A kit for detecting polymorphism of human MTHFR and MTRR genes in a single reaction well

Abstract

The application discloses a kit for simultaneously detecting human MTHFR and MTRR gene polymorphism in a single reaction hole, and belongs to the technical field of detection kits, and comprises a box body, a plurality of moving grooves and placing grooves are symmetrically arranged at the top and inner part of the box body at equal intervals, the number of the moving grooves and the placing grooves is the same and the positions of the moving grooves and the placing grooves are one-to-one corresponding, and two warning cover shells are symmetrically arranged in the moving grooves; the warning cover shells are arranged on the two sides of a negative control tube, a positive control tube, a PCR reaction mixed liquid tube and a primer and probe mixed liquid tube; when the negative control tube, the positive control tube, the PCR reaction mixed liquid tube or the primer and probe mixed liquid tube is damaged in the transportation diagram, the two warning cover shells can be closed due to the weight loss, so that the warning effect is achieved, the problem that a detection personnel does not know the damage situation and contacts the negative control tube, the positive control tube, the PCR reaction mixed liquid tube or the primer and probe mixed liquid tube and influences the same is avoided, and the use safety is improved.

Description

Technical Field

[0001] This invention belongs to the field of detection kit technology, specifically relating to a detection kit for simultaneously detecting human MTHFR and MTRR gene polymorphisms in a single reaction well. Background Technology

[0002] In the fields of life sciences and medicine, the association between gene polymorphism and human health and disease has always been a hot topic. Among them, the polymorphisms of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene and the methionine synthase reductase (MTRR) gene occupy an important position in this research field.

[0003] The MTHFR gene is located at chromosome lp36.3, is 19.3 kb in length, and has 12 exons. The MTHFR it encodes plays a key role in the folate-methionine cycle metabolic pathway, catalyzing the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, providing methyl raw materials for the synthesis of substances in the body and the methylation of nucleic acids and proteins, while maintaining a low level of homocysteine ​​in the body. The common mutation site A1298C in the MTHFR gene causes the encoded amino acid to change from glutamate to alanine, resulting in decreased enzyme activity and thus affecting folate metabolism.

[0004] The MTRR gene is also crucial in the folate metabolism pathway. The common mutation site A66G in the MTRR gene can affect the activity of methionine synthase, thereby interfering with the metabolism of homocysteine.

[0005] Numerous studies have confirmed that MTHFR and MTRR gene polymorphisms are closely linked to a variety of diseases, including cardiovascular and cerebrovascular diseases and reproductive health.

[0006] In terms of cardiovascular and cerebrovascular diseases, when the activity of enzymes related to folic acid metabolism is low, even if folic acid is supplemented at the normal level, the body's folic acid level may still be insufficient, leading to the accumulation of homocysteine ​​in the body, causing hyperhomocysteinemia, which in turn increases the risk of cardiovascular and cerebrovascular diseases, such as systemic atherosclerosis and thrombosis. Mild to moderate elevation of homocysteine ​​levels can increase the risk of death from cardiovascular disease by 4 to 6 times and is an independent risk factor for coronary heart disease.

[0007] In terms of reproductive health, MTHFR and MTRR gene polymorphisms are closely related to diseases such as recurrent miscarriage and fetal neural tube defects. For example, the TT genotype at the C677T site of the MTHFR gene is significantly associated with low sperm concentration and high DNA fragmentation rate in male infertility patients, and may be a high-risk gene for male infertility.

[0008] Furthermore, these gene polymorphisms have also been found to be associated with the occurrence, development, and prognosis of neurological diseases and tumors.

[0009] Currently, there are many methods for detecting MTHFR and MTRR gene polymorphisms, among which polymerase chain reaction-restriction fragment length polymorphism PCR-RFLP is widely used.

[0010] Chinese Patent Application No. 202223533154.6 discloses a real-time fluorescence quantitative PCR detection kit for human MTHFR gene, comprising a box body with a first and a second box body symmetrically arranged on the top of the box body. The sides of the first and second box bodies are connected to the box body by sliding rods within a sliding groove. The first and second box bodies are connected by movable limiting hooks that slide out of or insert into a limiting groove. In use, the limiting hooks are moved to disengage from the limiting grooves, and the first and second box bodies are pushed in the opposite direction. The sliders of the first and second box bodies move to their maximum position within the sliding groove of the box body, thereby opening the first and second box bodies to the maximum extent.

[0011] If the internal test tubes of the above-mentioned real-time quantitative PCR detection kit for human MTHFR gene are damaged during transportation, the testing personnel will not know in advance and may accidentally touch the damaged test tubes to come into contact with the reagents, which may affect the reagents and result in low safety.

[0012] Therefore, a detection kit for simultaneously detecting human MTHFR and MTRR gene polymorphisms in a single reaction well was designed to address the aforementioned issues. Summary of the Invention

[0013] To address the problems mentioned in the background section, this invention provides a detection kit for simultaneously detecting human MTHFR and MTRR gene polymorphisms in a single reaction well, featuring improved safety and convenience, low detection cost, and high throughput.

[0014] To achieve the above objectives, the present invention provides the following technical solution: a detection kit for simultaneous detection of human MTHFR and MTRR gene polymorphisms in a single reaction well, comprising: a box body, wherein a plurality of moving slots and placement slots are equally spaced at the top and inside of the box body, the number of moving slots and placement slots being the same and their positions corresponding one-to-one; two warning covers are symmetrically arranged inside the moving slots; a support plate is arranged inside the placement slot directly below the two warning covers; an elastic lifting mechanism is assembled between the support plate and the bottom of the placement slot; a follow-up driving mechanism is assembled between the support plate and the two warning covers; and two support rods are symmetrically arranged at the top of the support rods along the central axis. The support rod is located between two warning covers and extends above the moving slot. The top of the two support rods is fixed with a rubber ring. On one side edge, the two rubber rings are respectively clamped inside the negative control tube and the positive control tube. In the middle, several rubber rings are respectively clamped inside the PCR reaction mixture tubes. On the other side edge, several rubber rings are respectively clamped inside the primer and probe mixture tubes. The box body is provided with a box cover. A locking mechanism is installed between the box cover and the box body. The inner top wall of the box cover is fixed with clamping blocks corresponding to the positions of the negative control tube, the positive control tube, the PCR reaction mixture tube, and the primer and probe mixture tube.

[0015] Furthermore, the elastic lifting mechanism includes two first springs symmetrically fixed to the bottom of the placement slot, with the top of the first springs fixed to the bottom of the support plate.

[0016] Furthermore, when the negative control tube, positive control tube, PCR reaction mixture tube, and primer and probe mixture tube are securely clamped in the rubber ring, the support plate descends to its lowest point due to gravity, and the first spring is in a compressed state.

[0017] Furthermore, the follow-up drive mechanism includes two first racks symmetrically fixed to the bottom end of the support plate, four drive shafts connected to the inside of the placement groove via bearings, two first limiting rods symmetrically fixed to the inside of the placement groove, and two movable frames symmetrically fixed to the two warning covers. The two first racks are located on the side away from each other of the two first springs. The four drive shafts are symmetrically arranged in pairs on the side away from each other of the two first racks. Gears are fixedly sleeved on the drive shafts. The gears on the side closer to the first racks mesh with the first racks. The two gears on the same side mesh with each other. The two first limiting rods are located on the side of the drive shafts on the same side away from the first racks and are arranged perpendicular to the two first racks. The two movable frames are respectively opened and sleeved on the two first limiting rods. The two movable frames are respectively fixedly connected to the side closer to each other of the two movable frames. The gears on the side closer to the second racks mesh with the second racks.

[0018] Furthermore, when the negative control tube, positive control tube, PCR reaction mixture tube, and primer and probe mixture tube are damaged, the support plate rises to its highest point due to the loss of gravity, that is, when it is in contact with the bottom of the two warning covers, the two warning covers move to a closed state.

[0019] Furthermore, when the two warning covers move to the closed state, the moving frame does not disengage from the first limit rod.

[0020] Furthermore, the number of PCR reaction mixture tubes is the same as the number of primer and probe mixture tubes.

[0021] Furthermore, the locking mechanism includes two second limiting rods symmetrically fixed to the inner wall of the box cover and four locking holes opened on the box cover. The second limiting rods are located above the box body. A movable frame is sleeved on the second limiting rod. A second spring is elastically connected between the box cover and the movable frame outside the second limiting rod. Two locking blocks are symmetrically arranged on the two movable frames away from each other and away from the side wall. The four locking blocks correspond one-to-one with the four locking holes and are respectively inserted into the four locking holes.

[0022] Furthermore, when the locking block squeezes open the box cover, the movable frame does not disengage from the second limiting rod.

[0023] Furthermore, a positioning mechanism is assembled between the support plate and the placement slot. The positioning mechanism includes a support ring plate connected to the center of the support plate via a bearing, two guide rods symmetrically fixed to the bottom end of the support plate, and a fixed seat fixed to the bottom wall of the placement slot. The two support rods are symmetrically fixed to the top of the support ring plate, and a screw is fixed to the bottom end of the support ring plate. A movable seat is connected to the screw via a transmission nut. The two guide rods are located outside the screw and inside the two first springs. The movable seat is fitted with the two guide rods through an opening. The position of the movable seat corresponds to the position of the fixed seat. Two magnets are symmetrically fixed inside the movable seat and the fixed seat, respectively. The magnets on the movable seat and the fixed seat have opposite polarities.

[0024] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention provides warning covers on both sides of the negative control tube, positive control tube, PCR reaction mixture tube, and primer and probe mixture tube. When the negative control tube, positive control tube, PCR reaction mixture tube, or primer and probe mixture tube is damaged during transport, the weightlessness will cause the two warning covers to close, thus serving as a warning and preventing testing personnel from unknowingly touching the negative control tube, positive control tube, PCR reaction mixture tube, or primer and probe mixture tube, which could affect its operation and improve safety.

[0025] 2. The present invention is equipped with a positioning mechanism that can position the support plate when removing undamaged negative control tubes, positive control tubes, PCR reaction mixture tubes, and primer and probe mixture tubes, thereby preventing the two warning covers from closing and improving the ease of removal.

[0026] 3. The primer and probe mixture in the tube of this invention can simultaneously detect human MTHFR and MTRR gene polymorphisms through a single reaction well, and has the characteristics of low detection cost and high detection throughput. Attached Figure Description

[0027] Figure 1 This is a perspective view of the present invention; Figure 2 This is a partial cross-sectional view of the present invention; Figure 3 This is a partial vertical sectional view of the present invention; Figure 4 This is a schematic diagram of the elastic lifting mechanism and positioning mechanism of the present invention; Figure 5 This is a schematic diagram of the follower drive mechanism of the present invention; Figure 6 For the present invention Figure 3 Enlarged view of point A in the middle; Figure 7 This is a schematic diagram of the locking mechanism of the present invention; In the diagram: 1. Box body; 2. Box lid; 3. Negative control tube; 4. Positive control tube; 5. PCR reaction mixture tube; 6. Primer and probe mixture tube; 7. Warning cover; 8. Compression block; 9. Moving slot; 10. Placement ring with rubber ring; 11. Support rod; 12. Support plate; 13. Placement slot; 101. The first spring; 201. First rack; 202. Gear; 203. Drive shaft; 204. First limit rod; 205. Second rack; 206. Moving frame; 301. Movable frame; 302. Locking block; 303. Locking hole; 304. Second spring; 305. Second limit rod; 401. Support ring plate; 402. Fixed seat; 403. Magnet; 404. Screw; 405. Movable seat; 406. Guide rod. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1 This invention provides the following technical solution: a detection kit for simultaneously detecting human MTHFR and MTRR gene polymorphisms in a single reaction well, comprising: a box body 1, wherein a plurality of moving slots 9 and placement slots 13 are equally spaced at the top and inside of the box body 1, the number of moving slots 9 and placement slots 13 being the same and their positions corresponding one-to-one; two warning covers 7 are symmetrically arranged inside the moving slots 9; a support plate 12 is arranged inside the placement slots 13 directly below the two warning covers 7; an elastic lifting mechanism is assembled between the support plate 12 and the bottom of the placement slots 13; a follow-up driving mechanism is assembled between the support plate 12 and the two warning covers 7; and two support rods 11 are symmetrically arranged at the top of the support rods 11 along the central axis. 1 is located between two warning covers 7 and extends above the moving slot 9. The top of the two support rods 11 is fixed with rubber ring placement rings 10. On one side edge, the two rubber ring placement rings 10 respectively clamp negative control tubes 3 and positive control tubes 4. In the middle, several rubber ring placement rings 10 respectively clamp several PCR reaction mixture tubes 5. On the other side edge, several rubber ring placement rings 10 respectively clamp several primer and probe mixture tubes 6. The box body 1 is provided with a box cover 2. The box cover 2 is equipped with a locking mechanism between the box body 1 and the box body 2. The inner top wall of the box cover 2 is fixed with clamping blocks 8 corresponding to the positions of negative control tubes 3, positive control tubes 4, PCR reaction mixture tubes 5 and primer and probe mixture tubes 6.

[0030] In this embodiment, please refer to the appendix. Figure 1-3When using the single-well detection kit for simultaneous detection of human MTHFR and MTRR gene polymorphisms, release the locking mechanism and remove the lower lid 2. At this point, the negative control tube 3, positive control tube 4, PCR reaction mixture tube 5, and primer and probe mixture tube 6 can be removed as they lose pressure. The negative control tube 3, positive control tube 4, PCR reaction mixture tube 5, and primer and probe mixture tube 6, which were not damaged during transport, remain in their original positions due to gravity and await removal. The negative control tube 3, positive control tube 4, PCR reaction mixture tube 5, or primer and probe mixture tube 6, which were damaged during transport, lose their internal liquid and thus lose gravity. After the lid 2 is removed and the clamping force of the clamping block 8 is lost, the lower support plate 1... 2. Driven by the elastic lifting mechanism, the tube rises, causing the damaged negative control tube 3, positive control tube 4, PCR reaction mixture tube 5 or primer and probe mixture tube 6, as well as part of the follow-up driving mechanism, to rise. The rise of part of the follow-up driving mechanism drives other structures of the follow-up driving mechanism, causing the two corresponding warning covers 7 to move towards each other until the two warning covers 7 merge and stop. At this time, the damaged negative control tube 3, positive control tube 4, PCR reaction mixture tube 5 or primer and probe mixture tube 6 are covered by the two corresponding warning covers 7, which can remind the testing personnel that the negative control tube 3, positive control tube 4, PCR reaction mixture tube 5 or primer and probe mixture tube 6 at this position is damaged, avoiding contact with the tube and causing damage to the tube. The testing personnel took out the undamaged negative control tube 3, positive control tube 4, PCR reaction mixture tube 5, and primer and probe mixture tube 6. First, they opened the caps of the negative control tube 3 and positive control tube 4, and poured the PCR reaction mixture from the two PCR reaction mixture tubes 5 and the primer and probe mixture from the two primer and probe mixture tubes 6 into the negative control tube 3 and positive control tube 4 respectively, and mixed them evenly. After PCR amplification, fluorescence analysis was performed to verify the effectiveness of the test kit. After verifying the effectiveness of the test kit, pour the PCR reaction mixture from tube 5 and the primer and probe mixture from tube 6 into a test tube containing the DNA sample and mix them thoroughly. After PCR amplification, perform fluorescence analysis to determine the polymorphism of the MTHFR and MTRR genes in the DNA sample. The above PCR reaction mixture includes Taq DNA polymerase, dNTP mixture, 10× PCR buffer, MgCl2, UNG enzyme and deionized water; The primer and probe information for the A1298C site of the MTHFR gene in the primer and probe mixture is as follows: Forward primer: 5'-CTGAAGGAGAAGGTGTCTGCGGGA-3' Reverse primer: 5'-AGGACGGTGCGGTGAGAGTG-3' Wild-type probe 1: FAM-5'-ACGCCCGGCTCCATG-3'-MGB Mutant probe 1: VIC-5'-ACGCCCGGCTCCCTG-3'-MGB Wild-type probe 2: ROX-5'-TACGCCCGGCTCCATG-3'-MGB Mutant probe 2: CY5-5'-TACGCCCGGCTCCCTG-3'-MGB The primer and probe information for the A66G site of the MTRR gene is as follows: Primer and probe information Forward primer: 5'-TGAAGGAGAAGGTGTCTGCGGGA-3' Reverse primer: 5'-AGGACGGTGCGGTGAGAGTG-3' Wild-type probe 1: FAM-5'-CCTGAAGCACGCGGCTG-3'-MGB Mutant probe 1: VIC-5'-CCTGAAGCACACGGCTG-3'-MGB Wild-type probe 2: ROX-5'-TCCTGAAGCACGCGGCTG-3'-MGB Mutant probe 2: CY5-5'-TCCTGAAGCACACGGCTG-3'-MGB.

[0031] Specifically, the elastic lifting mechanism includes two first springs 101 symmetrically fixed to the bottom of the placement slot 13, with the top of the first spring 101 fixed to the bottom of the support plate 12.

[0032] In this embodiment, please refer to the appendix. Figure 4 The support plate 12 is raised and lowered under the elastic force of the two first springs 101.

[0033] Specifically, when the negative control tube 3, positive control tube 4, PCR reaction mixture tube 5, and primer and probe mixture tube 6 are intact and locked inside the rubber ring 10, the support plate 12 descends to its lowest point due to gravity, and the first spring 101 is in a compressed state.

[0034] In this embodiment, please refer to the appendix. Figure 4It can ensure that the negative control tube 3, positive control tube 4, PCR reaction mixture tube 5, and primer and probe mixture tube 6, which are not damaged during transportation, remain in their original positions due to gravity and wait to be removed, and that the negative control tube 3, positive control tube 4, PCR reaction mixture tube 5, or primer and probe mixture tube 6, which are damaged during transportation, lose their gravity due to the loss of their internal liquid. After the lid 2 is removed and the clamping force of the clamping block 8 is lost, the lower support plate 12 rises under the drive of the elastic lifting mechanism.

[0035] Specifically, the follow-up drive mechanism includes two first racks 201 symmetrically fixed to the bottom end of the support plate 12, four drive shafts 203 connected to the inside of the placement groove 13 via bearings, two first limit rods 204 symmetrically fixed to the inside of the placement groove 13, and two movable frames 206 symmetrically fixed to the two warning covers 7. The two first racks 201 are located on the side away from each other of the two first springs 101, and the four drive shafts 203 are symmetrically arranged in pairs on the side away from each other of the two first racks 201. The drive shafts 203 are fixedly sleeved on the two drive shafts 203. There is a gear 202, the gear 202 on the side closer to the first rack 201 meshes with the first rack 201, and two gears 202 on the same side mesh with each other. Two first limiting rods 204 are located on the same side of the drive shaft 203 away from the first rack 201 and are perpendicular to the two first racks 201. Two movable frames 206 are respectively opened and sleeved on the two first limiting rods 204. The two movable frames 206 are respectively fixed to the side closer to each other, and the gear 202 on the side closer to the second rack 205 meshes with the second rack 205.

[0036] In this embodiment, please refer to the appendix. Figure 3 and 5 The follow-up drive mechanism is driven by the support plate 12 rising to drive the two first racks 201 to rise. During the rise of the two first racks 201, the two meshing gears 202 are driven to rotate. The two meshing gears 202 drive the other two meshing gears 202 to rotate. The other two meshing gears 202 drive the two meshing second racks 205 to rotate. The two second racks 205 drive the two moving frames 206 to move towards each other on the two first limit rods 204. The two moving frames 206 drive the two warning covers 7 to move towards each other, so as to realize the follow-up function of the two warning covers 7 and the support plate 12.

[0037] Specifically, when the negative control tube 3, positive control tube 4, PCR reaction mixture tube 5, and primer and probe mixture tube 6 are damaged, the support plate 12 rises to its highest point due to the loss of gravity, that is, when it is in contact with the bottom of the two warning covers 7, the two warning covers 7 move to the closed state.

[0038] In this embodiment, please refer to the appendix. Figure 3 and5 It can provide a warning function to cover and cover damaged negative control tubes 3, positive control tubes 4, PCR reaction mixture tubes 5, or primer and probe mixture tubes 6 during transportation.

[0039] Specifically, when the two warning covers 7 are moved to the closed state, the moving frame 206 does not disengage from the first limit rod 204.

[0040] In this embodiment, please refer to the appendix. Figure 5 This is to prevent the movable frame 206 from detaching from the first limit rod 204 and affecting its operation.

[0041] Specifically, the number of PCR reaction mixture tubes 5 is the same as the number of primer and probe mixture tubes 6.

[0042] In this embodiment, please refer to the appendix. Figure 1 This facilitates the detection of human MTHFR and MTRR gene polymorphisms simultaneously in a single reaction well using a detection kit.

[0043] Specifically, the locking mechanism includes two second limiting rods 305 symmetrically fixed to the inner wall of the lid 2 and four locking holes 303 opened on the lid 2. The second limiting rods 305 are located above the box body 1. A movable frame 301 is sleeved on the second limiting rod 305. A second spring 304 is elastically connected between the lid 2 and the movable frame 301 outside the second limiting rod 305. Two locking blocks 302 are symmetrically arranged on the two movable frames 301 away from each other and away from the side wall. The four locking blocks 302 correspond one-to-one with the four locking holes 303 and are respectively inserted into the four locking holes 303.

[0044] In this embodiment, please refer to the appendix. Figure 3 , 6 7. The locking mechanism is formed by pressing either of the two locking blocks 302 on both sides. The two locking blocks 302 pressed on both sides drive the two connected movable frames 301 to move towards each other on the two second limit rods 305. The two movable frames 301 drive the other two locking blocks 302 to move in the same direction until the locking blocks 302 pressed on both sides disengage from the locking holes 303. At this time, the four locking blocks 302 lock the four locking holes 303 respectively, completing the separation of the box cover 2 from the box body 1.

[0045] Specifically, when the locking block 302 squeezes open the box cover 2, the movable frame 301 does not disengage from the second limit rod 305.

[0046] In this embodiment, please refer to the appendix. Figure 7 This is to prevent the movable frame 301 from detaching from the second limit rod 305 and affecting its operation.

[0047] Example 2 The difference between this embodiment and Embodiment 1 is that: Specifically, a positioning mechanism is assembled between the support plate 12 and the placement groove 13. The positioning mechanism includes a support ring plate 401 connected to the center of the support plate 12 via bearings, two guide rods 406 symmetrically fixed to the bottom of the support plate 12, and a fixed seat 402 fixed to the bottom wall of the placement groove 13. The two support rods 11 are symmetrically fixed to the top of the support ring plate 401. A screw 404 is fixed to the bottom of the support ring plate 401. A movable seat 405 is connected to the screw 404 via a transmission nut. The two guide rods 406 are located outside the screw 404 and inside the two first springs 101. The movable seat 405 is fitted onto the two guide rods 406 through an opening. The position of the movable seat 405 corresponds to the position of the fixed seat 402. Two magnets 403 are symmetrically fixed inside the movable seat 405 and the fixed seat 402, respectively. The magnets 403 on the movable seat 405 and the magnets 403 on the fixed seat 402 have opposite polarities.

[0048] In this embodiment, please refer to the appendix. Figure 3 and 4 During transportation, the undamaged negative control tube 3, positive control tube 4, PCR reaction mixture tube 5, and primer and probe mixture tube 6 remain in their original positions due to gravity and await removal. Before removal, rotate the undamaged negative control tube 3, positive control tube 4, PCR reaction mixture tube 5, or primer and probe mixture tube 6. This rotation will cause the rubber ring placement ring 10 to rotate (the clamping force between the rubber ring placement ring 10 and the placement tube is sufficient to drive the rotation). 10 drives the two support rods 11 to rotate, the two support rods 11 drive the support ring plate 401 to rotate, the support ring plate 401 drives the screw 404 to rotate, during the rotation of the screw 404, the movable seat 405 moves up and down on the screw 404 under the guidance of the two guide rods 406 until it can no longer rotate and stops. At this time, the movable seat 405 is attached to the fixed seat 402, and the upper and lower magnets 403 adsorb and fix the position of the support plate 12. During the removal of the undamaged negative control tube 3, positive control tube 4, PCR reaction mixture tube 5 or primer and probe mixture tube 6, the corresponding two warning covers 7 will not merge and will not affect the removal.

[0049] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A detection kit for simultaneously detecting human MTHFR and MTRR gene polymorphisms in a single reaction well, characterized in that, include: The box body (1) has several moving slots (9) and placement slots (13) evenly spaced at its top and inside. The number of moving slots (9) and placement slots (13) are the same and their positions correspond one-to-one. Two warning covers (7) are symmetrically arranged inside the moving slots (9). A support plate (12) is arranged inside the placement slots (13) directly below the two warning covers (7). An elastic lifting mechanism is assembled between the support plate (12) and the bottom of the placement slots (13). A follow-up driving mechanism is assembled between the support plate (12) and the two warning covers (7). Two support rods (11) are symmetrically arranged at the top of the support rods (11) along the central axis. The two support rods (11) are located between the two warning covers (7) and extend above the moving slots (9). A rubber ring is fixed to the top of the support rod (11). Two rubber rings (10) on one side edge are respectively clamped inside the negative control tube (3) and positive control tube (4). Several rubber rings (10) in the middle are respectively clamped inside the PCR reaction mixture tube (5). Several primer and probe mixture tubes (6) are respectively clamped inside the rubber rings (10) on the other side edge. A box cover (2) is provided outside the box body (1). A locking mechanism is installed between the box cover (2) and the box body (1). A clamping block (8) is fixed to the top wall of the box cover (2) at the positions corresponding to the negative control tube (3), positive control tube (4), PCR reaction mixture tube (5) and primer and probe mixture tube (6). The elastic lifting mechanism includes two first springs (101) symmetrically fixed to the bottom of the placement slot (13), and the top of the first springs (101) is fixed to the bottom of the support plate (12); When the negative control tube (3), positive control tube (4), PCR reaction mixture tube (5) and primer and probe mixture tube (6) are not damaged and are locked in the rubber ring (10), the support plate (12) drops to the lowest point due to gravity and the first spring (101) is in a compressed state. The follow-up drive mechanism includes two first racks (201) symmetrically fixed to the bottom end of the support plate (12), four drive shafts (203) connected to the inside of the placement groove (13) via bearings, two first limiting rods (204) symmetrically fixed to the inside of the placement groove (13), and two movable frames (206) symmetrically fixed to the two warning covers (7). The two first racks (201) are located on the side away from each other of the two first springs (101), and the four drive shafts (203) are symmetrically arranged in pairs on the side away from each other of the two first racks (201). Gears are fixedly sleeved on the drive shafts (203). 202), the gear (202) on the side closer to the first rack (201) meshes with the first rack (201), the two gears (202) on the same side mesh with each other, the two first limiting rods (204) are located on the same side of the drive shaft (203) away from the first rack (201) and are perpendicular to the two first racks (201), the two moving frames (206) are respectively opened and sleeved on the two first limiting rods (204), the two moving frames (206) are respectively fixed with the second rack (205) on the side closer to each other, the gear (202) on the side closer to the second rack (205) meshes with the second rack (205); The PCR reaction mixture tube (5) contains a PCR reaction mixture, which includes Taq DNA polymerase, dNTP mixture, 10× PCR buffer, MgCl2, UNG enzyme, and deionized water; the primer and probe mixture tube (6) contains a primer and probe mixture, wherein the primers and probes for the MTHFR gene A1298C site in the primer and probe mixture are: forward primer 5'-CTGAAGGAGAAGGTGTCTGCGGGA-3', reverse primer 5'-AGGACGGTGCGGTGAGAGTG-3', wild-type probe 1 is FAM-5'-ACGCCCGGCTCCATG-3'-MGB, mutant probe 1 is VIC-5'-ACGCCCGGCTCCCTG-3'-MGB, wild-type probe 2 is ROX-5'-TACGCCCGGCTCCATG-3'-MGB, and mutant probe 2 is... The primers and probes for the A66G site of the MTRR gene in the primer and probe mixture are: forward primer 5'-TGAAGGAGAAGGTGTCTGCGGGA-3', reverse primer 5'-AGGACGGTGCGGTGAGAGTG-3', wild-type probe 1 is FAM-5'-CCTGAAGCACGCGGCTG-3'-MGB, mutant probe 1 is VIC-5'-CCTGAAGCACACGGCTG-3'-MGB, wild-type probe 2 is ROX-5'-TCCTGAAGCACGCGGCTG-3'-MGB, and mutant probe 2 is CY5-5'-TCCTGAAGCACACGGCTG-3'-MGB.

2. The detection kit for simultaneous detection of human MTHFR and MTRR gene polymorphisms in a single reaction well according to claim 1, characterized in that: When the negative control tube (3), positive control tube (4), PCR reaction mixture tube (5), and primer and probe mixture tube (6) are damaged, the support plate (12) rises to its highest point due to the loss of gravity, that is, when it is in contact with the bottom of the two warning covers (7), the two warning covers (7) move to the closed state.

3. The detection kit for simultaneous detection of human MTHFR and MTRR gene polymorphisms in a single reaction well according to claim 2, characterized in that: When the two warning covers (7) are moved to the closed state, the moving frame (206) does not disengage from the first limit rod (204).

4. The detection kit for simultaneous detection of human MTHFR and MTRR gene polymorphisms in a single reaction well according to claim 3, characterized in that: The number of PCR reaction mixture tubes (5) is the same as the number of primer and probe mixture tubes (6).

5. The detection kit for simultaneous detection of human MTHFR and MTRR gene polymorphisms in a single reaction well according to claim 4, characterized in that: The locking mechanism includes two second limiting rods (305) symmetrically fixed to the inner wall of the box cover (2) and four locking holes (303) opened on the box cover (2). The second limiting rods (305) are located above the box body (1). A movable frame (301) is sleeved on the second limiting rod (305). A second spring (304) is elastically connected between the box cover (2) and the movable frame (301) outside the second limiting rod (305). Two locking blocks (302) are symmetrically arranged on the two movable frames (301) away from each other and away from the side wall. The four locking blocks (302) correspond one-to-one with the four locking holes (303) and are respectively inserted into the four locking holes (303).

6. The detection kit for simultaneous detection of human MTHFR and MTRR gene polymorphisms in a single reaction well according to claim 5, characterized in that: When the locking block (302) squeezes open the box cover (2), the movable frame (301) does not disengage from the second limit rod (305).

7. The detection kit for simultaneous detection of human MTHFR and MTRR gene polymorphisms in a single reaction well according to claim 6, characterized in that: A positioning mechanism is assembled between the support plate (12) and the placement groove (13). The positioning mechanism includes a support ring plate (401) connected to the center of the support plate (12) by bearings, two guide rods (406) symmetrically fixed to the bottom end of the support plate (12), and a fixing seat (402) fixed to the bottom wall of the placement groove (13). The two support rods (11) are symmetrically fixed to the top of the support ring plate (401). A screw (404) is fixed to the bottom end of the support ring plate (401). The screw (404) is connected to a transmission rod. The moving nut is connected to a movable seat (405). Two guide rods (406) are located outside the screw (404) and inside the two first springs (101). The movable seat (405) is fitted onto the two guide rods (406) with an opening. The position of the movable seat (405) corresponds to the position of the fixed seat (402). Two magnets (403) are symmetrically fixed inside the movable seat (405) and the fixed seat (402). The magnets (403) on the movable seat (405) and the magnets (403) on the fixed seat (402) have opposite polarities.

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