KR20180125814A - Pretreatment chamber for nucleic acid extraction, cartridge and nucleic acid extraction method using the same - Google Patents

Abstract

The present invention relates to a pretreatment chamber for nucleic acid extraction, a cartridge using the same, and a nucleic acid extraction method, which ensures simplified processes to extract nucleic acid from a sample and is to be applied to point of care testing (POCT). According to the present invention, the cartridge includes a chamber module, an air valve module, and a liquid valve module. The chamber module includes a pretreatment chamber for homogenization, cell destruction and purification by grinding of the sample to be introduced, and has a plurality of chambers for extracting nucleic acid from the sample. The air valve module and a fluid valve module installed at the upper and lower parts of the chamber module are interlocked with each other to control fluid movement between the plurality of chambers.

Description

[0001] The present invention relates to a pretreatment chamber for nucleic acid extraction, a cartridge using the same, and a nucleic acid extraction method using the nucleic acid extraction method.

The present invention relates to a nucleic acid analyzing apparatus and method, and more particularly, to a pretreatment chamber for extracting nucleic acid by pretreating a sample, a cartridge using the same, and a nucleic acid extracting method.

As the life expectancy of people has increased and their interest in health has increased, the importance of genetic analysis, in vitro diagnosis, and gene sequencing has been increasing, and their demand is also increasing.

Accordingly, a platform and a system capable of performing a large amount of inspection in a short time with a small amount of samples are being released. Microfluidic device platforms using microfluidic technologies such as microfluidics chips or lab-on-a-chip have attracted attention. The microfluidic device includes a plurality of microchannels and fine chambers designed to control and manipulate a small amount of fluid. By using the microfluidic device, the reaction time of the microfluidic fluid can be minimized, and the reaction of the microfluidic fluid and the measurement of the resultant fluid can be simultaneously performed. Such a microfluidic device can be manufactured by various methods, and various materials are used according to the production method.

For example, in order to accurately determine the presence of a specific nucleic acid or the amount of nucleic acid in a sample during gene analysis, it is necessary to sufficiently amplify the actual sample so as to be measurable after purification and extraction. Of the various gene amplification methods, polymerase chain reaction (PCR) is the most widely used. The fluorescence detection method is mainly used as a method for detecting nucleic acid amplified by PCR.

The PCR process involves a process of capturing cells from a biological sample, a step of disrupting the captured cells, a step of extracting nucleic acid from the cleaved column, and a series of steps of mixing the extracted nucleic acid with a PCR reagent. On the other hand, since the sample contains various impurities besides the cells to be extracted with the nucleic acid, a purification step is required to remove the impurities contained in the sample before extracting the nucleic acid from the sample.

In the past, purification, cell trapping, cell destruction, nucleic acid extraction, and nucleic acid amplification are sequentially performed on the sample, which is time consuming and low reproducibility.

Since the apparatuses for performing these processes require chambers for performing the respective processes, the structure is complicated, and the sample may be contaminated in the process of processing the sample.

Meanwhile, the contents described in this section merely provide background information on the present embodiment and do not constitute the prior art.

Korea Patent Publication No. 2014-0095342 (2014.08.01)

Accordingly, an object of the present invention is to provide a pretreatment chamber for nucleic acid extraction, which can simplify the nucleic acid extraction process through pretreatment of a sample, a cartridge using the same, and a nucleic acid extraction method.

Another object of the present invention is to provide a pretreatment chamber for nucleic acid extraction which collectively performs sample crushing, cell destruction and purification, a cartridge using the same, and a nucleic acid extracting method.

It is still another object of the present invention to provide a pretreatment chamber for nucleic acid extraction, which performs nucleic acid extraction and nucleic acid amplification in a batch, including pretreatment of a sample to be introduced, a cartridge using the same, and a nucleic acid extraction method.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments.

In order to accomplish the above object, the present invention provides a plasma processing apparatus comprising: a chamber body having an inner space through homogenization and cell destruction by pulverization of a sample to be charged; And a cup filter installed at a lower portion of the inner space of the chamber body for filtering and passing the purified solution containing the nucleic acid flowing out from the cells by cell destruction of the sample.

Wherein the cup filter includes a filter part formed to be sloped downward to be coupled to the inside of the chamber body and to filter and pass the purified solution containing the nucleic acid flowing out from the cells by cell destruction of the sample; And a cup portion connected to the filter portion and filtered and remaining debris moving and settling.

The pretreatment chamber for nucleic acid extraction according to the present invention may further include a pretreatment member including a pretreatment liquid, a magnet block and cell destruction particles contained in an inner space of the chamber body on the cup filter.

Wherein the chamber body includes: an upper body having an injection port through which the pretreatment member and the sample are injected; And a lower body connected to a lower portion of the upper body and having an inner diameter smaller than that of the upper body and having a discharge port for discharging the purified liquid at a lower portion thereof and to which the cup filter is coupled.

The magnet block of the pretreatment member may be located inside the lower main body.

The cell-destroying particles of the pretreatment member may be a glass bead.

The pore size of the filter portion may be larger than that of the cell-destroying particles.

The cup portion of the cup filter may be located at the center of the inner space of the chamber body. The filter portion of the cup filter extends upward from the upper end of the cup portion and is coupled to the inside of the chamber body. The filter portion is formed as a funnel-shaped inclined surface, and pores for passing the purifying solution are formed.

The present invention also provides a chamber module comprising a pre-treatment chamber for homogenization, cell disruption and purification by grinding of a sample to be introduced, the plurality of chambers for extracting nucleic acid from the sample; An air valve module installed on the chamber module for opening and closing the application of pressure required for fluid movement between the plurality of chambers; And a liquid valve module installed at a lower portion of the chamber module for opening and closing fluid movement between the plurality of chambers.

The pretreatment chamber of the chamber module contains a pretreatment member including a pretreatment liquid, a magnet block and cell destruction particles, and can discharge a primary purification solution containing nucleic acid after pulverization and cell disruption to a sample to be introduced.

The chamber module includes a separation chamber for separating the nucleic acid-containing second purification liquid by performing phase separation on the first purification liquid using the first purification liquid supplied from the pre-processing chamber, ; At least one cleaning chamber for supplying a cleaning liquid necessary for cleaning the secondary refining liquid; A dissolution chamber for supplying an effluent for separating the nucleic acid; Wherein the magnetic particles selectively adsorb the nucleic acid contained in the secondary purification liquid and the at least one cleaning chamber is provided with a binding reagent and magnetic particles, wherein when the secondary purification liquid is supplied from the separation chamber, A reaction chamber for supplying a washing liquid and washing the magnetic particles adsorbed by the nucleic acid and supplying the eluent from the elution chamber to separate the nucleic acid from the magnetic particles; A nucleic acid amplification reagent is provided, and a nucleic acid-containing eluate is supplied from the reaction chamber A nucleic acid amplification reagent chamber for receiving and mixing with the nucleic acid amplification reagent to generate a nucleic acid amplification mixture; And a PCR chamber for receiving the nucleic acid amplification mixture from the nucleic acid amplification reagent chamber and performing a nucleic acid amplification reaction.

The chamber module may further include a waste chamber through which the reagent and the cleaning liquid used in the reaction chamber are discharged.

The chamber module may further include a pump for applying air pressure to the air valve module.

The chamber module may include the pre-treatment chamber, the separation chamber, the cleaning chamber, the reaction chamber, and the elution chamber around the pump.

The nucleic acid amplification reagent chamber and the nucleic acid amplification chamber may be disposed between the pretreatment chamber and the elution chamber, and may protrude outwardly from the other plurality of chambers.

The present invention also relates to a method of preparing a sample, a pretreatment liquid, a magnetic block, and a cell destruction particle; The magnetic block is intermittently applied to the pretreatment chamber to move the magnet block to homogenize the sample, and the cell destruction particles move in association with the movement of the magnet block to break down the cells contained in the sample, Sample homogenization and cell disruption step; And applying a pressure such that the pretreatment liquid passes through a cup filter disposed in a lower portion of the pretreatment chamber to filter a primary purification solution containing a nucleic acid flowing out from the cell by cell destruction of the sample Thereby providing a nucleic acid extraction method using a cartridge.

Wherein the separation chamber of the cartridge having the separation reagent, which is performed after the filtering, is supplied with the first purification liquid from the pretreatment chamber and performs phase separation on the first purification liquid using the heat, Discharging the second purification liquid; A reaction chamber of a cartridge including a binding reagent and magnetic particles is supplied with a second purification liquid from the pretreatment chamber and the magnetic particles selectively adsorb the nucleic acid contained in the second purification liquid; Washing the nucleic acid-adsorbed magnetic particles by supplying the washing liquid from the washing chamber of the cartridge to the reaction chamber; Separating the nucleic acid from the magnetic particles by receiving the eluent from the elution chamber of the cartridge; A nucleic acid amplification reagent chamber of a cartridge having a nucleic acid amplification reagent is supplied with an eluate containing nucleic acid from the reaction chamber and mixed with the nucleic acid amplification reagent to generate a nucleic acid amplification mixture; And receiving the nucleic acid amplification mixture from the nucleic acid amplification reagent chamber of the nucleic acid amplification chamber of the cartridge.

Applying a magnetic force to the reaction chamber to fix the nucleic acid-adsorbed magnetic particles, which is performed after the washing step; And discharging the cleaning liquid from the reaction chamber to the waste chamber of the cartridge.

The step of separating the nucleic acid comprises the steps of: receiving the eluent from the elution chamber; And separating the nucleic acid from the magnetic particles with the supplied eluent by blocking the magnetic force acting on the reaction chamber.

Applying a magnetic force to the reaction chamber to immobilize the nucleic acid-separated magnetic particles, which is performed after the step of separating the nucleic acid; And discharging a nucleic acid-containing eluate from the reaction chamber into the nucleic acid amplification reagent chamber.

The cartridge according to the present invention can collectively perform crushing, cell disruption, purification, nucleic acid extraction and nucleic acid amplification on a sample to be injected.

The cartridge according to the present invention provides a basis for performing nucleic acid testing in-line through nucleic acid extraction and amplification.

Since the pretreatment chamber of the cartridge according to the present invention collectively performs crushing, cell destruction and purification on the sample to be injected, the nucleic acid extracting process can be simplified through pretreatment of the sample.

The pretreatment chamber of the cartridge according to the present invention has a structure in which a cup filter is coupled to the lower part of the chamber body. The chamber body on the upper part of the cup filter is subjected to pulverization and cell destruction, Since the purification is carried out, the purification liquid containing the nucleic acid extracted from the sample can be obtained quickly.

A magnetic force is intermittently applied to the chamber body from the outside after the sample is charged into the pretreatment liquid containing the magnet block and the cell destruction particles contained in the chamber body so that the magnet block can be moved in the pretreatment liquid of the chamber body, It is possible to simultaneously perform crushing and cell destruction on the sample while moving the cell destruction particles together.

Since the filter cup includes the funnel-shaped inclined filter portion and the cup portion at the center of the filter portion, the purified liquid containing the nucleic acid extracted from the sample moves to the outlet of the chamber body through the filter portion, The water moves to the cup portion at the center of the filter portion along the inclined filter portion and is settled. This prevents the residue, which has not passed through the filter portion, from obstructing the filtering by blocking the filter portion, so that the purification can be smoothly performed.

In addition, various effects other than the above-described effects can be directly or implicitly disclosed in the detailed description according to the embodiment of the present invention to be described later.

1 is a schematic view showing a nucleic acid extracting apparatus using a cartridge for nucleic acid extraction according to an embodiment of the present invention.
FIG. 2 is a perspective view showing the cartridge for nucleic acid extraction of FIG. 1; FIG.
3 is a top view showing the air valve module of FIG.
Figures 4 and 5 are plan views showing the liquid valve module of Figure 2;
Figure 6 is a view showing the pretreatment chamber of Figure 2;
7 is a flowchart illustrating a nucleic acid extraction method using a cartridge for nucleic acid extraction according to an embodiment of the present invention.
8 is a detailed flowchart of the preprocessing step of FIG.
FIGS. 9 to 11 are views showing respective detailed steps according to the preprocessing step of FIG.
FIG. 12 is a view showing a separation chamber according to the second purification step of FIG. 7; FIG.
13 is a detailed flowchart of the third purification step of FIG.
14 is a detailed flowchart of the nucleic acid separation step of FIG.

BRIEF DESCRIPTION OF THE DRAWINGS For a more complete understanding of the nature and advantages of the present invention, reference should be made to the following detailed description taken in conjunction with the accompanying drawings, in which:

In the following description and the accompanying drawings, detailed description of well-known functions or constructions that may obscure the subject matter of the present invention will be omitted. It should be noted that the same constituent elements are denoted by the same reference numerals as possible throughout the drawings.

The terms and words used in the following description and drawings are not to be construed in an ordinary sense or a dictionary, and the inventor can properly define his or her invention as a concept of a term to be described in the best way It should be construed as meaning and concept consistent with the technical idea of the present invention. Therefore, the embodiments described in the present specification and the configurations shown in the drawings are merely the most preferred embodiments of the present invention, and not all of the technical ideas of the present invention are described. Therefore, It is to be understood that equivalents and modifications are possible.

Also, terms including ordinal numbers such as first, second, etc. are used to describe various elements, and are used only for the purpose of distinguishing one element from another, Not used. For example, without departing from the scope of the present invention, the second component may be referred to as a first component, and similarly, the first component may also be referred to as a second component.

In addition, when referring to an element as being "connected" or "connected" to another element, it means that it can be connected or connected logically or physically. In other words, it is to be understood that although an element may be directly connected or connected to another element, there may be other elements in between, or indirectly connected or connected.

Also, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise. It is also to be understood that the terms such as " comprising "or" having ", as used herein, are intended to specify the presence of stated features, integers, It should be understood that the foregoing does not preclude the presence or addition of other features, numbers, steps, operations, elements, parts, or combinations thereof.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 is a schematic view showing a nucleic acid extracting apparatus using a cartridge for nucleic acid extraction according to an embodiment of the present invention. FIG. 2 is a perspective view showing the cartridge for nucleic acid extraction of FIG. 1; FIG.

Referring to FIGS. 1 and 2, a nucleic acid extracting apparatus 100 according to the present embodiment is a molecular diagnostic point-of-care testing (POCT) apparatus using a cartridge 10 for extracting nucleic acid, Pretreatment of the sample to be added, nucleic acid extraction / purification and nucleic acid amplification are performed collectively.

The nucleic acid extracting apparatus 100 may include a cartridge 10, magnetic force applying units 73 and 75, a pump driving unit 77, and heater units 76 and 79.

The cartridge 10 is loaded with a sample and is subjected to pretreatment, cell disruption, nucleic acid extraction / purification and nucleic acid amplification in a batch, and is used in a single use. The sample is a biosample requiring pretreatment, such as stool, tissue, and sputum. Other samples may be blood, urine, saliva, semen, spinal fluid, mucus, and the like.

The cartridge 10 includes a chamber module 31, an air valve module 33, and a liquid valve module 35. The chamber module 31 includes a plurality of chambers for extracting nucleic acid from a sample, including a pretreatment chamber 40 where homogenization, cell destruction and purification are performed by grinding the sample to be injected. The air valve module 33 is installed on the upper portion of the chamber module 31 and opens and closes the application of pressure required for fluid movement between the plurality of chambers. The liquid valve module 35 is installed at a lower portion of the chamber module 31 and opens and closes fluid movement between the plurality of chambers.

At this time, the plurality of chambers of the chamber module 31 are connected to the pretreatment chamber 40, the separation chamber 51, the cleaning chamber 53, the elution chamber 57, the reaction chamber 55, the nucleic acid amplification reagent chamber 59, And an amplification chamber 61. The chamber module 31 may further include a waste chamber 58 in which used reagents and debris are discarded. The chamber module 31 may be provided at its center with a pump 37 for applying an air pressure required for driving the air valve module 33. For example, the chamber module 31 includes a pretreatment chamber 40, a separation chamber 51, a cleaning chamber 53, a reaction chamber 55, a release chamber 57, A nucleic acid amplification reagent chamber 59 and a nucleic acid amplification chamber 61 may be provided. The nucleic acid amplification reagent chamber 59 and the nucleic acid amplification chamber 61 may be disposed between the pretreatment chamber 40 and the elution chamber 57 and may protrude outward with respect to the other plurality of chambers. The waste chamber 58 may be disposed between the other chambers and the pump 37.

The pump 37 is installed in the central part of the chamber module 31 and moves up and down by the pump driving part 77 to supply necessary pressures to the plurality of chambers. The chamber module 31 is formed with a pump hole 39 at a central portion thereof so that the pump 37 can move up and down to transmit the necessary pressure to the plurality of chambers. In the case of the present embodiment, the air pressure can be transferred to the plurality of chambers by the rise of the pump 37. [

The magnetic force applying portions 73 and 75 include a first magnetic force applying portion 73 and a second magnetic force applying portion 75. [

The first magnetic force applying unit 73 is installed on the outer side of the pre-processing chamber 40 and intermittently applies a magnetic force to the pre-processing chamber 40 to move the magnet block contained in the pre-processing chamber 40 to the pretreatment chamber 40 So that the pretreatment process can be performed smoothly. The first magnetic force applying unit 73 may be installed at a plurality of positions in order to smoothly move the magnet blocks contained in the pretreatment chamber 40 in the pretreatment chamber 40. For example, the first magnetic force applying unit 73 may include a first magnetic force applying unit 73a and a second magnetic force applying unit 73b.

The second magnetic force application unit 75 is installed outside the reaction chamber 55 and applies a magnetic force to the reaction chamber 55 to fix or fix the magnetic particles contained in the reaction chamber 55 to perform cleaning and nucleic acid elution So that the process can be performed smoothly.

The heater sections 76 and 79 include a first heater section 76 and a second heater section 79.

The first heater unit 76 is installed on the outer side of the separation chamber 51 and applies heat to the separation chamber 51 so that the separation process for the first purification solution supplied from the pretreatment chamber 40 can be smoothly performed. do.

The second heater unit 79 is installed outside the nucleic acid amplification chamber 61 and applies heat to the nucleic acid amplification chamber 61 so that the nucleic acid amplification reaction can be smoothly performed.

The pump driving unit 77 drives the pump 37 to apply air pressure to the air valve module 33 of the cartridge 10. As the pump driving unit 77, a stepping motor can be used.

The cartridge 10 according to this embodiment will now be described with reference to FIGS. 1 to 6. FIG. 3 is a plan view showing the air valve module 33 of FIG. Figs. 4 and 5 are plan views showing the liquid valve module 35 of Fig. And FIG. 6 is a view showing the pretreatment chamber 40 of FIG.

The cartridge 10 includes a chamber module 31, an air valve module 33 and a liquid valve module 37, as described above.

The chamber module 31 includes a pretreatment chamber 40, a separation chamber 51, a cleansing chamber 53, a dissolution chamber 57, a reaction chamber 55, a nucleic acid amplification reagent chamber 59, a nucleic acid amplification chamber 61, And a waste chamber 58. The pretreatment chamber 40, the separation chamber 51, the cleaning chamber 53, the reaction chamber 55 and the elution chamber 57 can be sequentially arranged around the pump 37 around the pump 37. [ The waste chamber 58 may be disposed between the other chambers and the pump 37.

The pretreatment chamber 40 contains a pretreatment member 49 including a pretreatment liquid 49a, a magnet block 49b and cell destruction particles 49c, And the primary purification liquid is discharged to the separation chamber 51.

This pretreatment chamber 40 includes a chamber body 41 and a cup filter 45 and may contain a pretreatment member 49 inside the chamber body 41 above the cup filter 45. The chamber body 41 has an internal space 44 in which homogenization and cell destruction are caused by pulverization of the sample to be injected. The cup filter 45 is installed in the lower part of the inner space 44 of the chamber body 41 and filters and passes the first purification solution containing the nucleic acid flowing out from the cells by the cell destruction of the sample.

The chamber main body 41 includes an upper main body 41a and a lower main body 41b. The upper body 41a is provided at its upper portion with a pretreatment member 49 and a charging port 42 through which a sample is charged. The lower main body 41b is connected to the lower portion of the upper main body 41a and has an inner diameter smaller than that of the upper main body 41a. The lower body 41b has a discharge port 43 through which the first purified liquid is discharged, and a cup filter 45 is coupled to the inside.

The reason why the inner diameter of the upper main body 41a is formed larger than the inner diameter of the lower main body 41b is that the introduction of the sample and the pretreatment member 49 through the inlet 42 can be facilitated.

The pretreatment member 49 includes a pretreatment liquid 49a, a magnet block 49b and a cell destruction particle 49c as described above

The pretreatment liquid 49a can be used without limitation as long as it is a general pretreatment liquid used for molecular diagnostics. When the sample is put into the pretreatment liquid 49a, 0.01 to 0.1 part by weight of a sample may be added to 100 parts by weight of the pretreatment liquid 49a. If the sample is added at less than 0.01 part by weight, the yield may be too low to be inefficient. If the sample is added in an amount exceeding 0.1 part by weight, the homogenization may not be performed smoothly. Therefore, the amount of the pretreatment liquid 49a and the amount of the sample can be adjusted within the aforementioned range.

Glass beads can be used as the cell-destroying particles 49c as a non-magnetic substance. As the material of the other cell-destroying particles 49c, silica, latex, polymeric materials may be used.

The magnet block 49b and the cell-destroying particles 49c are moved in the pretreatment liquid 49a by the magnetic force applied intermittently, and the sample is pulverized and homogenized. Furthermore, the cell-destroying particles 49c move in association with the movement of the magnet block 49b, thereby destroying the cells contained in the sample and allowing the nucleic acid to flow out.

Particles larger than the pores formed in the cup filter 45 are used to prevent the cell-destroying particles 49c from passing through the cup filter 45 in the first purification process. For example, the particle size of the cell-destroying particles 49c may be 50 占 퐉 or more.

The magnet block 49b may have a size that can be located inside the lower main body 41b.

The pretreatment member 49 is contained in the inner space 44 of the chamber body 41 on the cup filter 45. The cup filter 45 includes a filter portion 46 and a cup portion 47. The filter unit 46 is formed so that a portion of the filter unit 46 coupled to the inside of the chamber body 41 is inclined downward to filter and pass the first purification solution containing the nucleic acid flowing out from the cells by the cell destruction of the sample. The cup portion 47 is connected to the filter portion 46, and the filtered and remaining debris is moved and settled.

At this time, the cup portion 47 may be located at the center of the inner space 44 of the chamber body 41. The filter portion 46 extends upward from the upper end of the cup portion 47 and is coupled to the interior of the chamber body 41. The filter portion 46 is formed as a funnel-shaped inclined surface, and pores for passing the primary purification liquid are formed.

The debris that has not passed through the filter portion 46 moves into the cup portion 47 on the inclined surface of the filter portion 46 and is settled as the filter portion 46 is formed as an inclined surface toward the cup portion 47. Therefore, in the course of the primary purification, debris that has not passed through the filter unit 46 can block the pores of the filter unit 46, thereby preventing delay or blocking of the first purification.

On the other hand, although it is desirable to filter only the nucleic acid through the first purification, some debris passing through the filter unit 46 is also present. Therefore, the first purification solution contains nucleic acid, pretreatment solution 49a and debris.

The separation chamber 51 receives the first purification liquid from the pretreatment chamber 40 and performs secondary purification using heat. This separation chamber 51 is disposed adjacent to the pretreatment chamber 40 and contains a separation reagent. The separation chamber 51 receives the first purified liquid from the pretreatment chamber 40 and performs phase separation on the first purified liquid using the heat applied from the first heater unit 76. The separation chamber 51 discharges the nucleic acid-containing secondary purification liquid to the reaction chamber 55 by phase separation.

The separation reagent coagulates the protein when heat is applied. Therefore, when heat is applied after the first purification liquid is supplied to the separation chamber 51 containing the separation reagent, the debris containing the protein component flocculates and floats up, and the second purification solution is located below.

The separation chamber 51 discharges a part of the secondary purification liquid located below the agglomerated debris to the reaction chamber 55.

The cleaning chamber 53 is disposed between the separation chamber 51 and the reaction chamber 55 and contains the cleaning liquid necessary for cleaning the secondary purification liquid and supplies the cleaning liquid to the reaction chamber 55 to perform the third purification . For example, the cleaning chamber 53 may include a first cleaning chamber 53a containing the first cleaning liquid and a second cleaning chamber 53b containing the second cleaning liquid. The first cleaning liquid may include ethanol and water. The second cleaning liquid may be ethanol.

The third purification may be carried out by a first cleaning with the first cleaning liquid and a second cleaning with the second cleaning liquid. The reason for carrying out the cleaning in plural steps is to remove the remaining debris or reagent leaving only the nucleic acid in the second purification solution.

The reason why water is used together with ethanol in the first cleaning liquid is as follows. When the second purification liquid is supplied to the reaction chamber 55, the nucleic acid contained in the second purification liquid is adsorbed to the magnetic particles contained in the reaction chamber 55. In the course of nucleic acid adsorption, debris may be adsorbed to the magnetic particles along with the nucleic acid, or a nucleic acid to which the adsorption strength is weakly adsorbed may be present. Water has the property of separating the substance adsorbed by the magnetic particles from the magnetic particles. Therefore, by adding some water together with ethanol as the first washing liquid, it is possible to separate debris adsorbed on the magnetic particles or nucleic acids adsorbed weakly on the adsorption strength.

Then, the nucleic acid can be separated from the second purification solution by washing with the first washing solution and then with the second washing solution. The separated nucleic acid is adsorbed to the magnetic particles.

The elution chamber 57 is disposed between the reaction chamber 55 and the pretreatment chamber 40 and contains the eluent. The elution chamber 57 supplies the elution solution to the reaction chamber 55. Water may be used as the eluent. The eluent separates the nucleic acid adsorbed on the magnetic particles.

The reaction chamber 55 is disposed between the cleaning chamber 53 and the elution chamber 57, and performs third purification and nucleic acid separation (extraction). The reaction chamber 55 contains a binding reagent and magnetic particles.

First, the reaction chamber 55 performs secondary purification on the secondary purification liquid supplied from the separation chamber 51. That is, when the second purification liquid is supplied from the separation chamber 51 to the reaction chamber 55, the magnetic particles selectively adsorb the nucleic acid contained in the second purification liquid. The reaction chamber 55 discharges the binding reagent and the secondary purification liquid to the waste chamber 58 except for the magnetic particles to which the nucleic acid is adsorbed. The reaction chamber 55 receives the cleaning liquid from the cleaning chamber 53, cleans the nucleic acid-adsorbed magnetic particles, and discharges the waste particles to the waste chamber 58. At this time, before discharging the solution in the reaction chamber 55 to the waste chamber 58, the second magnetic force applying unit 75 applies a magnetic force to the reaction chamber 55 to fix the nucleic acid-adsorbed magnetic particles.

The reaction chamber 55 receives the eluent from the elution chamber 57, separates the nucleic acid from the magnetic particles, and discharges the eluted solution containing the nucleic acid to the nucleic acid amplification reagent chamber 59. The second magnetic force applying unit 75 applies a magnetic force to the reaction chamber 55 to fix the nucleic acid separated magnetic particles before discharging the eluate containing the nucleic acid to the nucleic acid amplification reagent chamber 59.

The nucleic acid amplification reagent chamber 59 contains a nucleic acid amplification reagent. The nucleic acid amplification reagent chamber 59 receives the nucleic acid-containing eluate from the reaction chamber 55 and mixes with the nucleic acid amplification reagent to generate a nucleic acid amplification mixture. The nucleic acid amplification reagent chamber 59 discharges the resulting nucleic acid amplification mixture to the nucleic acid amplification chamber 61. The nucleic acid amplification reagent may be provided in the nucleic acid amplification reagent chamber 59 in a lyophilized form. A plurality of nucleic acid amplification reagent chambers 59 may be provided. For example, the nucleic acid amplification reagent chamber 59 may include first to fourth nucleic acid amplification reagent chambers 59a, 59b, 59c and 59d.

The nucleic acid amplification chamber 61 receives the nucleic acid amplification mixture from the nucleic acid amplification reagent chamber 59 and then performs nucleic acid amplification reaction using the heat applied from the second heater unit 79. A plurality of nucleic acid amplification chambers 61 may be provided. A plurality of nucleic acid amplification chambers 61 form a nucleic acid amplification module 63. For example, the nucleic acid amplification chamber 61 may include first to fourth nucleic acid amplification chambers 61a, 61b, 61c and 61d.

The air valve module 33 opens and closes the application of air pressure required for fluid movement between the plurality of chambers, as shown in Fig. The air valve module 33 includes a plurality of valves 1 to 12 for opening and closing the application of air pressure and an air flow passage for connecting the plurality of valves 1 to 12 and the pump 37. At this time, the plurality of valves 1 to 12 may be electromagnet valves.

The air valve module 33 may include first to twelfth valves 1-12.

The first valve (1) opens and closes the application of air pressure to the pretreatment chamber (40).

The second and eighth valves (2, 8) open and close the application of air pressure to the separation chamber (51).

The third and ninth valves 3 and 9 open and close the application of the air pressure to the first cleaning chamber 53a.

The fourth and tenth valves 4 and 10 open and close the application of air pressure to the second cleaning chamber 53b.

The fifth and eleventh valves 5 and 11 open and close the application of air pressure to the reaction chamber 55.

The sixth and twelfth valves (6, 12) open and close the application of air pressure to the elution chamber (57).

The seventh valve 7 opens and closes the application of air pressure to the nucleic acid amplification reagent chamber 59.

The liquid valve module 35 opens and closes fluid movement between the plurality of chambers, as shown in Figs. The liquid valve module 35 includes a plurality of valves 13 to 28 for opening and closing fluid movement between a plurality of chambers and a liquid flow path for guiding fluid movement between the plurality of chambers. At this time, the plurality of valves 13 to 28 may be electromagnet valves.

The liquid valve module 35 may include the thirteenth through twenty-eighth valves 13-28.

The thirteenth valve (13) is installed in the pretreatment chamber (40).

The fourteenth valve (14) is installed in the separation chamber (51).

The fifteenth valve 15 is installed in the first cleaning chamber 53a.

The sixteenth valve 16 is installed in the second cleaning chamber 53b.

The seventeenth valve 17 is installed in the fluid channel between the reaction chamber 55 and the waste chamber 58.

The eighteenth valve 18 is installed in the reaction chamber 55.

The nineteenth valve (19) is installed in a liquid flow path between the reaction chamber (55) and the nucleic acid amplification reagent chamber (59).

The twentieth valve (20) is installed in the elution chamber (57).

And the twenty-first to twenty-eighth valves 21 to 28 are installed in a plurality of nucleic acid amplification reagent chambers 59a, 59b, 59c and 59d. At this time, the number of the nucleic acid amplification reagent chambers 59 is four, and two valves 21 to 28 are connected to each other.

Fluid movement between the plurality of chambers in accordance with the nucleic acid extraction process is achieved by interlocking the air valve module 33 and the liquid valve module 35. A detailed description will be given in the nucleic acid extraction method.

The nucleic acid extraction method using the cartridge 10 for nucleic acid extraction according to this embodiment will be described with reference to FIGS. 7 to 14. FIG.

7 is a flowchart illustrating a nucleic acid extraction method using a cartridge for nucleic acid extraction according to an embodiment of the present invention.

The nucleic acid extraction method according to this embodiment includes a pretreatment step (S10) including crushing, cell destruction and first purification of a sample, a second purification step (S20) using heat-induced phase separation, a third purification step using a cleaning liquid and magnetic particles A nucleic acid amplification mixture generation step (S60), a nucleic acid amplification chamber injection step (S70), and a nucleic acid amplification reaction step (S80). The nucleic acid amplification reaction step (S30), the nucleic acid separation step (S50) using the eluent and the magnetic particles,

[Pretreatment including primary purification]

First, in step S10, when a sample is introduced into the pretreatment chamber, pretreatment steps including crushing, cell destruction, and primary purification of the sample are collectively performed in the pretreatment chamber, and the first refining liquid is discharged to the separation chamber.

The preprocessing step according to step S10 will be described with reference to FIGS. 8 to 11. FIG. 8 is a detailed flowchart of the preprocessing step of FIG. FIGS. 9 to 11 are views showing respective detailed steps according to the preprocessing step of FIG.

9, in step S11, a pretreatment chamber 40 containing a sample 81 and a pretreatment member 49 is prepared. The sample 81 may be introduced after the pretreatment member 49 is immersed in the pretreatment chamber 40, for example. Or the pretreatment member 49 may be introduced after the sample 81 is introduced into the pretreatment chamber 40. Alternatively, the sample 81 and the pretreatment member 49 may be simultaneously introduced into the pretreatment chamber 40.

Next, as shown in FIG. 10, homogenization and cell destruction are performed on the sample in step S13. In other words, the first-first magnetic force application unit 73a and the second-magnetic force application unit 73b intermittently apply magnetic force to the pretreatment chamber 40 to move the magnet block 49b to homogenize the sample. The magnet block 49b is moved in the pretreatment liquid 49a by switching the magnetic force applied to the first magnetic force applying portion 73a and the first magnetic force applying portion 73b. In addition, the cell-destroying particles 49c move in association with the movement of the magnet block 49b to break down the cells contained in the sample so that the nucleic acid flows out.

On the other hand, additional heat may be applied so that homogenization and cell destruction of the sample can be performed more quickly.

Then, as shown in FIG. 11, the primary purification liquid 83 is filtered and discharged to the separation chamber in step S15. Pressure is applied through the pump 37 so that the pretreatment liquid passes through the cup filter 45 installed in the lower part of the pretreatment chamber 40 to remove the primary purification solution containing the nucleic acid flowing out from the cells by cell destruction of the sample (83).

The debris which has not passed through the filter portion 46 of the cup filter 45 travels on the inclined surface of the filter portion 46 to the cup portion 47 and is settled. Reference numeral 85 denotes a sediment formed of debris moved to the cup portion 47.

Hereinafter, the operation of the air valve module 33 and the liquid valve module 35 for moving the first refining liquid 83 from the pretreatment chamber 40 to the separation chamber will be described with reference to FIG. 3 and FIG.

The first valve 1, the eighth valve 8 and the thirteenth valve 13 are sequentially opened. Next, the pump 37 is operated to apply pressure to the pretreatment chamber 40 to move the first refining liquid to the separation chamber 51. When the movement of the first purification liquid is completed, the first valve 1, the eighth valve 8 and the thirteenth valve 13 are sequentially closed. Then, the operation of the pump 37 is turned off and the vent is performed.

[Secondary purification]

Next, as shown in FIG. 12, when the first purified liquid pretreated in step S20 is introduced into the separation chamber 51, second purification using heat is performed in the separation chamber 51, The liquid 86 is discharged to the reaction chamber. 12 is a view showing the separation chamber 51 according to the second purification step of FIG.

At this time, the first heater unit 76 can apply heat of 50 to 80 캜 to the separation chamber 51 for 3 to 30 minutes. The debris contained in the primary purification liquid is flocculated and floated up in the form of a float 87 by the heat applied to the separation chamber 51 and the relatively clean secondary purification liquid 86 is located below the float 87 do.

Here, the operation of the air valve module 33 and the liquid valve module 35 for moving the secondary refill liquid from the separation chamber 51 to the reaction chamber will be described with reference to FIGS. 3 to 5 as follows.

The second valve 2, the eighteenth valve 18, the eleventh valve 11 and the fourteenth valve 14 are sequentially opened. Next, the pump 37 is operated to apply pressure to the separation chamber 51 to transfer the second purification liquid to the reaction chamber 55. Next, the second valve 2, the eighteenth valve 18 and the eleventh valve 11 are sequentially closed. The second valve 2, the fourteenth valve 14 and the seventeenth valve 17 are opened to separate the two remaining liquids from the separation chamber 51 and the liquid chamber connecting the separation chamber 51 and the reaction chamber 55 And the tea refining liquid is discharged into the waste chamber 58. Next, the operation of the pump 37 is turned off and the vent is performed. Then, the second valve (2), the fourteenth valve (14) and the seventeenth valve (17) are closed.

[Third purification]

Next, in step S30, when the secondary purification liquid is introduced into the reaction chamber, the tertiary purification using the cleaning liquid and the magnetic particles is performed in the reaction chamber. The cleaning according to the third purification may be performed a plurality of times. In this embodiment, an example of carrying out twice cleaning is disclosed.

The third purification according to step S30 will be described with reference to FIG. 13 is a detailed flowchart of the third purification step of FIG.

In step S31, the secondary purification liquid is supplied to the reaction chamber from the separation chamber.

Next, in step S33, the magnetic particles contained in the reaction chamber selectively adsorb the nucleic acid contained in the second purification solution. The magnetic force can be switched and applied to the reaction chamber so that the magnetic particles can more effectively adsorb the nucleic acid. After the step of adsorbing the nucleic acid on the magnetic particles, a magnetic force is applied to the reaction chamber to fix the magnetic particles on which the nucleic acid is adsorbed. And the remaining solution except for the magnetic particles adsorbed by the nucleic acid can be discharged from the reaction chamber to the waste chamber. The application of the magnetic force to the reaction chamber is performed by the second magnetic force application unit.

In steps S35 to S45, the reaction chamber receives the cleaning liquid from the cleaning chamber, cleans the nucleic acid-adsorbed magnetic particles, and discharges the cleaning liquid to the waste chamber. In this embodiment, an example of carrying out twice cleaning is disclosed.

That is, in step S35, the first cleaning liquid is introduced into the reaction chamber, and then the first cleaning is performed by switching the magnetic force. Next, in step S37, a magnetic force is applied to the reaction chamber to fix the magnetic particles on which the first washed nucleic acid is adsorbed. In step S39, the first cleaning is completed by discharging the cleaning solution firstly washed in the reaction chamber to the waste chamber.

Next, in step S41, the second cleaning liquid is introduced into the reaction chamber, and then the second cleaning is performed by switching the magnetic force. Next, in step S43, a magnetic force is applied to the reaction chamber to fix the magnetic particles on which the second washed nucleic acid is adsorbed. Then, in step S39, the second cleaning is completed by discharging the cleaning solution secondarily washed in the reaction chamber to the waste chamber.

The operation of the air valve module 33 and the liquid valve module 35 for adsorbing nucleic acid by magnetic particles in the reaction chamber 55 according to step S35 will now be described with reference to FIGS.

A magnetic force is first applied to the reaction chamber 55 through the second magnetic force application unit so that the nucleic acid contained in the second purification solution is adsorbed on the magnetic particles. The magnetic particles are adsorbed by the switching magnetic force while moving in the mixed solution of the second purification solution and the binding reagent.

Next, when nucleic acid is adsorbed to the magnetic particles, a magnetic force is applied to the reaction chamber 55 to fix the magnetic particles.

Next, the fifth valve 5, the eighteenth valve 18 and the seventeenth valve 17 are opened and then the pump 37 is operated to discharge the solution remaining in the reaction chamber 55 to the waste chamber 58 do.

The seventeenth valve 17 is then closed and the pump 37 is turned off.

Then, the fifth valve 5 and the eighteenth valve 18 are sequentially closed to complete step S35.

The operation of the air valve module 33 and the liquid valve module 37 in the first cleaning step according to the steps S37 to S39 will be described with reference to FIGS. 3 to 5 as follows.

First, the magnetic particles to which the nucleic acid is adsorbed are fixed by the magnetic force applied by the second magnetic force application unit.

The third valve 3, the eighteenth valve 18, the eleventh valve 11, and the fifteenth valve 15 are sequentially opened. Next, the pump 37 is driven to supply the first cleaning liquid in the first cleaning chamber 53a to the reaction chamber 55. Then,

Next, the fifteenth valve 15, the eleventh valve 11, the eighteenth valve 18 and the third valve 3 are sequentially closed. Subsequently, the operation of the pump 37 is turned off and the vent is performed.

Next, the magnetic force is switched and applied to the reaction chamber 55 through the second magnetic force applying unit to clean the magnetic particles adsorbed by the nucleic acid. The magnetic particles which have been adsorbed by the nucleic acid are firstly cleaned while the magnetic particles move in the first cleaning liquid by the switching magnetic force.

Next, when the nucleic acid-adsorbed magnetic particles are first cleaned, a magnetic force is applied to the reaction chamber 55 to fix the magnetic particles.

Next, the fifth valve 5, the eighteenth valve 18 and the seventeenth valve 17 are opened and then the pump 37 is operated to move the first cleaning liquid remaining in the reaction chamber 55 to the waste chamber 58, .

The seventeenth valve 17 is then closed and the pump 37 is turned off.

Then, by sequentially closing the fifth valve (5) and the eighteenth valve (18), the primary cleaning is completed.

The operation of the air valve module 33 and the liquid valve module 35 in the second cleaning step according to the steps S41 to S45 will be described with reference to FIG. 3 to FIG. 5 as follows. The secondary cleaning is performed in the same manner as the primary cleaning. In the case of the second cleaning, there is a difference in that the second cleaning liquid is supplied to the reaction chamber 55 in the second cleaning chamber 53b to perform secondary cleaning.

First, the magnetic particles adsorbed by the nucleic acid by the first washing are fixed by the magnetic force applied by the second magnetic force applying unit.

The fourth valve 4, the eighteenth valve 18, the eleventh valve 11 and the sixteenth valve 16 are sequentially opened. Next, the pump 37 is driven to supply the second cleaning liquid in the second cleaning chamber 53b to the reaction chamber 55. Then,

Next, the sixteenth valve 16, the eleventh valve 11, the eighteenth valve 18 and the fourth valve 4 are sequentially closed. Subsequently, the operation of the pump 37 is turned off and the vent is performed.

Next, the magnetic force is switched and applied to the reaction chamber 55 through the second magnetic force applying unit to clean the magnetic particles adsorbed by the nucleic acid. The magnetic particles are moved in the second cleaning liquid by the switching magnetic force, and the magnetic particles adsorbed by the nucleic acid are secondly cleaned.

Next, when the nucleic acid-adsorbed magnetic particles are subjected to secondary washing, a magnetic force is applied to the reaction chamber 55 to fix the magnetic particles.

Next, the fifth valve 5, the eighteenth valve 18 and the seventeenth valve 17 are opened, and then the pump 37 is operated to transfer the second cleaning liquid remaining in the reaction chamber 55 to the waste chamber 58, .

The seventeenth valve 17 is then closed and the pump 37 is turned off.

Then, the fifth valve (5) and the eighteenth valve (18) are sequentially closed to complete the secondary cleaning.

[Nucleic Acid Separation]

Next, nucleic acid separation using an eluent and magnetic particles is performed in a reaction chamber in step S50. The eluted solution containing the separated nucleic acid is discharged from the reaction chamber 55 to the nucleic acid amplification reagent chamber 59.

The nucleic acid separation step in step S50 will be described with reference to FIG. Here, FIG. 14 is a detailed flowchart of the nucleic acid separation step of FIG.

First, in step S51, the effluent is supplied from the elution chamber 57 to the reaction chamber 55.

Subsequently, in step S53, a magnetic force is switched and applied to the reaction chamber 55 through the second magnetic force applying unit 75 to separate the nucleic acid from the magnetic particles adsorbed by the nucleic acid. The magnetic particles are separated from the magnetic particles while the magnetic particles move in the elution liquid by the switching magnetic force.

When the nucleic acid is separated from the magnetic particles, a magnetic force is applied to the reaction chamber 55 in step S55 to fix the magnetic particles. At this time, the nucleic acid separated from the magnetic particles is distributed in the eluent.

Here, the operation of the air valve module 33 and the liquid valve module 35 for nucleic acid separation according to the step S50 will be described with reference to FIG. 3 to FIG. 5 as follows.

First, the magnetic particles on which the nucleic acid is adsorbed by the secondary cleaning are fixed by the magnetic force applied by the second magnetic force applying portion.

Next, the sixth valve 6, the eighteenth valve 18, the eleventh valve 11 and the twentieth valve 20 are sequentially opened.

Next, the pump 37 is operated to supply the effluent from the elution chamber 57 to the reaction chamber 55.

Next, the twentieth valve 20, the eleventh valve 11, the eighteenth valve 18 and the sixth valve 6 are sequentially closed. Subsequently, the operation of the pump 37 is turned off and the vent is performed.

Subsequently, the magnetic force is switched to the reaction chamber 55 to separate the nucleic acid from the magnetic particles into the eluent.

When the nucleic acid is separated, a magnetic force is applied to the reaction chamber 55 to fix the magnetic particles.

[Generation of nucleic acid amplification mixture]

Next, in step S60, an eluate containing nucleic acid is injected into the nucleic acid amplification reagent chamber 59 from the reaction chamber 55, and mixed with the nucleic acid amplification reagent in the nucleic acid amplification reagent chamber 59 to generate a nucleic acid amplification mixture.

Here, the operation of the air valve module 33 and the liquid valve module 35 for generating the nucleic acid amplification mixture according to the step S60 will be described with reference to FIG. 3 to FIG. 5 as follows.

First, the magnetic particles separated from the nucleic acid are fixed to the reaction chamber 55 by the magnetic force applied by the second magnetic force applying unit.

Next, the fifth valve (5), the eighteenth valve (18), and the nineteenth valve (19) are sequentially opened. And then the 25 th to 28 th valves 25, 26, 27, 28 are opened.

The nucleic acid-containing eluate is sequentially passed through the first to fourth nucleic acid amplification reagent chambers 59a, 59b, 59c and 59d while sequentially turning on and off the 21st to 24th valves 21, 22, To produce a nucleic acid amplification mixture.

Namely, after the 24th valve 24 is opened, the pump 37 is operated to supply the nucleic acid-containing eluate to the first nucleic acid amplification reagent chamber 59a. When the nucleic acid-containing effluent is filled in the first nucleic acid amplification reagent chamber 59a, the operation of the pump 37 is turned off and the 24th valve 24 and the 28th valve 28 are closed. Whether or not the eluate containing the nucleic acid into the first nucleic acid amplification reagent chamber 59a is charged can be detected using an infrared sensor.

Next, after the 23rd valve 23 is opened, the pump 37 is operated to supply the nucleic acid-containing eluate to the second nucleic acid amplification reagent chamber 59b. When the second nucleic acid amplification reagent chamber 59b is filled with the nucleic acid-containing effluent, the operation of the pump 37 is turned off and the 23rd valve 23 and the 27th valve 27 are closed.

Next, after the 22nd valve 22 is opened, the pump 37 is operated to supply the nucleic acid-containing eluate to the third nucleic acid amplification reagent chamber 59c. When the third nucleic acid amplification reagent chamber 59c is filled with the nucleic acid-containing eluent, the operation of the pump 37 is turned off and the twenty-second valve 22 and the twenty-sixth valve 26 are closed.

Then, after opening the twenty-first valve 21, the pump 37 is operated to supply the nucleic acid-containing eluate to the fourth nucleic acid amplification reagent chamber 59d. When the nucleic acid-containing effluent is filled in the fourth nucleic acid amplification reagent chamber 59d, the operation of the pump 37 is turned off and the twenty-first valve 21 and the twenty-fifth valve 25 are closed.

The nucleic acid supplied to the first to fourth nucleic acid amplification reagent chambers 59a, 59b, 59c and 59d is mixed with a nucleic acid amplification reagent to form a nucleic acid amplification mixture.

[Nucleic acid amplification chamber injection]

Next, in step S70, the nucleic acid amplification chamber 61 receives the nucleic acid amplification mixture from the nucleic acid amplification reagent chamber 59. [

The operation of the air valve module 33 and the liquid valve module 35 for injecting the nucleic acid amplification mixture into the nucleic acid amplification chamber 61 according to the step S70 will now be described with reference to FIGS.

First, the seventh valve (7) is opened and then the pump (37) is driven.

Next, the 24th valve (24) and the 28th valve (28) are opened to supply and charge the nucleic acid amplification mixture of the first nucleic acid amplification reagent chamber (59a) to the first nucleic acid amplification chamber (61a). At this time, whether or not the nucleic acid amplification mixture is charged into the first nucleic acid amplification chamber 61a can be detected using an infrared sensor.

In the same manner, the nucleic acid amplification mixture of the second to fourth nucleic acid amplification reagent chambers 59b, 59c and 59d is supplied to and charged in the second to fourth nucleic acid amplification chambers 61b, 61c and 61d.

The 23rd valve 23 and the 27th valve 27 are opened to supply and supply the nucleic acid amplification mixture of the second nucleic acid amplification reagent chamber 59b to the second nucleic acid amplification chamber 61b.

Next, the 22nd valve (22) and the 26th valve (26) are opened to supply and charge the nucleic acid amplification mixture of the third nucleic acid amplification reagent chamber (59c) to the third nucleic acid amplification chamber (61c).

The 21st valve 21 and the 25th valve 25 are opened to supply and supply the nucleic acid amplification mixture of the fourth nucleic acid amplification reagent chamber 59d to the fourth nucleic acid amplification chamber 61d.

By turning off the operation of the pump 37, the nucleic acid amplification module 63 in which the nucleic acid amplification mixture is filled in the first to fourth nucleic acid amplification chambers 61a, 61b, 61c and 61d can be obtained.

[Nucleic acid amplification reaction]

In step S80, the nucleic acid amplification reaction is performed using the heat applied to the nucleic acid amplification chamber 61. The heat is applied to the second heater portion nucleic acid amplification chamber 61.

While the specification contains a number of specific implementation details, it should be understood that they are not to be construed as limitations on the scope of any invention or claim, but rather on the description of features that may be specific to a particular embodiment of a particular invention Should be understood. Certain features described herein in the context of separate embodiments may be implemented in combination in a single embodiment. Conversely, various features described in the context of a single embodiment may also be implemented in multiple embodiments, either individually or in any suitable subcombination. Further, although the features may operate in a particular combination and may be initially described as so claimed, one or more features from the claimed combination may in some cases be excluded from the combination, Or a variant of a subcombination.

Likewise, although the operations are depicted in the drawings in a particular order, it should be understood that such operations must be performed in that particular order or sequential order shown to achieve the desired result, or that all illustrated operations should be performed.

The present invention relates to a pretreatment chamber for nucleic acid extraction, a cartridge using the same, and a nucleic acid extraction method, and is used in a molecular diagnostic field inspection apparatus. The cartridge includes a pretreatment chamber, and performs batching, crushing, cell disruption, purification, nucleic acid extraction, and nucleic acid amplification on the sample to be injected.

Since the pretreatment chamber of the cartridge collectively performs crushing, cell disruption and purification on the sample to be injected, there is an advantage that the nucleic acid extraction process can be simplified through the pretreatment of the sample.

The cartridge according to the present invention provides a basis for performing nucleic acid testing in-line through nucleic acid extraction and amplification.

In addition, since the present invention is not only possible to be marketed or operated, but also can be practically and practically carried out, it is industrially applicable.

10: cartridge 31: chamber module 33: air valve module
35: liquid valve module 37: pump 39: pump hole
40: pretreatment chamber 41: chamber body 41a: upper body
41b: lower body 42: inlet 43: outlet
44: inner space 45: cup filter 46: filter part
47: cup portion 49: sample pretreatment member 49a: pretreatment liquid
49b: magnet block 49c: cell-destroying particle 51: separation chamber
53: cleaning chamber 53a: first cleaning chamber 53b: second cleaning chamber
55: reaction chamber 57: elution chamber 58: waste chamber
59: nucleic acid amplification reagent chamber 61: nucleic acid amplification chamber 63: nucleic acid amplification chip
73: First magnetic force applying section 73a: 1st magnetic force applying section
73b: 1-2 magnetic force application part 75: second magnetic force application part
76: first heater part 77: pump driving part 79: second heater part
81: Sample 83: Primary purification liquid 85: Precipitate
86: Second refinement liquid 87: Float
100: nucleic acid extraction device

Claims (20)

A chamber body having an inner space through which homogenization and cell destruction are performed by pulverization on a sample to be charged; And
A cup filter installed at a lower portion of the inner space of the chamber body for filtering and passing a purified solution containing nucleic acid flowing out from cells by cell destruction of the sample;
Wherein the nucleic acid extracting chamber comprises: [2] The apparatus of claim 1,
A filter unit formed in the chamber body to be coupled to the inside of the chamber so as to be inclined downward to filter and pass the purifying solution containing the nucleic acid flowing out from the cells by cell destruction of the sample; And
A cup portion connected to the filter portion and filtered and remaining debris is moved and settled;
Wherein the nucleic acid extracting chamber comprises: 3. The method of claim 2,
A pretreatment member including a pretreatment liquid, a magnet block, and cell destruction particles contained in an inner space of the chamber body on the cup filter;
Further comprising a pre-treatment chamber for nucleic acid extraction. 4. The apparatus according to claim 3,
An upper body having an inlet for receiving the pretreatment member and a sample; And
A lower body connected to a lower portion of the upper body and having an inner diameter smaller than that of the upper body and having a discharge port for discharging the purified liquid at a lower portion thereof,
Wherein the nucleic acid extracting chamber comprises: 5. The method of claim 4,
Wherein the magnet block of the pretreatment member is located inside the lower main body. The method of claim 3,
Wherein the cell destruction particles of the pretreatment member are glass beads. The method of claim 3,
Wherein the pore size of the filter portion is larger than the cell destruction particle size. 4. The apparatus of claim 3, wherein the cup filter
The cup portion being located at the center of the inner space of the chamber body,
Wherein the filter portion extends upward from an upper end of the cup portion and is coupled to the inside of the chamber body, the filter portion being formed as an inclined surface in the shape of a funnel, and having pores passing through the purifying solution. A chamber module including a plurality of chambers for extracting nucleic acid from the sample, including a pretreatment chamber for homogenization, cell disruption and purification by grinding of the sample to be added;
An air valve module installed on the chamber module for opening and closing the application of pressure required for fluid movement between the plurality of chambers; And
A liquid valve module installed at a lower portion of the chamber module for opening and closing fluid movement between the plurality of chambers;
Wherein the nucleic acid extracting cartridge further comprises: 10. The apparatus of claim 9, wherein the pre-
A cartridge for nucleic acid extraction containing a pretreatment liquid comprising a pretreatment liquid, a magnetic block and cell destruction particles, and discharging a first purification liquid containing nucleic acid after pulverization and cell destruction of a sample to be introduced. 11. The apparatus of claim 10,
A separation chamber provided with a separation reagent, which receives a first purification solution from the pretreatment chamber and performs phase separation on the first purification solution using heat to discharge a second purification solution containing the nucleic acid;
At least one cleaning chamber for supplying a cleaning liquid necessary for cleaning the secondary refining liquid;
A dissolution chamber for supplying an effluent for separating the nucleic acid;
Wherein the magnetic particles selectively adsorb the nucleic acid contained in the secondary purification liquid and the at least one cleaning chamber is provided with a binding reagent and magnetic particles, wherein when the secondary purification liquid is supplied from the separation chamber, A reaction chamber for supplying a washing liquid and washing the magnetic particles adsorbed by the nucleic acid and supplying the eluent from the elution chamber to separate the nucleic acid from the magnetic particles;
A nucleic acid amplification reagent chamber provided with a nucleic acid amplification reagent and supplied with an eluate containing nucleic acid from the reaction chamber and mixing with the nucleic acid amplification reagent to generate a nucleic acid amplification mixture; And
A nucleic acid amplification chamber for receiving the nucleic acid amplification mixture from the nucleic acid amplification reagent chamber and performing a nucleic acid amplification reaction;
Wherein the nucleic acid extracting cartridge further comprises: 12. The apparatus of claim 11, wherein the chamber module comprises:
A waste chamber through which reagents and cleaning liquid used in the reaction chamber are discharged;
Wherein the nucleic acid extracting cartridge further comprises: 13. The apparatus of claim 12, wherein the chamber module comprises:
A pump for applying air pressure to the air valve module;
Wherein the nucleic acid extracting cartridge further comprises: 14. The apparatus of claim 13, wherein the chamber module comprises:
Wherein the pretreatment chamber, the separation chamber, the cleaning chamber, the reaction chamber, and the elution chamber are disposed around the pump. 15. The method of claim 14,
Wherein the nucleic acid amplification reagent chamber and the nucleic acid amplification chamber are disposed between the pretreatment chamber and the elution chamber, and protrude outward with respect to the other plurality of chambers. Preparing a pretreatment chamber in the cartridge, the pretreatment chamber containing a sample, a pretreatment liquid, a magnet block, and a cell destruction particle;
The magnetic block is intermittently applied to the pretreatment chamber to move the magnet block to homogenize the sample, and the cell destruction particles move in association with the movement of the magnet block to break down the cells contained in the sample, Sample homogenization and cell disruption step; And
Applying a pressure such that the pretreatment liquid passes through a cup filter embedded in a lower portion of the pretreatment chamber to filter a primary purification solution containing a nucleic acid flowing out from cells by cell destruction of the sample;
Wherein the nucleic acid extracting method comprises the steps of: 17. The method of claim 16, further comprising:
The separation chamber of the cartridge having the separation reagent is supplied with the first purification solution from the pretreatment chamber and performing phase separation on the first purification solution using heat to discharge the second purification solution containing the nucleic acid;
A reaction chamber of a cartridge including a binding reagent and magnetic particles is supplied with a second purification liquid from the pretreatment chamber and the magnetic particles selectively adsorb the nucleic acid contained in the second purification liquid;
Washing the nucleic acid-adsorbed magnetic particles by supplying the washing liquid from the washing chamber of the cartridge to the reaction chamber;
Separating the nucleic acid from the magnetic particles by receiving the eluent from the elution chamber of the cartridge;
A nucleic acid amplification reagent chamber of a cartridge having a nucleic acid amplification reagent is supplied with an eluate containing nucleic acid from the reaction chamber and mixed with the nucleic acid amplification reagent to generate a nucleic acid amplification mixture; And
The nucleic acid amplification chamber of the cartridge being supplied with the nucleic acid amplification mixture from the nucleic acid amplification reagent chamber;
Wherein the nucleic acid extracting method further comprises: 18. The method of claim 17, further comprising:
Applying magnetic force to the reaction chamber to immobilize the magnetic particles on which the nucleic acid is adsorbed; And
Discharging the cleaning liquid from the reaction chamber to the waste chamber of the cartridge;
Wherein the nucleic acid extracting method further comprises: 19. The method of claim 18, wherein separating the nucleic acid comprises:
Receiving the eluent from the elution chamber by the reaction chamber; And
Blocking the magnetic force acting on the reaction chamber and separating the nucleic acid from the magnetic particles with the supplied eluent;
Wherein the nucleic acid extracting method comprises the steps of: 20. The method of claim 19, further comprising:
Applying magnetic force to the reaction chamber to fix magnetic particles separated from the reaction chamber; And
Discharging a nucleic acid-containing eluate from the reaction chamber into the nucleic acid amplification reagent chamber;
Wherein the nucleic acid extracting method further comprises:

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