A fully enclosed sample nucleic acid extraction and purification device and method

Abstract

The present invention belongs to the technical field of nucleic acid extraction, and in particular, relates to a fully enclosed sample nucleic acid extraction and purification device and method. In the present invention, the tube cover needs to be opened only when adding the sample, which ensures the closedness of the processing process; the sample and the lysis reagent are separated and can only be mixed under centrifugal state; the heating lysis and heating elution processes are all closed operations, which eliminates the evaporation problem. Combined with the principle of magnetic force moving magnetic beads, consumables (magnetic sleeves) and liquid transfer sample tips are saved. Only two sample tips are needed for the entire processing process, which saves 2 / 3 of the sample tips compared to the prior art. All operations of the present invention are carried out in a closed environment, avoiding the contamination problem of samples and nucleic acid extracts, and when handling samples with infectious risks, it has a good protective effect on operators and laboratory environments. The device and method of the present invention have fast processing speed, higher safety, low cost, and are suitable for manual and automated operations.

Description

Technical Field

[0001] The invention belongs to the technical field of nucleic acid extraction, and in particular relates to a fully enclosed sample nucleic acid extraction and purification device and method. Background Art

[0002] When nucleic acid is extracted from solid biological samples (including sample cotton swabs, exfoliated cell adhesives, cut clothing stains, ropes, etc.), an inevitable process is to lyse and centrifuge to remove the carrier and then absorb the lysate for subsequent processing. The current mainstream technology for this treatment process is to use a centrifugal cannula. Whether it is manual or automated, the inner tube containing the sample carrier and the outer tube containing the centrifugal recovery product need to be separated before the lysate can be taken out. Patent No. CN208260788U discloses a two-way test tube suitable for manual operation. After inversion, the centrifugal product can be transferred to another test tube. Although there is no need to take the cannula, the automated steps cannot be implemented, and the lid needs to be manually unscrewed to take out the lysate.

[0003] At present, the magnetic bead sample nucleic acid extraction and purification technology is mainly divided into the following two types:

[0004] One is the liquid transfer method, which combines the lysate with the magnetic beads, adsorbs the magnetic beads through a magnetic device, leaves the magnetic beads in place, transfers the waste liquid through a liquid treatment channel, and then adds new washing liquid to wash the magnetic beads, discards the waste liquid, and finally adds an elution liquid to elute the nucleic acid bound to the magnetic beads, and transfers the elution product as a nucleic acid sample. However, this method is time-consuming, uses a large amount of consumables, and requires heating during the final elution process, which has the risk of liquid evaporation and aerosol generation.

[0005] The other is the transfer magnetic bead method, which uses a magnetic rod and a magnetic sleeve to transfer the magnetic beads between different washing solutions and elution solutions. Although this method is time-consuming, it is also easy to cause cross contamination due to the elution heating, the magnetic sleeve oscillation or rotation mixing, and there is a risk of aerosol generation. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a fully enclosed sample nucleic acid extraction and purification device and method which saves time and effort, avoids pollution and is suitable for automated batch processing of samples.

[0007] The technical solution of the present invention to solve the above technical problems is as follows: a fully enclosed sample nucleic acid extraction and purification device, which is special in that it includes a closed sample lysis container and a sample purification container;

[0008] Wherein, one side of the sample lysis container is provided with a lysis liquid chamber, a sample chamber, an extraction liquid chamber and a binding liquid chamber in parallel inwardly; the upper parts of the lysis liquid chamber, the sample chamber, the extraction liquid chamber and the binding liquid chamber are sequentially connected through a gas channel; the lysis liquid chamber, the sample chamber and the binding liquid chamber are respectively provided with a filter I, a filter II and a filter III, and the filter I, the filter II and the filter III respectively divide the lysis liquid chamber, the sample chamber and the binding liquid chamber into an upper and a lower section; the lower section of the lysis liquid chamber is connected to the gas channel. The upper section of the sample chamber is connected to the upper section of the sample chamber through liquid channel I; the lower section of the sample chamber is connected to the extraction liquid chamber through liquid channel II; the lower section of the binding liquid chamber is connected to the extraction liquid chamber through liquid channel III; the top of the sample chamber is provided with an injection port extending out of the sample lysis container, the outer surface of the injection port is provided with an external thread, and the injection port is threadedly connected to a tube cover with an internal thread on the inner surface; the tops of the lysis liquid chamber and the binding liquid chamber are both sealed with tin foil; the top of the extraction liquid chamber is a puncturable heat-sealed film;

[0009] One side of the sample purification container is provided with a mixing chamber, a first washing chamber, a second washing chamber and an elution chamber which are connected to each other at the top side by side; the tops of the mixing chamber, the first washing chamber, the second washing chamber and the elution chamber are all puncturable heat-sealed films; the mixing chamber is provided with magnetic beads which can move under the action of external magnetic force.

[0010] Furthermore, the pore size of the filter I is 1-20 μm; the pore size of the filter II is 0.22-0.85 μm; and the pore size of the filter III is 0.22-0.85 μm.

[0011] The beneficial effect of adopting the above further solution is that the pore size of the filter is controlled to avoid clogging of each liquid channel.

[0012] Furthermore, the diameters of the liquid channels I, II and III are 0.5-3 mm.

[0013] The beneficial effect of adopting the above further solution is that the chambers are connected through the gas channel to ensure the balance of gas pressure, so that the liquid in each chamber is transferred through the liquid channel by relying on centrifugal force.

[0014] Furthermore, there are multiple lysis solution chambers.

[0015] Furthermore, the sample lysis container and the sample purification container are both made of transparent materials; preferably, the sample lysis container and the sample purification container are made into a sample lysis reagent card and a sample purification reagent card.

[0016] The second object of the present invention is to provide a method for extracting and purifying nucleic acid using the above-mentioned fully enclosed sample nucleic acid extraction and purification device, the steps of which are as follows:

[0017] (1) Mixing: adding lysis solution to the lysis solution chamber; adding binding solution to the binding solution chamber; inserting the solid biological sample to be extracted into the sample chamber and covering the tube cover; placing the sample lysis container in a centrifuge and performing low-speed centrifugation at a speed of 200-500 r / min. Under the action of centrifugal force, the lysis solution in the lysis solution chamber enters the sample chamber through liquid channel I until the lysis solution completely covers the solid biological sample, and then the centrifugation is stopped;

[0018] (2) Lysis: The sample lysis container is heated to 37-95°C to complete the lysis of the solid biological sample; the sample lysis container is then placed in a centrifuge and centrifuged at a speed of 2000-12000 r / min. Under the action of centrifugal force, the nucleic acid extract in the sample chamber enters the extraction chamber through liquid channel II, and at the same time, the liquid in the binding liquid chamber also enters the extraction chamber through liquid channel III;

[0019] (3) Purification: The nucleic acid extract in the extract chamber is taken out to the sample purification container by a pipette through the punctureable heat-sealed film at the top of the extract chamber. In the mixing chamber, the nucleic acid extract combines with the magnetic beads, and under the action of the external magnetic force, the magnetic beads are adsorbed and moved along the inner wall to the first washing chamber, the second washing chamber, and finally into the elution chamber, where they are heated and eluted at 37-70°C. The purified nucleic acid extract is obtained.

[0020] Further, when the magnetic beads are moved to the first washing chamber and the second washing chamber respectively, the external magnetic force is withdrawn, and the magnetic beads are oscillated and suspended three times in the first washing chamber and the second washing chamber respectively.

[0021] The characteristics and beneficial effects of the present invention are:

[0022] In the present invention, the tube cover only needs to be opened when adding the sample, which ensures the closedness of the processing process; the sample and the lysis reagent are separated and can only be mixed under centrifugal state; the heating lysis and heating elution processes are closed operations, which eliminates the evaporation problem. Combined with the principle of magnetic force moving magnetic beads, consumables (magnetic sleeves) and liquid transfer sample tips are saved. Only two sample tips are needed for the whole processing process, which saves 2 / 3 of the sample tips compared to the prior art. All operations of the present invention are carried out in a closed environment, avoiding the contamination problem of samples and nucleic acid extracts, and when handling samples with infectious risks, it has a good protective effect on operators and laboratory environments. The device and method of the present invention have fast processing speed, higher safety, low cost, and are suitable for manual and automated operations. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the structure of the sample lysis container;

[0024] Figure 2It is a schematic diagram of the structure of the sample purification container;

[0025] In the figure, 1, sample lysis container; 1-1, lysis liquid chamber; 1-2, sample chamber; 1-3, extraction liquid chamber; 1-4, binding liquid chamber; 1-5, gas channel; 1-6, filter I; 1-7, filter II; 1-8, filter III; 1-9, liquid channel I; 1-10, liquid channel II; 1-11, liquid channel III; 1-12, tube cover;

[0026] 2. Sample purification container; 2-1. Mixing chamber; 2-2. First washing chamber; 2-3. Second washing chamber; 2-4. Elution chamber; 2-5. Magnetic beads. DETAILED DESCRIPTION

[0027] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0028] A fully enclosed sample nucleic acid extraction and purification device, which is special in that it includes a closed sample lysis container 1 and a sample purification container 2;

[0029] Among them, one side of the sample lysis container is provided with a lysis liquid chamber 1-1, a sample chamber 1-2, an extraction liquid chamber 1-3 and a binding liquid chamber 1-4 in parallel inwardly; the upper parts of the lysis liquid chamber, the sample chamber, the extraction liquid chamber and the binding liquid chamber are connected in sequence through a gas channel 1-5; the lysis liquid chamber, the sample chamber and the binding liquid chamber are respectively provided with a filter I 1-6, a filter II 1-7 and a filter III 1-8, and the filter I, the filter II and the filter III respectively divide the lysis liquid chamber, the sample chamber and the binding liquid chamber into upper and lower sections; the lysis liquid chamber The lower section of the chamber is connected to the upper section of the sample chamber through liquid channel I1-9; the lower section of the sample chamber is connected to the extraction liquid chamber through liquid channel II1-10; the lower section of the binding liquid chamber is connected to the extraction liquid chamber through liquid channel III1-11; the top of the sample chamber is provided with an injection port extending out of the sample lysis container, the outer surface of the injection port is provided with an external thread, and the injection port is threadedly connected to a tube cover 1-12 with an internal thread on the inner surface; the tops of the lysis liquid chamber and the binding liquid chamber are both sealed with tin foil; the top of the extraction liquid chamber is a puncturable heat-sealed film;

[0030] One side of the sample purification container is provided with a mixing chamber 2-1, a first washing chamber 2-2, a second washing chamber 2-3 and an elution chamber 2-4 which are connected at the top inwardly side by side; the tops of the mixing chamber, the first washing chamber, the second washing chamber and the elution chamber are all puncturable heat-sealed films; the mixing chamber is provided with magnetic beads 2-5 which can move under the action of external magnetic force.

[0031] Further, the pore size of the filter I is 5 μm; the pore size of the filter II is 0.65 μm; the pore size of the filter III is 0.65 μm;

[0032] Furthermore, the diameters of the liquid channels I, II and III are 2 mm.

[0033] A method for extracting and purifying nucleic acid using the fully enclosed sample nucleic acid extraction and purification device, the steps are as follows:

[0034] (1) Mixing: adding lysis solution to the lysis solution chamber; adding binding solution to the binding solution chamber; inserting the solid biological sample to be extracted into the sample chamber and covering the tube with a cap; placing the sample lysis container in a centrifuge and performing low-speed centrifugation at a speed of 300 r / min. Under the action of centrifugal force, the lysis solution in the lysis solution chamber enters the sample chamber through liquid channel I until the lysis solution completely covers the solid biological sample, and then the centrifugation is stopped;

[0035] (2) Lysis: The sample lysis container is heated to 65°C to complete the lysis of the solid biological sample; the sample lysis container is then placed in a centrifuge and centrifuged at a speed of 4000 r / min. Under the action of centrifugal force, the nucleic acid extract in the sample chamber enters the extraction chamber through liquid channel II, and at the same time, the liquid in the binding liquid chamber also enters the extraction chamber through liquid channel III;

[0036] (3) Purification: The nucleic acid extract in the extract chamber is taken out to the sample purification container by a pipette through the punctureable heat-sealed film at the top of the extract chamber. In the mixing chamber, the nucleic acid extract combines with the magnetic beads, and under the action of the external magnetic force, the magnetic beads are adsorbed and moved along the inner wall to the first washing chamber, the second washing chamber, and finally into the elution chamber, where they are heated and eluted at 40°C. The purified nucleic acid extract is obtained.

[0037] Further, when the magnetic beads are moved to the first washing chamber and the second washing chamber respectively, the external magnetic force is withdrawn, and the magnetic beads are oscillated and suspended three times in the first washing chamber and the second washing chamber respectively.

[0038] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A fully enclosed sample nucleic acid extraction and purification device, characterized in that: Includes a closed sample lysis container and a sample purification container; Wherein, one side of the sample lysis container is provided with a lysis liquid chamber, a sample chamber, an extraction liquid chamber and a binding liquid chamber in parallel inwardly; the upper parts of the lysis liquid chamber, the sample chamber, the extraction liquid chamber and the binding liquid chamber are sequentially connected through a gas channel; the lysis liquid chamber, the sample chamber and the binding liquid chamber are respectively provided with a filter I, a filter II and a filter III, and the filter I, the filter II and the filter III respectively divide the lysis liquid chamber, the sample chamber and the binding liquid chamber into an upper and a lower section; the lower section of the lysis liquid chamber is connected to the gas channel. The upper section of the sample chamber is connected to the upper section of the sample chamber through liquid channel I; the lower section of the sample chamber is connected to the extraction liquid chamber through liquid channel II; the lower section of the binding liquid chamber is connected to the extraction liquid chamber through liquid channel III; the top of the sample chamber is provided with an injection port extending out of the sample lysis container, the outer surface of the injection port is provided with an external thread, and the injection port is threadedly connected to a tube cover with an internal thread on the inner surface; the tops of the lysis liquid chamber and the binding liquid chamber are both sealed with tin foil; the top of the extraction liquid chamber is a puncturable heat-sealed film; One side of the sample purification container is provided with a mixing chamber, a first washing chamber, a second washing chamber and an elution chamber connected to each other at the top side by side; the tops of the mixing chamber, the first washing chamber, the second washing chamber and the elution chamber are all puncturable heat-sealed films; the mixing chamber is provided with magnetic beads that can move under the action of external magnetic force; The pore size of the filter I is 1-20 μm; the pore size of the filter II is 0.22-0.85 μm; the pore size of the filter III is 0.22-0.85 μm; The diameters of the liquid channels I, II and III are 0.5-3 mm.

2. The fully enclosed sample nucleic acid extraction and purification device according to claim 1, characterized in that: The lysate chambers are multiple.

3. A method for extracting and purifying nucleic acid using the fully enclosed sample nucleic acid extraction and purification device according to claim 1 or 2, characterized in that: Here are the steps: (1) Mixing: adding lysis solution to the lysis solution chamber; adding binding solution to the binding solution chamber; inserting the solid biological sample to be extracted into the sample chamber and covering the tube cover; placing the sample lysis container in a centrifuge and performing low-speed centrifugation at a speed of 200-500 r / min. Under the action of centrifugal force, the lysis solution in the lysis solution chamber enters the sample chamber through liquid channel I until the lysis solution completely covers the solid biological sample, and then the centrifugation is stopped; (2) Lysis: The sample lysis container is heated to 37-95°C to complete the lysis of the solid biological sample; the sample lysis container is then placed in a centrifuge and centrifuged at a speed of 2000-12000 r / min. Under the action of centrifugal force, the nucleic acid extract in the sample chamber enters the extraction chamber through liquid channel II, and at the same time, the liquid in the binding liquid chamber also enters the extraction chamber through liquid channel III; (3) Purification: The nucleic acid extract in the extract chamber is taken out to the sample purification container by a pipette through the punctureable heat-sealed film at the top of the extract chamber. In the mixing chamber, the nucleic acid extract combines with the magnetic beads, and under the action of the external magnetic force, the magnetic beads are adsorbed and moved along the inner wall to the first washing chamber, the second washing chamber, and finally into the elution chamber, where they are heated and eluted at 37-70°C. The purified nucleic acid extract is obtained.

4. The method according to claim 3, characterized in that When the magnetic beads are moved to the first washing chamber and the second washing chamber respectively, the external magnetic force is withdrawn, and the magnetic beads are oscillated and suspended three times in the first washing chamber and the second washing chamber respectively.

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