WO2018169307A1

WO2018169307A1 - Microbial concentration method using diatomite and nucleic acid extraction method

Info

Publication number: WO2018169307A1
Application number: PCT/KR2018/002994
Authority: WO
Inventors: 신용; 조비
Original assignee: 울산대학교 산학협력단; 재단법인 아산사회복지재단
Priority date: 2017-03-15
Filing date: 2018-03-14
Publication date: 2018-09-20

Abstract

The present invention relates to a microbial concentration method using diatomite and a nucleic acid extraction method. A technique was developed that can achieve approximately 4-8 times the microbial concentration by using diatomite when compared with an existing prior, and a method was developed that can directly extracts nucleic acids from concentrated microorganisms by using dimethyl adipimidate (DMA) or the like. The microbial concentration and nucleic acid extraction techniques allowable in a single tube is expected to significantly reduce contamination coming from the outside, costs, time, complexity, and the like.

Description

Microbial enrichment method and nucleic acid extraction method using diatomaceous earth

The present invention relates to a method for enriching microorganisms and a method for extracting nucleic acids using diatomaceous earth, in particular diatomaceous earth modified with a silane compound.

Diatoms are unicellular algae that are distributed in various sizes from 2 μm to 2 mm and are representative phytoplankton commonly found on the earth. Diatoms account for 20-25% of the total primary production of land and 40% of marine biomass production, and have played an important role in ecosystems as marine producers for millions of years. Currently, microalgae have high photosynthetic efficiency relative to cell mass and are mainly used for the production of lipids, biofuels, and bioavailable resources through culture.

In addition, diatomaceous earth is a natural siliceous material which is a main component of diatomaceous cell membrane, and is known as bio-silica because it is calcined at 450 ° C. and composed of silica-rich silica on the surface. The size of the bio-silica is about 10-20 μm.

On the other hand, conventional methods for detecting microorganisms do not use the entire solution in more than 1 ml of the patient sample sample, using only a portion of the nucleic acid has been extracted and proceeded to detect 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.

Recently, researches are actively underway to utilize diatoms or diatomaceous earth as inorganic materials, bioenvironmental materials, nanoengineered materials or industrial application materials, but research on methods of using the materials for microbial enrichment or nucleic acid extraction is insufficient.

An object of the present invention is a composition for concentrating microorganisms comprising diatomaceous earth modified with a silane compound as an active ingredient, a method and kit for concentrating microorganisms using the same; A nucleic acid extracting composition comprising diatomaceous earth modified with a silane compound as an active ingredient, a nucleic acid extracting method and kit using the same; And it provides a composition for microbial enrichment and nucleic acid extraction, including a diatomaceous earth modified with a silane compound as an active ingredient, a method and kit for extracting nucleic acid from the concentrated microorganism at the same time as the microorganism concentration using the same.

In order to achieve the above object, the present invention provides a composition for concentrating microorganisms comprising diatomaceous earth modified with a silane compound as an active ingredient.

In addition, the present invention provides a method for concentrating microorganisms comprising contacting a sample containing microorganisms with diatomaceous earth modified with the silane compound.

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

The present invention also provides a nucleic acid extracting composition comprising diatomaceous earth modified with a silane compound as an active ingredient.

In addition, the present invention comprises the steps of (1) reacting by adding a diatomaceous earth modified with a silane compound to the nucleic acid sample to be extracted; And (2) provides a nucleic acid extraction method comprising the step of eluting the nucleic acid from the reaction.

In addition, the present invention comprises the steps of (1) adding and mixing the diatomaceous earth modified with the silane compound to the nucleic acid sample to be extracted; (2) dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS) and dimethyl 3,3'-dithiobis Reacting by adding one or more selected from the group consisting of dimethyl 3,3'-dithiobispropionimidate (DTBP); And (3) provides a nucleic acid extraction method comprising the step of eluting the nucleic acid from the reaction.

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

The present invention also provides a composition for microbial enrichment and nucleic acid extraction comprising diatomaceous earth modified with a silane compound as an active ingredient.

In addition, the present invention comprises the steps of (1) contacting the sample containing the microorganism to the diatomaceous earth modified with the silane compound to concentrate the microorganism; (2) dissolving the concentrated microorganism; And (3) eluting the nucleic acid from the lysate, and providing a method for extracting the nucleic acid from the concentrated microorganism at the same time as the microorganism is concentrated.

In addition, the present invention comprises the steps of (1) contacting the sample containing the microorganism to the diatomaceous earth modified with the silane compound to concentrate the microorganism; (2) dissolving the concentrated microorganism; (3) dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS) and dimethyl 3,3'-diti in the lysate Reacting by adding any one or more selected from the group consisting of dimethyl 3,3'-dithiobispropionimidate (DTBP); And (4) provides a method for extracting nucleic acid from the concentrated microorganism simultaneously with the concentration of the microorganism comprising eluting the nucleic acid from the reaction.

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

The present invention also provides the use of diatomaceous earth modified with a silane compound for microbial enrichment.

The present invention also provides the use of diatomaceous earth modified with silane compounds for nucleic acid extraction.

The present invention also provides the use of diatomaceous earth modified with silane compounds for microbial enrichment and nucleic acid extraction.

The present invention relates to a method for concentrating microorganisms and nucleic acid extraction method using diatomaceous earth, conventional methods for detecting microorganisms do not use the entire solution in more than 1 ml of the patient sample, extracting nucleic acids using only a part of them Detection has been performed using this. 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. Therefore, the present inventors have developed a technology capable of concentrating microorganisms about 4-8 times using existing diatomaceous earth, and extracting nucleic acids directly using dimethyl adipimidate (DMA). Developed methods. By enabling microbial enrichment and nucleic acid extraction techniques in one tube, it is expected to significantly reduce external contamination, cost, time and complexity.

1 shows the FTIR spectra of diatomaceous earth before (blue) and after (red) modification with APTES.

Figure 2 shows the results of experimental validation in the pathogen concentration process. D: diatomaceous earth, DA: diatomaceous earth modified with APTES.

Figure 3 is the result of optimizing the pathogen concentration experimental conditions by changing the modified diatomaceous earth concentration.

4 is a result of optimizing the pathogen concentration experimental conditions by varying the reaction time.

5 is a result of optimizing the pathogen concentration experiment conditions by varying the Brucella concentration.

6 shows the results of optimizing virus concentration experiment conditions using modified diatomaceous earth.

Figure 7 shows the results of experimental validation in the DNA extraction process. D: diatomaceous earth, DA: diatomaceous earth modified with APTES.

8 is a result of optimizing DNA extraction experiment conditions by varying the diatomaceous earth (DA) concentration modified with APTES.

9 is a result of optimizing the DNA extraction experiment conditions by varying the reaction time.

10 shows results of optimizing DNA extraction experiment conditions by varying HCT-116 cell concentration.

11 shows results of experimental validation in the RNA extraction process. D: diatomaceous earth, DA: diatomaceous earth modified with APTES.

12 is a result of optimizing RNA extraction experiment conditions by varying the concentration of diatomaceous earth (DA) modified with APTES.

Figure 13 is the result of optimizing the RNA extraction experimental conditions by varying the reaction time.

14 shows the results of optimizing RNA extraction experiment conditions by varying HCT-116 cell concentration.

15 shows the results of Q-PCR after concentrating pathogens and extracting DNA using a modified diatomaceous earth.

FIG. 16 shows experimental results of applying an amine-functionalized diatomaceous earth-based, dimethyl suberimidate assisted system (ADD system) based on RNA extraction from pathogen Brucella sample. (a) Ct values of RNA extracted according to the cell concentration serially diluted in PBS. The gray bars are the result of using the ADD system of the present invention, and the black bars are the result of using the solid phase extraction commercial kits used previously. A linear correlation was shown between Ct values and cell concentrations of RNA extracted through the ADD system (R ² = 0.979). (b) The Tm value of the extracted RNA was measured via RT-qPCR. (c) 10 ⁷ in the urine sample of Brucella CFU / mL concentration represents the Ct value of the RNA extracted with ADD systems and existing kits. (d) Results of testing relatively large volume samples for RNA extraction via the ADD system using HCT 116 cells at a concentration of 10 ³ cells / mL. No-template control (NTC) showed no signal. Error bars represent standard deviation of the mean of experiments repeated three times independently. The p-value was measured by Student t test comparing ADD and conventional kits in the same concentration sample. Pvalue <0.01, pvalue <0.001; p-value <0.01 indicates statistical significance and p-value <0.001 indicates statistically high significance.

The present invention provides a composition for concentrating microorganisms comprising diatomaceous earth modified with a silane compound as an active ingredient.

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), N- [3- (trimethoxysilyl) propyl] ethylenediamine (N- [3- (trimethoxysilyl) propyl] ethylenediamine), [3- (2-aminoethyl Amino) propyl] trimethoxysilane ([3- (2-aminoethylamino) propyl] trimethoxysilane; AEAPTMS) or 3-[(trimethoxysilyl) propyl] diethylenetriamine (3-[(trimethoxysilyl) propyl] diethylenetri amine; TMPTA), but is not limited thereto. Even more preferably, the silane compound is most suitable for (3-aminopropyl) triethoxysilane (APTES) described in the embodiment of the present invention.

Preferably, the microorganism may be any negatively charged microorganism such as bacteria, viruses or cells, and more preferably Brucella ovis or para influenza virus (PIV3) described in the embodiment of the present invention.

More preferably, the concentration of the microorganism may be 10 ⁰ cfu / ml to 10 ⁷ cfu / ml, but is not limited thereto.

The present invention also provides a method for concentrating microorganisms comprising contacting a sample containing microorganisms with diatomaceous earth modified with a silane compound.

[Formula 1]

Even more preferably, the silane compound is most suitable for (3-aminopropyl) triethoxysilane (APTES) described in the embodiment 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.

Preferably, the step of contacting the microorganism to the diatomaceous earth modified with the silane compound may be contacted for 20 to 60 minutes, but is not limited thereto.

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.

[Formula 1]

Preferably, the composition is dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS) and dimethyl 3,3'-dity It may further include any one or more selected from the group consisting of dimethyl propionimate (Dimethyl 3,3'-dithiobispropionimidate; DTBP).

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

[Formula 1]

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.

[Formula 1]

Preferably, the composition is dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS) and dimethyl 3,3'-dity It may further comprise the step of reacting by adding any one or more selected from the group consisting of dimethyl propionimate (Dimethyl 3,3'-dithiobispropionimidate; DTBP).

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

[Formula 1]

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 lysis buffer or a protease for lysing the cells in the sample to release the nucleic acid from inside the cell, and for adjusting the pH required for effective microbial concentration or nucleic acid extraction. Buffers and the like may be additionally included.

Preferably, the kit may be in the form of a well-plate, a tube, a column or a microfluidic chip, but the kit may be in the form of any commercially available form. It is possible. On the other hand, the well-plate may be manufactured without limitation, such as 96 wells, 384 wells.

The kit may be a single composition or the individual components of the composition for microbial enrichment or nucleic acid extraction may be stored separately for mixing before use. As such, the components of the composition for microbial enrichment or nucleic acid extraction in the kit of the present invention may be packaged separately, or may be packaged in one or more mixtures in any combination of components. Each of the individual components or one or more mixtures may be placed in different containers and may comprise the entire kit in a single container.

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.

<실시예 1> 3-아미노프로필트리에톡시실란(3-aminopropyltriethoxysilane; APTES)으로 규조토 개질Example 1 Modification of Diatomaceous Earth with 3-aminopropyltriethoxysilane (APTES)

As a method of modifying APTES in diatomaceous earth, 1g of diatomaceous earth powder was washed by dipping in distilled water for 30 minutes, then washed in 70% EtOH and 99% EtOH sequentially for 30 minutes, and then completely dried using Vaccum. Let's do it. The dried 500 mg of diatomaceous earth was put into 80 ml of 98% EtOH solution, and 2.4 ml of APTES was added to react at room temperature for 4 hours. After washing with distilled water, the final 50mg / ml concentration is used and stored.

The FTIR spectra of diatomaceous earth before (blue) and after (red) modification with APTES are shown in FIG. After modification with APTES, a strong peak appeared at 2987 nm, where the primary amine group appeared to be functionalized.

<실시예 2> 병원균 농축 공정Example 2 Pathogen Concentration Process

Brucella ovis was used as the test pathogen. For the experiment, 1 ml of 10 ⁶ cfu / ml Brucella solution was used.

1. 실험 타당성 확인1. Validation of experiment

1) 200 μl of sample was taken, DNA was extracted with Qiagen DNA Kit, and this was used as a control.

2) 1 ml of sample was each concentrated using diatomaceous earth (D) and diatomaceous earth (DA) modified with APTES. The concentration process is as follows.

3) 200 μl elution buffer was used to concentrate the pathogen. After centrifugation, the supernatant was extracted and DNA extracted with Qiagen DNA Kit.

4) As shown in Figure 2, Q-PCR results confirmed that the diatomaceous earth can concentrate the pathogen.

2. 실험 조건 최적화2. Optimize experimental conditions

Experiments were performed with varying modified diatomaceous earth concentrations. 40 μl, 80 μl, 120 μl and 200 μl of 50 mg / ml modified diatomaceous earth (DA) solution were used. Balanced with distilled water. For example, 160 μl of distilled water was added to 40 μl of the modified diatomaceous earth (DA) solution. 1 ml of 10 ⁴ cfu / ml Brucella solution was used. As shown in FIG. 3, the Q-PCR results showed the optimum effect when using 40 μl of 50 mg / ml of modified diatomaceous earth (DA) solution.

In addition, experiments were carried out with different reaction times. The reaction time was varied from 20 to 60 minutes. 1 ml of 10 ³ cfu / ml Brucella solution was used. As shown in FIG. 4, the Q-PCR results showed an optimal effect at a reaction time of 40 minutes.

In addition, experiments were performed by varying the Brucella concentration. Brucella concentrations were varied from 10 ⁰ cfu / ml to 10 ⁷ cfu / ml. As shown in FIG. 5, the Q-PCR results showed that the use of diatomaceous earth successfully concentrated the pathogens and was more effective than the control group Qiagen kit at low concentrations.

In addition, para-influenza virus (PIV3) concentration experiment was conducted using the same conditions. In a 2 ml patient sample containing para-influenza virus (PIV3), which does not know the exact concentration, when comparing the experiment with the Qiagen kit and the modified diatomaceous earth, the concentration of the modified diatomaceous earth was twice as high as before. It was confirmed (FIG. 6).

<실시예 3> DNA 추출 공정Example 3 DNA Extraction Process

HCT-116 cancer cells were used as test samples. For the experiment, 1 ml of 10 ⁵ cells / ml cell solution was used. 100 μl elution buffer was used for final DNA extraction.

1. DNA 추출 실험 타당성 확인1. Validation of DNA Extraction Experiment

2) DNA was extracted using diatomaceous earth (D) and diatomaceous earth (DA) modified with APTES, and dimethyl adipimidate (DMA) was added. The detailed extraction process is as follows.

i. Transfer 200 μl sample to a 2 ml microcentrifuge tube.

ii. 200 μl Lysis Buffer and 20 μl Protease K are added to the sample and mixed 10 times with a pipette. Be careful not to create air bubbles at this time.

iii. The sample is allowed to react at room temperature until the sample becomes clear (within 1 minute).

iv. Add 80 μl of 50 mg / ml DA solution to the 220 μl sample (Stage iii above) and mix 5 times with a pipette.

v. Add 100 μl of DMA to the mixture and mix 10 times with a pipette. Be careful not to create air bubbles at this time.

vi. The reaction is carried out at 56 ° C. for 20 minutes to bind DNA to the modified diatomaceous earth (DA). The mixture is well dispersed during the reaction time.

vii. The mixture is centrifuged for 1 minute using a microcentrifuge. Carefully remove the supernatant.

viii. Add 100 μl of PBS buffer and mix 10 times with a pipette.

ix. The mixture is centrifuged for 1 minute. Carefully remove the supernatant. Be careful not to create air bubbles at this time.

x. Rinse twice.

xi. Add 100 μl of elution buffer (pH 11) to the sample and mix 15 times with a pipette.

xii. Reaction at room temperature (within 1 minute).

xiii. The mixture is centrifuged for 1 minute. Using a pipette, transfer the supernatant to a new microtube.

3) As shown in FIG. 7, Q-PCR showed that diatomaceous earth (DA) modified with APTES was effective for DNA extraction.

2. DNA 추출 실험 조건 최적화2. Optimizing DNA Extraction Experiment Conditions

Experiments were performed with varying diatomaceous earth (DA) concentrations modified with APTES. 10-120 μL of 50 mg / ml DA solution was used. Balanced with distilled water. For example, 160 µl of distilled water was added to 40 µl of DA solution. 1 ml of 10 ⁵ cells / ml HCT-116 solution was used. As shown in FIG. 8, Q-PCR results showed an optimal effect on DNA extraction when 40 μl of 50 mg / ml DA solution was used.

In addition, experiments were carried out with different reaction times. The reaction time was varied from 5 to 35 minutes. 1 ml of 10 ⁵ cells / ml HCT-116 solution was used. As shown in FIG. 9, the Q-PCR results showed an optimal effect on DNA extraction at a reaction time of 20 minutes.

In addition, experiments were performed with varying HCT-116 cell concentrations. HCT-116 cell concentration was varied from 10 ⁰ cells / ml to 10 ⁷ cells / ml. As shown in FIG. 10, Q-PCR results showed that DNA extraction was effective even at low cell concentrations.

<실시예 4> RNA 추출 공정Example 4 RNA Extraction Process

HCT-116 cancer cells were used as test samples. For the experiment, 1 ml of 10 ⁵ cells / ml cell solution was used. 100 μl elution buffer was used for final RNA extraction.

1. RNA 추출 실험 타당성 확인1. Validation of RNA Extraction Experiment

1) 200 μl of sample was taken and RNA was extracted with Qiagen RNA Kit, which was used as a control.

2) RNA was extracted using diatomaceous earth (D) and diatomaceous earth (DA) modified with APTES, and dimethyl adipimidate (DMA) was added. It is similar to the DNA extraction process except for the following three parts.

i. After 20 μl of DNase, 20 μl of Protease K is treated.

ii. 40 μl of DA was used for RNA extraction.

iii. The reaction was stirred for 25 minutes at room temperature.

3) As shown in FIG. 11, Q-PCR results showed that diatomaceous earth (DA) modified with APTES was effective for RNA extraction.

2. RNA 추출 실험 조건 최적화2. Optimize RNA Extraction Experiment Conditions

Experiments were performed with varying diatomaceous earth (DA) concentrations modified with APTES. 10-120 μL of 50 mg / ml DA solution was used. Balanced with distilled water. For example, 160 µl of distilled water was added to 40 µl of DA solution. 1 ml of 10 ⁵ cells / ml HCT-116 solution was used. As shown in FIG. 12, the Q-PCR results showed an optimal effect on RNA extraction using 40 μl of 50 mg / ml DA solution.

In addition, experiments were carried out with different reaction times. The reaction time was varied from 5 to 35 minutes. 1 ml of 10 ⁵ cells / ml HCT-116 solution was used. As shown in FIG. 13, the Q-PCR results showed an optimal effect on RNA extraction at a reaction time of 25 minutes.

In addition, experiments were performed with varying HCT-116 cell concentrations. HCT-116 cell concentration was varied from 10 ¹ cells / ml to 10 ⁶ cells / ml. As shown in FIG. 14, Q-PCR results showed that RNA extraction was effective even at low cell concentrations.

<실시예 5> 한 튜브에서의 병원균 농축 및 DNA 추출 공정 Example 5 Pathogen Concentration and DNA Extraction Process in One Tube

Since diatomaceous earth was able to successfully concentrate pathogens and extract nucleic acids effectively, we designed an experiment to concentrate pathogens and extract DNA from one tube, without large equipment. Brucella ovis was used as the test pathogen. For the experiment, 1 ml of 10 ⁵ cfu / ml Brucella solution was used.

1) 100 μl of sample was taken, DNA was extracted with Qiagen DNA Kit, and this was used as a control.

2) 40 μl of a diatomaceous earth (DA) solution modified with 50 mg / ml of APTES was added to 1 ml of 10 ⁵ cfu / ml of Brucella Orbis sample. The reaction was shaken for 40 minutes. Mix well to prevent precipitates.

3) After centrifugation, all supernatant was removed. Washed twice with 1 ml distilled water.

5) 100 μl elution buffer was added. After reacting for 1 minute, it was settled slightly.

6) Add 100 μl elution buffer containing 20 μl Protease K to the mixture and mix 10 times with a pipette.

7) Add 100 μl of 100 mg / ml DMA to the mixture and mix 10 times with a pipette.

8) The reaction was stirred at 650 RPM for 20 minutes at 56 ° C. and mixed well every 5 minutes.

9) Sink and remove all supernatant. Wash twice with 200 μl PBS buffer (1 ×, pH 7.4).

10) Add 100 μl elution buffer and react for 1 minute at room temperature.

11) Transfer the supernatant containing the extracted DNA to a 1.5 ml tube.

As shown in FIG. 15, it was shown that even without large equipment, it was possible to successfully concentrate pathogens and extract DNA in one tube.

<< 실시예Example 6> 진단을 위해 6> for diagnosis 아민Amine 개질된Modified 규조토 기반 시스템(amine-functionalized, diatomaceous earth-based, Diatomaceous earth based systems (amine-functionalized, diatomaceous earth-based, dimethyldimethyl suberimidatesuberimidate assisted system; ADD system)의 적용 assisted system; Application of ADD system

The ADD system of the present invention was able to successfully extract RNA from biological samples. Compared with traditional analytical methods, the optimized protocol of the ADD system is expected to be useful for nucleic acid-based diagnostics. Human brucellosis, an endemic disease in many countries and regions, is a worldwide common infectious disease. We extracted RNA from Brucella bacteria using the ADD system. No RT signal could be detected from the RT control. As shown in Figure 16a, using the ADD system was able to detect even at very low Brucella concentration of 10 ⁰ CFU / mL, which was confirmed that the detection sensitivity is 100 times more improved than the existing kit. There was a high linear correlation between the Ct value of the RNA extracted through the ADD system and the pathogen cell concentration (R ² = 0.979). Fluorescent signals were found to be specific for Tm and no-template control (NTC) (FIG. 16B). In addition, the ADD system of the present invention further confirmed clinical utility using RNA extracted from 10 ⁵ CFU / mL Brucella samples derived from urine. Since Brucella is a transfection pathogen, we included the same concentration of Brucella in human urine and sheep urine samples. Although the overall Ct value of Brucella in urine was slower than PBS, it was confirmed that the RNA extracted using the ADD system was faster than the RNA extracted using the existing kit (FIG. 16c).

In addition, we have successfully extracted RNA from Salmonella and E. coli pathogens in addition to Brucella according to the same optimized protocol in the present invention. All RNA samples extracted by the ADD system showed faster Ct values compared to existing kits. In addition, we tested a relatively large volume of samples using the ADD system, increasing the detection limit by loading more samples. As a result of the RT-qPCR, using the ADD system was able to extract more RNA from a large volume (1 mL) sample compared to using the other system (Fig. The Ct value for the 1 mL sample derived RNA was about 2 cycles faster than the 200 μL sample, clearly indicating that the total amount of extracted RNA increased when the sample volume was large. Although the protocol was optimized for 200 μL samples, the ADD system could also be used for RNA extraction of large volume samples. Therefore, the ADD system proposed in the present invention can be usefully used for RNA extraction and pathogen detection based on nucleic acid amplification by RT-qPCR.

Claims

A composition for concentrating microorganisms comprising diatomaceous earth modified with a silane compound as an active ingredient.
The composition of claim 1, wherein the silane compound is a compound represented by the following Chemical 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 2, 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 3-aminopropyl (diethoxy) methylsilane, 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] diethylenetriamine; TMPTA) any one or more composition selected from the group consisting of microorganisms for concentration.
The composition of claim 1, wherein the microorganism is a bacterium, a virus or a cell.
A method for concentrating microorganisms comprising contacting a sample containing microorganisms with diatomaceous earth modified with a silane compound.
The method of claim 5, wherein the sample containing the microorganisms, feces, urine, tears, saliva, external secretions of the skin, external secretions of the respiratory tract, external secretions of the intestine, 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.
6. The method of claim 5, wherein the microorganism is a bacterium, a virus or a cell.
Microbial enrichment kit comprising the composition of any one of claims 1 to 4.
A composition for extracting nucleic acids comprising diatomaceous earth modified with a silane compound as an active ingredient.
10. The nucleic acid extracting composition according to claim 9, wherein the silane compound is a compound represented by the following Chemical 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 composition of claim 9, wherein the composition is dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS) and dimethyl 3,3 ′. -A composition for extracting nucleic acids, characterized in that it further comprises any one or more selected from the group consisting of didimethylbispropionimidate (Dimethyl 3,3'-dithiobispropionimidate; DTBP).
10. The nucleic acid extracting composition according to claim 9, wherein the nucleic acid is DNA or RNA.
(1) adding diatomaceous earth modified with a silane compound to the nucleic acid sample to be extracted and reacting; And

(2) eluting nucleic acid from the reaction.
(1) adding diatomaceous earth modified with a silane compound to the nucleic acid sample to be extracted and mixing;

(2) dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS) and dimethyl 3,3'-dithiobis Reacting by adding one or more selected from the group consisting of dimethyl 3,3'-dithiobispropionimidate (DTBP); And

(3) eluting nucleic acid from the reactant.
A nucleic acid extraction kit comprising the composition of any one of claims 9 to 12.
A composition for microorganism concentration and nucleic acid extraction comprising diatomaceous earth modified with a silane compound as an active ingredient.
The composition of claim 16, wherein the silane compound is a compound represented by the following Chemical 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 composition of claim 16 wherein the composition is dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS) and dimethyl 3,3 ′. -Dithiobipropionimide (Dimethyl 3,3'-dithiobispropionimidate; DTBP) composition for microorganism concentration and nucleic acid extraction, characterized in that it further comprises any one or more selected from the group consisting of.
The composition of claim 16, wherein the nucleic acid is DNA or RNA.
(1) concentrating the microorganisms by contacting a sample containing microorganisms with diatomaceous earth modified with a silane compound;

(2) dissolving the concentrated microorganism; And

(3) extracting nucleic acid from the concentrated microorganism simultaneously with concentrating the microorganism comprising eluting the nucleic acid from the lysate.
(1) concentrating the microorganisms by contacting a sample containing microorganisms with diatomaceous earth modified with a silane compound;

(2) dissolving the concentrated microorganism;

(3) dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS) and dimethyl 3,3'-diti in the lysate Reacting by adding any one or more selected from the group consisting of dimethyl 3,3'-dithiobispropionimidate (DTBP); And

(4) extracting the nucleic acid from the concentrated microorganism simultaneously with the concentration of the microorganism comprising eluting the nucleic acid from the reactant.
20. A microbial enrichment and nucleic acid extraction kit comprising the composition of any one of claims 16-19.
23. The microorganism of claim 22, wherein the kit is in the form of any one selected from the group consisting of a well-plate, a tube, a column, and a microfluidic chip. Concentration and Nucleic Acid Extraction Kit.