KR20220086489A - Integrated pretreatment kit for specimen collection tool for on-site molecular diagnosis and method of extracting nucleic acid using same - Google Patents

Abstract

Disclosed are a pre-processing kit integrated with a sample collection tool for on-site molecular diagnosis and a nucleic acid extraction method using the same. The pre-processing kit integrated with the sample collection tool for on-site molecular diagnosis includes: a case 100 including an inner space 110; an injection unit 200 located at the upper end of the case 100 adjacent to the inner space 110 and including an injection port 210 through which a specimen collection tool that has collected a specimen can pass; Located in the inner space 110, including a lysis buffer chamber 310 containing a lysis buffer (Lysis buffer), the lysis buffer unit 300 for dissolving the nucleic acid by decomposing the sample; a filtering unit 400 located at the lower end of the case 100 adjacent to the inner space 110 and including a membrane 410 for filtering the nucleic acid; a wash buffer unit 500 located in the inner space 110, including a wash buffer chamber 510 including a wash buffer, and washing the nucleic acids filtered by the membrane 410; and an elution buffer unit 600 located in the inner space 110, including an elution buffer chamber 610 containing an elution buffer, and eluting the washed nucleic acid to the outside. can do. The sample collection tool integrated pretreatment kit for on-site molecular diagnostics of the present invention and the nucleic acid extraction method using the same do not use professional machines such as centrifuges or pipettes, so they can be used by the general public, there is little contamination, and the cost is low.

Description

Specimen collection tool integrated pretreatment kit for on-site molecular diagnosis and nucleic acid extraction method using the same

The present invention relates to a pretreatment kit and a nucleic acid extraction method using the same, and more particularly, to a sample collection tool integrated pretreatment kit for on-site molecular diagnosis and a nucleic acid extraction method using the same.

Genome extraction is one of the most important tasks in experiments in molecular biology, biotechnology, biochemistry, and diagnostic laboratory medicine. This is because experimental results such as polymerase chain reaction and molecular diagnostics may vary depending on the quality of the extracted genome or nucleic acid.

The dielectric extraction method typically includes a dielectric extraction method using a filter and a method using magnetic beads.

In the genome extraction method using a filter, a centrifugal separator is essentially used to filter a sample.

In addition, in the method using magnetic beads, reagents are added through pipetting a predetermined number of times after separately adding beads, and the reagents left after fixing the beads inside by the magnetic force of an external magnet There is the inconvenience of having to proceed with the iterative process of removal.

That is, in the above-described genome extraction methods, procedures such as changing the buffer are repeated at each step, so that the procedure is quickly performed close to the subject (Ex. patient), and the clinical site immediate examination (Point) that can be used for diagnosis and treatment Of Care Testing (POCT), it is inappropriate.

In addition, since medical devices such as centrifuges are often equipped in large hospitals or laboratories, there is a practical limit to the extraction of genomes by the conventional method as described above.

An object of the present invention is to provide an integrated pre-treatment kit for on-site molecular diagnostics with low contamination and low cost, and a nucleic acid extraction method using the same, which can be used by the general public without using a professional machine such as a centrifuge or pipette. have.

According to one aspect of the present invention, the case 100 including the inner space (110); an injection unit 200 located at the upper end of the case 100 adjacent to the inner space 110 and including an injection port 210 through which a specimen collection tool that has collected a specimen can pass; Located in the inner space 110, including a lysis buffer chamber 310 containing a lysis buffer (Lysis buffer), the lysis buffer unit 300 for dissolving the nucleic acid by decomposing the sample; a filtering unit 400 located at the lower end of the case 100 adjacent to the inner space 110 and including a membrane 410 for filtering the nucleic acid; a wash buffer unit 500 located in the inner space 110, including a wash buffer chamber 510 including a wash buffer, and washing the nucleic acids filtered by the membrane 410; and an elution buffer unit 600 located in the inner space 110, including an elution buffer chamber 610 containing an elution buffer, and eluting the washed nucleic acid to the outside. A sample collection tool integrated pre-treatment kit 10 for on-site molecular diagnosis is provided.

In addition, the upper and lower ends of the lysis buffer chamber 310 may be destroyed by the injection of the specimen collection tool.

Also, the lysis buffer may be discharged to the outside of the case 100 through the membrane 410 .

In addition, the lysis buffer is guanidinium chloride (Gaunidinium chloride), tris (hydroxymethyl) aminomethane, triton X-100 (triton X-100), polysorbate 20 (tween), sodium chloride (NaCl), ethylene Diaminetetraacetic acid (ethylene-diamine-tetraacetic acid, EDTA), nonyl phenoxypolyethoxyethanol (nonyl phenoxypolyethoxyethanol), and sodium dodecyl sulfate (sodium dodecyl sulfate, SDS) may include at least one selected from the group consisting of .

Also, the wash buffer unit 500 may include a single or a plurality of wash buffer chambers 510 .

In addition, the wash buffer may include at least one selected from the group consisting of tris(hydroxymethyl)aminomethane, polysorbate 20 (tween 20), and water.

In addition, the sample collection tool integrated pretreatment kit further includes a separation unit 700 , the separation unit 700 includes a vertical wall 710 , and the vertical wall 710 divides the internal space 110 . It may be divided into a first inner space 111 and a second inner space 112 , and may be positioned between the first inner space 111 and the second inner space 112 .

In addition, the injection unit 200 is located at the upper end of the case 100 adjacent to the first inner space 111 , and the filter unit 400 is located at the lower end of the case 100 adjacent to the first inner space 111 . can be located in

In addition, the lysis buffer unit 300 may be located in the first internal space 111 , and the wash buffer unit 500 and the illusion buffer unit 600 may be located in the second internal space 112 , respectively. .

In addition, a wash buffer supply line 520 through which the wash buffer unit 500 supplies the wash buffer of the wash buffer chamber 510 to the first internal space 111 through the vertical wall 710 is additionally provided. may include

In addition, an illusion buffer supply line 620 through which the illusion buffer unit 600 supplies the illusion buffer of the illusion buffer chamber 610 to the first internal space 111 through the vertical wall 710 is additionally provided. may include

In addition, the elution buffer may include at least one selected from the group consisting of tris(hydroxymethyl)aminomethane, polysorbate 20 (tween 20), and water.

In addition, each of the wash buffer chamber 510 and the illusion buffer chamber 610 has elasticity and can be deformed by hand.

Also, the membrane 410 may be a silica membrane.

In addition, the pre-treatment kit integrated with the sample collection tool for on-site molecular diagnosis is corona virus, influenza virus, acquired immunodeficiency virus (HIV) virus, smallpox virus, foot-and-mouth disease virus, Ebola virus, dengue virus, Zika (Zika) ) may be for diagnosing one or more selected from the group consisting of virus, HPV virus, bacterial pneumonia, tuberculosis and gonorrhea.

According to another aspect of the present invention, it is a nucleic acid extraction method using the integrated pre-processing kit for on-site molecular diagnostics, and (a) injecting a sample collection tool from which a sample containing nucleic acid is collected into the injection port 210, and physically destroying the upper end of the lysis buffer chamber 310 with a sample collection tool, and immersing the sample in the lysis buffer to generate a lysis buffer in which nucleic acids are dissolved; (b) physically destroying the lower end of the lysis buffer chamber 310 with the sample collection tool to supply the lysis buffer in which the nucleic acid is dissolved to the internal space 110; (c) filtering the lysis buffer supplied to the inner space 110 with a membrane 410 to bind the nucleic acid to the membrane 410; (d) applying pressure to the wash buffer chamber 510 to discharge the wash buffer into the inner space 110 and passing it through the membrane 410 to wash the nucleic acid bound to the membrane 410; and (e) applying pressure to the elution buffer chamber 610 to discharge the elution buffer into the inner space 110, passing it through the membrane 410, and eluting the nucleic acid bound to the membrane 410 to elute the case 100 ) discharging to the outside; is provided a nucleic acid extraction method comprising.

In addition, each of the wash buffer chamber 510 and the illusion buffer chamber 610 has elasticity and can be deformed by hand.

Also, the membrane 410 may be a silica membrane.

In addition, the nucleic acid extraction method is corona virus, influenza virus, acquired immunodeficiency (HIV) virus, smallpox virus, foot-and-mouth disease virus, Ebola virus, dengue virus, Zika virus, HPV virus, It may be for diagnosing one or more selected from the group consisting of bacterial pneumonia, tuberculosis and gonorrhea.

In addition, the step (d) may be continuously repeated a single time or a plurality of times.

The sample collection tool integrated pretreatment kit for on-site molecular diagnostics of the present invention and the nucleic acid extraction method using the same do not use professional machines such as centrifuges or pipettes, so they can be used by the general public, there is little contamination, and the cost is low.

1 is a schematic diagram showing the structure of an integrated pretreatment kit 10 for on-site molecular diagnosis of the present invention.
2 is a schematic diagram sequentially showing a nucleic acid extraction method using the pre-treatment kit integrated with the sample collection tool for on-site molecular diagnosis of the present invention.
3A is quantitative PCR (qPCR) data for sample preparation of mucus-free bacterial cells using the integrated pretreatment kit using the lysis buffer of Examples 1-1 to 1-3.
3B is quantitative PCR (qPCR) data for sample preparation of bacterial cells with mucus using the integrated pretreatment kit using the lysis buffer of Examples 1-1 to 1-3.
4 shows the relative fluorescence unit (RFU) according to the volume of the elution buffer when preparing the integrated pretreatment kit using the lysis buffer of Example 1-1.

Since the present invention can apply various transformations and can have various embodiments, specific embodiments are illustrated and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

In addition, terms including ordinal numbers such as first, second, etc. to be used below may be used to describe various elements, but the elements are not limited by the terms. The above terms are used only for the purpose of distinguishing one component from another. For example, without departing from the scope of the present invention, a first component may be referred to as a second component, and similarly, a second component may also be referred to as a first component.

Also, when it is stated that a certain component is “on another component,” “formed on another component,” “located on another component,” or “stacked on another component,” the It should be understood that the other components may be formed by being directly attached to the front surface or one surface on the surface of the other components, positioned or stacked, but other components may be further present in the middle.

The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present application, terms such as “comprise” or “have” are intended to designate that a feature, number, step, operation, component, part, or combination thereof described in the specification exists, but one or more other features It should be understood that this does not preclude the existence or addition of numbers, steps, operations, components, parts, or combinations thereof.

1 is a schematic diagram showing the structure of an integrated pretreatment kit 10 for on-site molecular diagnosis of the present invention.

Hereinafter, with reference to FIG. 1, a sample collection tool integrated pretreatment kit 10 for on-site molecular diagnosis of the present invention will be described.

The present invention is a case 100; injection unit 200; Lysis buffer unit 300; filtering unit 400; Wash buffer unit 500; and an illusion buffer unit 600; provides an integrated pre-processing kit 10 for on-site molecular diagnosis, including a sample collection tool.

Case (100)

The sample collection tool integrated pretreatment kit 10 of the present invention may include a case 100 including an inner space 110 .

injection unit 200

The sample collection tool integrated pretreatment kit 10 of the present invention may include an injection unit 200 located at the upper end of the case 100 adjacent to the inner space 110 .

The injection unit 200 may include an injection port 210 through which a specimen collection tool that has collected a specimen may pass.

Lysis buffer unit 300

The sample collection tool integrated pre-processing kit 10 of the present invention is located in the inner space 110 and includes a lysis buffer chamber 310 containing a lysis buffer, and decomposes the sample to prepare nucleic acids. It may include a lysis buffer unit 300 for dissolving.

The upper and lower ends of the lysis buffer chamber 310 may be destroyed by the injection of the specimen collection tool.

The lysis buffer is guanidinium chloride (Gaunidinium chloride), tris (hydroxymethyl) aminomethane, triton X-100 (triton X-100), polysorbate 20 (tween 20), sodium chloride (NaCl), ethylene Diamine tetraacetic acid (ethylene-diamine-tetraacetic acid, EDTA), nonyl phenoxy polyethoxyethanol (nonyl phenoxypolyethoxyethanol) and sodium dodecyl sulfate (sodium dodecyl sulfate, SDS) may include at least one selected from the group consisting of, and , preferably guanidinium chloride (Gaunidinium chloride), tris (hydroxymethyl) aminomethane, triton X-100 (triton X-100) and polysorbate 20 (tween 20) may be included.

filtration unit 400

The sample collection tool integrated pretreatment kit 10 of the present invention may include a filtration unit 400 located at the lower end of the case 100 adjacent to the inner space 110 .

The filtering unit 400 may include a membrane 410 for filtering the nucleic acid.

The lysis buffer may be discharged to the outside of the case 100 through the membrane 410 .

The membrane 410 may be a silica membrane.

Wash buffer unit (500)

The sample collection tool integrated pretreatment kit 10 of the present invention is located in the inner space 110, includes a wash buffer chamber 510 containing a wash buffer, and the membrane 410 filtered It may include a wash buffer unit 500 for washing the nucleic acids.

The wash buffer unit 500 may include a single or a plurality of wash buffer chambers 510 .

The wash buffer may include at least one selected from the group consisting of tris(hydroxymethyl)aminomethane, polysorbate 20 (tween 20), and water.

Illusion buffer unit 600

The sample collection tool integrated pretreatment kit 10 of the present invention is located in the inner space 110, includes an elution buffer chamber 610 containing an elution buffer, and elutes the washed nucleic acid to the outside. It may include an illusion buffer unit 600 for discharging.

The elution buffer may include at least one selected from the group consisting of tris(hydroxymethyl)aminomethane and water.

Separator (700)

The sample collection tool integrated pretreatment kit 10 of the present invention may further include a separation unit 700 .

The separation unit 700 may include a vertical wall 710 .

The vertical wall 710 divides the inner space 110 into a first inner space 111 and a second inner space 112 , and the first inner space 111 and the second inner space 112 . can be located between

The injection unit 200 is located at the upper end of the case 100 adjacent to the first inner space 111 , and the filtering unit 400 is located at the lower end of the case 100 adjacent to the first inner space 111 . can be located

The lysis buffer unit 300 may be located in the first internal space 111 , and the wash buffer unit 500 and the illusion buffer unit 600 may be located in the second internal space 112 , respectively.

The wash buffer unit 500 further includes a wash buffer supply line 520 for supplying the wash buffer of the wash buffer chamber 510 to the first internal space 111 through the vertical wall 710 . can do.

The illusion buffer unit 600 further includes an illusion buffer supply line 620 for supplying the illusion buffer of the illusion buffer chamber 610 to the first internal space 111 through the vertical wall 710 . can do.

Each of the wash buffer chamber 510 and the illusion buffer chamber 610 has elasticity and can be deformed by hand.

The pre-treatment kit integrated with the sample collection tool for on-site molecular diagnosis is corona virus, influenza virus, acquired immunodeficiency virus (HIV) virus, smallpox virus, foot-and-mouth disease virus, Ebola virus, dengue virus, Zika It may be for diagnosing one or more selected from the group consisting of virus, HPV virus, bacterial pneumonia, tuberculosis and gonorrhea.

2 is a schematic diagram sequentially showing a nucleic acid extraction method using the pre-treatment kit integrated with the sample collection tool for on-site molecular diagnosis of the present invention.

Hereinafter, a nucleic acid extraction method using an integrated pretreatment kit for on-site molecular diagnostics of the present invention will be described with reference to FIG. 2 .

First, a sample collection tool from which a sample containing nucleic acid is collected is injected into the injection port 210, the upper end of the lysis buffer chamber 310 is physically destroyed with the sample collection tool, and the sample is immersed in the lysis buffer to produce a lysis buffer in which the nucleic acid is dissolved (step a).

Next, by physically destroying the lower end of the lysis buffer chamber 310 with the sample collection tool, the lysis buffer in which the nucleic acid is dissolved is supplied to the internal space 110 (step b).

Next, the lysis buffer supplied to the inner space 110 is filtered through the membrane 410 to bind the nucleic acid to the membrane 410 (step c).

The membrane 410 may be a silica membrane.

Next, pressure is applied to the wash buffer chamber 510 to discharge the wash buffer into the inner space 110 , and the wash buffer is passed through the membrane 410 to wash the nucleic acid bound to the membrane 410 (step d). .

Step (d) may be repeatedly performed singly or continuously a plurality of times.

Finally, by applying pressure to the elution buffer chamber 610, the elution buffer is discharged into the inner space 110, passed through the membrane 410 to elute the nucleic acid bound to the membrane 410, and the case 100 Discharge to the outside (step e).

Each of the wash buffer chamber 510 and the illusion buffer chamber 610 has elasticity and can be deformed by hand.

The nucleic acid extraction method is a corona virus, influenza virus, acquired immunodeficiency (HIV) virus, smallpox virus, foot-and-mouth disease virus, Ebola virus, dengue virus, Zika virus, HPV virus, bacteria It may be for diagnosing one or more selected from the group consisting of pneumonia, tuberculosis and gonorrhea.

[Example]

Hereinafter, the present invention will be described in more detail by way of examples. However, this is for illustrative purposes, and the scope of the present invention is not limited thereto.

Example 1: Preparation of an integrated pretreatment kit for on-site molecular diagnostics sample collection tool

Referring to FIG. 1 , a case 100 including a first inner space 111 and a second inner space 112 ; an injection unit 200 located at the upper end of the case 100 adjacent to the first inner space 111 and including an injection port 210 through which a specimen collection tool that has collected a specimen can pass; Located in the first inner space 111, including a lysis buffer chamber 310 containing a lysis buffer (Lysis buffer), the lysis buffer unit 300 for dissolving the nucleic acid by decomposing the sample; a filtering unit 400 located at the lower end of the case 100 adjacent to the first inner space 111 and including a membrane 410 for filtering the nucleic acids; It is located in the second inner space 112, and includes a wash buffer chamber 510 including a wash buffer, and a wash buffer unit 500 for washing the nucleic acids filtered by the membrane 410. ; an elution buffer unit 600 located in the second inner space 112, including an elution buffer chamber 610 including an elution buffer, and eluting the washed nucleic acid to the outside; and a separation unit 700 including a vertical wall 710 positioned between the first inner space 111 and the second inner space 112. An integrated pretreatment kit for on-site molecular diagnostics (10) was prepared.

At this time, the wash buffer unit 500 includes two wash buffer chambers 511 and 512 , and passes the wash buffers of each of the two wash buffer chambers 511 and 512 through the vertical wall 710 . Wash buffer supply lines 521 and 522 for supplying the first inner space 111 were included.

In addition, the illusion buffer unit 600 includes an illusion buffer supply line 620 for supplying the illusion buffer of the illusion buffer chamber 610 to the first internal space 111 through the vertical wall 710. .

One of the two wash buffers was a mixture of 10 mM Tris (pH 8.0) and 0.1% (v/v) Tween 20, the other was water, and the elution buffer was water.

The lysis buffer was used up to a range of 1,400 μL by increasing 700 μL, each of the two wash buffers by 1 mL, and the elution buffer from 100 μL to 100 μL.

By diversifying the types of the lysis buffer, a sample collection tool integrated pretreatment kit for on-site molecular diagnosis was prepared. The used lysis buffer is summarized in Table 1 below.

division Lysis Buffer Composition and Ingredients Example 1-1
(Buffer A) 800 mM guanidine hydrochloride
50 mM Tris pH 8.0
0.5 % (v/v) Triton X-100
1% (v/v) Tween 20 Example 1-2
(Buffer B) 20 mM Tris (pH 8)
25 mM NaCl
2.5 mM EDTA
0.5 % (v/v) Triton X-100
0.5 % (v/v) NP-40 Examples 1-3
(Buffer C) 1 M Tris-HCl (pH 7.2)
1M Triton X-100
10% (v/v) SDS

Example 2: Nucleic Acid Extraction

Referring to FIG. 2 , nucleic acids were extracted using the integrated pretreatment kit 10 for on-site molecular diagnosis prepared according to Example 1 in the following manner.

First, a sample collection tool from which a sample containing nucleic acid is collected is injected into the injection port 210, the upper end of the lysis buffer chamber 310 is physically destroyed with the sample collection tool, and the sample is immersed in the lysis buffer Thus, a lysis buffer in which the nucleic acid was dissolved was generated.

Next, by physically destroying the lower end of the lysis buffer chamber 310 with the sample collection tool, the lysis buffer in which the nucleic acid was dissolved was supplied to the first internal space 111 .

Next, the lysis buffer supplied to the first inner space 111 was filtered through the membrane 410 to bind the nucleic acid to the membrane 410 .

Next, pressure was applied to the wash buffer chamber 511 to discharge the wash buffer into the first inner space 111 , and passed through the membrane 410 to wash the nucleic acids bound to the membrane 410 . Thereafter, pressure was applied to the wash buffer chamber 512 to discharge the wash buffer into the first inner space 111 , and passed through the membrane 410 to wash the nucleic acid bound to the membrane 410 once more.

Finally, by applying pressure to the elution buffer chamber 610, the elution buffer is discharged into the first inner space 111, passed through the membrane 410 to elute the nucleic acid bound to the membrane 410, and the case ( 100) was discharged to the outside to extract nucleic acids.

[Test Example]

Test Example 1: Selection of the optimal lysis buffer

For the pre-treatment kit integrated with the sample collection tool for on-site molecular diagnosis, the lysis ability and polymerase chain reaction (PCR) inhibitory effect of the lysis buffer were evaluated for three types of lysis buffer. This is important because it is difficult to remove the lysis buffer from the final elution solution in an unequipped setup, unlike laboratory-scale systems. Therefore, the selection experiment of the lysis buffer (Examples 1-1 to 1-3) having a small inhibitory effect on the PCR reaction was carried out.

The optimal lysis buffer selection method was performed as follows. The three types of lysis buffers (Examples 1-1 to 1-3) were loaded with the same amount of bacteria and mucus, respectively, and the bacteria were lysed for 10 minutes. Then, different types of lysis buffers were filtered with a membrane. Nucleic acids dissolved in bacteria were bound to the membrane, and wash buffer 1 (a mixture of 10 mM Tris (pH 8.0) and 0.1% (v/v) Tween 20) and wash buffer 2 (water) were filtered through the membrane. The nucleic acid bound to the membrane was washed.Finally, the nucleic acid solution from which the bound nucleic acid was eluted by passing the membrane through the elution buffer chamber was quantified by quantitative PCR (qPCR).Biorard's 2X SsoAdvanced Universal SYBR Green Supermix as a qPCR master mix. was used, and a pair of primers capable of detecting a specific bacterial gene were added, and the eluted nucleic acid extraction solution was added, followed by reaction with a final volume of 20 uL under the following conditions (Denaturation: 95 ℃, 10 sec, Annealing: 61 ℃, 15 sec, Extension: 74 ℃, 30 sec, Cycles: 45)

Figure 3a is quantitative PCR (qPCR) data for the sample preparation of mucus-free bacterial cells using the integrated pretreatment kit using the lysis buffer of Examples 1-1 to 1-3, Figure 3b is Examples 1-1 to Quantitative PCR (qPCR) data for sample preparation of mucus-bearing bacterial cells using an integrated pretreatment kit using lysis buffer of 1-3. The quantitative PCR data were normalized to the lysis buffer of Example 1-3 (Buffer C).

According to FIG. 3a, when using a bacterial sample, it can be seen that the lysis buffers of Examples 1-1 to 1-3 exhibit similar lysis efficiency and PCR inhibitory effect.

According to FIG. 3b, when there is other biological interference such as nasal mucus, it can be confirmed that the lysis buffer of Example 1-1 (Buffer A) showed the highest dissolution efficiency and less inhibition of DNA binding to the silica membrane.

Therefore, it can be confirmed that the lysis buffer of Example 1-1 (Buffer A) based on guanidine hydrochloride shows the highest lysis efficiency and the minimum inhibitory effect in the biological interference sample.

Test Example 2: Volume Optimization of Elution Buffer

In order to improve the purity and quantity of the final elution, the effect of the volume of the elution buffer on the polymerase chain reaction (PCR) was confirmed.

The confirmation proceeded as follows. The same amount of bacteria and mucus were loaded into the lysis buffer of Example 1-1 (buffer A), and the bacteria were lysed for 10 minutes. Thereafter, the lysis buffer was filtered through a membrane to bind nucleic acids dissolved in bacteria to the membrane. Next, wash buffer 1 (a mixture of 10 mM Tris (pH 8.0) and 0.1% (v/v) Tween 20) and wash buffer 2 (water) were filtered through the membrane to wash the nucleic acids bound to the membrane. Finally, the nucleic acid solution from which the bound nucleic acid was eluted by passing different volumes of elution buffer through the membrane was quantified by quantitative PCR (qPCR). Biorad's 2X SsoAdvanced Universal SYBR Green Supermix was used as the qPCR master mix, and a pair of primers capable of detecting a specific bacterial gene was added, and the eluted nucleic acid extraction solution was added, followed by reaction with a final volume of 20 uL under the following conditions. (Denaturation: 95 ℃, 10 sec, Annealing: 61 ℃, 15 sec, Extension: 74 ℃, 30 sec, Cycles: 45)

4 shows the relative fluorescence unit (RFU) according to the volume of the elution buffer when an integrated pretreatment kit is prepared using the lysis buffer of Example 1-1.

According to FIG. 4, if the volume of the elution buffer is increased, the DNA concentration is decreased instead of the concentration of the interfering component is decreased, thereby increasing PCR efficiency. It can be seen that there is no significant increase.

In addition, it can be seen that the volume of the elution buffer shows the highest PCR efficiency at 300 μL and 1,100 μL.

Considering the size of the pretreatment kit integrated with the sample collection tool for on-site molecular diagnosis of the present invention, it is determined that the volume of the elution buffer is preferably 300 μL.

As mentioned above, although preferred embodiments of the present invention have been described, those of ordinary skill in the art can add, change, delete or The present invention may be variously modified and changed by addition, etc., and this will also be included within the scope of the present invention. For example, each component described as a single type may be implemented in a dispersed form, and likewise components described as distributed may also be implemented in a combined form. The scope of the present invention is indicated by the following claims rather than the above detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be interpreted as being included in the scope of the present invention. do.

10: Sample collection tool integrated pre-treatment kit for on-site molecular diagnosis
100: case
110: inner space
111: first internal space
112: second inner space
200: injection unit
210: inlet
300: Lysis buffer unit
310: Lysis buffer chamber
400: filter unit
410: membrane
500: wash buffer unit
510: wash buffer chamber
511: wash buffer chamber
512: wash buffer chamber
520: wash buffer supply line
521: wash buffer supply line
522: wash buffer supply line
600: Illusion buffer unit
610: Illusion Buffer Chamber
620: Illusion buffer supply line
700: separation unit
710: vertical wall

Claims (20)

Case 100 including an inner space (110);
an injection unit 200 located at the upper end of the case 100 adjacent to the inner space 110 and including an injection port 210 through which a specimen collection tool that has collected a specimen can pass;
Located in the inner space 110, including a lysis buffer chamber 310 containing a lysis buffer (Lysis buffer), the lysis buffer unit 300 for dissolving the nucleic acid by decomposing the sample;
a filtering unit 400 located at the lower end of the case 100 adjacent to the inner space 110 and including a membrane 410 for filtering the nucleic acid;
a wash buffer unit 500 located in the inner space 110, including a wash buffer chamber 510 including a wash buffer, and washing the nucleic acids filtered by the membrane 410; and
It is located in the inner space 110, and includes an elution buffer chamber 610 including an elution buffer, and an elution buffer unit 600 for eluting and discharging the washed nucleic acid to the outside;
An integrated pretreatment kit for on-site molecular diagnostics, including a sample collection tool (10). According to claim 1,
The sample collection tool integrated pretreatment kit, characterized in that the upper and lower ends of the lysis buffer chamber (310) can be destroyed by the injection of the sample collection tool. According to claim 1,
An integrated pretreatment kit for sample collection, characterized in that the lysis buffer is discharged to the outside of the case (100) through the membrane (410). According to claim 1,
The lysis buffer is guanidinium chloride (Gaunidinium chloride), tris (hydroxymethyl) aminomethane, triton X-100 (triton X-100), polysorbate 20 (tween), sodium chloride (NaCl), ethylenediamine tetraacetic acid (ethylene-diamine-tetraacetic acid, EDTA), nonyl phenoxypolyethoxyethanol (nonyl phenoxypolyethoxyethanol) and sodium dodecyl sulfate (sodium dodecyl sulfate, SDS) An integrated pre-processing kit for sample collection tools. According to claim 1,
The sample collection tool integrated pretreatment kit, characterized in that the wash buffer unit (500) includes a single or a plurality of wash buffer chambers (510). According to claim 1,
An integrated pretreatment kit for sample collection, wherein the wash buffer comprises at least one selected from the group consisting of tris(hydroxymethyl)aminomethane, polysorbate 20 (tween 20) and water. According to claim 1,
The sample collection tool integrated pre-processing kit further includes a separation unit 700,
The separation part 700 includes a vertical wall 710 , and the vertical wall 710 divides the internal space 110 into a first internal space 111 and a second internal space 112 , and the An integrated pretreatment kit for sample collection, characterized in that it is located between the first inner space (111) and the second inner space (112). 8. The method of claim 7,
The injection part 200 is located at the upper end of the case 100 adjacent to the first internal space 111 , and the filtering part 400 is located at the lower end of the case 100 adjacent to the first internal space 111 . An integrated pretreatment kit for sample collection tools, characterized in that it is located. 9. The method of claim 8,
The lysis buffer unit 300 is located in the first internal space 111,
The sample collection tool integrated pre-processing kit, characterized in that the wash buffer unit 500 and the illusion buffer unit 600 are respectively located in the second inner space (112). 10. The method of claim 9,
The wash buffer unit 500 further includes a wash buffer supply line 520 for supplying the wash buffer of the wash buffer chamber 510 to the first internal space 111 through the vertical wall 710 . A sample collection tool integrated pre-treatment kit, characterized in that. 10. The method of claim 9,
The illusion buffer unit 600 further includes an illusion buffer supply line 620 for supplying the illusion buffer of the illusion buffer chamber 610 to the first internal space 111 through the vertical wall 710 . A sample collection tool integrated pre-treatment kit, characterized in that. According to claim 1,
An integrated pretreatment kit for sample collection, wherein the elution buffer comprises at least one selected from the group consisting of tris(hydroxymethyl)aminomethane, polysorbate 20 (tween 20) and water. According to claim 1,
The wash buffer chamber 510 and the illusion buffer chamber 610 each have elasticity and can be deformed by hand. According to claim 1,
An integrated pretreatment kit for sample collection, characterized in that the membrane 410 is a silica membrane. According to claim 1,
The pre-treatment kit integrated with the sample collection tool for on-site molecular diagnosis is corona virus, influenza virus, acquired immunodeficiency virus (HIV) virus, smallpox virus, foot-and-mouth disease virus, Ebola virus, dengue virus, Zika An integrated pretreatment kit for sample collection, characterized in that it is for diagnosing one or more selected from the group consisting of virus, HPV virus, bacterial pneumonia, tuberculosis and gonorrhea. It is a nucleic acid extraction method using the pre-treatment kit integrated with the sample collection tool for on-site molecular diagnosis according to claim 1,
(a) injecting a sample collection tool from which a sample containing a nucleic acid is collected into the injection port 210, physically destroying the upper end of the lysis buffer chamber 310 with the sample collection tool, and placing the sample in the lysis buffer immersion to generate a lysis buffer in which the nucleic acid is dissolved;
(b) physically destroying the lower end of the lysis buffer chamber 310 with the sample collection tool to supply the lysis buffer in which the nucleic acid is dissolved to the internal space 110;
(c) filtering the lysis buffer supplied to the inner space 110 with a membrane 410 to bind the nucleic acid to the membrane 410;
(d) applying pressure to the wash buffer chamber 510 to discharge the wash buffer into the inner space 110 and passing it through the membrane 410 to wash the nucleic acid bound to the membrane 410; and
(e) applying pressure to the elution buffer chamber 610 to discharge the elution buffer into the inner space 110, passing it through the membrane 410, and eluting the nucleic acid bound to the membrane 410 to elute the case 100 discharging to the outside;
Nucleic acid extraction method comprising. 17. The method of claim 16,
The nucleic acid extraction method, characterized in that the wash buffer chamber (510) and the illusion buffer chamber (610) each has elasticity and can be deformed by hand. 17. The method of claim 16,
The nucleic acid extraction method, characterized in that the membrane (410) is a silica membrane. 17. The method of claim 16,
The nucleic acid extraction method is corona virus, influenza virus, acquired immunodeficiency (HIV) virus, smallpox virus, foot-and-mouth disease virus, Ebola virus, dengue virus, Zika virus, HPV virus, bacteria Nucleic acid extraction method, characterized in that for diagnosing one or more selected from the group consisting of pneumonia, tuberculosis and gonorrhea. 17. The method of claim 16,
Nucleic acid extraction method, characterized in that the step (d) is performed by repeatedly repeating a single or a plurality of times in succession.

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