A fritillaria cirrhosa identification device based on CRISPR / Cas12a technology

Abstract

The utility model discloses a fritillaria cirrhosa identification device based on CRISPR / Cas12a technology, its characterized in that: the device is by reagent box device (1) and reaction frame (2) two parts constitute, reagent box device (1) include the casing (3), and the casing (3) in sliding connection has three reagent boxes, and three reagent box respectively is RPA reaction reagent box (4), DNA purification reagent box (5) and CRISPR reaction detection reagent box (6), RPA reaction reagent box (4) is equipped with reagent bottle for carrying out RPA reaction in, DNA purification reagent box (5) is equipped with reagent bottle and material for DNA purification in, CRISPR reaction detection reagent box (6) is equipped with reagent bottle and material for carrying out CRISPR reaction in.

Description

Technical Field

[0001] This utility model relates to the field of reagent detection, and in particular to a device for identifying Fritillaria cirrhosa based on CRISPR / Cas12a technology. Background Technology

[0002] Fritillariae Cirrhosae Bulbus is the dried bulb of the Fritillariae Cirrhosae plant, belonging to the Liliaceae family. It possesses the effects of clearing heat and moistening the lungs, resolving phlegm and relieving cough, and dispersing nodules and reducing swelling, making it an excellent medicine for relieving cough and resolving phlegm. However, adulteration of Chinese medicinal materials and powders in the market is serious, severely affecting the safety of medication use. Frequent adulteration with Fritillariae Iliense, Fritillariae Zhejiangense, Fritillariae Pinghuai, and Fritillariae Tuberculata is necessary to distinguish its authenticity through testing.

[0003] CRISPR technology is an effective method for identifying genuine and counterfeit Fritillaria cirrhosa. It does not require high-end instruments and equipment; a water bath or metal bath is sufficient for the reaction, making it an accurate and convenient detection method. However, due to the numerous reaction steps and the cumbersome process, requiring multiple reaction kits to complete the reaction, its application and promotion are limited.

[0004] When using CRISPR technology to detect Fritillaria cirrhosa, test strips need to be placed into different 8-tube strips. There are specific requirements for the depth of the test strips immersed in the reagent in the 8-tube strips, and the test strips also need to maintain their current state for a certain period of time (usually 3-5 minutes). These requirements result in a large amount of labor and time being required for staff to conduct the tests, leading to relatively low work efficiency, a large workload for personnel, and time and effort.

[0005] Meanwhile, during the entire experimental operation, it is often necessary to put multiple 8-tube sets containing reagents into ice baths, water baths, or metal baths at the same time. This requires the operator to frequently insert the 8-tube sets one by one into the pre-set holes of the ice bath, water bath, or metal bath equipment, which is quite labor-intensive.

[0006] Therefore, a method or apparatus is needed to solve the above problems. Summary of the Invention

[0007] This invention addresses the aforementioned shortcomings of existing technologies by proposing a device that features a simple structure, ingenious design, and reasonable layout, enabling rapid, efficient, and labor-saving identification of Fritillaria cirrhosa using CRISPR / Cas12a technology.

[0008] The technical solution of this utility model is: a device for identifying Fritillaria cirrhosa based on CRISPR / Cas12a technology, characterized in that: the device consists of two parts: a reagent kit 1 and a reaction rack 2. The reagent kit device 1 includes a housing 3, within which three reagent kits are slidably connected. These three reagent kits are an RPA reaction kit 4, a DNA purification kit 5, and a CRISPR reaction detection kit 6. The RPA reaction kit 4 contains reagent vials for performing the RPA reaction; the DNA purification kit 5 contains reagent vials and materials for DNA purification; and the CRISPR reaction detection kit 6 contains reagent vials and materials for performing the CRISPR reaction. The reaction frame 2 includes a support plate 7, with telescopic support columns 8 connected to its four corners. Each telescopic support column 8 has a magnet 9 at its bottom. The support plate 7 has two rows of reaction holes 10, the specifications and dimensions of which are consistent with the 8-connecting pipe. The support plate 7 also has markings corresponding to the reaction holes 10, dividing the two rows of reaction holes 10 into row A and row B. Row A reaction holes 10 are sequentially marked as -, 1, 2, 3, 4, 5, 6, +, while row B reaction holes 10 are marked in order from 1 to 8. Two guide frames are symmetrically rotatably connected to the support plate 7. Each guide frame consists of a guide frame plate 11 and a connecting plate 12 connected to its two ends. The guide frame plate 11 has eight guide holes evenly distributed on it, while the connecting plate 12 is rotatably connected to the side end face of the support plate 7 via a rotating shaft. The side end face of the support plate 7 is provided with a limiting component that matches the connecting plate 12. The limiting component includes a fixed limiting block 13 and a movable limiting block 14. When the side of the connecting plate 12 contacts the fixed limiting block 13, the guide frame plate 11 is horizontally distributed. At this time, the eight guide holes on the guide frame plate 11 correspond one-to-one with the multiple reaction holes 10 below them and remain coaxial. The movable limiting block 14 is movably connected to the countersunk hole opened on the side end face of the support plate 7 by a first spring 15.

[0009] A clamping and correction mechanism is provided in the guide hole. The clamping and correction mechanism includes a fixing block 16 fixedly installed in the guide hole and a clamping block 17 that matches the fixing block 16. The clamping block 17 is connected to a connecting shaft 18. The connecting shaft 18 is movably inserted through the side wall of the guide plate 11. A second spring 19 is also sleeved on the connecting shaft 18. The second spring 19 is located between the clamping block 17 and the inner wall of the guide hole. An end 20 located outside the guide hole is also provided at the outer end of the connecting shaft 18.

[0010] Compared with the prior art, this utility model has the following advantages: This type of Fritillaria cirrhosa identification device based on CRISPR / Cas12a technology has a simple structure, ingenious design, and reasonable layout. It addresses many problems of traditional identification devices, such as cumbersome operation and low work efficiency, by designing a special structure. It categorizes and places the reagent bottles and materials for CRISPR reactions into different kits. It also features a specially structured reaction rack whose overall height can be adjusted to suit different needs and stably adheres to the water bath, ensuring a stable environment for the reaction. This rack can be placed directly onto an ice box or ice bath used during reaction system preparation, or into a 96-well metal bath, without removing the reaction tubes and transferring them to the water or metal bath. Furthermore, it incorporates a swingable guide frame with multiple guide holes. When the guide frame is in the working position, these guide holes correspond one-to-one with the reaction holes on the main body of the rack. A clamping and correction mechanism within these guide holes secures the test strips, reducing manual operation (eliminating the need for the operator to hold the test strip for a specific time) and increasing efficiency (allowing simultaneous operation on multiple test strips). This identification device is simple to manufacture and inexpensive, possessing numerous advantages and making it particularly suitable for widespread application in this field, with a very promising market prospect. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the reagent kit device in an embodiment of this utility model.

[0012] Figure 2 This is a front view of the reaction frame portion in an embodiment of this utility model.

[0013] Figure 3 This is a top view of the reaction frame portion in an embodiment of this utility model.

[0014] Figure 4 This is a top view of the guide frame plate portion in an embodiment of this utility model.

[0015] Figure 5 yes Figure 4 Enlarged view (section view) of part A in the image.

[0016] Figure 6 This is a structural schematic diagram of the connection between the connecting plate and the support plate in an embodiment of this utility model.

[0017] Figure 7 This is a schematic diagram of the structure of the movable limiting block in an embodiment of this utility model. Detailed Implementation

[0018] The specific embodiments of this utility model will be described below with reference to the accompanying drawings. Figures 1 to 7 As shown: A device for identifying Fritillaria cirrhosa based on CRISPR / Cas12a technology, which consists of two parts: a reagent kit 1 and a reaction frame 2. The reagent kit device 1 includes a housing 3, within which three reagent kits are slidably connected. These three reagent kits are an RPA reaction kit 4, a DNA purification kit 5, and a CRISPR reaction detection kit 6. The RPA reaction kit 4 contains reagent vials for performing the RPA reaction; the DNA purification kit 5 contains reagent vials and materials for DNA purification; and the CRISPR reaction detection kit 6 contains reagent vials and materials for performing the CRISPR reaction. The reaction frame 2 includes a support plate 7, with telescopic support columns 8 connected to its four corners. Each telescopic support column 8 has a magnet 9 at its bottom. The support plate 7 has two rows of reaction holes 10, the specifications and dimensions of which are consistent with the 8-connecting pipe. The support plate 7 also has markings corresponding to the reaction holes 10, dividing the two rows of reaction holes 10 into row A and row B. Row A reaction holes 10 are sequentially marked as -, 1, 2, 3, 4, 5, 6, +, while row B reaction holes 10 are marked in order from 1 to 8. Two guide frames are symmetrically rotatably connected to the support plate 7. Each guide frame consists of a guide frame plate 11 and a connecting plate 12 connected to its two ends. The guide frame plate 11 has eight guide holes evenly distributed on it, while the connecting plate 12 is rotatably connected to the side end face of the support plate 7 via a rotating shaft. The side end face of the support plate 7 is provided with a limiting component that matches the connecting plate 12. The limiting component includes a fixed limiting block 13 and a movable limiting block 14. When the side of the connecting plate 12 contacts the fixed limiting block 13, the guide frame plate 11 is horizontally distributed. At this time, the eight guide holes on the guide frame plate 11 correspond one-to-one with the multiple reaction holes 10 below them and remain coaxial. The movable limiting block 14 is movably connected to the countersunk hole opened on the side end face of the support plate 7 by a first spring 15.

[0019] A clamping and correction mechanism is provided in the guide hole. The clamping and correction mechanism includes a fixing block 16 fixedly installed in the guide hole and a clamping block 17 that matches the fixing block 16. The clamping block 17 is connected to a connecting shaft 18. The connecting shaft 18 is movably inserted through the side wall of the guide plate 11. A second spring 19 is also sleeved on the connecting shaft 18. The second spring 19 is located between the clamping block 17 and the inner wall of the guide hole. An end 20 located outside the guide hole is also provided at the outer end of the connecting shaft 18.

[0020] The working process of the Fritillaria cirrhosa identification device based on CRISPR / Cas12a technology in this embodiment of the utility model is as follows: The RPA reaction kit 4 is provided with a shock-absorbing foam pad, which has multiple grooves that match the shape of the reagent bottles. The grooves contain reagent bottles for primers, reagent bottles for buffer solutions, reagent bottles for nuclease-free water, reagent bottles for reactive enzymes, reagent bottles for magnesium acetate, and reagent bottles for positive control reagents. The instruction manual is also placed in the grooves. The DNA purification kit 5 also includes a shock-absorbing foam pad with multiple grooves that conform to the shape of the reagent bottle and filter column. These grooves hold multiple reagent bottles containing buffer solution and the filter column. The CRISPR reaction detection kit 6 also includes a shock-absorbing foam pad with multiple grooves that conform to the shape of the reagent bottles. These grooves hold reagent bottles containing the Cas12a reaction enzyme, the guide RNA, the reaction buffer, the ssDNA, and the nuclease-free water. The CRISPR reaction detection kit 6 also includes test strips.

[0021] When conducting identification experiments on Fritillaria cirrhosa, different reaction kits can be selected according to different reaction steps. During operation, reaction rack 2 can be used, which can be used in ice bath, water bath and metal bath without removing the reaction tube.

[0022] In the initial state, the connecting plates 12 in both guide frames are horizontally distributed. When the CRISPR reaction is tested, the tester moves the connecting plate 12, causing it to swing from a horizontal state to a vertical state, until the side of the connecting plate 12 contacts the fixed limiting block 13. At the same time, after the constraint of the connecting plate 12 is removed, the movable limiting block 14 will also pop outward under the action of the first spring 15. At this time, the two sides of the connecting plate 12 are pressed together by the fixed limiting block 13 and the movable limiting block 14 respectively, thereby locking the connecting plate 12 in the current vertical state. In this state, the guide frame plate 11 is horizontal, and the eight guide holes on it correspond one-to-one with the eight reaction holes 10 below it. After passing the test strip through the guide hole, place it into the reaction hole 10 and clamp it using the clamping and correction mechanism inside the guide hole. When clamping, pay attention to adjusting the depth of the bottom of the test strip immersed in the detection liquid in the 8-tube. The test strip is fixed by this guide frame and clamping and correction mechanism. This method can effectively save labor and allow multiple test strips to be tested at one time, which can effectively improve work efficiency. At the same time, there is no need to worry about the test strip tipping over due to its excessive length. When using the clamping and correction mechanism to clamp the test strip, simply hold the end 20 and pull the connecting shaft 18 outwards. The clamping block 17 will separate from the fixing block 16, and the second spring 19 will be compressed. Then, place the test strip into the gap between the fixing block 16 and the clamping block 17. After adjusting the test strip to a suitable height, release the end 20. Under the action of the second spring 19, the clamping block 17 will move towards the fixing block 16 and clamp the test strip. After the test, pull all the test strips out of the guide hole, press the movable limit block 14 to avoid the movement trajectory of the connecting plate 12, and at the same time, turn the connecting plate 12 in the opposite direction to return it from the vertical state to the horizontal state. At this time, there is no obstruction above all the reaction holes 10, so the 8-tube can be taken out from the reaction holes 10. When using the reaction rack 2 in this embodiment of the present invention to conduct multiple 8-tube water baths or metal baths, the distance between the support plate 7 and the bottom surface of the water bath or metal bath device can be adjusted according to the length of the 8-tubes. Specifically, the length of the four telescopic support columns 8 is adjusted. At the same time, since each telescopic support column 8 is equipped with a magnet 9 at its bottom, the magnet 9 can make the reaction rack 2 be adsorbed in the device as a whole, preventing it from moving or flipping during the experiment.

Claims

1. A device for identifying Fritillaria cirrhosa based on CRISPR / Cas12a technology, characterized in that: The device consists of two parts: a reagent kit (1) and a reaction rack (2). The kit device (1) includes a housing (3), within which three kits are slidably connected. These three kits are an RPA reaction kit (4), a DNA purification kit (5), and a CRISPR reaction detection kit (6), respectively. The RPA reaction kit (4) contains reagent bottles for performing the RPA reaction, the DNA purification kit (5) contains reagent bottles and materials for DNA purification, and the CRISPR reaction detection kit (6) contains reagent bottles and materials for performing the CRISPR reaction. The reaction frame (2) includes a support plate (7), with telescopic support columns (8) connected to the four corners of the support plate (7), and a magnet (9) is provided at the bottom of each telescopic support column (8). The support plate (7) has two rows of reaction holes (10), the specifications and dimensions of which are consistent with the 8-pipe connection. The support plate (7) is also marked with corresponding marks to the reaction holes (10), which divide the two rows of reaction holes (10) into row A and row B. The reaction holes (10) in row A are marked as -, 1, 2, 3, 4, 5, 6, +, and the reaction holes (10) in row B are marked in the order from 1 to 8. Two guide frames are symmetrically rotatably connected to the support plate (7). Each guide frame consists of a guide frame plate (11) and a connecting plate (12) connected to both ends thereon. The guide frame plate (11) has eight guide holes evenly distributed on it, while the connecting plate (12) is rotatably connected to the side end face of the support plate (7) via a rotating shaft. The side end face of the support plate (7) is provided with a limiting component that matches the connecting plate (12). The limiting component includes a fixed limiting block (13) and a movable limiting block (14). When the side of the connecting plate (12) contacts the fixed limiting block (13), the guide frame plate (11) is horizontally distributed. At this time, the eight guide holes on the guide frame plate (11) correspond one-to-one with the multiple reaction holes (10) below it and remain coaxial. The movable limiting block (14) is movably connected to the countersunk hole opened on the side end face of the support plate (7) by the first spring (15).

2. The Fritillaria cirrhosa identification device based on CRISPR / Cas12a technology according to claim 1, characterized in that: A clamping and correction mechanism is provided in the guide hole. The clamping and correction mechanism includes a fixed block (16) fixedly installed in the guide hole and a clamping block (17) that matches the fixed block (16). The clamping block (17) is connected to the connecting shaft (18). The connecting shaft (18) is movably inserted in the side wall of the guide frame plate (11). At the same time, a second spring (19) is also sleeved on the connecting shaft (18). The second spring (19) is located between the clamping block (17) and the inner wall of the guide hole. The outer end of the connecting shaft (18) is also provided with an end (20) located outside the guide hole.

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