Manual centrifugal simple device for nucleic acid detection pretreatment

Abstract

The utility model discloses a manual centrifugal simple device for nucleic acid detection pretreatment, and relates to the field of nucleic acid detection, a cylindrical fixed seat is fixedly connected in a first shell, the center of the cylindrical fixed seat coincides with the center of the first shell, the fixed seat is provided with a first clamping groove, a second clamping groove and a third clamping groove along the radial direction, the first clamping groove is provided with a lysate pipe, and the second clamping groove is provided with an enzyme-free water pipe; a nucleic acid purification column tube is mounted in the third clamping groove; the center of the upper surface of the fixing base extends in the vertical direction to form a protruding ring, the center coincides with the center of the upper surface of the fixing base, the center of the inner wall of the second shell is fixedly connected with a fixing column, the fixing column is fixedly inserted into the protruding ring, the first shell and the second shell form a closed protective shell, and two rope penetrating holes symmetrical about the center of the protective shell are formed in the protective shell. The first shell, the fixing base, the fixing column and the second shell are sequentially communicated through the rope penetrating holes, a pull rope penetrates through each rope penetrating hole, and low-cost and simple separation and extraction of nucleic acid in a detection sample can be achieved.

Description

TECHNICAL FIELD

[0001] The utility model relates to the field of nucleic acid detection, and specifically relates to a simple manual centrifugal device for nucleic acid detection pretreatment. BACKGROUND

[0002] As an important method in the field of pathogen detection, nucleic acid detection in molecular biology detection has the characteristics of strong detection capacity and fast detection speed. Before nucleic acid detection, a series of pretreatments are often required for the sample, including removing proteins and other substances that affect the nucleic acid detection reaction. During this process, various conditions such as temperature, time, and pH value need to be strictly controlled to ensure that the nucleic acid in the sample is efficiently separated, so that the nucleic acid can be analyzed and detected by subsequent equipment, and finally the conclusion information of whether the target pathogen nucleic acid is in the sample is obtained.

[0003] Nucleic acid pretreatment is one of the key steps to ensure the accuracy and reliability of nucleic acid detection. This process provides high-precision, high-purity nucleic acid templates, providing a reliable foundation for subsequent nucleic acid detection. Since in the rapid detection at home, residents detect themselves at home through rapid detection tools, the required method needs to be simple, fast, and does not require professional operation. Therefore, developing a simple, fast, and low-cost pretreatment method is conducive to promoting the application of nucleic acid detection in rapid detection at home.

[0004] Currently, when designing nucleic acid detection pretreatment devices, it is basically based on the two commonly used methods of magnetic bead method and membrane filtration method. The magnetic bead method uses the adsorption of magnetic beads to nucleic acid under certain conditions. After the adsorption of nucleic acid is completed under certain liquid conditions, the nucleic acid is purified by washing, and then released under new liquid conditions. This method has the advantages of high extraction purity and fast speed, but it also has high requirements for equipment. This problem hinders the application of this method in small batch samples, especially in rapid detection at home. The other method is membrane filtration method, which uses the characteristics of different adsorption capacities of silica gel membrane to nucleic acid in different liquid environments to separate and extract nucleic acid in the sample. This method is suitable for small batch samples, but it needs a centrifuge, which hinders its application in rapid detection at home. UTILITY MODEL CONTENT

[0005] In view of the deficiencies in the prior art, the utility model provides a simple manual centrifugal device for nucleic acid pretreatment, which can realize low-cost and simple separation and extraction of nucleic acid in the detection sample.

[0006] The utility model is realized by the following technical solutions:

[0007] A kind of simple device for manual centrifugation of nucleic acid detection pretreatment, including fixed seat and the first shell and second shell of being spherical bowl;

[0008] The cylindrical fixed seat is fixedly connected in the first shell, the center of fixed seat coincides with the center of first shell, first clamping groove, second clamping groove and third clamping groove are opened along the radial direction of fixed seat, first clamping groove is used to install lysate pipe, second clamping groove is used to install enzyme-free water pipe, and third clamping groove is used to install nucleic acid purification column pipe;

[0009] The center of the upper surface of the fixed seat is provided with a convex ring extending in the vertical direction, the center of the convex ring coincides with the center of the upper surface of the fixed seat, the inner wall of the second shell is fixedly connected with a fixed column inserted into the convex ring, the first shell and the second shell form a closed protective shell, two rope holes symmetrically arranged about the center of the protective shell are formed on the protective shell, the rope holes sequentially communicate the first shell, the fixed seat, the fixed column and the second shell, and one pull rope is arranged in each rope hole.

[0010] Further improvement of the utility model lies in that:

[0011] The first shell and the second shell are identical in shape, and the opening of the first shell and the opening of the second shell are arranged in contact.

[0012] The first clamping groove, the second clamping groove and the third clamping groove are all T-shaped, the inner side end is wider than the outer side end, and the outer side end and the upper and lower ends are all communicated with the outside.

[0013] The inner side ends of the first clamping groove, the second clamping groove and the third clamping groove are identical in distance from the convex ring.

[0014] The center axes of the first clamping groove and the second clamping groove coincide and pass through the center of the fixed seat.

[0015] The first clamping groove and the second clamping groove are both one, and the third clamping groove is two, the center axes of the two third clamping grooves coincide and pass through the center of the fixed seat.

[0016] Four fourth clamping grooves in T-shaped structure are further arranged along the radial direction on the fixed seat, the inner side end is wider than the outer side end, the outer side end and the upper and lower ends of the fourth clamping groove are all communicated with the outside, the first clamping groove, the second clamping groove and the two third clamping grooves form a cross-shaped structure, the length of the wide end of the first clamping groove, the second clamping groove and the third clamping groove is identical, and the four fourth clamping grooves are evenly arranged on the fixed seat.

[0017] The center of the inner wall of the first shell is fixedly connected with a first fixed column, the fixed column fixedly connected with the center of the inner wall of the second shell is a second fixed column, a first convex ring is arranged on the center of the lower surface of the fixed seat extending in the vertical direction, the convex ring on the center of the upper surface of the fixed seat is a second convex ring, and the first fixed column is fixedly inserted into the first convex ring.

[0018] The inner and outer diameters of the first and second convex rings are the same, and the diameters of the first and second fixed columns are the same.

[0019] The first and second outer shells and the first and second fixed columns are provided with first and second through holes, the second outer shell and the second fixed column are provided with third and fourth through holes, and the fixed seat is provided with fifth and sixth through holes, the centers of the first, third and fifth through holes are aligned to form one rope passing hole, and the centers of the second, fourth and sixth through holes are aligned to form another rope passing hole.

[0020] The inner diameters of the first, third, fifth, second, fourth and sixth through holes are the same.

[0021] Compared with the prior art, the manual centrifugal simple device has the following beneficial technical effects:

[0022] The first and second outer shells are in the shape of a spherical bowl and can form a closed protective shell to provide a light-proof environment for the internal reagent, the fixed seat is in the shape of a cylinder and the center of the fixed seat coincides with the center of the first outer shell, so that the entire simple device can be kept relatively stable, the first clamping groove can be used to install the lysate pipe, the second clamping groove can be used to install the enzyme-free water pipe, and the third clamping groove can be used to install the nucleic acid purification column pipe, the center of the upper surface of the fixed seat is provided with a convex ring extending in the vertical direction and coinciding with the center of the upper surface of the fixed seat, and the center of the inner wall of the second outer shell is fixedly connected with a fixed column that is inserted and matched with the convex ring, so that the fixed column can be fixedly inserted in the convex ring, the rope passing holes are sequentially connected with the first outer shell, the fixed seat, the fixed column and the second outer shell, and a pull rope can be inserted to form a pull whistle, the entire device can be quickly rotated to realize centrifugation during the separation and washing process, and the centrifugal force required for the column membrane method to separate and purify nucleic acid is provided. Since the entire first and second outer shells, the fixed column and the fixed seat can be made of plastic, the entire device is not only light but also low in cost, and manual centrifugation can be conveniently achieved. Before use, the lysate pipe, the enzyme-free water pipe and the nucleic acid purification column pipe are installed in the corresponding clamping grooves. During the pretreatment, the sample can be inserted into the lysate and stirred to completely release the nucleic acid, then the nucleic acid purification column pipe is added for extraction, and then the enzyme-free water is added for centrifugation, so that the nucleic acid is finally enriched in the nucleic acid extraction liquid in the lower pipe of the nucleic acid purification column pipe. While reducing air resistance and effectively improving the rotation speed, the entire protective shell can prevent the pipes in the clamping grooves from being separated and hurting people, and effectively reduces the need for special instruments such as a centrifuge or a suction filter pump, effectively promoting the application of the method in family rapid detection. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1a A top view of the manual centrifugal simple device without a pull rope.

[0024] Figure 1b The main view of the manual centrifugal simple device without the pull rope.

[0025] Figure 2 The separated component view of the manual centrifugal simple device without the pull rope.

[0026] Figure 3 The schematic view of installing various tubes after removing the pull rope and the first shell of the manual centrifugal simple device.

[0027] Figure 4 The structure schematic view of the fixed seat.

[0028] Figure 5 The Figure 4 The nucleic acid purification column pipe schematic view of installing two nucleic acid purification column pipes.

[0029] Figure 6 The schematic view of showing the internal structure after installing two nucleic acid purification column pipes and removing the pull rope and part of the first shell of the manual centrifugal simple device.

[0030] In the figure: 1 - first shell, 2 - second shell, 11 - first through hole, 12 - second through hole, 13 - first fixed column, 21 - third through hole, 22 - fourth through hole, 23 - second fixed column, 3 - fixed seat, 31 - fifth through hole, 32 - sixth through hole, 33 - first clamping groove, 34 - second clamping groove, 35 - third clamping groove, 36 - fourth clamping groove, 37 - first convex ring, 38 - second convex ring, 4 - lysis solution pipe, 5 - enzyme-free water pipe, 6 - freeze-dried reagent bead reaction tube, 7 - nucleic acid purification column pipe. DETAILED DESCRIPTION

[0031] The utility model will be further explained in detail in combination with the drawings and specific embodiments, which is the explanation of the utility model but not the limitation.

[0032] The utility model discloses a nucleic acid detection pretreatment's manual centrifugal simple device can be used to store reaction reagent and carry out the centrifugal of nucleic acid extraction process, including first shell 1 and second shell 2, and fixed seat 3 and two pull ropes, as Figure 1a And Figure 1b As shown, the shape of first shell 1 and second shell 2 is all ball bowl, shape and size are same, the opening of first shell 1 and the opening of second shell 2 are contacted. Figure 6 As shown, the fixed seat 3 is fixed in the center of first shell 1, and the centers coincide, and the fixed seat 3 is cylindrical. Figure 4As shown, as an example, a first clamping groove 33, a second clamping groove 34 and two third clamping grooves 35 are radially formed, the central axes of the two third clamping grooves 35 coincide and pass through the center of the fixing base 3, the first clamping groove 33 is used to install the lysate pipe 4 (i.e. the pipe containing lysate), the second clamping groove 34 is used to install the enzyme-free water pipe 5 (i.e. the pipe containing enzyme-free water), and the third clamping groove 35 is used to install the nucleic acid purification column pipe 7 (i.e. the pipe containing the nucleic acid purification column, the lower part of the nucleic acid purification column faces the pipe bottom), as shown in Figure 5 and Figure 6 As shown, only two nucleic acid purification column pipes 7 are installed during centrifugation.

[0033] In order to further install the second shell 2, a convex ring is vertically extended on the center of the upper surface of the fixing base 3, the center of the convex ring coincides with the center of the upper surface of the fixing base 3, and a fixed column is fixedly connected to the center of the inner wall of the second shell 2, which is inserted into the convex ring in a plug-in manner, so that the fixed column can be fixedly inserted into the convex ring, thereby forming a closed protective shell with the first shell 1 and the second shell 2. Finally, two rope passing holes are formed on the protective shell, which are symmetrically arranged about the center of the protective shell, and the two rope passing holes are sequentially communicated with the first shell 1, the fixing base 3, the fixed column and the second shell 2. One pulling rope is inserted into each rope passing hole, so that the entire simple device can form a whistle, and the entire device can be quickly rotated by manually pulling the pulling rope. Since the first shell 1, the second shell 2, the fixed column and the fixing base 3 are made of plastic, they are light and low in cost, and can be easily manually centrifuged.

[0034] Specifically, the first clamping groove 33, the second clamping groove 34 and the third clamping groove 35 are all T-shaped, and the inner side end is wider than the outer side end. The outer side end, the upper end and the lower end of each clamping groove are communicated with the outside, so that the lysate pipe 4, the enzyme-free water pipe 5 and the nucleic acid purification column pipe 7 can be installed in a horizontal manner, as shown in Figure 3 The openings of the pipes are clamped in the inner side end of the corresponding clamping groove. When the clamping grooves are formed, the distance between the inner side end of the first clamping groove 33, the second clamping groove 34 and the third clamping groove 35 and the convex ring is the same, and the central axes of the first clamping groove 33 and the second clamping groove 34 coincide and pass through the center of the fixing base 3.

[0035] In addition, in order to facilitate the storage of the freeze-dried reagent bead reaction tube 6 and meet the needs of subsequent reactions, as shown in Figure 3As shown, four fourth clamping grooves 36 in T-shaped structure are radially formed on the fixing base 3, the inner side is wider than the outer side, the outer side and the upper and lower ends of the fourth clamping groove 36 are in communication with the outside, and the inner side is wider than the outer side, the first clamping groove 33, the second clamping groove 34 and the two third clamping grooves 35 form a cross-shaped structure, the length of the wide end of the first clamping groove 33, the second clamping groove 34 and the third clamping groove 35 is the same, and the four fourth clamping grooves 36 are uniformly arranged on the fixing base 3, so as to form a cross-shaped structure, so that the slot on the fixing base 3 presents a certain symmetry, which is convenient for keeping relatively stable during rotation. Of course, in order to further reduce the weight of the fixing base 3, a symmetric V-shaped structure is further formed on both sides of each fourth clamping groove 36, which is in communication with the outside while leaving a certain supporting part with the corresponding clamping groove.

[0036] In order to further make the entire simple device have a detachable function, a first fixed column 13 is fixedly connected to the center of the inner wall of the first shell 1. In order to facilitate description, the fixed column fixedly connected to the center of the inner wall of the second shell 2 mentioned earlier is a second fixed column 23. The first protruding ring 37 vertically extends from the center of the lower surface of the fixing base 3, and the protruding ring mentioned earlier on the upper surface of the fixing base 3 is a second protruding ring 38. The first fixed column 13 is fixedly inserted into the first protruding ring 37. Since the inner and outer diameters of the first protruding ring 37 and the second protruding ring 38 are the same, the diameters of the corresponding first fixed column 13 and the second fixed column 23 are the same.

[0037] Two rope passing holes need to be specifically formed on the first shell 1, the first fixed column 13, the second shell 2, the second fixed column 23 and the fixing base 3, as shown in Figure 2 and Figure 4 The first shell 1 and the first fixed column 13 are regarded as a whole, and the first through hole 11 and the second through hole 12 are formed thereon. The second shell 2 and the second fixed column 23 are regarded as a whole, and the third through hole 21 and the fourth through hole 22 are formed thereon. The fixing base 3 is provided with the fifth through hole 31 and the sixth through hole 32. Further, the centers of the first through hole 11, the third through hole 21 and the fifth through hole 31 are aligned, and one of the rope passing holes can be formed. The centers of the second through hole 12, the fourth through hole 22 and the sixth through hole 32 are aligned, and the other rope passing hole can be formed. Specifically, the inner diameters of the first through hole 11, the third through hole 21, the fifth through hole 31, the second through hole 12, the fourth through hole 22 and the sixth through hole 32 are the same, so that the pull rope can swing uniformly.

[0038] It should be noted that before use, the lysis solution pipe 4, the enzyme-free water pipe 5, the reagent bead reaction pipe 6 and the nucleic acid purification column pipe 7 are installed in the corresponding clamping grooves.

[0039] The utility model relates to a simple manual centrifugal device for nucleic acid detection pretreatment, taking the nucleic acid extraction process of the new coronavirus as an example, the specific pretreatment, that is, the operation process of extracting the nucleic acid of the new coronavirus is as follows:

[0040] Step 1, open the first shell 1 of the simple manual centrifugal device (hereinafter referred to as the device), and remove the lysate pipe 4, the enzyme-free water pipe 5, the freeze-dried reagent bead reaction pipe 6 and the nucleic acid purification column pipe 7 fixedly installed on the fixing seat 3.

[0041] Step 2, insert the sample (nasal swab or throat swab) into the lysate and stir to completely release the nucleic acid.

[0042] Step 3, take two 200ul lysates from the lysate pipe 4, add them to two nucleic acid purification column pipes 7, install the two nucleic acid purification column pipes 7 in the fixing seat 3, and install the first shell 1 in place.

[0043] Step 4, rotate and pull the pull rope, so that the device rotates quickly, and the rotating speed reaches the highest, and the steps 3-4 are repeated 3-4 times.

[0044] Step 5, open the first shell 1 of the device, remove the nucleic acid purification column pipe 7, and pour the waste lysate extracted from the lower part of the nucleic acid purification column pipe 7 into the waste liquid cylinder.

[0045] Step 6, add 50ul enzyme-free water to the upper part of the nucleic acid purification column pipe obtained in step 5 from the enzyme-free water pipe 5, and centrifuge according to step 4, so as to wash the nucleic acid on the previous nucleic acid purification column, and finally enrich the nucleic acid in the nucleic acid extraction liquid in the lower pipe of the nucleic acid purification column pipe 7.

[0046] The standard plasmid nucleic acid extraction liquid extracted by the device is detected by ultramicro ultraviolet spectrophotometer, and the absorbance at 260nm is 3.102 OD, 2.992 OD and 2.904 OD respectively. The nucleic acid concentration is 141ng / ul, 136ng / ul and 132ng / ul respectively. Compared with the data 176.25ng / ul, 172.15ng / ul and 160.98ng / ul finally obtained by the general nucleic acid extraction kit combined with the high-speed centrifuge method, the extraction capacity is about 80% of the above-mentioned kit centrifuge method, which meets the experimental requirements.

[0047] As a supplementary step, 25ul of nucleic acid extraction liquid can be added to the aforementioned freeze-dried reagent bead reaction pipe 6 for subsequent reaction to detect whether there is a new coronavirus in the sample.

[0048] The subsequent reaction is a conventional reaction, and generally comprises: after the temperature of the reaction system reaches a preset temperature, nucleic acid incubation is performed, fluorescence in the sample is excited, specific waveband fluorescence intensity in the fluorescence is analyzed, whether the fluorescence is the required fluorescence is confirmed through the spectrum value, when it is judged that the fluorescence of the target waveband is detected, and the detected fluorescence intensity value obviously shows an upward trend with time, and the fluorescence intensity value is greater than 5 times the initial background fluorescence intensity value, it is indicated that the detection target contains the target nucleic acid.

[0049] It should be noted that the number and shape of the clamping grooves of the manual centrifugal simple device are only examples, and the number of the clamping grooves can be determined according to actual production and use needs, and is not a specified number.

Claims

1. A manual centrifugal simple device for nucleic acid detection pretreatment, characterized in that, It comprises a fixed seat (3) and a first shell (1) and a second shell (2) which are all spherical bowl-shaped. The first shell (1) is fixedly connected with a cylindrical fixed seat (3) in it, the center of the fixed seat (3) coincides with the center of the first shell (1), the fixed seat (3) is provided with a first clamping groove (33), a second clamping groove (34) and a third clamping groove (35) along the radial direction, the first clamping groove (33) is used for installing a lysate pipe (4), the second clamping groove (34) is used for installing an enzyme-free water pipe (5), and the third clamping groove (35) is used for installing a nucleic acid purification column pipe (7). The center of the upper surface of the fixed seat (3) is provided with a convex ring extending in the vertical direction, the center of the convex ring coincides with the center of the upper surface of the fixed seat (3), the inner wall of the second shell (2) is fixedly connected with a fixed column which is insertedly matched with the convex ring, the fixed column is fixedly inserted in the convex ring, the first shell (1) and the second shell (2) form a closed protective shell, two rope passing holes which are symmetric about the center of the protective shell are formed in the protective shell, the rope passing holes sequentially communicate the first shell (1), the fixed seat (3), the fixed column and the second shell (2), and one pull rope is passed through each rope passing hole.

2. The manual centrifugal simple device for pre-treatment of nucleic acid detection according to claim 1, characterized in that, The first shell (1) and the second shell (2) are the same in shape, the opening of the first shell (1) and the opening of the second shell (2) are arranged in contact.

3. The manual centrifugal simple device for pre-treatment of nucleic acid detection according to claim 1, characterized in that, The first clamping groove (33), the second clamping groove (34) and the third clamping groove (35) are all T-shaped, the inner side end is wider than the outer side end, and the outer side end and the upper and lower ends are all communicated with the outside.

4. The manual centrifugal simple device for pre-treatment of nucleic acid detection according to claim 3, characterized in that, The inner side ends of the first clamping groove (33), the second clamping groove (34) and the third clamping groove (35) are the same in distance from the convex ring.

5. The manual centrifugal simple device for pre-treatment of nucleic acid detection according to claim 4, characterized in that, The center axes of the first clamping groove (33) and the second clamping groove (34) coincide and pass through the center of the fixed seat (3).

6. The manual centrifugal simple device for pre-treatment of nucleic acid detection according to claim 5, characterized in that, The first clamping groove (33) and the second clamping groove (34) are both one, and the third clamping groove (35) is two, the center axes of the two third clamping grooves (35) coincide and pass through the center of the fixed seat (3). The fixed seat (3) is further provided with four fourth clamping grooves (36) of T-shaped structure along the radial direction, the inner side end is wider than the outer side end, the outer side end and the upper and lower ends of the fourth clamping groove (36) are all communicated with the outside, the first clamping groove (33), the second clamping groove (34) and the two third clamping grooves (35) form a cross-shaped structure, the wide ends of the first clamping groove (33), the second clamping groove (34) and the third clamping groove (35) are the same in length, and the four fourth clamping grooves (36) are uniformly arranged on the fixed seat (3).

7. The manual centrifugal simple device for pre-treatment of nucleic acid detection according to claim 1, characterized in that, The center of the inner wall of the first shell (1) is fixedly connected with a first fixed column (13), the fixed column fixedly connected with the center of the inner wall of the second shell (2) is a second fixed column (23), the center of the lower surface of the fixed seat (3) is provided with a first convex ring (37) extending in the vertical direction, the convex ring at the center of the upper surface of the fixed seat (3) is a second convex ring (38), and the first fixed column (13) is fixedly inserted in the first convex ring (37).

8. The manual centrifugal simple device for pre-treatment of nucleic acid detection according to claim 7, characterized in that, The inner and outer diameters of the first convex ring (37) and the second convex ring (38) are the same, and the diameters of the first fixed column (13) and the second fixed column (23) are the same.

9. The manual centrifugal simple device for pre-treatment of nucleic acid detection according to claim 8, characterized in that, The first shell (1) and the first fixed column (13) are provided with a first through hole (11) and a second through hole (12), the second shell (2) and the second fixed column (23) are provided with a third through hole (21) and a fourth through hole (22), the fixed seat (3) is provided with a fifth through hole (31) and a sixth through hole (32), the centers of the first through hole (11), the third through hole (21) and the fifth through hole (31) are aligned to form one rope passing hole, and the centers of the second through hole (12), the fourth through hole (22) and the sixth through hole (32) are aligned to form another rope passing hole.

10. The manual centrifugal simple device for pre-treatment of nucleic acid detection according to claim 9, characterized in that, The first through hole (11), the third through hole (21), the fifth through hole (31), the second through hole (12), the fourth through hole (22) and the sixth through hole (32) have the same inner diameter.

Images