Implant flow type cell sample preparation device and use method thereof

Abstract

The invention relates to an implant flow type cell sample preparation device and a use method thereof, the device comprises the following components in split design: a sample tube, the bottom surface of the sample tube is a plane, and the inner bottom surface of the sample tube is provided with a plurality of grooves; a plurality of sample tube holes for accommodating sample tubes are formed in the sample box; the sample processor comprises a plate and a plurality of processing pipes arranged on the plate, each processing pipe comprises a pipe column and a plurality of protrusions located at the outer bottom of the pipe column and matched with the grooves, and a hollow area is arranged on the side wall, close to the protrusions, of the pipe column and provided with a filter screen. According to the device, the processor is designed to be a plate, meanwhile, a plurality of processing pipes are arranged on one side of the plate, filter screens and protrusions are arranged at the bottoms of the processing pipes, grooves matched with the protrusions are formed in the bottoms of sample pipes, the depth of the grooves is larger than the height of the protrusions, it is guaranteed that processed blades can be broken but not extruded, in addition, processing liquid can be directly added or sucked out, and the processing efficiency is improved. The sample preparation efficiency is greatly improved.

Description

Technical Field

[0001] The invention relates to a plant flow cell sample preparation device and a use method thereof, belonging to the technical field of plant ploidy detection. Background Art

[0002] Ploidy breeding is an important research topic in plant breeding. In recent years, with the gradual maturity of mutagenesis technology, plant polyploidy breeding has gradually entered the era of large-scale creation. How to quickly, accurately and efficiently identify the ploidy of mutagenic materials has become a limiting factor. Flow cytometry (FCM) is widely used in plant ploidy identification.

[0003] The leaves collected for plant ploidy detection are mainly taken from the wild and the field. In order to avoid DNA degradation caused by high temperature during transportation, which will affect the test results, young leaves should be selected in the morning or evening when collecting in the wild and the field, put into a ziplock bag, remove the air, and immediately put into an ice box filled with ice cubes, and bring it back to the room for flow testing as soon as possible.

[0004] At present, the main preparation steps of plant cell nucleus suspension are as follows: put the young leaves to be tested into a disposable culture dish or a glass culture dish, add the dissociation solution, and then quickly chop the sample with a double-sided blade, and then filter it into the sample tube with a filter, add the staining solution to the filtrate, and incubate it at low temperature to obtain the cell nucleus suspension. However, plant materials such as cotton contain more phenolic substances, which are very easy to oxidize and affect the peak. In order to prevent the oxidation of the sample, polyvinyl pyrrolidone (PVP) needs to be added to the dissociation solution.

[0005] Due to the complexity of the operation and the limitation of the operation requirements, it is not possible to test a large number of samples at the same time, and the efficiency is very low. Therefore, improving the preparation method of the suspension is of great significance to improving the efficiency of sample detection. Summary of the invention

[0006] In view of the above technical problems, the present invention provides a plant flow cytometer sample preparation device and a method for using the same. In the device, a sample processor is designed as a plate, and a plurality of processing tubes are arranged on one side of the plate. A filter screen and a protrusion are arranged at the bottom of the processing tube. The bottom of the sample tube is arranged in a groove matched with the protrusion. The depth of the groove is greater than the height of the protrusion, ensuring that the protrusion and the groove are spaced a certain distance apart. In this way, leaves frozen with liquid nitrogen can be broken but not squeezed after being processed by the processor. At the same time, lysis solution can be directly added or processing solution can be sucked out through the processing tube. In addition, multiple processing tubes can be processed and filtered at the same time, thereby greatly improving the sample preparation efficiency.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A plant flow cell sample preparation device includes the following components of split design:

[0009] A number of sample tubes, each having a flat bottom surface and a number of grooves disposed on its inner bottom surface;

[0010] A sample box, wherein the sample box is provided with a plurality of sample tube holes for accommodating the sample tubes;

[0011] A sample processor includes a plate and a plurality of processing tubes arranged on the plate, wherein the processing tubes include a tube column and a plurality of protrusions located at the outer bottom of the tube column and adapted to the plurality of grooves, a hollow area is arranged on the side wall of the tube column close to the protrusions, and the hollow area is equipped with a filter.

[0012] In the plant flow cell sample preparation device, preferably, a plurality of the grooves are arranged in rows on the inner bottom surface of the sample tube, and two adjacent grooves in each row are in contact with each other.

[0013] In the plant flow cytometer sample preparation device, preferably, a plurality of the grooves are discretely distributed on the inner bottom surface of the sample tube.

[0014] In the plant flow cytometer sample preparation device, preferably, the shape of the groove includes any one of a semicircular shape, a conical shape, an elliptical shape or a cylindrical shape.

[0015] In the plant flow cell sample preparation device, preferably, the sample box comprises a sample box rack, the sample box rack is provided with three transverse plates, and the sample tube holes are provided on two adjacent transverse plates.

[0016] In the plant flow cytometer sample preparation device, preferably, the sample tube further comprises a sample tube cover, and the sample tube cover is detachably buckled on the sample tube, and the sample tube cover is arranged individually or in a row or array.

[0017] The plant flow cytometer sample preparation device preferably further comprises a gasket, which is detachably disposed around the four sides of the sample box.

[0018] In the plant flow cell sample preparation device, preferably, the plate is detachably connected to the processing tube.

[0019] In the plant flow cell sample preparation device, preferably, the plate is provided with a plate protrusion, and the opening end surface of the processing tube is provided with a processing tube groove matched with the plate protrusion.

[0020] A second aspect of the present invention provides a method for using the plant flow cytometer sample preparation device, comprising the following steps:

[0021] Collecting leaves and placing them into the sample tube, covering the sample tube, and refrigerating or freezing the tube;

[0022] During the test, the sample tube is taken out, the cover of the sample tube is removed, the lower part of the sample tube is clamped with an anti-scalding clamp and frozen in liquid nitrogen, and the sample processor is frozen in liquid nitrogen at the same time, the sample tube and the sample processor are taken out, and the sample processor is quickly inserted into the sample tube and pressed to the bottom, and the process is repeated several times, and the gasket is placed;

[0023] Add the lysate into the sample processor and let it stand, during which the sample processor can be moved up and down to mix;

[0024] Remove the gasket, aspirate the lysate and transfer it to the sample loading tube matched with the instrument, add dye, and finally test on the instrument.

[0025] The present invention adopts the above technical solution, which has the following advantages:

[0026] 1. The present invention designs the processor into a plate, and arranges a plurality of processing tubes on one side of the plate. A filter screen and a protrusion are arranged at the bottom of the processing tube, and a groove matched with the protrusion is arranged at the bottom of the sample tube. In this way, while the leaves are crushed, it is convenient to add reagents and absorb filtrate. In addition, multiple processing tubes process and filter at the same time, which greatly improves the sample preparation efficiency.

[0027] 2. The present invention designs the processing tube and the plate to be detachable. When a small number of blades need to be tested, the processing tube can be disassembled for easy operation.

[0028] 3. The device of the present invention is suitable for long-term storage and large amounts of samples, and is simple to operate and highly efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A schematic diagram of a sample tube provided in one embodiment of the present invention;

[0030] Figure 2 A schematic diagram of a sample box provided for this embodiment of the present invention;

[0031] Figure 3 A schematic diagram of a sample processor provided for this embodiment of the present invention;

[0032] Figure 4 A schematic diagram of a processing tube in a sample processor provided in this embodiment of the present invention;

[0033] Figure 5 for Figure 4 A top view of

[0034] Figure 6 This is a schematic diagram of the sample tube being placed in the sample box;

[0035] Figure 7 for Figure 6 Side view of

[0036] The marks in the figure are as follows:

[0037] 1-sample tube, 101-groove, 102-sample tube cover; 2-sample box, 201-sample tube hole, 202-sample box rack, 203-cross plate; 3-sample processor, 301-plate, 302-processing tube, 3021-tube column, 3022-protrusion, 3023-filter. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary persons in the art without creative work are within the scope of protection of the present invention.

[0039] Unless otherwise defined, the technical or scientific terms used in the present invention shall have the usual meanings understood by persons with ordinary skills in the field to which the present invention belongs. The words "first", "second", "third", "fourth" and similar terms used in the present invention do not indicate any order, quantity or importance, but are only used to distinguish different components. Words such as "include" or "comprise" and the like mean that the elements or objects appearing before the word include the elements or objects listed after the word and their equivalents, without excluding other elements or objects. Words such as "connect" or "connected" and the like are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect.

[0040] For ease of description, spatially relative terms may be used herein to describe the relationship of one element or feature relative to another element or feature as shown in the figures, such as "inside", "outside", "inner side", "outer side", "below", "above", etc. Such spatially relative terms are intended to include different orientations of the device in use or operation in addition to the orientation depicted in the figures.

[0041] At present, the main preparation steps of plant cell nucleus suspension are as follows: put the young leaves to be tested into a disposable culture dish or a glass culture dish, add the dissociation solution, and then quickly chop the sample with a double-sided blade, and then filter it into the sample tube with a filter, add the staining solution to the filtrate, and incubate it at low temperature to obtain the cell nucleus suspension. However, plant materials such as cotton contain more phenolic substances, which are very easy to oxidize and affect the peak. In order to prevent the oxidation of the sample, polyvinyl pyrrolidone (PVP) needs to be added to the dissociation solution.

[0042] Due to the complexity of the operation and the limitation of the operation requirements, it is not possible to test a large number of samples at the same time, and the efficiency is very low. Therefore, improving the preparation method of the suspension is of great significance to improving the efficiency of sample detection.

[0043] Based on the above problems, the present invention provides a plant flow cytometer sample preparation device and a use method thereof. In the device of the present invention, a processor is designed as a plate, and a plurality of processing tubes are arranged on one side of the plate. A filter screen and a protrusion are arranged at the bottom of the processing tube, and the bottom of the sample tube is arranged in a groove matched with the protrusion. The depth of the groove is greater than the height of the protrusion, so that the protrusion and the groove are spaced a certain distance apart. In this way, the leaves frozen by liquid nitrogen can be better broken after being processed by the processor but are not subjected to excessive squeezing. At the same time, lysis solution can be directly added or processing solution can be sucked out through the processor. In addition, multiple filters are processed and filtered at the same time, thereby greatly improving the sample preparation efficiency.

[0044] like Figure 1-3 As shown, the plant flow cell sample preparation device provided by the present invention includes the following components of split design:

[0045] A plurality of sample tubes 1, each having a flat bottom surface and a plurality of grooves 101 disposed on the inner bottom surface thereof;

[0046] A sample box 2, wherein the sample box 2 is provided with a plurality of sample tube holes 201 for accommodating the sample tubes 1;

[0047] The sample processor 3 includes a plate 301 and a plurality of processing tubes 302 arranged on the plate 301. The processing tubes 302 include a tube column 3021 and a plurality of protrusions 3022 located at the outer bottom of the tube column 3021 and adapted to the plurality of grooves 101 (the depth of the groove 101 is greater than the height of the protrusion 3022, so that the leaves frozen by liquid nitrogen are brittle, and the leaves are broken by the external force of the liquid nitrogen flow and the curvature of the bottom of the groove 101). A hollow area is provided on the side wall of the tube column 3021 close to the protrusion 3022, and the hollow area is equipped with a filter 3023. Four filter screens 3023 with a length of 0.5 cm and a width of 0.3 cm are distributed in the hollow area, and the filter screen density is 50 um.

[0048] Furthermore, a number is engraved on the tube wall of the sample tube 1 (not shown in the figure) or a number is engraved next to the sample tube hole 201 of the sample box 2 (not shown in the figure) to facilitate the management of the samples.

[0049] Further, a plurality of grooves 101 are arranged in a row on the inner bottom surface of the sample tube 1, and two adjacent grooves 101 in each row are in contact with each other. Alternatively, a plurality of grooves 101 are discretely distributed on the inner bottom surface of the sample tube 1. The present invention does not specifically limit the dispersion mode of the grooves 101, and any distribution mode belongs to the protection scope of the present invention. In some preferred embodiments of the present invention, the shape of the groove 101 includes any one of a semicircular, conical, elliptical or cylindrical shape.

[0050] Furthermore, if Figure 2 As shown, the sample box 2 includes a sample box frame 202, and three horizontal plates 203 are arranged on the sample box frame 202. The three horizontal plates 203 are arranged in an upper, middle and lower interval manner, and the sample tube holes 201 are arranged on the upper two horizontal plates 203. The sample tube holes 201 are arranged in an array on the horizontal plates 203, and preferably, the sample tube holes 201 are arranged on the horizontal plates 203 in 5 rows and 10 columns.

[0051] like Figure 3 As shown, the plate 301 in the processing tube 302 of the present invention is detachably connected to the processing tube 302. Specifically, a plate protrusion is provided on the plate 301, and a processing tube groove matching the plate protrusion is provided on the open end surface of the processing tube 302. The tube column 3021 is cylindrical, 1.5 cm high and 1.3 cm in diameter.

[0052] In a preferred embodiment of the present invention, Figure 1 As shown, the sample tube 1 also includes a soft sample tube cover 102 (e.g., a rubber soft cover), which is detachably buckled on the sample tube 1. The sample tube 1 has a height of 1.5 cm and a diameter of 1.5 cm. The sample tube cover 102 is arranged individually or in a row or array. The sample tube cover 102 is arranged in a row or array, so that after the sample tube 1 is placed in the sample tube hole 201 of the sample box 2, the sample tube cover 102 can be quickly buckled.

[0053] Furthermore, the plant flow cytometer sample preparation device of the present invention also includes a gasket, which is a soft open type and detachably surrounds the sample box 2, thereby increasing the space between the sample tube 1 and the sample processor 3, so that the dissociation solution and the sample are fully mixed.

[0054] The second aspect of the present invention also provides a method for using the plant flow cell sample preparation device, comprising the following steps: 2 Put the cotton leaves into sample tube 1 according to the serial number, cover the sample tube cap 102, put them into a foam box with ice packs and bring them back to the room and store them in a refrigerator at 4℃. For long-term storage: quick freeze them in liquid nitrogen at -196℃ for 10 seconds and transfer them to a refrigerator at -80℃ for storage.

[0055] Number the sample tubes of the test instrument in advance, take out the sample tube 1 from the refrigerator, remove the sample tube cover 102, use the anti-scalding clamp to clamp the lower and middle part of the sample tube 1 in -196℃ liquid nitrogen for 10 seconds, and at the same time, freeze the sample processor 3 in -196℃ liquid nitrogen for 10 seconds. After taking out the sample tube 1 and the sample processor 3, quickly insert the sample processor 3 into the sample tube 1 and press it to the bottom, which can be repeated 2-3 times, and place the gasket.

[0056] Use a pipette or a pipette gun to add the lysate into the sample processor 3 and let it stand for 5 minutes. During this time, move it up and down 2-3 times to mix it.

[0057] Remove the gasket, use a pipette or a pipette gun to transfer the lysate to the sample tube of the test instrument, add dye, and finally test on the instrument.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A plant flow cell sample preparation device, characterized in that: It includes the following components of split design: A plurality of sample tubes (1), wherein the bottom surface of the sample tube (1) is a plane, and a plurality of grooves (101) are arranged on the inner bottom surface thereof; A sample box (2), wherein the sample box (2) is provided with a plurality of sample tube holes (201) for accommodating the sample tubes (1); A sample processor (3) comprises a plate (301) and a plurality of processing tubes (302) arranged on the plate (301), wherein the processing tubes (302) comprise a tube column (3021) and a plurality of protrusions (3022) located at the outer bottom of the tube column (3021) and adapted to the plurality of grooves (101), wherein the depth of the groove (101) is greater than the height of the protrusion (3022), and a hollow area is arranged on the side wall of the tube column (3021) close to the protrusion (3022), and the hollow area is equipped with a filter screen (3023).

2. The plant flow cytometer sample preparation device according to claim 1, characterized in that: A plurality of the grooves (101) are distributed in a row on the inner bottom surface of the sample tube (1), and two adjacent grooves (101) in each row are in contact with each other.

3. The plant flow cytometer sample preparation device according to claim 1, characterized in that: A plurality of grooves (101) are discretely distributed on the inner bottom surface of the sample tube (1).

4. The plant flow cell sample preparation device according to claim 2 or 3, characterized in that: The shape of the groove (101) includes any one of a semicircular shape, a conical shape, an elliptical shape or a cylindrical shape.

5. The plant flow cytometer sample preparation device according to claim 4, characterized in that: The sample box (2) comprises a sample box frame (202), three transverse plates (203) are arranged on the sample box frame (202), and the sample tube holes (201) are arranged on two adjacent transverse plates (203).

6. The plant flow cytometer sample preparation device according to claim 5, characterized in that: The sample tube (1) further comprises a sample tube cover (102), wherein the sample tube cover (102) is detachably buckled onto the sample tube (1), and the sample tube cover (102) is arranged individually or in a row or array.

7. The plant flow cytometer sample preparation device according to claim 6, characterized in that: It also comprises a gasket, which is detachably arranged around the four sides of the sample box (2).

8. The plant flow cytometer sample preparation device according to claim 7, characterized in that: The plate (301) is detachably connected to the processing tube (302).

9. The plant flow cytometer sample preparation device according to claim 8, characterized in that: The plate (301) is provided with a plate protrusion, and the opening end surface of the processing tube (302) is provided with a processing tube groove that matches the plate protrusion.

10. A method for using the plant flow cytometric sample preparation device according to any one of claims 7 to 9, characterized in that: The steps include: Collecting leaves and placing them into the sample tube (1), covering the sample tube cover (102), and refrigerating or freezing them; During the test, the sample tube (1) is taken out, the sample tube cover (102) is removed, the lower middle part of the sample tube (1) is clamped with an anti-scalding clamp and frozen in liquid nitrogen, and the sample processor (3) is simultaneously frozen in liquid nitrogen, the sample tube (1) and the sample processor (3) are taken out, the sample processor (3) is quickly inserted into the sample tube (1) and pressed to the bottom, and the process is repeated several times, and the gasket is placed; Add the lysate into the sample processor (3) and let it stand, during which the sample processor (3) can be moved up and down to mix; Remove the gasket, aspirate the lysate and transfer it to the sample loading tube matched with the instrument, add dye, and finally test on the instrument.

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