assigned to each experimental group. The HBV DNA was extracted from the blood collected from the blood samples.

Example  2. Gold nanoparticles  Preparation of Polymer ( GNP Conjugation )

Prepare gold nanoparticles (GNP) and concentrated gold nanoparticles (cGNP) in a 1.5 mL microcentrifuge tube. cGNP was prepared by centrifuging GNP at 4 ° C, 12,000 rpm for 20 minutes, discarding 90% of the supernatant, and mixing the remaining solution (vortexing or tapping). Oligonucleotide DNA, a GNP probe, was added to the two groups at 1, 2, 3, and 5 μM, and then incubated at room temperature for 16 hours. Add 1x potassium phosphate buffer (pH 7) to the tube in 1/10 volume, mix well by pipetting and tapping (without vortexing).

Then, the cells were incubated at room temperature for 24 hours. After centrifugation at 12,000 rpm for 20 minutes at 4 DEG C, the supernatant was removed and 1x potassium phosphate buffer (pH 7) was added. When the supernatant was completely removed, it was accompanied with GNP, which is an oil-quality precipitant, and was carefully removed as much as possible, and then the same amount of 1x potassium phosphate buffer (pH 7) was added. Mix well by pipetting and tapping (without vortexing).

The GNP was rinsed by repeating centrifugation at 12,000 rpm at 4 DEG C for 20 minutes, removing the supernatant, and adding 1x potassium phosphate buffer (pH 7).

After transferring to a 96-well plate, the absorbance was measured in a wavelength range of 450 to 700 nm. As a control, GNP and cGNP were prepared and the absorbance was measured in the same conditions as in the experimental group.

2-1: GNP  How the probe is activated and Buffer  According to composition GNP  Polymerization results

The GNP probe was activated by reduction using DTT (dithiothreitol; A in FIG. 4) or TCEP (tris (2-carboxyethyl) phosphine; When DTT was used, it was post-treated with ethyl acetate. KPB or Tris buffer of pH 7.0 or 7.5 was also used. The absorbance was measured according to the activation method and buffer composition of each of the GNP probes as described above to confirm the GNP polymerization results (FIG. 4). As a result, it was confirmed that the efficiency was the best when TCEP activated pH 7.0 potassium phosphate buffer solution was used.

2-2: GNP Wow GNP  Polymerization efficiency according to probe concentration

The concentration of the GNP probe was adjusted to 1 or 2 using GNP stock solution (FIG. 5A), GNP (cGNP; FIG. 5B) concentrated 10 times, potassium phosphate buffer (pH 7.0) and TCEP-activated oligonucleotide DNA. , 3, and 5 μM, and absorbance (520 to 530 nm) was measured to confirm the GNP polymerization efficiency (FIG. 5). As a result, when the GNP probe was combined, it was confirmed that the peaks were shifted (absorbance shift) compared with the case where the GNP probes were not combined.

Example  3. Magnetic bead particle  Preparation of Polymer ( MP - probe conjugation )

The magnetic beads in powder form were dissolved in 200 μL of suspend buffer (10 ⁶ cells / μL) and mixed with amine-modified oligonucleotide DNA (100 ng / μL). The mixture was thoroughly mixed by vortexing or pipetting, followed by reaction at 50 ° C overnight (shaking; 1350 rpm). The tube containing the sample was placed in a magnetic separator and magnetic beads were separated for 3 minutes. The supernatant was transferred to a new tube and washed 5 times. The beads were resuspended in distilled water so that the magnetic beads became 1 mg / mL and stored at 4 ° C.

3-1: Shaking incubator rpm  Depending on speed MP  Polymerization efficiency

The MP polymerization efficiency was measured by varying the rpm rate of the shaking incubator at 300, 650, and 1350 rpm. C1 means the result before the reaction, and C2 means the result after reacting at each rpm. As a result, it was confirmed that the binding efficiency was high when the rpm rate of the shaking incubator was 650 to 1350 rpm (Table 1).

Sample ID Nucleic acid concentration unit A260 A280 A260 / 280 A260 / 230 Coupling Efficiency (%) C1 16.4 ng / μl 0.490 0.307 1.60 0.65 300rpm
(C2, 8hr) 7.3 ng / μl 0.146 0.099 1.47 1.76 70.20 650rpm
(C2, 8hr) 1.2 ng / μl 0.024 0.014 1.69 0.47 95.10 1350rpm
(C2, 8hr) 0.7 ng / μl 0.02 0.004 5.41 0.42 95.91

(A: absorbance, unit nm)

3-2: Depending on the type of cleaning solution MP  Polymerization efficiency

The MP polymerization efficiency was measured by varying the wash solution. As a result, compared with the kit (BcMag ^TM Quick Oligo-DNA Conjugation Kit, Bioclon Inc., USA), DW and potassium phosphate buffer were suitable as washing solutions, but PBS was not suitable (Table 2).

Sample ID Nucleic acid concentration unit A260 A280 260/280 260/230 pre-bound MP solution 24.2 ng / μl 0.483 0.292 1.65 1.25 Kit 3 ng / μl 0.06 0.018 3.39 0.05 D.W 2.4 ng / μl 0.049 0.02 2.38 0.39 PBS (pH 7.4) 29.7 ng / μl 0.595 0.049 12.22 0.05 Potassium phosphate
The buffer (pH 7) 2.7 ng / μl 0.053 0.024 2.19 0.53

Example  4. Target binding - sandwich formation ( Sandwich Formation )

The reaction rates of GNP polymer and MP polymer prepared in Examples 2 and 3 were determined (volume: volume, number of particles: number of particles). The GNP coupled with the probe and the probe-bound MP were mixed at a ratio and reacted at 95 ° C for 5 minutes in a heat block. Similar to the preparation of the MP polymer, the hybridization temperature was set at 50 DEG C and reacted in a shaking incubator (1350 rpm) for 1 hour. For comparison, the supernatant from the separator was transferred to a new tube. 10 [mu] L of 1xKPB (pH 7.0) was added to the beads (Edgar D. Goluch et al., Lab on a Chip , 6: 1293-1299, 2006).

The concentration of 82 bp target DNA was measured using a DNA chip (Agilent High Sensitivity DNA Analysis Kit (82 mer), Agilent Technology, USA), and only the concentration of the target DNA was specifically measured using Nanodrop, The results of the assay and nanodrop measurements were compared (Table 3).

Target DNA concentration /
Hybridization temperature Nano drop (ng) Agilent High Sensitivity DNA Analysis Kit (82mer) Input Combination Input Combination 0 μM / 25 ° C. 0 0 0 0 0.5 μM / 25 ° C. 16.04 14.08 16.04 13.41 1 μM / 25 ° C. 31.547 18.687 31.547 22.967 0 μM / 50 ° C. 0 0 0 0 0.5 μM / 50 ° C. 16.04 14.34 16.04 7.31 1 μM / 50 ° C. 31.547 18.157 31.547 17.587

Example  5. DNA  Detection - silver detection Silver enhancement )

In Example 4, the sandwich-formulated product (50 μM) was added to the tube bottom in 50 mL. (Silver enhancing detection solution contained in Silver Enhancement Kit (SE100, Sigma-Aldrich, USA)) were mixed and reacted at a ratio of 1: 1. The silver detection results were confirmed by measuring the electrical resistance using an electrode.

5-1: Synthesis HBV  Evaluation of Silver Detection Potential for Silver Growth

The probes of Example 1 were used to evaluate whether the sandwich formation of Example 4 and the silver enhancement detection of Example 5 were possible through the synthetic target oligonucleotides. As a result, it was confirmed that the bioassay was possible by measuring the simple resistance value (FIG. 6).

5-2: Synthesis HBV  The results of silver detection

In order to evaluate the silver detectability of the synthetic HBV gene, the silver detection was confirmed by varying the temperature to 25 ° C or 50 ° C. As a result, it was confirmed that the resistance value with respect to the control group was larger when silver detection test was conducted at 50 캜, and that stable results could be obtained (Fig. 7).

5-3: oligonucleotide target DNA Silver Enhancement Detection Result Using

The results of detection of silver enhancement were confirmed by varying the target DNA concentration to 1, 0.5, 0.1, 0 μM and the temperature at 25 ° C or 50 ° C. As a result, it was confirmed that the detection of silver enhancement was more affected by the concentration of the target DNA than the temperature (FIG. 8).

5-4: Result of silver detection by comparison standard

The reaction was carried out at 25 캜 or 95 캜 for 25 minutes to confirm the silver enhancement detection result. The efficiency of the silver enhancement detection was compared based on the amount of the solution containing beads (A and B in FIG. 9) or the number of beads (C and D in FIG. 9). As a result, it was confirmed that the efficiency at 95 ° C was higher than that at low temperature. In addition, it was confirmed that the efficiency was higher when the number of beads was compared with each other (FIG. 9).

5-5: Plasmid target DNA Evaluation of the pre-metamorphic process

As in Example 1, when the plasmid DNA was used in the form of a plasmid target DNA, the plasmid DNA had a double helix structure, so that the double helix structure was disassembled at the step of binding with the probe to form a single helix structure. Therefore, the usefulness of the pre-denaturation process of the plasmid target DNA was evaluated. As a result, since the experimental group and the control group showed a considerable difference when they were subjected to pre-denaturation, experiments were possible at various concentrations, and the usefulness of the pre-denaturation procedure was confirmed (FIG. 10).

5-6: Plasmid target DNA  Concentration and silver detection results

The concentration of plasmid target ^{DNA, 10 0 (30μg / μl} ), 10 -1 (3μg / μl) 10 -2 (300ng / μl), 10 -3 (30ng / μl), 10 -4 (3ng / μl), ^{10 -5 (300pg / μl),} 10 -6 (30pg / μl), 10 -7 (3pg / μl), 10 -8 (300fg / μl), in contrast to 95 ℃ to 10 ^-9 (30fg / μl) The reaction was carried out for 25 minutes. As a result, it was confirmed that the resistance value when the plasmid target DNA concentration was 10 ^-7 or more was low as compared with the control group (Fig. 11). This indicates that the concentration of the plasmid target DNA can be detected up to 10 &lt; ^{-7 &} gt ;.

While the present invention has been described with reference to exemplary embodiments, those skilled in the art will appreciate that various changes and modifications can be made thereto without departing from the scope of the invention, . In addition, many modifications may be made to adapt a particular situation and material to the teachings of the invention without departing from the essential scope thereof. Accordingly, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention be construed as including all embodiments falling within the scope of the appended claims.

<110> SK TELECOM CO., LTD. <120> Non-Amplification Method for DNA Detection Using Gold          Nanoparticle and Magnetic Bead Particle <130> SP-53774 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> HBV <400> 1 ctgctcaagg aacctctatg tttccctctt gttgctgtac aaaaccttcg gacggaaact 60 gcacttgtat tcccatccca tc 82 <210> 2 <211> 1280 <212> DNA <213> Unknown <220> <223> HBV DNA <400> 2 agattacccg gaccttgaca ggctccggcc gccatggcgg ccgcgggaat tcgatttgct 60 ggtggctcca gttccggaac aataaaccct gttccgacta ctgcctcacc catatcgtca 120 atcttcttga ggactgggga ccctgcaccg aacatggaga gcacaacatc aggattccta 180 ggacccctgc tcgtgttaca ggcggggttt ttcttgttga caagaatcct cacaataccg 240 cagagtctag actcgtggtg gacttctctc aattttctag ggggagcacc aaagtgtcct 300 ggccaaaatt cgcagtcccc aacctccaat cactcaccaa cctcttgtcc tccaatttgt 360 cctggctatc gctggatgtg gctgcggcgt tttatcatat tcctcttcat cctgctgcta 420 tgcctcatct tcttgttggc tcttctggac taccaaggta tgttgcccgt ttgtcctcta 480 cttccagaaa catcaactac cagcacggga ccatgcaaga cctgcacgag tcctgctcaa 540 ggaacctcta tgtttccctc ttgttgctgt acaaaacctt cggacggaaa ctgcacttgt 600 attcccatcc catcatcctg ggctttcgca agattcctat gggagtgggc ctcagtccgt 660 ttctcctgac tcagtttact agtgccattt gttcagtggt tcgtagggct ttcccccact 720 gtttggcttt cagttatatg gatgatgtgg tattgggggc caagtctgta caacatcttg 780 agtcccttta tacctctatt accaattttc ttttgtcttt gggtatacat ttgaccccca 840 ataaaactaa acgttggggc tactccctca atttcatggg atatgtaatt ggaagttggg 900 gtactttacc acaggaacat attgtactta aaatcaagca atgttttcga aaactgcctg 960 taaatcgacc tattgattgg aaagtatgtc aaagaattgt gggtcttttg ggctttgctg 1020 cgcttttaca caatgtggct atcctgcctt gatgctttat atgcatgtat acatcaaagc 1080 agctttcact ttctcgccac tacaagcttt ctatcactag tgatcgcgcg ctgcagtcga 1140 ccatatggga gagctcccac gcgtgatgca tagctgagtt tccatatttc acttaatagc 1200 tgggcgtaat ccatggctac cggtcttgtg tggatgttat ccgctccaaa ttcagcaaca 1260 attcagacgc ggagccataa 1280 <210> 3 <211> 1257 <212> DNA <213> Unknown <220> <223> HBV DNA <400> 3 ggctcccggg ccgccatggc ggccgcggga attcgatttg ctggtggctc cagttccgga 60 acagtaaacc ctgttccgac tactgcctca cccatatcgt caatcttctc gaggactggg 120 gaccctgcac cgaacatgga gagcacaaca tcaggattcc taggacccct gctcgtgtta 180 caggcggggt ttttcttgtt gacaagaatc ctcacaatac cacagagtct agactcgtgg 240 tggacttctc tcaattttct aggggaagca cccgcgtgtc ctggccaaaa ttcgcagtcc 300 ccaacctcca atcactcacc aacctcttgt cctccaattt gtcctggcta tcgttggatg 360 tgtctgcggc gttttatcat attcctcttc atcctgctgc tatgcctcat cttctcgttg 420 gtctctctgg actaccaagg tatgttgccc gtttgtcctc tacttccagg aacatcaact 480 accagcacgg gaccatgcaa gacctgcacg attcctgctc aaggaacctc tatgtttccc 540 tcttgttgct gtacaaaacc ttcggacgga aactgcactt gtattcccat cccatcatcc 600 tgggctttcg caagattcct atgggagtgg gcctcagtcc gtttctcctg gttcagttta 660 ctagtgccat ttgttcagtg gttcgcaggg ctttccccca ctgtttggct ttcagttata 720 tggatgatgt ggtattgggg gccaagtctg tacaacatct ggagtccctt tttacctcta 780 ttaccaattt tcttttgtct ttgggtatac atttgacccc taataaaacc aaacgctggg 840 gctactccct taacttcatg ggatatgtaa ttggaagttg gggtacttta ccacaggaac 900 atattgtatt aaaaatcaag caatgttttc ggaaactgcc tgtaaataga cctgttgatt 960 ggaaagtatg tcaagaattg tggtcttttg gctttgctgc cccttttaca catgtggcta 1020 tctgctaatg ctttatatac atgtatacat ctagcagctt cactttctcg ccactacagc 1080 ttctatcact atgatcgcgg cgctgcagtc gacatatggg agagctccca acgcgtgatg 1140 catagctgag atctatatgt cacttaatag ctgcgtatca tggtcaaagc cgttctggtg 1200 gatggtatcc gctccaattc acaacacaat cagagccggg agcacataag gtgtagc 1257 <210> 4 <211> 1272 <212> DNA <213> Unknown <220> <223> HBV DNA <400> 4 tgtggccaag tcgcatgctc cggccgccat ggcggccgcg ggaattcgat ttgctggtgg 60 ctccagttcc ggaacagtaa accctgttcc gacttctgcc tcacccatat cgtcaatctt 120 ctcgaggact ggggaccctg cacagaacat ggagagcaca acatcaggat tcctaggacc 180 cctgctcgtg ttacaggcgg ggtttttctt gttgacaaga atcctcacaa taccgcagag 240 tctagactcg tggtggactt ctctcaattt tccaggggga gcacccaggt gtcctggcca 300 aaattcgcag tccccaacct ccaatcactc accaacctct tgtcctccaa cttgtcctgg 360 ctatcgctgg atgtgtctgc ggcgttttat catattcctc ttcatcctgc tgctatgcct 420 caccttcttg ttggttcttg tggactaccg aggtatgttg cccgtttgtc ctctacttcc 480 aggaacatca actaccagca cgggaccctg caagacctgc acgagtcctg ctcaaggaac 540 ctctatgttt ccctcttgtt gctgtacaaa accttcggac ggaaactgca cttgtattcc 600 catcccatca tcctgggctt tcgcaagatt cctatgggag tgggcctcag tccgtttctc 660 atggctcagt ttcttagtgc catttgttca gtggttcgca gggctttccc ccactgtttg 720 gctttcagtt atgtggatga tgtggtattg ggggccaagt ctgtacaaca tcttgagtcc 780 ctttttacct ctattaccaa ttttcttttg tctttgggta tacatttgaa ccctaataaa 840 accaaacgtt ggggctattc ccttaacttc atgggatatg taattggaag ttggggtact 900 ttaccacagg aacatattgt actaaaaatc aagcaatgtt ttcggaaact gcctgtaaat 960 agacctattg attggaaagt atgtcaaaga attgtgggtc tcttgggctt tgctgcccct 1020 ttacacagtg tgctatcctg ccttgatgct ttatatgcat gtatacagct aaacagcttt 1080 cacttttcgc actacagctt tctatcacta gtgaattcgc gcgctgcagt cgacatatgg 1140 gaaagctcca cgcgtgatgc atagctgagt atttctatag tgtcacttaa atagctgcgt 1200 atcatgttct acgatctgtt catgttatcc gctcaatccc acaacaatcg accggaagca 1260 ataaaagtgt ta 1272 <210> 5 <211> 1251 <212> DNA <213> Unknown <220> <223> HBV DNA <400> 5 agggcgagtc gcagctccgg ccgccatggc ggccgcggga attcgatttg ctggtggctc 60 cagttccgga acagtaaacc ctgttccgac tactgcctca cccatatcgt caatcttctc 120 gaggactggg gaccctgcac cgaacatgga gaacacaaca tcaggattcc taggacccct 180 gctcgtgtta caggcggggt ttttctcgtt gacaagaatc ctcacaatac cacagagtct 240 agactcgtgg tggacttctc tcaattttct agggggagca cccacgtgtc ctggccaaaa 300 ttcgcagtcc ccaacctcca atcactcacc aacctcttgt cctccaactt gtcctggcta 360 tcgctggatg tgtctgcggc gttttatcat attcctcttc atcctgctgc tatgcctcat 420 cttcttgttg gttcttctgg actaccaagg tatgttgccc gtttgtcctc tacttccagg 480 aacatcaact accagcacgg gaccatgcaa gacctgcacg actcctgctc aaggaacctc 540 tatgtttccc tcttgttgct gtacaaaacc ttcggacgga aactgcactt gtattcccat 600 cccatcgtcc tgggctttcg caagattcct atgggagtgg gcctcagtcc gtttctcatg 660 gctcagttta ctagtgccat ttgttcagtg gttcgtaggg ctttccccca ctgtttggct 720 ttcggttatg tggatgatgt ggtattgggg gcgaagtctg tacaacatct tgagtccctt 780 tttacctcta ttaccaattt tcttttgtct ttgggtatac atttgaaccc taataaaacc 840 aaacgttggg gctactccct taacttcatg ggatatgtaa ttggaagttg gggtacttta 900 ccacaggaac atattgtact aaaaatcaag caatgttttc ggaaactgcc tgtaaataga 960 cctattgatt ggaaagtatg tcaaagaatt gtgggtcttt tggctttgct gccccgttta 1020 cacaatgtgg ctatcctgcc ttgatgcctt tatatgcatg tatacagcta gcagctttca 1080 ctttctcgca cttacagact ttctatcact agtgatcgcg gcggctgcaa gtcgacatat 1140 gggagaagct ccacgcgttg atgcatagcc tggagtatct cttacgttca ctaaatagct 1200 tcgcgtatca tggtccatag cccgttcctg ctggtggaaa aatgggttaa t 1251 <210> 6 <211> 1269 <212> DNA <213> Unknown <220> <223> HBV DNA <400> 6 tgggcgagtc gcatgctccg gccgccatgg cggccgcggg aattcgattt ggctccagtt 60 ccggaacagt aaaccctgtt ccgactactg cctcacccat atcgtcaatc ttctcgagga 120 ctggggaccc tgcacagaac atggagagca caacatcagg attcctagga cccctgctcg 180 tgttacaggc ggggtttttc ttgttgacaa gaatcctcac aataccacag agtctagact 240 cgtggtggac ttctctcaat tttctagggg gagctcccac gtgtcctggc caaaattcgc 300 agtccccaac ctccaatcac tcaccaacct cttgtcctca aatttgtcct ggctatcgct 360 ggatgtgtct gcggcgtttt atcatattcc tcttcatcct gctgctatgc ctcatcttct 420 tgttggttct tctggactac caaggtatgt tgcccgtttg tcctctactt ccaggaacgt 480 caactaccag cacgggacca tgcaagacct gcacgattcc tgctcaagga acctctatgt 540 ttccctcttg ttgctgtaca aaaccttcgg acggaaactg cacttgtatt cccatcccat 600 catcctgggc tttcgtaaga ttcctatggg agtgggcctc agtccgtttc tcctgactca 660 gtttactagt gccatttgtt cagtggttcg tagggctttc ccccactgtt tggctttcag 720 ttatatggat gatgtggtat tgggggccaa gtctgtacaa catcttgagt ccctttttac 780 ctctattacc aattttcttt tgtctttggg tatacatttg aaccctaata aaaccaaacg 840 ttggggctac tcccttaact tcatgggata tgtaattgga agttggggta ctttaccaca 900 gt; tgattggaaa gtatgtcaaa gaattgtggg tcttttgggc tttgctgccc cttttacaca 1020 atgtgctatc ctgccttgat gctttgtatg catgtataca agctaagcag ctttcacttt 1080 ctccgccact acaagctttc taatcactat gaatccgcgc tgcagtcgac catatgggaa 1140 agcttcccac gcgtgaagca tagcctgaaa atctcataag tacctaaata gctggcgatc 1200 cactgctacc gttctctgtg gagatgttat cggctccaaa tcaccacaat ccagatcggg 1260 agacaataa 1269 <210> 7 <211> 1282 <212> DNA <213> Unknown <220> <223> HBV DNA <400> 7 aggcgaagtc gcatgctccg gccgccatgg cggccgcggg aattcgattt gctggtggct 60 ccagttccgg aacagtaaac cctgttccga ctactgcctc acccatatcg tcaatcttct 120 cgaggactgg ggaccctgca ccgaacatgg agagcataac atcaggattc ctaggacccc 180 tgctcgtgtt acaggcgggg tttttcttgt tgacaagaat cctcacaata ccacagagtc 240 tagactcgtg gtggacttct ctcaattttc tagggggagc acccacgtgt cctggccaaa 300 attcgcagtc cccaacctcc aatcactcac caacctcttg tcctccaatt tgtcctggct 360 atcgctggat gtgtctgcgg cgttttatca tattcctctt catactgctg ctatgcctca 420 tcttcttgtt ggttcttctg gactaccaag gtatgttgcc cgtttgtcct ctacttccag 480 gaacatcaac taccagcacg ggaccatgca agacctgcac gactcctgct caaggaacct 540 ctatgtttcc ctcttgttgc tgtacaaaac cttcggacgg aaactgcact tgtattccca 600 tcccatcatc ctgggctttc gcaagattcc tatgggagtg ggcctcagtc cgtttctcat 660 ggctcagttt actagtgcca tttgttcagt ggttcgcagg gctttccccc actgtttggc 720 tttcggttat gtggatgatg tggtattggg ggccaagtct gtacaacatc ttgagtccct 780 ttttacctct attaccaatt ttcttttgtc tttgggtata catttgaacc ctaataaaac 840 caaacgttgg ggctactccc ttaacttcat gggatatgta attggaagtt ggggtacttt 900 accacaagaa catattgtat taaaaatcaa acaatgtttt cgaaaactgc ctgtaaatag 960 acctattgat tggaaagtct gtcaaagaat tgtgggtctt ttgggctttg ctgccctttt 1020 acacaatgtg gctatcctgc ctgatgcctt tatatgcatg tatacaatct aagcagcttt 1080 cactttctcg cacttacagc tttctatcac tatgaattcc cgccgcctgc agtcgaacat 1140 atgggagagc tccccaccgc gtggatgcat agctgagaaa ttctaaggta actaatagct 1200 ggcgatacat gcctaatccg gttcttgtga aatgttatcg ctccattcgc catcgaccgg 1260 agcataaagg tgagagcctc cg 1282 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> DNA probe <400> 8 tttttttttt gacgagttcc ttggagatac 30 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> DNA probe <400> 9 tgaacataag ggtagggtag tttttttttt 30

Claims (16)

A non-amplified DNA detection method comprising the steps of:
(a) preparing a DNA probe complementary to the target DNA;
(b) binding a complementary DNA probe to the gold nanoparticle (GNP) with the target DNA prepared in step (a);
(c) binding a DNA probe complementary to the target DNA prepared in step (a) to magnetic bead particles (MP);
(d) inducing DNA binding in the form of a sandwich formation of the target DNA and the substance prepared in the steps (b) and (c); And
(e) detecting the target DNA using silver enhancement.
The non-amplified DNA detection method according to claim 1, wherein the target DNA is a viral infectious disease gene.
The non-amplified DNA detection method according to claim 1, wherein the target DNA is HBV (hepatitis B virus) DNA of SEQ ID NO: 1.
3. The method according to claim 1, further comprising pre-denaturing the target DNA prior to step (a) when the target DNA is in the form of a plasmid DNA. Way.
5. The method according to claim 4, characterized in that the target DNA is pre-denatured at 95 ° C.
The non-amplified DNA detection method according to claim 1, wherein the gold nanoparticles in step (b) are concentrated gold nanoparticles (cGNP).
The non-amplified DNA detection method according to claim 1, wherein the DNA probe complementary to the target DNA of step (b) comprises SEQ ID NO: 8.
The non-amplified DNA detection method according to claim 1, wherein a thiol group is labeled at the end of a DNA probe complementary to the target DNA of step (b).
The non-amplified DNA detection method according to claim 1, wherein the concentration of the DNA probe complementary to the target DNA of step (b) is 1 to 5 μM.
2. The method according to claim 1, wherein the DNA probe complementary to the target DNA of step (b) is activated using DTT (dithiothreitol) or TCEP (tris (2-carboxyethyl) phosphine) Way.
2. The method according to claim 1, wherein an amine group is labeled at the end of a DNA probe complementary to the target DNA of step (c).
The non-amplified DNA detection method according to claim 1, wherein the DNA probe complementary to the target DNA of step (c) comprises SEQ ID NO: 9.
The non-amplified DNA detection method according to claim 1, wherein a shaking incubator is used to increase the reaction efficiency of the step (b) or (c).
14. The method according to claim 13, characterized in that the speed of the shaking incubator is from 300 to 1350 rpm.
The method according to claim 1, wherein the silver detection of step (e) is performed at a temperature of 50 캜.
The non-amplified DNA detection method according to claim 1, wherein the concentration of the target gene is 10 ^-7 or more when detecting silver enhancement in step (e).

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