WO2019078638A2 - Method for enriching pathogen, using homobifunctional imidoester - Google Patents

Abstract

The present invention relates to a method for enriching pathogens, using a homobifunctional imidoester group. A pathogen-enriching method and a nucleic acid isolation method using a homobifunctional imidoester compound (DMA, DMP, DMS) according to the present invention can rapidly extract a low amount of pathogens contained in a sample without using a particular device and can be thus utilized as an in-situ diagnostic method. Being capable of concurrent pathogen enrichment and nucleic acid extraction in one tube or chip, the method has advantages over conventional methods in terms of high efficiency, excellent time and cost saving, and convenient usage.

Description

Method of Concentration of Pathogens Using Homologous Functional Imidoesters

The present invention relates to a method for concentrating a pathogen using a homobifunctional imidoester (HI) compound, and more particularly, to a method for concentrating a pathogen by using dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP ) Or dimethyl suberimidate (DMS). ≪ / RTI >

Nucleic acid is an important analytical tool for identifying disease states, and DNA biomarkers, such as single nucleotide polymorphism (SNP), mutation or DNA methylation, And provides an important clue to providing a great opportunity for prognosis and surveillance as well as diagnosing and monitoring the condition of the disease during the early stages of the disease.

Since nucleic acids such as DNA are present at very low physiological concentrations compared to other components such as proteins (e.g., tens of nanograms of DNA versus a few tens of micrograms of protein per microliter of whole blood), DNA is effectively extracted from clinical samples Preconcentration is very important for subsequent processes such as amplification and detection. In the case of methylated DNA, this problem is even more important.

Conventional methods for detecting microorganisms can not use the entire solution in a patient sample, and nucleic acid is extracted using only a part of them, and detection using the method has been carried out. There is no big problem in the case of a large amount of microorganisms, but in the case of a small amount of microorganisms, there is a problem in controlling the additional infectious diseases because it can not be detected accurately. It is necessary to study the concentration method for using all the microorganisms as much as possible in the sample Do.

In recent years, the use of high purity purified nucleic acids has been increasing in diverse fields such as biotechnology, diagnostic medicine, pharmacology, and metabolism, and efforts have been made to isolate nucleic acids more rapidly and neatly from various biological samples .

However, the most significant part of the method for separating nucleic acids to date is a carrier that specifically adsorbs only nucleic acid from various kinds of substances contained in the cell lysis solution such as genomic DNA, plasmid DNA, messenger RNA, protein, Have focused on the research and development of nucleic acid adsorbing materials.

Therefore, in order to isolate nucleic acid more rapidly and naturally, it is necessary to develop a technology capable of separating only desired nucleic acid rapidly from cell debris particles, protein denaturation aggregates, and various other cell decomposing materials.

It is an object of the present invention to provide a composition for concentrating a pathogen comprising a homologue 2 functional imidoester compound, a method for concentrating pathogens using the composition, and a kit; A composition for concentrating pathogen and nucleic acid extract containing the same type 2 functional imidoester compound, a method for concentrating pathogen and nucleic acid extraction using the same, and a kit.

In order to accomplish the above object, the present invention provides a composition for concentrating a pathogen comprising a compound represented by the following formula (1).

[Chemical Formula 1]

Wherein, X is (CH ₂₎ and _n,

n is an integer of 4 to 10;

The present invention also provides a kit for concentrating a pathogen comprising the composition.

In addition, the present invention provides a method for manufacturing a semiconductor device, comprising: a first step of introducing an amine group into an object to modify the object; And a second step of bringing the modified object into contact with a sample containing pathogens and a compound represented by the following formula (1).

[Chemical Formula 1]

Wherein, X is (CH ₂₎ and _n,

n is an integer of 4 to 10;

The present invention also provides a composition for the enrichment of a pathogen and a nucleic acid extract comprising a compound represented by the following formula (1).

[Chemical Formula 1]

Wherein, X is (CH ₂₎ and _n,

n is an integer of 4 to 10;

In addition, the present invention provides a kit for concentrating pathogen and nucleic acid extract comprising the composition.

In addition, the present invention provides a method for manufacturing a semiconductor device, comprising: a first step of introducing an amine group into an object to modify the object; A second step of contacting the modified object with a sample containing a pathogen and a compound represented by the following formula 1 to concentrate the pathogen; A third step of separating the nucleic acid from the enriched pathogen; A fourth step of forming a complex between the separated nucleic acid and the compound; And a fifth step of extracting the nucleic acid by treating the eluted buffer with an object on which the complex is formed, and extracting the nucleic acid from the enriched pathogen concurrently with the enrichment of the pathogen.

[Chemical Formula 1]

Wherein, X is (CH ₂₎ and _n,

n is an integer of 4 to 10;

The method of concentrating the pathogen and extracting the nucleic acid using the homologous type 2 functional imidoester compound (DMA, DMP, DMS) according to the present invention can quickly extract a small amount of pathogen contained in the sample without using special equipment, And it is possible to simultaneously concentrate the pathogen in a single tube or chip and to extract the nucleic acid. Thus, the method is more efficient than the conventional method, has excellent time and cost savings, and can be easily used.

FIG. 1 is a schematic diagram of a thin film device of the present invention for extracting nucleic acid and enriching pathogen using the same type 2 functional imidoester compound, and analyzing pathogen enrichment and nucleic acid samples.

FIG. 2 is a graph showing the results of (A) concentration and nucleic acid extraction of a pathogen (Escherichia coli), (B) capture efficiency of a pathogen, (C) concentration and nucleic acid extraction according to a pathogen concentration, Concentration and nucleic acid extraction results.

FIG. 3 shows the result of (A) concentration of pathogen and nucleic acid extraction in a sample of a parainfluenza infected sample and (B) concentration of a pathogen and nucleic acid extraction in a sample of a patient suffering from herpes zoster infection using the same type 2 functional imidoester compound.

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention have developed a method for concentrating a small amount of pathogen contained in a sample and extracting nucleic acid from the concentrated pathogen. The method of concentrating the pathogen and extracting the nucleic acid of the present invention is simpler and less expensive than the conventional method At the same time, it is possible to concentrate the pathogen and to extract the nucleic acid, and it is also possible to carry out immediate on-the-spot diagnosis without using large equipment.

The present invention provides a composition for concentrating a pathogen comprising a compound represented by the following formula (1).

[Chemical Formula 1]

Wherein, X is (CH ₂₎ and _n,

n is an integer of 4 to 10;

The pathogen is a microorganism and the microorganism may be, but is not limited to, a virus, a bacterium, a fungus, a protozoa, a ricketta or a spirotherte.

The present invention also provides a kit for concentrating a pathogen comprising the composition.

In addition, the present invention provides a method for manufacturing a semiconductor device, comprising: a first step of introducing an amine group into an object to modify the object; And a second step of bringing the modified object into contact with a sample containing pathogens and a compound represented by the following formula (1).

[Chemical Formula 1]

Wherein, X is (CH ₂₎ and _n,

n is an integer of 4 to 10;

The object of the first step may be a solid or solid support, for example, but not limited to, a thin film device, a magnetic bead, a ring resonator, or a nanoparticle. .

The object of the first step may be modified with a silane compound. Preferably, the silane compound may be a compound represented by the following formula (2), but is not limited thereto.

(2)

Wherein R ¹ to R ³ may be the same or different and are any one of C 1 to C 4 alkyl or C 1 to C 4 alkoxy, R ⁴ is amino (C 1 to C 10) alkyl, 3- (2 amino (C1 to C4) alkylamino] (C1 to C4) alkyl or 3- [2- (2-amino (C1 to C4) alkylamino) (C1 to C4) alkylamino] (C1 to C4) alkyl.

More preferably, the silane compound is selected from the group consisting of (3-aminopropyl) triethoxysilane (APTES), (3-aminopropyl) trimethoxysilane, Aminomethyl) triethoxysilane, (2-aminoethyl) triethoxysilane, (4-aminobutyl) triethoxysilane ((4-aminomethyl) triethoxysilane, -aminobutyl) triethoxysilane, (5-aminopentyl) triethoxysilane, (6-aminohexyl) triethoxysilane, 3-aminopropyl (3-aminopropyl (diethoxy) methylsilane (APDMS), N- [3- (trimethoxysilyl) propyl] ethylenediamine, N- [3- (Trimethoxysilyl) propyl] diethylenetriamine, [3- (2-aminoethylamino) propyl] trimethoxysilane ([3- (2-aminoethylamino ) propyl] trimethoxyl AEAPTMS) and 3 - [(trimethoxysilyl) propyl] diethylenetriamine (TMPTA), but not limited to, Specify.

The sample containing the pathogenic agent may be used as a sample of a subject suspected of being infected with a pathogen such as feces, urine, tears, saliva, external secretion of skin, external secretion of the respiratory tract, external secretion of the intestinal tract, external secretion of the digestive tract, Spinal fluid, lymph fluid, body fluid, and tissue.

The present invention also provides a composition for the enrichment of a pathogen and a nucleic acid extract comprising a compound represented by the following formula (1).

[Chemical Formula 1]

Wherein, X is (CH ₂₎ and _n,

n is an integer of 4 to 10;

The pathogen is a microorganism and the microorganism may be, but is not limited to, a virus, a bacterium, a fungus, a protozoa, a ricketta or a spirotherte.

The nucleic acid may be DNA or RNA.

In addition, the present invention provides a kit for concentrating pathogen and nucleic acid extract comprising the composition. The kit may further include a buffer and the like necessary for effective nucleic acid extraction.

In addition, the present invention provides a method for manufacturing a semiconductor device, comprising: a first step of introducing an amine group into an object to modify the object; A second step of contacting the modified object with a sample containing a pathogen and a compound represented by the following formula 1 to concentrate the pathogen; A third step of separating the nucleic acid from the enriched pathogen; A fourth step of forming a complex between the separated nucleic acid and the compound; And a fifth step of extracting the nucleic acid by treating the eluted buffer with an object on which the complex is formed, and extracting the nucleic acid from the enriched pathogen concurrently with the enrichment of the pathogen.

[Chemical Formula 1]

Wherein, X is (CH ₂₎ and _n,

n is an integer of 4 to 10;

The pathogen is a microorganism and the microorganism may be, but is not limited to, a virus, a bacterium, a fungus, a protozoa, a ricketta or a spirotherte.

The object of the first step may be, but is not limited to, a thin film device, a magnetic bead, a ring resonator, or a nanoparticle.

The object of the first step may be modified with a silane compound. Preferably, the silane compound may be a compound represented by the following formula (2), but is not limited thereto.

(2)

Wherein R ¹ to R ³ may be the same or different and are any one of C 1 to C 4 alkyl or C 1 to C 4 alkoxy, R ⁴ is amino (C 1 to C 10) alkyl, 3- (2 amino (C1 to C4) alkylamino] (C1 to C4) alkyl or 3- [2- (2-amino (C1 to C4) alkylamino) (C1 to C4) alkylamino] (C1 to C4) alkyl.

More preferably, the silane compound is selected from the group consisting of (3-aminopropyl) triethoxysilane (APTES), (3-aminopropyl) trimethoxysilane, Aminomethyl) triethoxysilane, (2-aminoethyl) triethoxysilane, (4-aminobutyl) triethoxysilane ((4-aminomethyl) triethoxysilane, -aminobutyl) triethoxysilane, (5-aminopentyl) triethoxysilane, (6-aminohexyl) triethoxysilane, 3-aminopropyl (3-aminopropyl (diethoxy) methylsilane (APDMS), N- [3- (trimethoxysilyl) propyl] ethylenediamine, N- [3- (Trimethoxysilyl) propyl] diethylenetriamine, [3- (2-aminoethylamino) propyl] trimethoxysilane ([3- (2-aminoethylamino ) propyl] trimethoxyl AEAPTMS) and 3 - [(trimethoxysilyl) propyl] diethylenetriamine (TMPTA), but not limited to, Specify.

The sample containing the pathogenic agent may be used as a sample of a subject suspected of being infected with a pathogen such as feces, urine, tears, saliva, external secretion of skin, external secretion of the respiratory tract, external secretion of the intestinal tract, external secretion of the digestive tract, Spinal fluid, lymph fluid, body fluid, and tissue.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

FIG. 1 shows a method of concentrating a pathogen and extracting nucleic acid using a homologous type 2 functional imidoester (HI; DMA, DMP, DMS) compound in the thin film device of the present invention, and comprises three steps of sample culture, washing and elution. The surface of the thin film device is modified with an amine using the amine-related solution, and the hydrophobic thin film device is modified to be hydrophilic by the modification.

When a nucleic acid sample, elution buffer and HI solution (DMA, DMP, DMS) are injected onto the modified thin film device, a cross-linking mechanism between the nucleic acid and HI by interaction of the amino group of the nucleic acid with the bi- And DNA can be extracted from the sample by forming a complex between the nucleic acid and HI.

Example 1 Preparation and Pretreatment of a Homogeneous Functional Imidoester (HI) Based Thin Film Device

1) Fabrication of thin film device

A low cost thin film device for use as a microfluidic device (closed device) was fabricated using a CO ₂ laser cutter (VLS3.50 (610 x 305 mm); Universal Laser Systems, Scottsdale, AZ) The thin film device comprises an upper thin film and a lower thin film, and a microfluidic chamber interposed between the upper thin film and the lower thin film, wherein the thin film device is combined with a homobifunctional imidoester (HI) for pathogen enrichment And a microchannel chamber of a single microchannel.

In contrast to the Qiagen kit (open device), the microfluidic chamber of the device is based on a confinement type device to prevent contamination caused by the open device. During the washing and elution steps, the reaction sample remains in the microfluidic chamber of the enclosed device to reduce contamination. Repeated rapid expansion and contraction in the flow cross-sectional area can create microvortices in liquid sample injection.

To concentrate the pathogen from the sample, 36 slot-type microwells in the chambers were connected at expansion and contraction ratios of 1: 5.6 and 5.6: 1, respectively. The microfluidic chip was designed using AutoCAD (Autodesk, Inc., San Rafael, Calif.) And printed with a laser cutter used in the production of prototyping devices with the advantages of low cost, simplicity and speed.

To fabricate a 3D disposable chip consisting of three layers, a 300 micron thick double-sided tape (Fig. 1) was used as an inner layer using a laser cutter (10.6 mu C CO ₂ laser source with a power range of 10 W to 50 W) Three layers consisting of two 100 μm thick films (Kemafoil hydrophilic film, HNW-100, COVEME, Italy) were prepared as outer layers. The outer layer was attached to the permanent adhesive side of the top and bottom of the inner layer to produce a 3D disposable chip for the HI reaction. The height of the microfluidic chamber was about 300 mu m, and the total volume was set to 300 mu l.

To control the sample flow in the microchannel, the tubing adapter was made by attaching a 3 mm thick cast acrylic sheet (MARGA CIPTA, Indonesia) to one side of the double-sided tape and cutting and perforating with a laser cutter. The fabricated adapters were attached to the inlet and outlet of the 3D disposable chip, respectively. Then, pre-cut Tygon tubing (AAC02548; Cole-Parmer, Vernon Hills, Ill.) Was placed in the adapter hole and sealed using thermally stable epoxy at 120 占 폚.

2) Pretreatment of thin film device

To facilitate the use of thin film devices for nucleic acid extraction, plastic cartridges were fabricated using a laser cutter. Plastic cartridges (top and bottom) serve to hold 3D disposable chips during analysis; Length 105 mm, width 60 mm, height 10 mm. The layout of each plastic component was designed using AutoCAD. The structure was patterned on a transparent acrylonitrile butadiene styrene (ABS) sheet using a milling machine. The chip was mounted on the lower plastic part and then assembled with the upper plastic part using four wrench bolts to build the device.

Finally, to use HI as a thin film device for pathogen enrichment and as a non-chaotropic reagent, a surface modification protocol was performed. Briefly, in order to produce amine groups on the inner surface of the 3D disposable chip, the inner surface was first treated with an oxygen plasma (Covance Model, Femtoscience) for 10 minutes to change the characteristics of the inner surface from hydrophobic to hydrophilic, Was immersed in an aqueous solution of 2% 3-aminopropyl triethoxysilane (APTES, Sigma-Aldrich) at 65 ° C for 60 minutes and then thoroughly washed with deionized water. After cleaning, to clean the thin film device, the cleaned thin film device was quickly dried under a nitrogen stream to modify the thin film device to an amine.

The water contact angle measurement of the amine-modified thin film device using Drop Shape Analyzer (DSA100, KRUSS, Germany) showed that the hydrophilicity of the thin film device changed significantly with temperature and incubation time. After the thin film device was silanized with APTES at 65 DEG C for 60 minutes, the hydrophilicity of the thin film surface was increased (about 30 to 40 DEG C). The device can be stored at room temperature until use.

Example 2: HI / thin film sample analysis

The analytical conditions and reactions for the enrichment of pathogens were optimized by using the thin film device of the present invention (simple and label-free pathogen enrichment via homobifunctional imidoesters using a microfluidic (SLIM)). HI can capture nucleic acids through complex formation on the surface of thin films. Therefore, in order to compare the binding ability of pathogens, it is preferable to use dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP) Experiments were performed using various HIs such as dimethyl suberimidate (DMS). All HI reagents were purchased from Sigma-Aldrich (St. Louis, Mo.).

In order to carry out the above reaction, 2 ml of sample from Severe fever with thrombocytopenia syndrome (SFTS) was sampled by swab method and 1 ml of E. coli culture sample was used, and 100 mg / Ml HI solution, and then injected into the device at a rate of 100 μl / min using a syringe pump (KD Scientific, MA). The device was incubated at room temperature for 10 minutes Lt; / RTI &gt; The pathogen enriched by HI (DMA, DMP, DMS) was collected in a few minutes using elution buffer (10 mM sodium bicarbonate, pH <10.6, flow rate: 50 μl / min).

In order to compare the concentration ability in the above experiment, nucleic acids were extracted with a QIAamp (R) DNA mini kit or a QIAamp (R) viral RNA mini kit (Qiagen, Germany) and experiments were conducted according to the protocol provided by the manufacturer.

In order to perform pathogen enrichment and nucleic acid extraction using the thin film device of the present invention, reference is made to previously reported papers (Scientific reports, 5, 14127, Analytical Chemistry, 89 (14), 7502-7510) The nucleic acid was extracted.

The eluted nucleic acid sample contained 10 μl of proteinase K, 10 μl of DNase (RNA only), 10 μl of self-lysis buffer (100 mM Tris-HCl (pH 8.0), 10 mM ethylenediaminetetraacetic acid, 1% Sodium sulfate, and 10% Triton X-100) and 100 μl of HI (100 mg / ml). After incubating the device for 20 min at 56 ° C (for DNA) or at room temperature (for RNA), the device was washed with PBS to remove debris from the sample. The nucleic acid was extracted in a few minutes using elution buffer (pH> 10.6). The amount and purity of the extracted nucleic acid were analyzed by measuring the ratio of the optical density of the sample at 260 nm and 280 nm using Nano Drop (Thermo Fisher Scientific, USA).

Example 3 Concentration of Pathogen (Escherichia coli) Using HI and Nucleic Acid Extraction

In order to confirm the concentration of the pathogen using the thin film device, the basic characteristics of the device were analyzed in a culture sample of Escherichia coli (ATCC 25922). The colony forming unit (CFU) of E. coli was calculated using standard plate agar (PCA; BD difco) and 1 ml of E. coli sample (10 ⁴ CFU ㎖ ^-1 ) The reagents (DMA, DMP, DMS) were evaluated.

The rodA gene was amplified from the extracted DNA using Qiagen kit and thin film device. The PCR conditions included an initial denaturation step at 95 占 폚 for 15 minutes; 30 seconds at 95 占 폚, 30 seconds at 60 占 폚, 30 seconds at 72 占 폚, 40 cycles; And a final elongation step of 7 minutes at 72 &lt; 0 &gt; C. 5 μl of DNA was amplified to a total volume of 25 μl containing 10 × PCR buffer (Qiagen), 5 mM MgCl ₂ , 0.25 mM dioxinucleotide triphosphate, 25 pmol each primer and 1 unit Taq DNA polymerase (Qiagen) .

Real-time PCR was performed with reference to the AriaMx real-time PCR instrument protocol (Agilent technologies). PCR conditions were 95 ° C for 15 min; 10 sec at 95 占 폚, 20 sec at 60 占 폚, 20 sec at 72 占 폚, 40 cycles; And a cooling step at 40 DEG C for 30 seconds. 5 μl of DNA was amplified to a total volume of 20 μl containing 10 μl of 2X Brillient III SYBR Green qPCR maste mix, 25 pmol of each primer and DI water. The SYBR Green signal of the amplified product was obtained using AriaMx real-time PCR (Agilent technologies), and the detailed sequence of the primer set is shown in Table 1 below.

2A and 2B, in order to concentrate the pathogen, 1 ml of Escherichia coli sample was mixed with 300 μl of HI solution (100 mg ㎖ ^-1 ) and then injected into the apparatus. The mixture was centrifuged at a flow rate of 100 μl / min at room temperature Lt; / RTI &gt; The enriched samples were extracted by using Qiagen DNA kit and real-time PCR was performed to compare the efficiency of pathogen enrichment. As a result, when the pathogen was concentrated using HI (DMA, DMS, DMP), the detection sensitivity was improved by decreasing the Ct value compared to the case of extracting the nucleic acid without the concentration step by the conventional Qiagen kit method. It was confirmed that the concentration efficiency was the most excellent. HI reagent concentration (100 mg ㎖ ^-1 ), HI reagent type (DMP) and incubation time (20 min) were optimized for pathogen enrichment.

After optimizing conditions for pathogen enrichment, the efficiency of pathogen enrichment and nucleic acid extraction by the thin membrane device (SLIM) of the present invention was analyzed using 1 ml of Escherichia coli (10 ¹ to 10 ⁴ CFU ml ^-1 ). As a result, referring to FIG. 2C, it was confirmed that the DNA was amplified from the enriched E. coli and the Ct value was increased by continuous dilution. When using the thin film device of the present invention, E. coli samples at all concentrations exhibited reduced Ct values compared to conventional Qiagen kits, indicating that DNA enrichment efficiency is excellent. The detection limit was also confirmed to be 10 times better than the conventional Qiagen kit.

Next, it was analyzed whether the thin film device of the present invention is capable of concentrating the pathogen and extracting nucleic acid simultaneously on the same platform. One-step process (pathogen enrichment and nucleic acid extraction on one chip) and two-step process (pathogen enrichment and nucleic acid extraction on two separate chips, respectively) were compared to existing Qiagen kits. As a result, referring to FIG. 2D, it was confirmed that the Ct value decreased in the on-step process, the detection sensitivity was improved, and pathogen enrichment and extraction could be simultaneously performed in one chip (or tube).

Example 4: Enrichment of pathogen (virus) and nucleic acid extraction using SFTS patient sample

A total of five swab samples were received from SFTS patients, and the non-pharyngeal swab / inhalant was used as part of the clinical trial. Dacron swabs pre-loaded with viral transport media were used to aseptically clean the surfaces frequently contacted by the patient or medical practitioner. In the case of the QIAamp Viral RNA mini kit, 140 μl was used in 2 ml of the swab method. In the case of using the thin film apparatus, 2 ml of the whole sample was used to extract the viral RNA. Samples were eluted with 60 μl of elution buffer and the eluted DNA was stored at -20 ° C. until use.

Next, the S portion of the SFTS virus was amplified from the eluted RNA using a Qiagen kit and a thin-film device. One-step reverse transcriptase (RT) PCR is an initial denaturation step at 50 ° C for 30 minutes; 30 sec at 95 占 폚, 30 sec at 60 占 폚, 30 sec at 72 占 폚, 50 cycles; And a final elongation step at 72 [deg.] C for 10 minutes.

5 ul of RNA was amplified to a total volume of 25 ul containing 5X OneStep RT-PCR buffer (Qiagen), 0.25 mM dioxinucleotide triphosphate, 25 pmol each primer and 1 unit OneStep RT-PCR Enzyme mix (Qiagen) . Quantitative RT-PCR was performed with reference to the AriaMx real-time PCR instrument protocol (Agilent technologies).

5 [mu] l of RNA was amplified to a total volume of 20 [mu] l containing 10 [mu] l of 2X Brillient III SYBR Green qPCR maste mix, 25 pmol each primer and DI water. PCR conditions were: 30 min at 50 캜, 10 min at 95 캜; 10 sec at 95 占 폚, 20 sec at 60 占 폚, 20 sec at 72 占 폚, 50 cycles; And a cooling step at 40 DEG C for 30 seconds. The SYBR Green signal of the amplified product was obtained using AriaMx real-time PCR (Agilent technologies), and the detailed sequence of the primer set is shown in Table 1 above.

Referring to Table 2, in the conventional Qiagen method, viruses were not detected in the samples (samples 1, 4 and 5) of the SFTS patients. On the other hand, in the case of using the thin film device of the present invention, And nucleic acid extraction are possible at the same time.

Example 5: Concentration of pathogen (virus) and nucleic acid extraction using paraffin (hPIV-3) patient and herpes zoster (HZ) patient sample

Between May and June 2016, swab specimens were obtained from the surface in contact with the patient during parainfluenza (hPIV-3) development. In the case of the QIAamp Viral RNA mini kit, 140 μl was used in 2 ml of the swab method. In the case of using the thin film apparatus, 2 ml of the whole sample was used to extract the viral RNA. Samples were eluted with 60 μl of elution buffer and the eluted DNA was stored at -20 ° C. until use.

Next, the S portion of the SFTS virus was amplified from the eluted RNA using a Qiagen kit and a thin-film device. One-step reverse transcriptase (RT) PCR is an initial denaturation step at 50 ° C for 30 minutes; 30 sec at 95 占 폚, 30 sec at 60 占 폚, 30 sec at 72 占 폚, 50 cycles; And a final elongation step at 72 [deg.] C for 10 minutes.

5 ul of RNA was amplified to a total volume of 25 ul containing 5X OneStep RT-PCR buffer (Qiagen), 0.25 mM dioxinucleotide triphosphate, 25 pmol each primer and 1 unit OneStep RT-PCR Enzyme mix (Qiagen) . Quantitative RT-PCR was performed with reference to the AriaMx real-time PCR instrument protocol (Agilent technologies).

5 [mu] l of RNA was amplified to a total volume of 20 [mu] l containing 10 [mu] l of 2X Brillient III SYBR Green qPCR maste mix, 25 pmol each primer and DI water. PCR conditions were: 30 min at 50 캜, 10 min at 95 캜; 10 sec at 95 占 폚, 20 sec at 60 占 폚, 20 sec at 72 占 폚, 50 cycles; And a cooling step at 40 DEG C for 30 seconds. The SYBR Green signal of the amplified product was obtained using AriaMx real-time PCR (Agilent technologies), and the detailed sequence of the primer set is shown in Table 1 above.

As a result, referring to FIG. 3A, in the conventional Qiagen method, viruses were not detected in samples (S4, S5, S10, S12) of some hPIV-3 patients. On the other hand, when using the thin film device of the present invention, It was confirmed that the virus can be concentrated and nucleic acid extracted at the same time, so that it can be used for virus detection.

Next, the thin film device of the present invention was applied to a saliva sample of a herpes zoster (HZ) patient. Chickenpox and HZ are known to be caused by varicella-zoster (VZV), and HZ rashes are generally thought to be sufficient for clinical diagnosis, but HZ and HZ mimic diseases such as herpes simplex virus , It is necessary to analyze saliva and plasma samples. On the other hand, in the detection of VZV, saliva DNA (88%) has been reported to have a much higher PCR sensitivity than plasma DNA (28%). However, it is painful to collect saliva samples from plasma samples from HZ patients.

Accordingly, in the present invention, a thin film device was applied to analyze the concentration efficiency of two samples using saliva and plasma samples. First, 1 ml saliva samples were used to extract VZV enriched and viral DNA, and 10 saliva samples were selected by real time PCR. All positive samples identified using the ORF62 region of VZV showed initial Ct values. On the other hand, it was confirmed that none of the negative samples showed positive results by the thin film device of the present invention. This is consistent with previous studies demonstrating that saliva samples are more useful for VZV detection.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims (23)

A composition for concentrating a pathogen comprising a compound represented by the following formula [Chemical Formula 1]

Wherein, X is (CH ₂₎ and _n, n is an integer of 4 to 10; The composition according to claim 1, wherein the pathogen is a microorganism. The composition according to claim 2, wherein the microorganism is a virus, a bacterium, a fungus, a protozoan, a ricketta or a spiroheta. A kit for enriching pathogens comprising the composition of any one of claims 1 to 3. A first step of introducing an amine group into an object to modify it; And A second step of contacting a sample containing pathogens on the modified object with a compound represented by the following formula 1; Lt; RTI ID = 0.0 &gt; of: &lt; [Chemical Formula 1]

Wherein, X is (CH ₂₎ and _n, n is an integer of 4 to 10; 6. The method according to claim 5, wherein the object of the first step is any one of a thin film device, a magnetic bead, a ring resonator, and a nanoparticle. The method of claim 5, wherein the object of the first step is modified with a silane compound. 8. The method of concentrating a pathogen according to claim 7, wherein the silane compound is a compound represented by the following formula (2) (2)

Wherein R ¹ to R ³ may be the same or different and are any one of C 1 to C 4 alkyl or C 1 to C 4 alkoxy, R ⁴ is amino (C 1 to C 10) alkyl, 3- (2 amino (C1 to C4) alkylamino] (C1 to C4) alkyl or 3- [2- (2-amino (C1 to C4) alkylamino) (C1 to C4) alkylamino] (C1 to C4) alkyl. The method of claim 8, wherein the silane compound is selected from the group consisting of (3-aminopropyl) triethoxysilane (APTES), (3-aminopropyl) trimethoxysilane, (1-aminomethyl) triethoxysilane, (2-aminoethyl) triethoxysilane, (4-aminobutyl) triethoxysilane (( 4-aminobutyl) triethoxysilane, (5-aminopentyl) triethoxysilane, (6-aminohexyl) triethoxysilane, 3-aminopropyl 3-aminopropyl (diethoxy) methylsilane (APDMS), N- [3- (trimethoxysilyl) propyl] ethylenediamine, N- [3 Propyl] diethylenetriamine), [3- (2-aminoethylamino) propyl] trimethoxysilane ([3- (2- aminoethylamino) propyl] trimethoxysilane; AEAPTMS) and 3 - [(trimethoxysilyl) propyl] diethylenetriamine (3 - [(trimethoxysilyl) propyl] diethylenetriamine]. 6. The method according to claim 5, wherein the sample containing the pathogen is selected from the group consisting of feces, urine, tears, saliva, external secretion of the skin, external secretion of the respiratory tract, external secretion of the intestinal tract, Wherein the method is any one selected from the group consisting of plasma, serum, blood, spinal fluid, lymph fluid, body fluids, and tissues. A composition for concentrating a pathogen and extracting nucleic acid comprising a compound represented by the following formula (1): [Chemical Formula 1]

Wherein, X is (CH ₂₎ and _n, n is an integer of 4 to 10; 12. The composition of claim 11, wherein the pathogen is a microorganism. 13. The composition according to claim 12, wherein the microorganism is a virus, a bacterium, a fungus, a protozoa, a ricketta or a spirotherte. 12. The composition of claim 11, wherein the nucleic acid is DNA or RNA. A kit for enriching pathogen enrichment and nucleic acid extraction comprising the composition of any one of claims 11 to 14. A first step of introducing an amine group into an object to modify it; A second step of contacting the modified object with a sample containing a pathogen and a compound represented by the following formula 1 to concentrate the pathogen; A third step of separating the nucleic acid from the enriched pathogen; A fourth step of forming a complex between the separated nucleic acid and the compound; And A fifth step of extracting the nucleic acid by treating the eluted buffer with an object on which the complex is formed; A method for extracting a nucleic acid from the enriched pathogen simultaneously with enriching a pathogen comprising: [Chemical Formula 1]

Wherein, X is (CH ₂₎ and _n, n is an integer of 4 to 10; 17. The method of claim 16, wherein the pathogen is a microorganism, and simultaneously concentrating the pathogen and extracting the nucleic acid from the concentrated pathogen. 18. The method according to claim 17, wherein the microorganism is a virus, a bacterium, a fungus, a protozoa, a ricketta or a spirotherte, and simultaneously extracting the nucleic acid from the enriched pathogen. 17. The method of claim 16, wherein the object of the first step is one of a thin film device, a magnetic bead, a ring resonator, or a nanoparticle. A method for extracting nucleic acid from a pathogen. 17. The method of claim 16, wherein the object of the first step is modified with a silane compound, and the nucleic acid is extracted from the enriched pathogen concurrently with the enrichment of the pathogen. 21. The method according to claim 20, wherein the silane compound is a compound represented by the following general formula (2): (2)

Wherein R ¹ to R ³ may be the same or different and are any one of C 1 to C 4 alkyl or C 1 to C 4 alkoxy, R ⁴ is amino (C 1 to C 10) alkyl, 3- (2 amino (C1 to C4) alkylamino] (C1 to C4) alkyl or 3- [2- (2-amino (C1 to C4) alkylamino) (C1 to C4) alkylamino] (C1 to C4) alkyl. The method of claim 21, wherein the silane compound is selected from the group consisting of (3-aminopropyl) triethoxysilane (APTES), (3-aminopropyl) trimethoxysilane, (1-aminomethyl) triethoxysilane, (2-aminoethyl) triethoxysilane, (4-aminobutyl) triethoxysilane (( 4-aminobutyl) triethoxysilane, (5-aminopentyl) triethoxysilane, (6-aminohexyl) triethoxysilane, 3-aminopropyl 3-aminopropyl (diethoxy) methylsilane (APDMS), N- [3- (trimethoxysilyl) propyl] ethylenediamine, N- [3 Propyl] diethylenetriamine), [3- (2-aminoethylamino) propyl] trimethoxysilane ([3- (2- aminoethylamino) propyl] trimethoxysilane; AEAPTMS) and 3 - [(trimethoxysilyl) propyl] diethylenetriamine (3 - [(trimethoxysilyl) propyl] diethylenetriamine; TMPTA). A method for extracting nucleic acid from a pathogen. 18. The method of claim 16, wherein the sample containing the pathogen is selected from the group consisting of feces, urine, tears, saliva, external secretions of the skin, external secretions of the respiratory tract, external secretions of the intestinal tract, Wherein the nucleic acid is any one selected from the group consisting of plasma, serum, blood, spinal fluid, lymph fluid, body fluids, and tissues.

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