WO2018038549A1

WO2018038549A1 - Method for concentrating microorganism or extracting nucleic acid using dtbp

Info

Publication number: WO2018038549A1
Application number: PCT/KR2017/009254
Authority: WO
Inventors: 신용
Original assignee: 울산대학교 산학협력단; 재단법인 아산사회복지재단
Priority date: 2016-08-24
Filing date: 2017-08-24
Publication date: 2018-03-01

Abstract

The present invention relates to a method for concentrating microorganisms and extracting nucleic acid using DTBP. A technology capable of concentrating microorganisms using DTBP has been developed, and also a method capable of extracting nucleic acid directly from the concentrated microorganisms has been developed. As the technologies of concentrating microorganisms and extracting nucleic acid can be implemented in one tube or chip, it is expected to significantly reduce contamination from the outside, costs, time, complexities, and the like.

Description

Microbial Concentration or Nucleic Acid Extraction Method Using DTBP

The present invention relates to a method for concentrating microorganisms or extracting nucleic acids using 3,3'-dithiobispropionimate.

Nucleic acids are an important analytical tool for identifying disease states, and DNA biomarkers, such as single nucleotide polymorphism (SNP), mutations or DNA methylation, help researchers find the cause of cancer and disease during the early stages of the disease. Diagnosing and observing the condition of the doctor also provides important clues in providing great opportunities for prognosis and monitoring.

Nucleic acids such as DNA are present at very low physiological concentrations compared to other components such as proteins (eg, tens of nanograms of DNA per microliter of whole blood versus tens of micrograms of protein), effectively extracting and preliminary DNA from clinical samples. Concentration is very important for subsequent processes such as amplification and detection.

Existing methods for detecting microorganisms have not used the entire solution in 1-2 ml of patient samples, and only a part of them has been used to extract nucleic acids and perform detection using the same. For large amounts of microorganisms, this is not a big problem, but for small amounts of microorganisms, it is not possible to detect them correctly, which leads to problems in controlling additional infectious diseases. Research is needed.

In recent years, as the use of highly purified nucleic acids has increased in various fields such as biotechnology, diagnostic medicine, pharmacy medicine, and metabolic medicine, efforts have been continuously made to separate nucleic acids from various biological samples more quickly and purely. .

However, the most advanced part of the nucleic acid separation method to date is that the carrier specifically absorbs the nucleic acid only from various kinds of substances contained in the cell lysis solution such as genomic DNA, plasmid DNA, messenger RNA, protein, and cell debris particles. The focus of almost all research, including related technologies, has been limited to research and development of substances that adsorb nucleic acids.

Accordingly, in order to separate nucleic acids more quickly and purely, the development of a technology capable of rapidly separating only desired nucleic acids from cell debris particles, protein denatured aggregates, and various other cell lysates is urgently needed.

An object of the present invention is a composition for concentrating microorganisms comprising 3,3'-dithiobispropionimidate (Dimethyl 3,3'-dithiobispropionimidate; hereinafter 'DTBP') as an active ingredient, a method and kit for concentrating microorganisms using the same; A nucleic acid extracting composition comprising DTBP as an active ingredient, a nucleic acid extracting method and kit using the same; And a composition for concentrating microorganisms and extracting nucleic acids comprising DTBP as an active ingredient, a method and kit for extracting nucleic acids from the concentrated microorganisms simultaneously with the concentration of microorganisms using the same.

In order to achieve the above object, the present invention provides a composition for concentrating microorganisms comprising DTBP as an active ingredient.

The present invention also provides a microbial enrichment kit comprising the composition.

The present invention also provides a method for concentrating microorganisms comprising contacting a sample containing microorganisms with the DTBP.

The present invention also provides a nucleic acid extracting composition comprising DTBP as an active ingredient.

The present invention also provides a nucleic acid extraction kit comprising the composition.

In addition, the present invention comprises the steps of modifying by introducing an amine group to the object (first step); Injecting a nucleic acid sample and DTBP onto the modified object and forming a complex between the nucleic acid and the DTBP (second step); And it provides a nucleic acid extraction method comprising the step of extracting the nucleic acid by processing the elution buffer to the object formed with the complex (third step).

The present invention also provides a composition for microorganism concentration and nucleic acid extraction comprising DTBP as an active ingredient.

The present invention also provides a microbial enrichment and nucleic acid extraction kit comprising the composition.

In addition, the present invention comprises the steps of modifying by introducing an amine group to the object (first step); Concentrating the microorganism by contacting the sample containing the microorganism with DTBP on the modified object (second step); Separating nucleic acid from the concentrated microorganism (third step); Forming a complex between the separated nucleic acid and the DTBP (fourth step); And it provides a method for extracting the nucleic acid from the concentrated microorganism at the same time the concentration of the microorganism comprising the step of extracting the nucleic acid by the treatment of the elution buffer solution to the object formed complex.

The present invention relates to a method for concentrating a microorganism or extracting nucleic acids using DTBP, and the present inventors have developed a technology capable of concentrating microorganisms using DTBP, and also a method for extracting nucleic acids directly from concentrated microorganisms. By enabling microbial enrichment and nucleic acid extraction techniques in a single tube or chip, it is expected to significantly reduce external contamination, cost, time and complexity.

1 is a schematic view of the present invention DTBP / Thin film Sample analysis.

2 is an exploded view showing the configuration of a thin film device.

3 is a diagram showing the DNA extraction efficiency according to the DTBP concentration change.

4 is a diagram illustrating end-point PCR (a) and real-time PCR (b) of DNA extracted from a colon cancer cell line (HCT116 cell line; hereinafter 'HCT116').

5 is a diagram illustrating end-point PCR (a) and real-time PCR (b) of DNA extracted from a breast cancer cell line (MCF cell line; hereinafter 'MCF').

FIG. 6 shows PCR results of DNA extracted from Brucella Ovis. FIG. M: DNA marker, Q: DNA sample extracted by the existing Qiagen method, DTBP: DNA sample extracted using the DTBP method of the present invention, N: negative control (negative control).

7 is a diagram illustrating a method of extracting RNA (a) and a method of simultaneously extracting RNA and cDNA (b). (a) The experiment was performed by repeating the same sample four times. L: DNA marker, N: negative control. (b) The experiment was performed by repeating the same sample twice. L: DNA marker.

8 shows the results of pathogen (E. coli) concentration analysis using DTBP.

Figure 9 shows the results of the nucleic acid extraction analysis at the same time the concentration of pathogens (E. coli) in one chip using DTBP.

10 shows the results of pathogen (PIV3-RNA virus) enrichment analysis using DTBP. P: positive control, N: negative control, L: 50 bp DNA ladder, samples 1 to 4: DNA samples extracted by conventional Qiagen method, samples 1-1 to 4-1 : DNA sample concentrated and extracted using DTBP method. In case of sample 1, it was diagnosed as a negative method in the conventional method, but it was confirmed as positive in sample 1-1 with DTBP concentration. For

samples

2 and 3, both the conventional method and the DTBP-concentrated samples were positive for samples, and for sample 4, the samples 4-1 with DTBP-concentrated samples were significantly increased. It was confirmed.

The present invention provides a composition for concentrating microorganisms comprising DTBP as an active ingredient.

Preferably the microorganism may be any negatively charged microorganism such as bacteria, viruses or cells.

The present invention also provides a microbial enrichment kit comprising the composition. The kit may further include a buffer solution for adjusting pH required for effective microbial concentration.

In addition, the present invention comprises the steps of modifying by introducing an amine group to the object (first step); And contacting a sample containing microorganisms with DTBP on the modified object (second step).

In the present invention, the object may be one modified with a silane compound on the surface. Preferably, the silane compound may be a compound represented by the following Chemical Formula 1, but is not limited thereto.

[Formula 1]

Wherein R ¹ to R ³ may each be the same or different, either C1 to C4 alkyl or C1 to C4 alkoxy, R ⁴ is amino (C1 to C10) 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 (3-aminopropyl) triethoxysilane (APTES), (3-aminopropyl) trimethoxysilane ((3-aminopropyl) trimethoxysilane), (1 -(Aminomethyl) triethoxysilane ((1-aminomethyl) triethoxysilane), (2-aminoethyl) triethoxysilane ((2-aminoethyl) triethoxysilane), (4-aminobutyl) triethoxysilane ((4- aminobutyl) triethoxysilane), (5-aminopentyl) triethoxysilane, (6-aminohexyl) triethoxysilane, (6-aminohexyl) triethoxysilane, 3-aminopropyl (diethoxy Methylsilane (3-aminopropyl (diethoxy) methylsilane; APDMS), N- [3- (trimethoxysilyl) propyl] ethylenediamine (N- [3- (trimethoxysilyl) propyl] ethylenediamine), [3- (2- Aminoethylamino) propyl] trimethoxysilane ([3- (2-aminoethylamino) propyl] trimethoxysilane; AEAPTMS) or 3-[(trimethoxysilyl) propyl] diethylenetriamine (3-[(trimethoxysilyl) propyl] diethy lenetriamine; TMPTA), but is not limited thereto. Even more preferably the silane compound is most suitably (3-aminopropyl) triethoxysilane (APTES) or 3-aminopropyl (diethoxy) methylsilane (APDMS) described in the examples of the present invention.

Preferably, the sample containing the microorganism is feces, urine, tears, saliva, external secretions of the skin, external secretions of the respiratory tract, external secretions of the intestinal tract, external secretions of the digestive tract, plasma, Serum, blood, spinal fluid, lymph, body fluids and tissues, but are not limited thereto.

The present invention also provides a nucleic acid extracting composition comprising DTBP as an active ingredient. Preferably, the nucleic acid may be DNA or RNA, but is not limited thereto.

The present invention also provides a nucleic acid extraction kit comprising the composition. The kit may further include a lysis buffer or a protease for lysing the cells in the sample to release the nucleic acid from inside the cell, and a buffer for pH adjustment required for effective nucleic acid extraction. It may additionally be included.

Preferably the object may be one modified with a silane compound on the surface. More preferably, the silane compound may be a compound represented by the following Chemical Formula 1, but is not limited thereto.

[Formula 1]

Even more preferably the silane compound is most suitably (3-aminopropyl) triethoxysilane (APTES) or 3-aminopropyl (diethoxy) methylsilane (APDMS) described in the examples of the present invention.

Preferably, the nucleic acid may be DNA or RNA, but is not limited thereto.

The present invention also provides a microbial enrichment and nucleic acid extraction kit comprising the composition. The kit may further include a buffer for effective microbial concentration and pH adjustment necessary for nucleic acid extraction, etc., and a lysis buffer or protease for lysing the cells in the sample to release the nucleic acid from inside the cell. And the like may further be included.

[Formula 1]

In the present invention, the "object" may be any one of a thin film device, a magnetic bead, a ring resonator, and a nanoparticle, but is not limited thereto. More preferably, the object is an upper thin film formed in each of the inlet and outlet holes described in the embodiment of the present invention; A lower thin film spaced apart from the upper thin film; The inlet end and the outlet end are formed so that the microchannels communicating in correspondence with the inlet and outlet holes of the upper thin film are formed inside, and the injection passage communicating with the inlet of the microchannel is adjacent to the inlet end. A micro channel chamber disposed between the upper thin film and the lower thin film; And sealing means for sealing each side of the upper thin film and the lower thin film to seal the micro channel chamber.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

Referring to Figure 1, the nucleic acid analysis according to the present invention DTBP / Thin film Sample analysis, which is a nucleic acid analysis using DTBP in a thin film device, includes three steps of sample elution / culture, washing and elution, and is performed without centrifugation. For example, the thin film device is modified via 3-aminopropyltriethoxysilane (APTES) as the silane compound, which converts the hydrophobic thin film device into hydrophilic.

The nucleic acid sample, the elution buffer, and the DTBP solution are injected onto the modified thin film device. The cross-linking mechanism between the amino acid of the nucleic acid and the DTBP by interaction with the bifunctional amine reactor of the DTBP may be used to form a complex between the nucleic acid and the DTBP and extract DNA from the sample.

In this case, since the DTBP has a structure of Formula 2 and includes di-functional imidoesters and disulfide bonds, the DTBP forms a reversible crosslinking structure to form an amino reactive crosslinking agent for cells, proteins and nucleic acids. Was used. Rather than interacting with DTBP and proteins, fast and strong interactions between DTBP and nucleic acids allow for highly efficient nucleic acid extraction from nucleic acid samples.

[Formula 2]

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

<< 실시예Example 1> 박막 장치 제작 및 전처리 1> thin film device fabrication and pretreatment

1. Thin film device fabrication

The thin film device of the present invention was manufactured using a laser cutting machine (Universal Laser Systems, Scottsdale, USA) (see Fig. 2 (a)). First, the thin film device comprises an upper thin film and a lower thin film, and a microfluidic chamber inserted between the upper thin film and the lower thin film, the microfluidic chamber being connected to each other by flow paths in the chamber to extract DNA from a nucleic acid source. It consists of slot-type microwells.

In order to fabricate the microfluidic chamber, the microfluid chamber design was cut by a laser cutting machine on a 300 µm thick double-sided tape (100 µm thick polyester film sandwiched between 100 µm thick double-sided tape) to manufacture a microfluidic chamber. It was. Thin films (top and bottom) were cut to the same dimensions as the microfluidic chamber using a laser cutting machine.

Inlets and outlets, which are through holes, were manufactured in the upper thin film. The laser cutting thin films (top and bottom) were adhered to the surfaces of the upper and lower portions of the laser cutting microfluidic chamber, respectively, using a permanent adhesive. The height of the microfluidic chamber was about 300 μm and the total volume was 300 μl (300 μl amount, 8.4 cm × 3.7 cm).

A tubing adapter for injecting a nucleic acid source was prepared by attaching a cast acrylic sheet (MARGA CIPTA, Indonesia) having a thickness of 3 mm to one side of a double-sided tape, and cutting and drilling with a laser cutting machine. The manufactured tubing adapter was attached to the inlet and the outlet of the microfluidic chamber, respectively. The pre-cut Tygon tubing (AAC02548; Cole-Parmer, Vernon Hills, USA) was then placed in the hole of the adapter and sealed with epoxy.

The thin film device manufactured as described above has the advantage of being capable of processing various nucleic acid samples (100 μl, 300 μl, and 500 μl).

2. Thin film device pretreatment

For DNA analysis using the thin film device, the inside of the thin film device was treated with oxygen plasma for 10 minutes, and the plasma treated thin film device was treated with 2% 3-aminopropyltriethoxysilane (APTES) for 10 to 60 minutes at 65 ° C. D) was immersed in an aqueous solution containing), and then washed thoroughly with deionized water. After cleaning, the thin film device was quickly dried under a stream of nitrogen to modify the thin film device with amine to cure the thin film device.

The water contact angle of the amine-modified thin film device using the Drop Shape Analyzer (DSA100, KRUSS, Germany) showed that the hydrophilicity of the thin film device changed considerably with temperature and incubation time. After silanization of the thin film device with APTES for 10 minutes at 65 ° C., the thin film surface hydrophilicity was increased (about 30-40 ° C.).

<< 실시예Example 2> 2> DTBPDTBP /Thin film Sample 분석/ Thin film Sample analysis

In the present invention, a nucleic acid analysis method in which dimethyl 3,3'-dithiobispropionimate (DTBP) was applied to the previously prepared thin film device was named DTBP, and DTBP analysis was performed in the following experiment.

That is, an optimized assay solution was first prepared to extract DNA using a thin film device (300 μl amount, 8.4 cm × 3.7 cm) modified with amine. The optimized assay solution was prepared by mixing elution buffer containing 100 mM tris-hydrochloric acid (pH 8.0), 10 mM EDTA, 1% SDS, 10% Triton X-100 with DTBP (100 mg / ml) As a nucleic acid analysis sample, 100 μl of each sample derived from cells, bacteria, blood or urine was mixed with 200 μl of the assay solution.

The mixed mixture of the mixed nucleic acid assay sample and the assay solution is introduced into the upper substrate inlet of the thin film device modified with amine, and the mixed solution is moved into the microfluidic chamber to bind two amine groups and DNA of DTBP. DNA was modified by combining the modified amine group with DNA to form a complex. At this time, the thin film device was placed in any one of a thermoelectric cooler (TEC) including an incubator or a controller (Alpha Omega Instruments) maintained at a constant temperature (56 ° C) for 20 minutes to sufficiently extract DNA from the nucleic acid analysis sample. .

After washing with PBS buffer to remove foreign substances in DTBP-DNA complex, DNA was extracted using elution buffer (10 mM sodium bicarbonate, pH 10.6). After measuring the amount and purity of the extracted DNA, the optical density ratio of the sample at 260 nm (DNA) and 280 nm (protein) was determined using Enspire Multimode Plate Reader (PerkinElmer). In order to compare the conventional DNA extraction and DTBP analysis of the present invention, it was analyzed using a QIAmp DNA mini kit according to a known method (Qiagen, Hilden, Germany).

As shown in FIG. 3, as a result of confirming the binding force for each DTBP concentration, the DNA binding efficiency was confirmed to be the highest when the DTBP concentration was 100 mg / ml.

<< 실시예Example 3> 3> DTBPDTBP 분석을 이용한 Analysis 진핵세로포부터의From eukaryotic cells DNA 추출 DNA extraction

Plastic of high glucose Dulbecco's modified eagle medium (DMEM, DMEM Life Technology) supplemented with 10% fetal calf serum (FCS) in a humidified incubator at 37 ° C in a 5% CO ₂ atmosphere After culturing two eukaryotic cells, HCT116 and MCF, in a culture plate, DNA was extracted from eukaryotic cells in the same manner as in Example 1, but proteinase K, a proteinase, was extracted to extract genomic DNA Treated. And, for comparison, DNA was extracted from eukaryotic cells using a QIAmp DNA mini kit. HCT116 and MCF used in this example were purchased from the US ATCC (https://www.atcc.org).

End-point PCR and real time PCR were performed to confirm the amount and purity of DNA. Forward and reverse primers of some genes (Actin) were synthesized to a normal length of about 24 base pairs. End-point PCR was performed at 95 ° C. for 15 minutes at the initial denaturation step; 45 cycles totaling 45 seconds at 95 ° C., 45 seconds at 59 ° C. (Actin), and 45 seconds at 72 ° C .; And 72 ° C., final extension for 10 minutes. 5-10 μl of DNA was added to 1 × PCR buffer (Qiagen), 2.5 mM magnesium chloride (MgCl ₂ ), 0.25 mM deoxynucleotide triphosphate, 25 pmol of each primer, and 1 unit of Taq DNA polymerase. Amplified at a total volume of 25 μl.

For real time-PCR analysis, the following steps were modified to the method provided in LightCycler 2.0 (Roche Diagnostics) as follows. 5-10 μl of DNA is mixed with 4 μl of LightCycler FastStart DNA Master, 25 pmol of each primer, 2 μl of 1 × PCR buffer (Qiagen, Hilden, Germany), 2.5 mM magnesium chloride (MgCl ₂ ), 0.25 mM deoxy Amplification was performed at a total volume of 20 μl containing nucleotide triphosphate, and distilled water. After the first pretreatment at 95 ° C. for 10 minutes, a total of 50 cycles were performed with 10 cycles at 95 ° C., 30 seconds at 59 ° C. (Actin), and 10 seconds at 72 ° C., followed by cooling at 40 ° C. for 30 seconds. I went through the steps. Amplified product with SYBR green signal was performed in LightCycler 2.0 (Roche Diagnostics).

4 and 5, the end-point PCR (FIGS. 4A and 5A) and real-time PCR (FIGS. 4B and 5B) are extracted using DTBP. As a result of end-point PCR of DNA, HCT116 was extracted about 1.3 times and MCF was about 2.6 times higher than that of Qiagen product, and real-time PCR result was not significantly different from Qiagen product. there was.

<< 실시예Example 4> 4> DTBPDTBP 분석을 이용한 박테리아 세포로부터의 DNA 추출 DNA Extraction from Bacterial Cells Using Assays

In order to confirm the performance of DTBP analysis in bacterial cells, PCR-based DNA amplification was performed using DNA extracted using DTBP analysis. All primers are Escherichia coli, Mycobacterium abscessus, Mycobacterium gordonae and Salmonella Strains (Salmonella Typhimurium, Salmonella Typhimurium) Commercial primers from Salmonella Newport, and Salmonella Saintpaul were used. Commercial primers of the strains used in this example were purchased from the US IDT (https://www.idtdna.com).

For the optimization reaction, elution buffer containing 100 mM tris-hydrochloric acid (pH 8.0), 10 mM EDTA, 1% SDS, 10% Triton X-100 and 20 mg / ml of lysozyme DTBP (50 mg / ml) Mixed with. General PCR was performed to validate the DTBP method of the present invention. E. coli XL1 blue strain (purchased from Korea Microbiological Resource Center; https://kctc.kribb.re.kr) was inoculated into 50 μg / ml of tetratracycline and Luria-Bertani (LB) medium. and it was cultured for one day at 37 ℃ with shaking conditions ^{(shaking condition), 10 3 ~10} 7 colony forming units; the sample (CFU colony forming unit) was used for testing. Escherichia coli, Mycobacterium abscessus, Mycobacterium gordonae and Salmonella strains cultured for use in DTBP assays and Qiagen kit assays ( Bacterial DNA was extracted from Salmonella Typhimurium, Salmonella Newport, Salmonella Saintpaul, and Brucella Ovis. The strain used in this example was purchased from the Korea Microbial Resources Center (https://kctc.kribb.re.kr).

For genetic analysis of bacterial genes, 2 μl of DNA extracted from DTBP assay and Qiagen kit assay was added to 1 × PCR buffer (Qiagen, Hilden, Germany), 2.5 mM magnesium chloride (MgCl ₂ ), Amplification at 95 ° C. for 15 minutes using a total volume of 25 μl comprising 0.25 mM deoxynucleotide triphosphate, 25 pmol of each primer, and 1 unit of Taq DNA polymerase; 30 seconds at 95 ° C, 30 seconds at 60 ° C (Mycobacterium abscessus, Mycobacterium gordonae, Salmonella strains and Brucella Ovis) A total of 45 cycles of 30 seconds at 72 ° C. in one cycle; And 72 ° C., final extension step for 7 minutes. PCR amplifications were visualized by gel electrophoresis to separate PCR products on 2% agarose gels containing ethidium bromide (EtBr) (Sigma-Aldrich). The gel was visualized using the Gel Doc System (Bio-Rad). Measurement of DNA concentration and purity was performed with a UV spectrometer (Perkin-Elmer).

As a result of PCR analysis with DNA extracted from Brucella Ovis using DTBP, it was confirmed that there is no significant difference from Qiagen products (FIG. 6).

<< 실시예Example 5> 5> DTBPDTBP 분석을 이용한 인간 Human with analysis 체액로부터의From body fluids DNA 추출 DNA extraction

In order to verify the DTBP analysis ability with human body fluids, 200 μl of body fluid samples (whole blood and urine) were introduced into the thin film device to extract DNA. First, the elution buffer containing proteinase K and DTBP and the bodily fluid sample were introduced into the previously prepared thin film device, and then moved to the microchannel chamber to form a complex between the DNA and the DTBP in the bodily fluid sample. Example 2 DNA was extracted in the same manner as. At this time, the elution buffer and the bodily fluid sample were introduced into two different inlets at a flow rate of 1.5 ml / hr for 10 minutes using a syringe pump (KD Scientific, MA), and the cartridge was extracted for 20 minutes for extraction and purification of DNA. Incubated at 56 ° C. In addition, the flow rate of the inlet for inflow of PBS buffer by the syringe pump was increased to 4 ml / hr for 10 minutes. Finally, the extracted DNA was eluted with 100 μl of elution buffer. In addition, 200 μl of whole blood or urine was used for genomic DNA extraction using the QIAmp DNA mini kit (Hilden, Germany) for comparison. All extracted DNA was determined for DNA concentration and purity by UV spectrophotometer (Perkin-Elmer).

<< 실시예Example 6> 6> DTBPDTBP 분석을 이용한 RNA 추출 RNA extraction using analysis

RNA was extracted using DTBP analysis in the same manner as in Example 2, and RNA extraction was confirmed by amplification of 18S rRNA after cDNA synthesis from the outside as shown in FIG.

In addition, RNA extraction and cDNA synthesis could be performed on a single thin film apparatus through DTBP analysis as shown in FIG. 7 (b), and it was confirmed that this method was well developed through amplification of 18S rRNA later.

<< 실시예Example 7> 7> DTBP를DTBP 이용한 병원균(대장균) 농축 및 핵산 추출 Concentrate Pathogens (E. Coli) and Extract Nucleic Acids

The previously produced thin film device was surface treated with 3-aminopropyl (diethoxy) methylsilane (APDMS) at 65 ° C. for 1 hour. In order to concentrate the pathogen, 1 to 2 ml of the pathogen sample was injected with DTBP (100 mg / ml) and concentrated at room temperature at a flow rate of 100 µl / min. The concentrated sample was suspended in 100 μl. In this example, E. coli was used as a pathogen sample. Meanwhile, DNA was extracted and compared with the Qiagen DNA Kit.

As a result, when the concentration was carried out by DTBP, it was confirmed that the RT-PCR Ct value was shortened closer to the absolute value (absolute) than the case of nucleic acid extraction without the concentration step by the existing Qiagen method (Fig. 8).

On the other hand, when one path of the present invention (E. coli) concentration and nucleic acid extraction is carried out at the same time, it was confirmed that the Ct value is much shorter than the other method, similar to the absolute value (absolute). That is, the method according to the present invention was able to effectively concentrate the pathogen in the 1-2 mL, and at the same time it was confirmed that the nucleic acid extraction efficiency is also high (Fig. 9).

<< 실시예Example 8> 8> DTBP를DTBP 이용한 병원균( Pathogens used PIV3PIV3 -RNA 바이러스) 농축 -RNA virus) enrichment

In order to confirm the efficiency of detecting pathogens (viruses) of the method according to the present invention, a comparison experiment with a Qiagen kit, which is a widely used method, was performed on four samples suspected of PIV3-RNA virus infection.

In other words, each sample was taken 1 ml and extracted with Qiagen kit to test PIV3-RNA virus detection (

lanes

1, 2, 3, 4 of Figure 10) and concentrated in DTBP according to the present invention after extraction PIV3-RNA When virus detection was tested (lanes 1-1, 2-1, 3-1, 4-1 in Figure 10) were compared.

As a result, when the PIV3-RNA virus was concentrated in a 1 ml sample using the DTBP method, the sample 1 was negatively diagnosed by the conventional method, but was positive after the DTBP concentration according to the present invention. In case of

samples

2 and 3, the positive signal was confirmed, and sample 4 was able to confirm that the intensity of the band (intensity) of the previously positive sample was significantly increased (FIG. 10).

As mentioned above, although this invention was demonstrated by the limited embodiment and drawing, this invention is not limited by this, The person of ordinary skill in the art to which this invention belongs, Of course, various modifications and variations are possible within the scope of equivalents of the claims to be described.

Claims

Dimethyl 3,3'-dithiobispropionimidate (Dimethyl 3,3'-dithiobispropionimidate; DTBP) as an active ingredient for a composition for concentrating microorganisms.
The composition of claim 1, wherein the microorganism is a bacterium, a virus or a cell.
A microbial enrichment kit comprising the composition of claim 1.
Introducing and modifying an amine group into the object (first step); And

Contacting a sample containing microorganisms with DTBP on the modified object (second step).
The method of claim 4, wherein the object is any one of a thin film device, a magnetic bead, a ring resonator, and a nanoparticle.
The method of claim 4, wherein the object is a microorganism concentration method characterized in that the surface is modified with a silane compound.
The method of claim 6, wherein the silane compound is a microorganism concentration method, characterized in that the compound represented by the formula (1).

[Formula 1]

Wherein R 1 to R 3 may each be the same or different, either C1 to C4 alkyl or C1 to C4 alkoxy, R 4 is amino (C1 to C10) 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 7, wherein the silane compound is (3-aminopropyl) triethoxysilane (APTES), (3-aminopropyl) trimethoxysilane ((3-aminopropyl) trimethoxysilane), (1-aminomethyl) triethoxysilane ((1-aminomethyl) triethoxysilane), (2-aminoethyl) triethoxysilane, (4-aminobutyl) triethoxysilane (( 4-aminobutyl) triethoxysilane), (5-aminopentyl) triethoxysilane, (6-aminohexyl) triethoxysilane, 3-aminopropyl Diethoxy) methylsilane (3-aminopropyl (diethoxy) methylsilane; APDMS), N- [3- (trimethoxysilyl) propyl] ethylenediamine (N- [3- (trimethoxysilyl) propyl] ethylenediamine), [3- ( 2-aminoethylamino) propyl] trimethoxysilane ([3- (2-aminoethylamino) propyl] trimethoxysilane; AEAPTMS) and 3-[(trimethoxysilyl) propyl] diethylenetriamine (3-[(trimethoxysilyl) propyl] diethylenetri amine; TMPTA) microbial enrichment method, characterized in that any one or more selected from the group consisting of.
The method of claim 4, wherein the sample containing the microorganism is feces, urine, tears, saliva, external secretions of the skin, external secretions of the respiratory tract, external secretions of the intestinal tract, external secretions of the digestive tract, A method for concentrating microorganisms, characterized in that any one selected from the group consisting of plasma, serum, blood, spinal fluid, lymph, body fluid and tissue.
Nucleic acid extraction composition comprising DTBP as an active ingredient.
The nucleic acid extracting composition of claim 10, wherein the nucleic acid is DNA or RNA.
A nucleic acid extraction kit comprising the composition of claim 10.
Introducing and modifying an amine group into the object (first step);

Injecting a nucleic acid sample and DTBP onto the modified object and forming a complex between the nucleic acid and the DTBP (second step); And

The nucleic acid extraction method comprising the step of extracting the nucleic acid by treating the object with the complex formed in the elution buffer (third step).
The nucleic acid extracting method of claim 13, wherein the object is any one of a thin film device, a magnetic bead, a ring resonator, and a nanoparticle.
The method of claim 13, wherein the object is modified with a silane compound on its surface.
The method of claim 15, wherein the silane compound is a nucleic acid extraction method, characterized in that the compound represented by the formula (1).

[Formula 1]

Wherein R 1 to R 3 may each be the same or different, either C1 to C4 alkyl or C1 to C4 alkoxy, R 4 is amino (C1 to C10) 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 16, wherein the silane compound is (3-aminopropyl) triethoxysilane (APTES), (3-aminopropyl) trimethoxysilane ((3-aminopropyl) trimethoxysilane), (1-aminomethyl) triethoxysilane ((1-aminomethyl) triethoxysilane), (2-aminoethyl) triethoxysilane, (4-aminobutyl) triethoxysilane (( 4-aminobutyl) triethoxysilane), (5-aminopentyl) triethoxysilane, (6-aminohexyl) triethoxysilane, (6-aminohexyl) triethoxysilane, 3-aminopropyl Diethoxy) methylsilane (3-aminopropyl (diethoxy) methylsilane; APDMS), N- [3- (trimethoxysilyl) propyl] ethylenediamine (N- [3- (trimethoxysilyl) propyl] ethylenediamine), [3- ( 2-aminoethylamino) propyl] trimethoxysilane ([3- (2-aminoethylamino) propyl] trimethoxysilane; AEAPTMS) and 3-[(trimethoxysilyl) propyl] diethylenetriamine (3-[(trimethoxysilyl) propyl] diethylenetr nucleic acid extraction method, characterized in that any one or more selected from the group consisting of iamine;
Microbial enrichment and nucleic acid extraction composition comprising DTBP as an active ingredient.
The composition of claim 18, wherein the microorganism is a bacterium, a virus or a cell.
The composition of claim 18, wherein the nucleic acid is DNA or RNA.
21. A microbial enrichment and nucleic acid extraction kit comprising the composition of any one of claims 18-20.
Introducing and modifying an amine group into the object (first step);

Concentrating the microorganism by contacting the sample containing the microorganism with DTBP on the modified object (second step);

Separating nucleic acid from the concentrated microorganism (third step);

Forming a complex between the separated nucleic acid and the DTBP (fourth step); And

A method of extracting nucleic acid from the concentrated microorganism at the same time as the concentration of the microorganism comprising the step of extracting the nucleic acid by treating the object with the complex formed in the elution buffer (step 5).
23. The method of claim 22, wherein the object is modified with a silane compound on a surface thereof, and at the same time the nucleic acid is extracted from the concentrated microorganism.
24. The method of claim 23, wherein the silane compound is a compound represented by the following Chemical Formula 1, wherein the nucleic acid is concentrated and the nucleic acid is extracted from the concentrated microorganism at the same time.

[Formula 1]

Wherein R 1 to R 3 may each be the same or different, either C1 to C4 alkyl or C1 to C4 alkoxy, R 4 is amino (C1 to C10) 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 24, wherein the silane compound is (3-aminopropyl) triethoxysilane (APTES), (3-aminopropyl) trimethoxysilane ((3-aminopropyl) trimethoxysilane), (1-aminomethyl) triethoxysilane ((1-aminomethyl) triethoxysilane), (2-aminoethyl) triethoxysilane, (4-aminobutyl) triethoxysilane (( 4-aminobutyl) triethoxysilane), (5-aminopentyl) triethoxysilane, (6-aminohexyl) triethoxysilane, 3-aminopropyl Diethoxy) methylsilane (3-aminopropyl (diethoxy) methylsilane; APDMS), N- [3- (trimethoxysilyl) propyl] ethylenediamine (N- [3- (trimethoxysilyl) propyl] ethylenediamine), [3- ( 2-aminoethylamino) propyl] trimethoxysilane ([3- (2-aminoethylamino) propyl] trimethoxysilane; AEAPTMS) and 3-[(trimethoxysilyl) propyl] diethylenetriamine (3-[(trimethoxysilyl) propyl] diethylenetr iamine; TMPTA) at the same time as the microorganisms characterized in that at least one selected from the group consisting of extracting nucleic acids from the concentrated microorganisms.
The method of claim 22, wherein the sample containing the microorganism is fecal, urine, tear, saliva, external secretion of the skin, external secretion of the respiratory tract, external secretion of the intestinal tract, external secretion of the digestive tract, A method for extracting nucleic acid from the concentrated microorganism simultaneously with the microorganism, characterized in that any one selected from the group consisting of plasma, serum, blood, spinal fluid, lymph, body fluid and tissue.