TWM477925U - Sample extraction device and kit - Google Patents

Description

Sample extraction device and kit

The present invention relates to an extraction device, and more particularly to a sample extraction device, a suction member, a fluid control device, and a housing set.

With the development of biotechnology and the decoding of genetic material, more and more biologically relevant laboratories or hospitals and even forensic tests, etc., frequently use the nucleic acids in the sample to conduct experiments or inspections. There are many methods for extracting and purifying nucleic acids. At present, the most common methods are divided into three categories: column extraction, magnetic bead extraction and reagent extraction. The reagent extraction method is divided into two categories, organic solvent extraction and non-organic. Solvent extraction method. Various extraction methods have their advantages and disadvantages. However, the column extraction method is the safest, simplest and most effective way to operate.

The extraction process using the column extraction method can be further divided into two types, one is a column centrifugal extraction method, and the other is a column vacuum extraction method. Figure 1 is a conventional column-centrifugal extraction method. First, the treated sample contained in the microcentrifuge tube 10 (such as breaking the cell with an anionic detergent to release the nucleic acid and denaturation of the protein) is transferred to a In the purification tube 20, the purification tube 20 can be generally divided into three parts, an upper neck portion 201, an intermediate tube portion 202, and a lower tip portion 204; the bottom portion of the intermediate tube portion 202 has a purification membrane 203, and the lower tip portion 204 has The passage of the passage allows the liquid to flow out. Usually, the sample is sucked from the sample in the microcentrifuge tube 10 by a pipette, and then injected into the purification tube 20 from above the purification tube 20. The purification tube 20 is jacketed with a waste liquid pipe 30, which is integrated into one body. The double tube is centrifuged in a centrifuge. Since the nucleic acid is negatively charged, it is bound to the positively charged purification membrane 203 and adsorbed thereon, while other impurities penetrate the purification membrane 203 and pass through the purification tube due to the centrifugal force. The passage of the lower tip portion of 20 flows into the waste liquid pipe 30. This step is referred to as a "binding step". Next, the cleaning liquid is added to the purification tube 20 and then centrifuged again to cause the impurities on the purification membrane 203 to be centrifuged to increase. The purity of the nucleic acid, this step is referred to as a "cleaning step"; finally, the purification tube 20 carrying the nucleic acid on the purified membrane 203 is transposed into the collection tube 40, and the purified membrane 203 is changed by adding a specific salinity and pH. The electrical properties are repeated again, and the nucleic acid is separated from the purification membrane 203 to flow out, and the nucleic acid is collected in the collection tube 40. This step is called a "collection step." Column centrifugation requires centrifugation at least three times, the process is complicated and the extraction time is elongated by waiting for centrifugation. Although there is an automated column centrifugal extraction machine on the market, since the centrifugal equipment must be provided in the machine, the machine is usually large in size, and the shortcomings of its extraction are not overcome.

Another type of column vacuum extraction method is shown in Figure 2, and the principle is similar to the column centrifugal extraction method, except that the steps of "combination step" and "cleaning step" are to use vacuum suction instead of centrifugation, which will directly The purification tube 20 is inserted into the pressure cell 50, and the negative pressure is applied by the gas pressure to cause the liquid to flow out of the purification tube 20 and directly enter the liquid collection bottle (not shown), but in the final "collection step", the nucleic acid must be collected by centrifugation. To the collection tube 40. Therefore, even for an automated machine, a centrifugal device must be placed in it.

In summary, although many manufacturers have developed various column extraction sets and automated machines on the market, the principle and extraction process are similar to the above-mentioned prior art, and their existence is large and time consuming. Longer deficiencies have not been overcome, and therefore there is a need in the art for a new way and machine to overcome these shortcomings.

In order to solve the problem that the above-mentioned extraction of nucleic acid is time-consuming and labor-intensive, and the size of the automated machine is too large, the creator unexpectedly imagines that the conventional purification tube can be used to directly use the air pressure in the purification tube. It only uses the lower end of the purification tube to pump and discharge the sample, the cleaning solution, the extract, etc., and does not need to use the centrifuge at all, and does not need to inject the sample, the cleaning solution or the extract from the neck of the purification tube. The nucleic acid can be efficiently extracted. Therefore, the present inventors have provided a device for pneumatically extracting nucleic acids.

In one embodiment, a suction member for use in a tubular member is provided, comprising: a first upper portion, an intermediate portion communicating below the first upper portion and having an inner diameter smaller than an inner diameter of the first upper portion, and communicating with a suction portion below the intermediate portion and having an inner diameter smaller than an inner diameter of the intermediate portion, whereby the first upper portion or the intermediate portion is fitted against an outer circumference of the column member, and a specific liquid is sucked into the tube via the suction portion Column component.

Furthermore, wherein the periphery of the first upper portion has a plurality of ribs that are radially present, whereby when the suction member is supported by the plurality of ribs, gas can pass through the gap between the plurality of ribs flow.

In another embodiment, a sample extraction device is provided, comprising the suction member, further comprising: a tubular member having a second upper portion, a second lower portion, and being sandwiched between the second upper portion and the second lower portion a second long post portion communicating with the second upper portion and the second lower portion, and a semipermeable membrane positioned within the second long post portion to leave the desired material out of the sample on the semipermeable membrane; a third upper portion, a third lower portion, a third long column portion sandwiched between the third upper portion and the third lower portion and communicating with the third upper portion and the third lower portion, and the third long column a filter film in the portion, whereby the third upper portion and the third long column portion cooperate to abut the inner circumference of the second upper portion or the second long post portion of the column member, and are prevented by the filter film Specific liquid by the third The lower portion overflows excessively toward the third upper portion.

Furthermore, wherein the first upper portion of the suction member has a plurality of ribs that are radially present, whereby when the suction member is supported by the plurality of ribs, gas can pass between the plurality of ribs The gap flows.

Furthermore, the column member further has a cover portion extending from the second upper portion and an O-ring disposed above the semi-permeable membrane, the cover portion sealing the opening of the second upper portion, the O-ring system To fix the semipermeable membrane.

Furthermore, the third long column portion of the connecting member has a knurling for clamping the filter film, and an inner diameter of the third upper portion is larger than an inner diameter of the third long column portion, the third length The inner diameter of the column portion is larger than the inner diameter of the third lower portion, whereby the third lower portion and the third long column portion cooperate to abut the inner circumference of the second upper portion or the second long column portion of the column member.

In another embodiment, a fluid control device for use with the suction member described above is provided, comprising: a tubular body, a push rod disposed in the tubular body, and an attachment member connected to the lower portion of the tubular body, wherein the The connecting member has a third upper portion, a third lower portion, and a third long column portion sandwiched between the third upper portion and the third lower portion and communicating with the third upper portion and the third lower portion, whereby the third portion The lower portion and the third long column portion cooperate to abut the inner portion of the second upper portion or the long column portion of the column member.

Furthermore, the inner diameter of the third upper portion of the connecting member is larger than the inner diameter of the third long column portion, and the inner diameter of the third long column portion is larger than the inner diameter of the third lower portion, thereby the third The lower portion and the third long column portion cooperate to abut the inner circumference of the second upper portion or the second long column portion of the column member.

In still another embodiment, a sample extraction device is provided in combination with the foregoing The accommodating set includes: a component receiving portion, a column member accommodating portion adjacent to the accommodating member accommodating portion, and a suction member accommodating portion adjacent to the column member accommodating portion, adjacent to the suction One or more heating sleeves of the component receiving portion, and one or more buffer sleeves adjacent to the one or more heating sleeves or the suction member receiving portion and accommodating a specific buffer, thereby The component that is placed in the corresponding accommodating portion, the column member, and the suction member are sequentially assembled and sequentially carried out in the one or more buffer sleeves or the one or more heating sleeves. Corresponding to extraction related actions.

Furthermore, the height of the one or more heating sleeves or the one or more buffer sleeves is slightly larger than the height of the intermediate portion of the suction member plus the suction portion, thereby avoiding the inability to absorb the one or more Heating the liquid in the cannula or the one or more buffer sleeves.

Furthermore, the one or more buffer sleeves are provided with aluminum foil, and respectively accommodate enzyme, cytolysis buffer, bonding buffer, prewash buffer, rinse buffer, post wash buffer, or wash. The buffer is extracted to perform the corresponding extraction-related actions in sequence.

Furthermore, wherein some of the one or more buffer sleeves contain only air to volatilize the residual organic solvent.

Furthermore, wherein the one or more heating sleeves are arranged separately to be separately heated to increase the heating efficiency and accelerate the evaporation rate of the organic solvent.

Furthermore, the receiving part receiving portion, the column member receiving portion, or the suction member receiving portion respectively have circular openings of different sizes to accommodate corresponding connecting members, column members, or Pipette parts.

Furthermore, the column component receiving portion further includes an elongated opening for receiving the cover portion of the column member.

10‧‧‧Microcentrifuge tube

100‧‧‧Inspector casing

110‧‧‧Product casing

120‧‧‧buffer casing

150‧‧‧ accommodating kit

152‧‧‧ Should be connected to the component housing

154‧‧‧Pipe component storage

156‧‧‧Sucking parts accommodation

158‧‧‧heating casing

20‧‧‧purification tube

201‧‧‧ upper neck

202‧‧‧Intermediate body

203‧‧‧Purified membrane

204‧‧‧The tip

205‧‧‧ 盖部

206‧‧O ring

30‧‧‧Waste tube

40‧‧‧ Collection tube

50‧‧‧pressure box

60‧‧‧pressure equipment

70‧‧‧Fluid control device

701‧‧‧ tube body

702‧‧‧Put

703‧‧‧Needle assembly

80‧‧‧ pipetting straw

802‧‧‧ first upper

804‧‧‧ middle part

806‧‧‧Drawing Department

808‧‧‧ ribs

90‧‧‧Adapter

901‧‧‧ filter

902‧‧‧ third upper

904‧‧‧The third long column

906‧‧ Third lower part

908‧‧·grain

Figure 1 shows a flow chart for the extraction of nucleic acids by conventional column-column extraction.

Figure 2 shows a flow chart for extracting nucleic acids by conventional column vacuum extraction.

Fig. 3 is a schematic view showing an embodiment of the pneumatic pressure extraction nucleic acid device of the present invention.

Fig. 4 is a schematic view showing another embodiment of the pneumatic pressure extraction nucleic acid device of the present invention.

Figure 5 is a schematic view of a fluid control device of one embodiment of the present invention.

Figure 6 is a schematic view of a fluid control device of another embodiment of the present invention.

Figure 7 is a schematic diagram of the accommodating set used in the automated machine for the pneumatic extraction of nucleic acids.

Figure 8 is a schematic view of a housing kit used in conjunction with a sample extraction device in one embodiment of the present invention.

The terms "a" and "an" are used herein to mean one or more than one (ie, at least one) of the grammatical objects herein.

The term "column component" or "purification tube" as used herein is a column of the art for purifying nucleic acids, which generally comprises an upper neck (also referred to herein as "the second upper portion". ""), an intermediate tube body (also referred to herein as "second long column portion"), a semi-permeable membrane and a lower tip (also referred to herein as "second lower portion") having a through hole It can be used to discharge liquid, but the purification tube in the present invention is not limited to this structure, as long as the purification tube is a through-hole tube having a semi-permeable membrane, that is, within the scope of the present invention, the material of the tube body may be plastic such as Polypropylene, polystyrene, polycarbonate or polyvinyl chloride, or biodegradable materials. The semipermeable membrane is also known in the art, and the material can be cerium oxide, and the surface charge is positively charged, and can be combined with the sample. The negatively charged nucleic acid is electrically coupled and is subjected to the electrical properties of the extracting liquid to release the nucleic acid; in the present invention, the "semipermeable membrane" is also referred to as "purified membrane", so that the solution can be passed through the interior thereof, that is, When there is a pressure difference between the space in contact with one side of the semipermeable membrane and the other surface of the semipermeable membrane, the solution may flow from the high pressure space to the low pressure space to penetrate the semipermeable membrane; or, when the half When the membrane is subjected to centrifugal force, the solution can penetrate the semipermeable membrane in the direction of centrifugal force, but the nucleic acid acts on the membrane with the semipermeable membrane; and the semipermeable membrane used in the present invention passes through the membrane and The interaction between nucleic acids to adsorb nucleic acids, wherein the nucleic acid and the semipermeable membrane are mutually attracted by polarity or electrical properties. In a preferred embodiment of the present invention, the semipermeable membrane is a porous membrane having a hydrophilic group. (also known as hydrophilic membrane), and it is presumed that the hydrophilic group of the nucleic acid and the hydrophilic group of the semipermeable membrane attract each other by changing the polarity of the surrounding; the semipermeable membrane having a hydrophilic group as referred to herein means The porous membrane material itself has a hydrophilic group ( Polyhydroxyethyl acrylate, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acid, polymethacrylic acid, polyoxyethylene, cellulose acetate, cellulose acetate saponified, cellulose acetate mixture, etc. It is preferable to use an organic polymer having a hydroxyl group, or to treat or coat a material constituting the porous film to introduce a hydrophilic group, and any organic or inorganic material is suitable as a material of the porous film. It may be a combination of a plurality of porous membranes; in the porous membrane, the pore diameter of the membrane is preferably 0.2 μm or more, and the thickness is preferably 10 μm to 500 μm, more preferably 50 μm to 250 μm, in order to allow the solution to pass therethrough. In addition, in the present invention, the upper end and the lower end of the purification tube are defined as the lower end of the general purification tube structure, and the upper end is far from the purification membrane position, but the creation is not limited thereto. The one that is joined to the pressure device is called the upper end of the purification tube, and the one used to suck the liquid is called the lower end of the purification tube.

The term "pressure equipment capable of applying positive or negative gas pressure" is used herein. Where the device can provide positive or negative gas pressure, the simplest device such as a cartridge can provide positive or negative gas pressure by pumping or pressing the push rod in the manifold; or a fluid control device as in the prior art a pipette, a pump or a pneumatic cylinder comprising a pneumatic valve and a pressure regulator capable of being controlled to generate a positive or negative gas pressure, wherein the pressure of the solution to penetrate the semipermeable membrane is from 10 kPa to 300 kPa, preferably From 40 kPa to 200 kPa, the most preferred pressure value is 60 kPa.

The term "cleaning fluid" (or "buffer") as used herein, includes any liquid of the prior art that can be used to clean a purification tube, including but not limited to water, alcohol or others that do not alter the electrical or polar properties of the semipermeable membrane. a buffer, that is, the cleaning liquid capable of washing out impurities in the nucleic acid mixture solution, the impurities being adsorbed on the semipermeable membrane together with the nucleic acid, and in this respect, the cleaning liquid only causes the impurities to be on the semipermeable membrane Rathering, without nucleating the nucleic acid, in order to achieve this, since the nucleic acid is poorly soluble in the aqueous organic solution, the use of suitable aqueous organic solutions is within the scope of this creation, such as alcohols, including methanol, Ethanol, isopropanol, n-propanol or butanol, preferably ethanol.

The term "extraction solution" (or "elution buffer") as used herein encompasses any liquid of the prior art that can be used to dissociate nucleic acids from the semipermeable membrane, including polar or electrical properties of the semipermeable membrane. a liquid in which the nucleic acid is dissociated, for example, a buffer containing 1.2 M NaCl, 50 mM 3-(N-morpholine)propanesulfonic acid (MOPS), 15% ethanol, and pH 8.0; A buffer containing 0.5 M ammonium acetate, 10 mM magnesium acetate (pH 7.5) and 1.5 mM EDTA; or a buffer containing 1.25 M NaCl, 50 mM Tris-HCl pH 8.5 and 15% isopropanol, and the like.

There is a technical prejudice in the technical field of the present invention, that is, those skilled in the art of the present invention generally believe that it is necessary to provide a nucleic acid-containing sample from the upper end of the purification tube to bind the nucleic acid to the semipermeable membrane. After that, the residual sample is from the other end of the purification tube (pure The lower end of the tube is discharged; however, this creation overcomes the technical bias, and before the purification tube is provided with the sample having the nucleic acid, the one end of the purification tube (the upper end of the purification tube) is combined with the pressure device, so that the nucleic acid is extracted. All liquid materials to be used (including samples with nucleic acids, residual samples with nucleic acid adsorbed by semipermeable membrane, cleaning solution, waste cleaning solution, eluent or nucleic acid-encapsulated extract) are only purified. The other end of the tube (lower end of the purification tube) is in and out. The advantage of using the created component and the accommodating set is that since all the liquid material is only in and out of one end of the purification tube, in addition, since the sample having the nucleic acid is taken up through the semipermeable membrane by the lower end of the purification tube, the nucleic acid is first Combined with the semipermeable membrane, when the purification tube is subjected to positive air pressure and the sample is discharged, the sample is again passed through a semipermeable membrane and discharged at the lower end of the purification tube, if any residue is not translucent. The membrane-bound nucleic acid may have a chance to combine with the semipermeable membrane. Therefore, the binding step of one operation in the present creation has actually repeated the binding step twice; the washing step and the collecting step are also the same principle. In the cleaning or collection step of one operation in this creation, the cleaning and collection steps have actually been repeated twice, so the amount of extracted nucleic acid is increased and the efficiency is higher. In addition, during the extraction process, the positive/negative gas pressure provided by the pressure device can be repeatedly used, so that the liquid used for the extraction can repeatedly pass through the semi-permeable membrane, thereby further improving the efficiency of nucleic acid binding, washing or elution, and effectively increasing the extraction amount of the nucleic acid. Compared with the prior art, all the reaction liquids pass through the semipermeable membrane at one time, and the liquid is drained or pumped away, but the method of the present invention can increase the extraction amount and improve the extraction efficiency.

Furthermore, with the components and housing kits of the present invention, all liquid materials used can be "absorbed/extracted in situ", that is, where the liquid material in the extraction process is sucked, it can be discharged back to it. Collect in a suction or other desired container without using a special waste container. Therefore, if a row of purified hydrazine is used, and when the nucleic acid is extracted, all the effluent is returned to its original position in the purified sputum, and the purified sputum after use can be directly discarded. Discarding is not only convenient but also reduces the risk of cross-contamination.

First, referring to Fig. 3, a schematic diagram of one embodiment of a pneumatic pressure extraction nucleic acid device is created. The pneumatic extraction nucleic acid device comprises a purification tube 20 and a pressure device 60 that can apply a positive or negative gas pressure. The purification tube 20 is an embodiment of a column member. The upper end of the purification tube 20 is hermetically joined to the pressure device 60, and the purification tube is penetrated from top to bottom, and is an upper neck portion 201 (ie, a second upper portion) and an intermediate tube portion 202 (ie, a second long column). a purification membrane 203 (ie, a semipermeable membrane) and a lower tip 204 (ie, a second lower portion), the purification membrane 203 being located within the intermediate tubular portion 202, the lower tip portion 204 having a through hole. With the apparatus, a "binding step" is first performed: the pressure device 60 is turned on to apply a negative air pressure to the purification tube 20, and the lower portion 204 of the purification tube 20 sucks a sample containing nucleic acid to pass the sample through the purification membrane. 203, the nucleic acid is electrically or polarly bonded to the purification membrane 203; the pressure device 60 is turned on to apply positive pressure to the purification tube 20, and the residual sample is passed through the purification membrane 203 in the opposite direction, and then the purification tube is passed through the purification tube. The tip 204 exits below 20, and this step can be repeated one to many times as needed. Wherein, the nucleic acid in the sample is one of the expected materials bound to the purified membrane, and the expected material may also be different as needed, and proteins, polypeptides, sugars, and esters may also be used as the expected materials.

Then, the "collection step" is performed: the pressure device 60 is turned on to apply a negative air pressure to the purification tube 20, and the extract liquid is sucked by the lower tip portion 204 of the purification tube 20, and the extract liquid is passed through the purification membrane 203 to change the The purified membrane 203 is electrically or polarized to separate the nucleic acid from the purification membrane 203 and dissolved in the extract; the pressure device 60 is turned on to apply positive pressure to the purification tube 20, and the nucleic acid-dissolved solution is re-inverted in the opposite direction. After passing through the purification membrane 203, it is discharged and collected from the lower tip 204 of the purification tube 20, and the collection step can be repeated one to many times as needed. The user can further perform one or more cleaning steps after the bonding step is completed as needed.

According to the pneumatic pressure extraction nucleic acid device of the present invention, the purification tube 20 can utilize various purification tubes existing in various prior art, and the purification tubes of different brands on the market can be utilized in the present creation. Furthermore, in order to prevent the sample in the purification tube 20 from being absorbed by the negative air pressure, it is flushed onto the pressure device 60 to cause pollution. As shown in the figure, a filter membrane 901 may be further disposed between the upper end of the purification tube 20 and the pressure device 60. The adapter 90 is in airtight engagement with the upper end of the purification tube 20 (i.e., the upper neck 201) and the pressure device 60.

In addition, in order to facilitate the handling of the liquid or the collection of the liquid, the tip portion 204 of the purification tube 20 may be further provided with a pipetting pipe 80 that is hermetically joined thereto, whereby the pipette 80 can be utilized by the purification pipe 20. Aspirate a small amount of liquid or collect the purified sample into a microcentrifuge tube. The pipetting pipette 80 is an embodiment of the suction member.

In a preferred embodiment of the present invention, the upper end structure of the pipette is a set body, and the sleeve body can hermetically cover the intermediate pipe body of the purification pipe portion to the lower end to fit the purification pipe. Moreover, the structure of the pipette and the purification pipe joint is versatile with the purification tube of different brands, and can be widely applied to various types of purification tubes, such as but not limited to the sleeve of the pipette and The structure of the joint of the purification tube is made of rubber material to make it elastic structure, and can be joined with different purification tubes; or the sleeve portion has two different slopes, and the first layer is close to the middle tube of the purification tube The slope of the second layer is relatively steep near the lower end of the purification tube, and the two layers have different slopes to make it suitable for gas-tight fitting in various purification tubes of various brands on the market.

In addition, in order to make the adapter 90 and the structure of the pipette 80 and the purification pipe joint with the purification pipe of different brands, the adapter 90 and the pipette 80 can be used. The structure of the joint of the purification tube 20 is made of a rubber material to make it an elastic structure, and the The purification tube 20 of the same specification is joined. In this creation, the nucleic acid can be extracted by a pneumatic method because the components must be hermetically joined. The manner in which the components are hermetically joined may be any conventional technique, such as the addition of an O-ring, and is not limited.

Fig. 4 is a schematic view showing another embodiment of the pneumatic pressure extraction nucleic acid device of the present invention. The structure of the extraction apparatus shown in Fig. 4 is basically the same as that of the extraction apparatus shown in Fig. 3, but there are still several differences to accommodate different operational requirements. The differences are detailed below. The purification tube 20 further has a lid portion 205 extending outward from the upper neck portion 201 and an O-ring 206 disposed above the purification membrane 203. The lid portion 205 is used to seal the opening above the upper neck portion 201 to avoid other processing. The purification tube 20 is contaminated. The O-ring 206 is used to fix the purification membrane 203 to avoid the positional change of the purification membrane 203 during the extraction treatment or other treatment.

The adapter 90 is an embodiment of the component to be connected. The adapter 90 further has a third upper portion 902, a third elongated post portion 904, and a third lower portion 906. Specifically, the filter membrane 901 is located within the third long column portion 904, and the third long column portion 904 is sandwiched between the third upper portion 902 and the third lower portion 906 and with the third upper portion 902 and the third lower portion 906. The third lower portion 906 and the third long post portion 904 are engaged to abut the inner circumference of the neck portion 201 or the intermediate tube portion 202 of the purification tube 20, and the solution in the sample is prevented from being pre-washed by the filter 901. A specific liquid such as a liquid, a post-wash buffer, or an elution buffer overflows excessively from the third lower portion 906 toward the third upper portion 902 during the extraction process.

In the preferred embodiment, the third long post portion 904 of the adapter 90 has a score 908 for holding the filter 901, and the inner diameter of the third upper portion 902 is greater than the inner diameter of the third long post portion 904. The inner diameter of the third long post portion 904 is larger than the inner diameter of the third lower portion 906, so that the third lower portion 906 and the third long post portion 904 can better fit against the neck 201 or the middle of the purification tube 20 The inner circumference of the tubular body portion 202. In a more preferred embodiment, there is substantially no need to provide an upper portion of the filter 901 for fixing the filter 901. ring.

As can be seen from the drawings, in the preferred embodiment of the present invention, the pipetting pipe 80 includes a first upper portion 802, an intermediate portion 804, and a suction portion 806 from top to bottom, wherein the intermediate portion 804 is communicated with the first upper portion 802 and The inner diameter is smaller than the inner diameter of the first upper portion 802; and the suction portion 806 is communicated with the inner portion 804 below and the inner diameter is smaller than the inner diameter of the intermediate portion 804, and the inner diameter away from the one end of the intermediate portion 804 is closer to the intermediate portion 804. The inner diameter of one end is also small, that is, the suction portion 806 is tapered in a direction away from the intermediate portion 804 to draw liquid. In particular, the first upper portion 802 can be a set of bodies that can be airtightly engaged from the intermediate tubular body portion 202 to the lower pointed portion 204 of the purified tubular portion. Accordingly, the pipetting pipette 80 can be widely applied to various types of purification tubes, and even if it is combined with the purification tube 20, it can be combined without slipping separation. That is, the first upper portion 802 and the intermediate portion 804 cooperate to abut against the outer circumference of the purification tube 20, and the specific liquid such as the pre-wash buffer, the post-wash buffer, or the elution buffer can be sucked to the purification tube 20 via the suction portion 806. In order to carry out extraction related processing.

Furthermore, the first upper portion 802 of the pipetting pipe 80 is provided with a plurality of ribs 808 that are radially formed, so that when the pipetting pipe 80 is supported by a plurality of ribs 808 in a housing set or the like described later. In the upper case, the gas can flow through the gap between the plurality of ribs, thereby preventing the receiving sleeve or the like from being hermetically covered by the pipetting pipe 80 when sucking the specific liquid, thereby causing the receiving set or The case where the internal pressure of the container is less than the ambient pressure and the pipette 80 cannot be smoothly moved.

Next, please refer to FIG. 5, which is a schematic diagram of another embodiment of the pneumatic pressure extraction nucleic acid device of the present invention. It can be seen that the pneumatic extraction nucleic acid device can be a fluid control device 70, which is a pressure element that can apply positive or negative gas pressure, or can be a needle. cylinder. The fluid control device 70 includes a tube body 701, a push rod 702 disposed in the tube body 701, and a needle assembly 703 detachably coupled to the tube body 701. The push rod 702 of the tube body 701 is characterized. A purification membrane 203 electrically coupled to the nucleic acid is provided below. According to the device of the present invention, the fluid control device 70 can directly use the suction and the pushing of the push rod 702 to perform the "combination step", the "cleaning step" and the "collection step"; in detail, First, the needle assembly 703 or the pipetting pipe 80 is used to suck the sample by the negative air pressure generated when the push rod 702 is pulled up. When the sample enters the tube body 701 through the needle, the sample passes through the purified film 203 from bottom to top. The negatively charged nucleic acid in the sample is electrically or polarly bound to the purification membrane 203, and the residual sample is discharged from the needle through the purification membrane 203 by pushing the positive air pressure generated by the push rod 702 from top to bottom. The "binding step" is completed; then, the pusher 702 is pulled up again, and the cleaning liquid is sucked by the needle assembly 703, and the cleaning liquid passes through the purification film 203 from bottom to top, so that the impurities on the purification film 203 are cleaned. The purity of the nucleic acid can be increased, and the positive air pressure generated by the push rod 702 can be pushed again. The cleaning liquid is discharged from the top to the bottom through the purified film 203, and then the needle is discharged to complete the "cleaning step", which can be repeated one or more times according to requirements. Times Finally, the pusher 702 is pulled up again, and the extract is sucked by the needle, and the extract passes through the purified membrane 203 from bottom to top, so that the extract of the special salinity and pH changes the electricity of the purified membrane 203. Sexual or polar, the nucleic acid is flushed from the semipermeable membrane and dissolved in the extract, and the push rod 702 is pushed to pass the nucleic acid-dissolved extract from the top to the bottom through the purified membrane 203 and then discharged through the needle. Collected in the collection tube to complete the "collection step."

FIG. 6 shows another embodiment of the fluid control device 70. The difference from the embodiment of Fig. 5 is that the purification membrane 203 is not provided, and the needle assembly 703 is substantially replaced by the adapter 90. Specifically, the fluid control device 70 includes a tubular body 701, a push rod 702 disposed in the tubular body 701, and a adaptor 90 detachably or integrally formed in communication with the lower portion of the tubular body 701. The adapter 90 further has a third upper portion 902, a third elongated post portion 904, and a third lower portion 906. The third long post portion 904 is sandwiched between the third upper portion 902 and the third lower portion 906 and communicates with the third upper portion 902 and the third lower portion 906. Therefore, the third lower portion 906 and the third long post portion 904 are mated. The inner circumference of the neck 201 or the intermediate tube portion 202 above the purification tube 20 is purified. In a preferred embodiment, the inner diameter of the third upper portion 902 is greater than the inner diameter of the third long post portion 904, and the inner diameter of the third long post portion 904 is greater than the inner diameter of the third lower portion 906, thereby making the third lower portion The 906 and the third long post portion 904 can better fit against the inner circumference of the neck portion 201 or the intermediate tube portion 202 above the purification tube 20. With the device of the present invention, in combination with the purification tube 20 and the pipette pipette 80, the effect of extracting the desired material can be easily achieved as in the foregoing embodiment. Moreover, such a configuration allows the flow control device 70 to be discarded after use, without the need for re-cleaning, increasing the likelihood of sample contamination.

Another aspect of the present invention is an automated machine for pneumatic extraction of nucleic acids (not shown) designed by the method of the present invention, comprising a robot arm, one or more sets of cartridges, and a console, and The console includes a housing set 150. In a preferred embodiment of the present invention, the automated machine may further comprise a heating module and/or an oscillator to selectively oscillate or heat the sample during operation to improve nucleic acid purification. Yield.

Figure 7 is a schematic view of a housing kit 150 for purifying a desired material in an embodiment of the present invention. The housing kit 150 includes: an upper neck portion, an intermediate tube portion, and a half-permeable membrane. And a purification tube 20 at the tip, a filter adapter 90, a pipette 80, a hole plate for accommodating the sample (referred to as the sample sleeve 100 for short), at least one for accommodating a buffer tray (abbreviated as buffer sleeve 120); and a well plate (referred to as product sleeve 110) for accommodating the extracted product.

In addition, in an embodiment of the present creation, the accommodating set includes: (a) one having a semi-permeable membrane purification tube, (b) a filter membrane adapter, (c) a pipette pipette, (d) a well plate for accommodating the sample, and (e) at least one for accommodating a well plate for a buffer; and (f) a well plate for accommodating the product after extraction. The components (a) to (c) in the above kit may be integrally formed. In a preferred embodiment of the present invention, the accommodating kits are integrally arranged in the form of a row of purified crucibles.

The automatic machine for extracting nucleic acid according to the present invention has a cylinder on the robot arm, and a tip end in front of the cylinder for combining the adapter 90, the purification tube 20 and the pipette 80 in the accommodation set 150 ( From the top to the bottom), when the three are combined (hereinafter referred to as the assembly), the cartridge can be airtightly engaged with the assembly, and a negative air pressure is provided to cause the pipette pipette 80 to suck the sample casing in the receiving sleeve 150. The sample in 100 is in the purification tube, so that the nucleic acid in the sample reacts with the semi-permeable membrane in the sample or the polarity, and the adapter 90 can prevent the sample from rushing to the cartridge and avoid contamination, when the reaction is completed. The cartridge is given a positive air pressure, and the purification tube 20 directly discharges the residual sample into the sample sleeve 100 in the housing kit 150 via the pipette 80 (this is a "binding step"), wherein, in the bonding step, The sample is repeatedly passed through the purification tube 20 one or more times to increase the amount of nucleic acid bound to the semipermeable membrane; then, the robot arm mechanically drives the assembly to the upper end of the orifice plate filled with the cleaning liquid in the housing set 150 ( That is, the buffer sleeve 120) provides a negative air pressure to make the pipette 8 0 sucking the cleaning liquid into the purification tube 20, after cleaning, the cylinder provides a positive air pressure, and the cleaning liquid is discharged into the same orifice plate that was originally sucked out (this is a "cleaning step"), and the cleaning step can be repeated one to several times; When the washing step is finished, the purification tube 20 may be further heated by a heating mechanism to evaporate the ethanol in the cleaning liquid, and/or the semi-permeable membrane may be dried by supplying a positive/negative pressure to the cylinder by a plurality of times; finally, the machine The arm mechanically drives the assembly to the upper end of the orifice plate (ie, the buffer sleeve 120) filled with the liquid in the accommodating sleeve 150, and the cartridge provides a negative air pressure to allow the pipette to draw the extract into the purification tube 20. The nucleic acid that binds the electrical/polarity to the semipermeable membrane is eluted into the extract, and then the cartridge provides positive air pressure, and the extract is extracted. Discharge into the same orifice plate that was originally sucked out, or the robot arm can mechanically drive the assembly into the product sleeve 110 of the accommodating sleeve 150 to collect the purified nucleic acid (this is a "collection step"), which The collecting step can be repeated one or more times, that is, the same extract is repeatedly passed through the semipermeable membrane one or more times, so that the extraction amount of the nucleic acid can be effectively improved.

The accommodating sleeve 150 can be two separate components, and can also form an integrally formed component, which is not limited. In addition, the buffer sleeves 120 in the housing set 150 can be different in number according to requirements, but the arrangement is optimal according to the steps in the process of extracting nucleic acids, which are sequentially cell lysate, washing solution, alcohol, water. Or the extract liquid, whereby when the dispenser drives the assembly to move, the nucleic acid extraction can be carried out in a stepwise step by step.

In addition, since the nucleic acid-containing sample usually contains a lysis buffer to break the cell membrane of the cells contained in the sample to release the nucleic acid, the buffer contains a large amount of surfactant, so when After the completion of the step of the step, when the residual sample is discharged, if it is discharged into the empty orifice plate, a large amount of air bubbles are easily generated and the operation is unfavorable. Therefore, in a preferred embodiment, the accommodation set 150 may include An orifice plate (not shown) for accommodating the residual sample, wherein the orifice plate contains an aqueous solution, the aqueous solution can absorb air bubbles, and the residual sample is discharged to the orifice plate without generating bubbles. Here, the amount of the aqueous solution is not limited, but is preferably from 100 to 500 μl, more preferably from 200 to 400 μl, most preferably 300 μl.

Furthermore, after the "binding step" is completed, there may be a residual sample on the semipermeable membrane. Therefore, if the cleaning solution is directly sucked, more bubbles are likely to be generated, so that the aqueous solution can be first absorbed and the semipermeable membrane can be absorbed. The remaining sample is cleaned, and the permeability of the semipermeable membrane is also increased, so that the cleaning liquid of the next cleaning step can better pass through the semipermeable membrane, thereby improving the cleaning efficiency. Therefore, the accommodating sleeve 150 is improved. The method may include: an orifice plate (not shown) containing an aqueous solution in the buffer sleeve Before the tube 120, the amount of the aqueous solution is not limited, but is preferably from 100 to 500 μl, more preferably from 200 to 400 μl, most preferably 300 μl.

Figure 8 shows another embodiment of a containment kit 150 that is used in conjunction with the aforementioned sample extraction device. The accommodating sleeve 150 may include the following elements arranged in a juxtaposed arrangement: the component receiving portion 152, the column member accommodating portion 154 adjoining the component accommodating portion 152, and the suction of the adjacent column member accommodating portion 154 a component accommodating portion 156, one or more heating sleeves 158 adjoining the suction member accommodating portion 156, and one or more adjacent to the one or more heating sleeves 158 or the suction member accommodating portion 156 and containing a specific buffer Multiple buffer sleeves 120. Thereby, the component, the column component, and the suction component sequentially placed in the corresponding accommodating portion are sequentially assembled and sequentially in one or more buffer sleeves 120 or one or more heating sleeves 158. Perform corresponding extraction related actions. In addition, the open end of the buffer sleeve 120 has an aluminum foil seal which is pierced by a needle or the like during processing, thereby preventing contamination of the specific buffer contained in the buffer sleeve 120.

In one embodiment, the height of the one or more heating sleeves 158 or one or more buffer sleeves 120 is slightly greater than the height of the intermediate portion of the suction member plus the suction portion such that the suction member is placed in the heating sleeve 158. Or the buffer sleeve 120 does not touch the bottom of the heating sleeve 158 or the buffer sleeve 120, thereby avoiding the inability to draw one or more heating sleeves 158 or one or more buffer sleeves 120. The liquid in it. In addition, in an embodiment, the heating sleeve 158 can be the same structure as the buffer sleeve 120, and can also be used to accommodate the buffer.

In one embodiment, one or more buffer sleeves 120 can house an enzyme, a cytolytic buffer, a binding buffer, a prewash buffer, a wash buffer, a post wash buffer, or an elution buffer, respectively. In order to perform corresponding extraction-related actions in sequence. In addition, several buffer sleeves 120 may contain only gas without additional buffer for drying treatment to allow purification in purification tube 20. Alcohol or other organic solvents on film 203 are more readily volatilized to avoid interference with subsequent processing. In a preferred embodiment, the receiving kit 150 can include 10 buffer sleeves for sequentially accommodating the enzyme, the cytolysis buffer, the binding buffer, the prewash buffer, the three tube rinse buffer, and the like. Wash buffer, gas, or elution buffer for sequential extraction.

In one embodiment, one or more heating sleeves 158 are spaced apart to heat separately at a temperature of about 65 ° C to increase heating efficiency and speed up the evaporation rate of organic solvents such as alcohol or elution buffer to avoid interference. Follow-up processing. In a preferred embodiment, the receiving kit 150 can include two heating sleeves 158 for removing alcohol or eluting solution, respectively. Among them, the removal of the elution solution can increase the yield of the final product.

In an embodiment, the component receiving portion 152, the column member accommodating portion 154, or the absorbing member accommodating portion 156 respectively have circular openings of different sizes to accommodate corresponding connecting members and column members. Or pick up parts. In another embodiment, the column member receptacle 154 further includes an elongated opening for receiving the cover portion 205 of the tubular member.

The following examples should not be construed as unduly limiting the creation. Modifications and variations of the embodiments discussed herein may be made without departing from the spirit and scope of the invention, and still fall within the scope of the present invention.

[Examples] Purification of gDNA of fish liver

Front work

All reagents were used in the QIAamp DNA Reagent Kit (Qiagen Taiwan Co. Ltd.), containing tissue lysate, cell lysate, cleaning solution I, cleaning solution II, rushing a liquid extracting protein and proteinase K, and a purification tube, wherein the semipermeable membrane in the purification tube is a silica membrane.

Fish liver of about 5x5mm size was taken, placed in a 2ml screw cap test tube, 800μl phosphate buffer saline (PBS) and a 5mm steel ball were added to homogenize the tissue homogenizer for 30 seconds. Next, 800 μl of the cell lysate was added, mixed with a shaker, and then 80 μl of proteolytic enzyme K was added and mixed using a shaker. The sample was incubated at 60 ° C for 30 minutes to dissolve the sample. Thereafter, 800 μl of ethanol was added and shaken with a shaker for 30 seconds. After centrifugation at 13,000 rpm for 3 minutes, 200 μl of the supernatant was taken into each of four sample tubes, which were the sample of Example 1, the sample of Example 2, the sample of Comparative Example 1, and the sample of Comparative Example 2.

Example 1

First, the upper neck of the purification tube is hermetically joined to a fluid control device, and the lower tip of the purification tube is hermetically joined to a pipette.

Combining step: pulling the push rod of the fluid control device, the sample of the sample 1 in the sample tube of the first embodiment is sucked through the pipette, and the lower tip of the purification tube is passed through the purification membrane, and then sucked to the middle tube body. Next, the fluid control device pusher is pushed down to discharge the residual sample that is not combined with the purified membrane, and the nucleic acid is electrically coupled to the purified membrane.

The cleaning step: pulling the push rod of the fluid control device, sucking 500 μl of the cleaning solution I through the pipette, passing the lower tip of the purification tube through the purification membrane, sucking it to the intermediate tube body, and then pushing the fluid control device The rod is pushed down to discharge the cleaning liquid I which has been cleaned of the purified membrane. Similarly, the cleaning step is performed again, and 750 μl of the cleaning solution II is sucked through the pipette, passed through the purification film by the lower tip of the purification tube, and then taken up to the intermediate tube body, and then the fluid control device is pushed under the rod. The cleaning liquid II that has been cleaned of the purified membrane is pushed out.

Post-cleaning step: Pull the plunger of the fluid control device, draw 800 μl of 100% alcohol through the pipette, pass the lower tip of the purification tube through the purification membrane, and then pipette it to the middle. The body portion, and then the fluid control device pusher is pushed down to discharge the alcohol that has been cleaned of the purified membrane; this step is repeated twice. Next, the fluid control device is aspirated and pushed at least 15 times to volatilize the alcohol remaining in the purification tube.

The collecting step: pulling the push rod of the fluid control device, sucking 200 μl of the extract liquid through the pipette, passing the lower tip of the purification tube through the purification membrane, sucking it to the middle tube body, and letting it stand for 3 minutes, then The fluid control device pusher is pushed down, and the nucleic acid-dissolved extract is discharged into a 1.5 ml microcentrifuge tube.

Example 2

The procedure of Example 2 was repeated as in Example 1, except that the procedure of Example 1 was repeated, and only the specimen was replaced with the specimen of Example 2.

Comparative example 1

Nucleic acid extraction was performed according to the QIAamp DNA Reagent Kit product data sheet. Binding step: The sample of Comparative Example 1 was pipetted into a purification tube using a pipette, and the test system was injected from above the purification tube. Next, a waste liquid tube is installed under the purification tube, and the two tubes are centrifuged in a centrifuge at a rotational speed of 13,000 rpm for 2 minutes, and the residual sample is centrifuged through the purification membrane to the waste liquid tube through the channel of the lower tip of the purification tube. After centrifugation, the waste liquid in the waste liquid tube is drained, and the nucleic acid is electrically coupled to the purified membrane, and the purification tube is placed back into the waste liquid tube.

Washing step: 500 μl of the cleaning solution I is sucked into the purification tube by means of a micro-dispenser, and the cleaning solution I is injected from above the purification tube. The two tubes are centrifuged together in a centrifuge at a rotational speed of 13000 rpm for 1 minute. The cleaning solution I is centrifuged through the passage of the lower end of the purification tube to the waste liquid tube through the purification membrane. After the centrifugation, the waste liquid in the waste liquid tube is drained. The purification tube is then placed back into the waste tube. The same cleaning step is performed again, and 750 μl of the cleaning solution II is sucked into the purification tube, and the cleaning solution II is supplied from the purification tube. Square injection. The two tubes are centrifuged at 13,000 rpm for 1 min in a centrifuge, and the washing solution II is centrifuged through the passage of the lower end of the purification tube to the waste liquid tube through the purification membrane. After the centrifugation is completed, the waste liquid in the waste liquid tube is drained, and the purification tube is further set. Return to the waste pipe.

Post-washing step: Centrifuge the two kits at 13,000 rpm for an additional three minutes to ensure complete removal of the wash solution.

Collection procedure: Remove the waste tube and replace it with a 1.5 ml microcentrifuge tube. Using a micro-dispenser, 200 μl of the extract was pipetted into a purification tube, and the extract was injected from the top of the purification tube to the middle tube body and allowed to stand for 3 minutes. Then, the two kits were placed in a centrifuge at a rotational speed of 13,000 rpm. Centrifuge for 1 minute to obtain the nucleic acid-dissolved extract into a 1.5 ml microcentrifuge tube.

Comparative example 2

The procedure of Comparative Example 2 was repeated in the same manner as in Comparative Example 1, except that the sample was replaced with the sample of Example 2.

The results of the extracted gDNA of the above Examples 1 and 2 and Comparative Examples 1 and 2 are summarized in Table 1 below.

As can be seen from the above Table 1, the DNA concentration extracted by the method and apparatus of the present invention is similar to the DNA concentration collected by the conventional method, and the method is relatively simple.

In addition, the method and apparatus of the present invention can be applied to a purification tube set for extracting nucleic acids of different brands.

Next, in order to compare the effects of using the hydrophilic film and the hydrophobic film on the method of the present creation, and in order to compare the effects of using the device of the present creation and the conventional centrifugal method, further use a semi-permeable membrane of different brands and utilize the device of the present creation. And nucleic acid extraction experiments were performed using conventional centrifugation.

Example 3

The procedure and the sample of Example 3 were the same as in Example 1, except that the semipermeable membrane of the purification tube was replaced with a hydrophilic cerium oxide film of PALL Corporation (product name: Glass Fiber Media), and the membrane pore diameter was 1.0 μm; The procedure of Example 1 was carried out in the apparatus of the present invention for nucleic acid extraction using a positive and negative gas pressure of 60 kPa/-60 kPa, and the collection step was repeated twice.

Example 4

The procedure of Example 4 was the same as that of Example 1, except that the semipermeable membrane of the purification tube was replaced with a hydrophilic nitrocellulose membrane of PALL (product name: BioTrace), and the membrane pore diameter was 0.2 μm; The procedure was carried out in the apparatus of the present invention for nucleic acid extraction using a positive and negative gas pressure of 60 kPa/-60 kPa, and the collection step was repeated twice.

Example 5

The procedure and the sample of Example 5 were the same as in Example 1, except that the semipermeable membrane of the purification tube was replaced with a hydrophilic nitrocellulose membrane (product name: Mixed Cellulose Esters) of Advantec Co., Ltd., and the membrane pore diameter was 0.2 μm; The procedure of 1 is to perform nucleic acid extraction in the apparatus of the present invention, using a positive and negative gas pressure of 60 kPa/-60 kPa, and the collection step is repeated twice.

Example 6

The procedure and the sample of Example 6 were the same as in Example 1, except that the semipermeable membrane of the purification tube was replaced with a hydrophilic cellulose acetate membrane (product name: Cellulose Acetate) of Advantec Co., Ltd., and the membrane pore diameter was 0.45 μm; The procedure of 1 is to perform nucleic acid extraction in the apparatus of the present invention, using a positive and negative gas pressure of 60 kPa/-60 kPa, and the collection step is repeated twice.

Comparative example 3

The procedure and the sample of Comparative Example 3 were the same as in Comparative Example 1, and the procedure of Comparative Example 1 was repeated, and only the semipermeable membrane used in the purification tube used was replaced with the semipermeable membrane used in Example 3.

Comparative example 4

The procedure and the sample of Comparative Example 4 were the same as in Comparative Example 1, and the procedure of Comparative Example 1 was repeated, and only the semipermeable membrane used in the purification tube used was replaced with the semipermeable membrane used in Example 4.

Comparative Example 5

The procedure and the sample of Comparative Example 5 were the same as in Comparative Example 1, and the procedure of Comparative Example 1 was repeated, and only the semipermeable membrane used in the purification tube used was replaced with the semipermeable membrane used in Example 5.

Comparative Example 6

The procedure and the sample of Comparative Example 6 were the same as in Comparative Example 1, and the procedure of Comparative Example 1 was repeated, and only the semipermeable membrane used in the purification tube used was replaced with the semipermeable membrane used in Example 6.

The results of the extracted gDNA of the above Examples 3 to 6 and Comparative Examples 3 to 6 are summarized in Table 2 below.

It can be seen from Table 2 that the amount of nucleic acid extracted by the device of the present invention is higher than that of the prior art, because the liquid material is only used by the lower end of the purification tube when the device of the present invention is used, and therefore, In the step, the positive/negative pressure provided by the pressure device can be repeatedly used, so that the extract liquid can repeatedly pass through the semi-permeable membrane, thereby improving the efficiency of nucleic acid extraction and effectively increasing the extraction amount of the nucleic acid. Compared with the method of using centrifugation, all the reaction liquids pass through the semipermeable membrane at one time, and the liquid is eliminated, and the method and the device of the present invention can be used to increase the extraction amount and improve the extraction efficiency.

Comparative Example 7A

The procedure and sample of Comparative Example 7A were the same as in Example 1, except that the semipermeable membrane of the purification tube was replaced with the hydrophobic carboxylated polyvinylidene fluoride (product name: Fluoro Trans G) of PALL Company, and the membrane pore diameter was 0.2 μm. The procedure of Example 1 was repeated, but the nucleic acid extraction was carried out in the apparatus of the present invention, using a positive and negative gas pressure of 60 kPa/-60 kPa, and the collection step was repeated twice.

Comparative Example 8A

The procedure and sample of Comparative Example 8A were the same as in Example 1, except that the semipermeable membrane of the purification tube was replaced with a hydrophobic nylon of PALL Co., Ltd. (product name: Hydrolon), and the membrane pore diameter was 1.2 μm; the procedure of Example 1 was repeated. , but in the device of this creation for nucleic acid extraction, the positive and negative The air pressure was 60 kPa / -60 kPa and the collection step was repeated twice.

Comparative Example 9A

The procedure and sample of Comparative Example 9A were the same as in Example 1, except that the semipermeable membrane of the purification tube was replaced with a hydrophobic polyether oxime (product name: Supor-450PR) of PALL Co., Ltd., and the membrane pore diameter was 0.45 μm; The procedure of 1 is to perform nucleic acid extraction in the apparatus of the present invention, using a positive and negative gas pressure of 60 kPa/-60 kPa, and the collection step is repeated twice.

Comparative Example 10A

The procedure and sample of Comparative Example 10A were the same as in Example 1, except that the semipermeable membrane of the purification tube was replaced with a hydrophobic polytetrafluoroethylene (product name: Supported PTFE) of Advantec Co., Ltd., and the membrane pore diameter was 0.45 μm; The procedure of 1 is to perform nucleic acid extraction in the apparatus of the present invention, using a positive and negative gas pressure of 60 kPa/-60 kPa, and the collection step is repeated twice.

Comparative Example 7B

The procedure and the sample of Comparative Example 7B were the same as in Comparative Example 1, and the procedure of Comparative Example 1 was repeated, and only the semipermeable membrane used in the purification tube used was replaced with the semipermeable membrane used in Example 7A.

Comparative Example 8B

The procedure and the sample of Comparative Example 8B were the same as in Comparative Example 1, and the procedure of Comparative Example 1 was repeated, and only the semipermeable membrane used in the purification tube used was replaced with the semipermeable membrane used in Example 8A.

Comparative Example 9B

The procedure and the sample of Comparative Example 9B were the same as in Comparative Example 1, and the procedure of Comparative Example 1 was repeated, and only the semipermeable membrane used in the purification tube used was replaced with the semipermeable membrane used in Example 9A.

Comparative Example 10B

The procedure and sample of Comparative Example 10B were the same as in Comparative Example 1, and the comparison of Comparative Example 1 was repeated. In the procedure, only the semipermeable membrane in the purification tube used was replaced with the semipermeable membrane used in Example 10A.

The results of the extracted gDNA of Comparative Examples 7A to 10A and Comparative Examples 7B to 10B above are summarized in Table 3 below.

As can be seen from Table 3, the use of a hydrophobic semipermeable membrane is incapable of attracting the hydrophilicity of the nucleic acid to change the polarity of the surrounding, so whether using the method and apparatus of the present invention or using conventional column centrifugal extraction There is no good extraction efficiency.

In summary, this creation overcomes the technical bias. In the prior art, the sample must be added from the upper end of the purification tube, and the purification tube is first cleaned before the purification tube is provided with the sample having the nucleic acid. The upper end of the tube is combined with the pressure device so that all liquid material used to extract the nucleic acid is only in and out of the other end of the purification tube (lower end of the purification tube). In this way, use this The creation method, device or automated machine can easily use the method of providing positive and negative pressure in all steps of extracting nucleic acid, and the liquid is sucked/discharged from the same end of the purification tube, which not only simplifies the extraction step, but also allows the liquid to be repeated. The result of improving the extraction efficiency is achieved by the semi-permeable membrane, and the automated machine can eliminate the need to configure the centrifuge, and only needs to be equipped with a device capable of providing a pressure difference, thereby achieving the effect of reducing the volume.

20‧‧‧purification tube

201‧‧‧ upper neck

202‧‧‧Intermediate body

203‧‧‧Purified membrane

204‧‧‧The tip

205‧‧‧ 盖部

206‧‧O ring

60‧‧‧pressure equipment

80‧‧‧ pipetting straw

802‧‧‧ first upper

804‧‧‧ middle part

806‧‧‧Drawing Department

808‧‧‧ ribs

90‧‧‧Adapter

901‧‧‧ filter

902‧‧‧ third upper

904‧‧‧The third long column

906‧‧ Third lower part

908‧‧·grain

Claims (15)

The suction member used for the pipe string member includes: a first upper portion, an intermediate portion connected to the lower portion of the first upper portion and having an inner diameter smaller than an inner diameter of the first upper portion, and an inner diameter portion below the intermediate portion and having an inner diameter smaller than The suction portion of the inner diameter of the intermediate portion, whereby the first upper portion or the intermediate portion is fitted against the outer circumference of the column member, and the specific liquid is sucked to the column member via the suction portion. The suction member according to claim 1, wherein the periphery of the first upper portion has a plurality of ribs that are radially formed, whereby the gas can pass through the plurality of ribs when the suction member is supported by the plurality of ribs The ribs flow in a gap between each other. A sample extraction device comprising the suction member of claim 1, further comprising: a tubular member having a second upper portion, a second lower portion, being sandwiched between the second upper portion and the second lower portion, and the second upper portion and a second long post portion communicating with the second lower portion, and a semipermeable membrane positioned within the second long post portion to leave the intended material out of the sample on the semipermeable membrane; and an attachment member having a third upper portion a third lower portion, a third long column portion sandwiched between the third upper portion and the third lower portion and communicating with the third upper portion and the third lower portion, and a filter located inside the third elongated column portion Thereby, the third lower portion or the third long column portion cooperates to abut the inner circumference of the second upper portion or the second long post portion of the column member, and the specific liquid is prevented from being the third by the filter film The lower portion overflows excessively toward the third upper portion. The sample extraction device of claim 3, wherein a periphery of the first upper portion of the suction member has a plurality of radially extending ribs, whereby when the suction member is supported by the plurality of ribs, the gas The plurality of ribs may flow through the gap between each other. The sample extraction device of claim 3, wherein the column member further has a cover portion extending from the second upper portion and an O-ring disposed above the semi-permeable membrane, the cover portion sealing the second upper portion The opening of the O ring is used to fix the semipermeable membrane. The sample extraction device of claim 3, wherein the third long column portion of the connecting member has a knurling for clamping the filter film, and an inner diameter of the third upper portion is larger than the third long column portion An inner diameter of the third long column portion that is larger than an inner diameter of the third lower portion, whereby the third lower portion or the third long post portion cooperates to abut the second upper portion or the first portion of the column member The inner perimeter of the two long columns. A fluid control device used in combination with the suction member according to claim 1, comprising: a pipe body, a push rod disposed in the pipe body, and an attachment member connected to the lower portion of the pipe body, wherein the component to be connected has a third upper portion, a third lower portion, and a third long column portion sandwiched between the third upper portion and the third lower portion and communicating with the third upper portion and the third lower portion, whereby the third lower portion or the third portion The long post portion cooperates to abut the inner circumference of the column member. The fluid control device according to claim 7, wherein an inner diameter of the third upper portion of the abutting member is larger than an inner diameter of the third long column portion, and an inner diameter of the third long column portion is larger than the third lower portion The inner diameter, whereby the third lower portion or the third long post portion cooperates to abut the inner circumference of the tubular member. A accommodating set used in combination with the sample extracting device of claim 3, comprising: an attached component accommodating portion, a column member accommodating portion adjacent to the accommodating member accommodating portion, adjacent to the column member a suction member accommodating portion of the portion, one or more heating sleeves adjacent to the suction member accommodating portion, and a one of the one or more heating sleeves or the suction member accommodating portion and accommodating the specific buffer Or more buffer sleeves, whereby the docking member, the column member, and the suction member sequentially disposed in the corresponding receiving portion are sequentially assembled and sequentially in the one or more buffer sleeves A corresponding extraction-related action is performed in the tube or the one or more heating tubes. The accommodating kit of claim 9, wherein a height of the one or more heating sleeves or the one or more buffer sleeves is slightly larger than a height of the intermediate portion of the suction member plus the suction portion, This avoids the inability to aspirate the liquid in the one or more heating sleeves or the one or more buffer sleeves. The accommodating kit of claim 10, wherein the one or more buffer sleeves are provided with aluminum foil, and respectively contain enzyme, cytolysis buffer, bonding buffer, prewash buffer, and rinsing buffer. liquid, The post-wash buffer or the elution buffer is used to perform the corresponding extraction-related actions in sequence. The accommodating kit of claim 11, wherein the one or more buffer sleeves are only 10, respectively, sequentially accommodating the enzyme, the cytolysis buffer, the binding buffer, the prewash buffer, and the third Tube rinse buffer, post wash buffer, gas, or elution buffer for sequential extraction. The accommodating kit of claim 12, wherein the one or more heating sleeves are only two for separately removing alcohol or eluting solution, and are separately arranged for heating separately to improve heating efficiency and speed up Organic solvent evaporation rate. The accommodating set according to claim 13, wherein the accommodating member accommodating portion, the column member accommodating portion, or the absorbing member accommodating portion respectively have circular openings of different sizes to accommodate corresponding ones. Should be connected to components, column components, or suction components. The accommodating kit of claim 14, wherein the column member housing further comprises an elongated opening for receiving the cover of the column member.