QPCR (quantitative polymerase chain reaction) detection kit and detection method for fungal pathogens

Abstract

The invention belongs to the technical field of biochemical detection, and discloses a qPCR (quantitative polymerase chain reaction) detection kit for fungal pathogens and a detection method.The qPCR detection kit for the fungal pathogens comprises a packaging box, a placement box is arranged in the packaging box, and placement grooves are formed in the top end of the placement box at equal intervals; a swinging assembly is fixedly installed at the position, located in the containing groove, of one end of the containing box and comprises a positioning column fixedly installed in the containing box, a rotating plate is rotationally connected to the outer side of the positioning column, two limiting rings are fixedly installed on one end face of the rotating plate, and a concave disc is rotationally connected to the interior of one limiting ring; the swing of the cylinder is controlled through simple manual operation, the swing control difficulty is changed, and the mode of manual swing in the prior art is changed, so that the labor intensity of personnel is reduced, and the consistency and accuracy of each operation are ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical detection, and in particular relates to a fungal pathogen qPCR detection kit and a detection method. Background Art

[0002] The clinical importance of fungal infections in pediatric and neonatal wards. Children and neonates are vulnerable to fungal pathogens due to their relatively weak immune systems, especially multidrug-resistant fungi in hospital environments. These infections not only endanger the life and health of children, but are also associated with higher hospital stays and medical costs. Therefore, early diagnosis and accurate pathogen identification are essential for developing effective treatment strategies. Secondly, the application of qPCR technology in pathogen detection. Real-time fluorescence quantitative polymerase chain reaction (qPCR) is currently an effective method for detecting pathogenic microorganisms, especially nucleic acid viruses and bacteria. Compared with traditional pathogen detection methods, qPCR has higher sensitivity and specificity, and can provide accurate test results in a short time. Especially for pathogens with complex structures or frequent mutations, qPCR can also provide quantitative analysis, which helps to accurately monitor the dynamic changes of infection and treatment response. Finally, the limitations of existing detection methods on the market are one of the driving forces for the development of qPCR detection kits and detection methods specifically for pediatric and neonatal patient groups. The currently used detection methods are not convenient for reacting samples and reactants, which may not be able to identify certain fungal pathogens in a timely and accurate manner. In addition, existing methods may not be suitable for rapid bedside testing or require complex laboratory equipment, limiting their widespread and efficient application in clinical settings. Therefore, designing an easy-to-use, highly sensitive and specific qPCR kit is crucial to improving the diagnostic efficiency and treatment effectiveness of fungal infections in pediatric and neonatal wards. Summary of the invention

[0003] The present invention aims at the problem that it is not convenient to react samples and reactants in the prior art, so that some fungal pathogens may not be identified in a timely and accurate manner. In addition, the prior art may not be suitable for rapid bedside detection or require complex laboratory equipment. The present invention proposes the following technical solutions: A fungal pathogen qPCR detection kit comprises a packaging box, wherein a placement box is arranged inside the packaging box, and placement grooves are equidistantly arranged on the top of the placement box; One end of the placement box is fixedly installed at a position inside the placement groove, and the swing assembly includes a positioning column fixedly installed inside the placement box, the outer side of the positioning column is rotatably connected with a rotating plate, and one end face of the rotating plate is fixedly installed with two limiting rings, one of the limiting rings is rotatably connected with a concave disk, and a worm wheel is fixedly installed at the bottom end of the concave disk, and a worm is meshingly connected to the outer side of the worm wheel. A positioning block is symmetrically rotatably connected to the outer side of the worm and the positioning block is fixedly installed at the bottom end of the limiting ring, and a gear is fixedly installed on one end of the worm, an arc ring is sleeved on the outer side of the gear, and the arc ring is fixedly installed inside the placement groove of the placement box.

[0004] As a preferred embodiment of the above technical solution, a tooth groove is provided at the bottom end of the inner wall of the arc-shaped ring, and the outer ring of the gear is meshedly connected to the inside of the tooth groove.

[0005] As a preferred embodiment of the above technical solution, limit plates are symmetrically installed on the back of the positioning column, one end of one of the limit plates is bonded with a rubber pad, and a vertical plate is fixedly installed inside the rotating plate.

[0006] As a preferred embodiment of the above technical solution, a clamping plate is integrally formed at one end of the positioning column, a manual telescopic rod is embedded and installed at the top end of the inner wall of the rotating plate, and a clamping block is clamped and installed at the bottom end of the manual telescopic rod.

[0007] As a preferred embodiment of the above technical solution, a triangular groove is provided on the top of the clamping disk, and the inner side of the triangular groove and the outer surface of the clamping block are in contact with each other.

[0008] As a preferred embodiment of the above technical solution, a spring rod is symmetrically embedded in one end surface of the rotating plate, a same moving bar is fixedly installed between one end surfaces of the two spring rods, a cylinder is fixedly installed at one end of the moving bar, and a rectangular block is clamped and installed on the outer side of the cylinder.

[0009] As a preferred embodiment of the above technical solution, one end face of the rectangular block is rotatably connected to a rotating column, and the center line of the rotating column and the center line of the limiting ring are in the same vertical plane.

[0010] As a preferred embodiment of the above technical solution, a groove is formed on one end surface of the rotating plate, and the spring rod, the moving bar, the cylinder and the rectangular block are all located inside the groove.

[0011] The present invention also provides a detection method applicable to a fungal pathogen qPCR detection kit, comprising the following steps: Step 1: Sample preparation: Collect samples according to the test object, such as blood, saliva, swab samples, etc.; Step 2: Nucleic acid extraction: Use a special nucleic acid extraction kit to extract the nucleic acid in the sample; Step 3: Preparation of qPCR reaction system: According to the instructions, the extracted nucleic acid and fluorescent quantitative PCR premix and other reagents are mixed in a certain proportion, and stirred and mixed by a rocking component to prepare a qPCR reaction solution; Step 4: qPCR reaction: inject the prepared reaction solution into the qPCR reaction tube, use the qPCR instrument to react and collect the fluorescence signal; Step 5: Result analysis: Analyze the fluorescence signal obtained from the qPCR reaction; Step 6: Waste disposal: After completing the test, properly dispose of used test kits, consumables and other items in accordance with the laboratory's safety management requirements to ensure the hygiene of the laboratory environment.

[0012] The beneficial effects of the present invention are: (1) The swing of the cylinder can be controlled by simple manual operation, which reduces the difficulty of swing control and changes the manual swing method in the prior art. This method reduces the labor intensity of personnel, and the mechanized operation method reduces the influence of human factors, ensuring the consistency and accuracy of each operation; (2) By swinging the cylinder back and forth, the enzyme substance and the sample can be evenly mixed, which helps to improve the thoroughness and accuracy of the reaction. Even mixing helps to ensure the accuracy of each test result and reduce false positive or false negative results caused by uneven mixing; (3) It is convenient to clamp and fix the cylinder, making the installation between the cylinder and the clamping disk more stable, preventing the cylinder and the clamping disk from separating, and further improving the safety of the cylinder during mixing. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The schematic diagram of the structure of a fungal pathogen qPCR detection kit in Example 1 is shown; Figure 2 The internal structure schematic diagram of a fungal pathogen qPCR detection kit in Example 1 is shown; Figure 3 What is shown is a schematic diagram of the structure of the placement slot in Example 1; Figure 4 The figure shows a schematic diagram of the installation structure of the swing assembly in Example 1; Figure 5 The figure shows a schematic diagram of the installation structure of the spring rod in Example 1; Figure 6 The figure shows a schematic diagram of the installation structure of the worm in Example 1; Figure 7 The figure shows a schematic diagram of the installation structure of the card receiving plate in the embodiment 1; Figure 8What is shown is a schematic diagram of the installation structure of the rubber pad in Example 1.

[0014] In the figure: 1. packaging box; 2. placement box; 3. placement slot; 4. swing assembly; 41. positioning column; 42. rotating plate; 43. limiting ring; 44. concave plate; 45. worm wheel; 46. worm; 47. positioning block; 48. gear; 49. arc ring; 410. vertical plate; 411. limiting plate; 412. rubber pad; 413. clamping plate; 414. manual telescopic rod; 415. clamping block; 416. spring rod; 417. moving bar; 418. cylinder; 419. rectangular block; 420. rotating column. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments.

[0016] Example 1: The present invention provides a fungal pathogen qPCR detection kit and detection method, such as Figures 1 to 8 As shown, it comprises: a packing box 1, a placing box 2 is arranged inside the packing box 1, and placing grooves 3 are equidistantly provided on the top of the placing box 2; One end of the placement box 2 is fixedly installed with a swing assembly 4 at a position inside the placement groove 3, and the swing assembly 4 includes a positioning column 41 fixedly installed inside the placement box 2, and a rotating plate 42 is rotatably connected to the outer side of the positioning column 41, and two limiting rings 43 are fixedly installed on one end face of the rotating plate 42, one of the limiting rings 43 is rotatably connected to a concave disk 44 inside, and a worm gear 45 is fixedly installed at the bottom end of the concave disk 44, and a worm 46 is meshingly connected to the outer side of the worm gear 45, and a positioning block 47 is symmetrically rotatably connected to the outer side of the worm 46, and the positioning block 47 is fixedly installed at the bottom end of the limiting ring 43, and a gear 48 is fixedly installed at one end of the worm 46, and an arc ring 49 is sleeved on the outer side of the gear 48 and the arc ring 49 is fixedly installed inside the placement groove 3 of the placement box 2.

[0017] like Figure 4 and Figure 5 As shown, a tooth groove is provided at the bottom end of the inner wall of the arc ring 49, and the outer ring of the gear 48 is meshedly connected to the inside of the tooth groove; Through the cooperation between the tooth groove and the gear 48 , the gear 48 can generate self-rotation when it moves along the inside of the arc circle 49 , thereby changing the difficulty of self-rotation of the gear 48 .

[0018] like Figure 5 and Figure 6 As shown, the back of the positioning column 41 is symmetrically installed with a limit plate 411, one end of one of the limit plates 411 is bonded with a rubber pad 412, and a vertical plate 410 is fixedly installed inside the rotating plate 42; When the rotating plate 42 rotates and fits with the limiting plate 411 , the vertical plate 410 blocks the rotating plate 42 , thereby further limiting the rotation angle of the rotating plate 42 .

[0019] like Figure 4 , Figure 5 , Figure 6 and Figure 7 As shown, a clamping plate 413 is integrally formed at one end of the positioning column 41, a manual telescopic rod 414 is embedded and installed at the top of the inner wall of the rotating plate 42, a clamping block 415 is clamped and installed at the bottom of the manual telescopic rod 414, and a triangular groove is opened at the top of the clamping plate 413, and the inner side of the triangular groove and the outer surface of the clamping block 415 are in contact with each other; When a person pulls the manual telescopic rod 414 and the manual telescopic rod 414 is stretched, the clamping block 415 is driven to move. When the clamping block 415 moves, it enters the triangular groove and contacts the clamping plate 413, so that the rotating plate 42 is limited to prevent the rotating plate 42 from swinging.

[0020] like Figure 4 , Figure 5 and Figure 6 As shown, a spring rod 416 is symmetrically embedded and installed on one end surface of the rotating plate 42, and a same moving bar 417 is fixedly installed between one end surfaces of the two spring rods 416. A cylinder 418 is fixedly installed on one end of the moving bar 417, and a rectangular block 419 is clamped and installed on the outer side of the cylinder 418. A rotating column 420 is rotatably connected to one end surface of the rectangular block 419, and the center line of the rotating column 420 and the center line of the limiting ring 43 are in the same vertical plane; The moving bar 417 is driven to move by the tension of the spring rod 416. When the moving bar 417 moves, it drives the rectangular block 419 to move through the cylinder 418. When the rectangular block 419 moves, it drives the rotating column 420 to fit the outer side of the mixing cylinder. At this time, the mixing cylinder is clamped and fixed without affecting the rotation of the mixing cylinder.

[0021] like Figure 4 , Figure 5 and Figure 6 As shown, a groove is provided on one end surface of the rotating plate 42, and the spring rod 416, the movable bar 417, the cylinder 418 and the rectangular block 419 are all located inside the groove, which can facilitate the placement of the spring rod 416, the movable bar 417, the cylinder 418 and the rectangular block 419, thereby reducing the difficulty of placing the spring rod 416, the movable bar 417, the cylinder 418 and the rectangular block 419.

[0022] The present invention also provides a detection method applicable to a fungal pathogen qPCR detection kit, comprising the following steps: Step 1: Sample preparation: Collect samples according to the test object, such as blood, saliva, swab samples, etc.

[0023] Step 2: Nucleic acid extraction: Use a special nucleic acid extraction kit to extract the nucleic acid in the sample.

[0024] Step 3: Preparation of qPCR reaction system: According to the instructions, the extracted nucleic acid and reagents such as fluorescent quantitative PCR premix are mixed in a certain proportion, and stirred and mixed by the rocking component 4 to prepare a qPCR reaction solution.

[0025] Step 4: qPCR reaction: Inject the prepared reaction solution into the qPCR reaction tube, and monitor the changes in the fluorescence signal through the qPCR instrument to determine the presence and concentration of the target gene.

[0026] Step 5: Result analysis: Analyze the fluorescent signal obtained from the qPCR reaction to determine whether the target gene exists in the sample.

[0027] Step 6: Waste disposal: After completing the test, properly dispose of used test kits, consumables and other items in accordance with the laboratory's safety management requirements to ensure the hygiene of the laboratory environment.

[0028] Working principle: During the actual use of the device, the medical staff collects the saliva from the child's oral cavity with a cotton swab, and the collected cotton swab head is placed in the cylinder, where it is mixed with the preservation solution in the cylinder. The cylinder cover is then closed, and the cylinder is placed in the limiting ring 43. At this time, the bottom end of the cylinder is clamped into the concave disk 44. The staff then pulls the manual telescopic rod 414, which drives the clamping block 415 to separate from the inside of the clamping disk 413, and then releases the moving bar 417. At this time, the moving bar 417 is driven to move by the tension of the spring rod 416. When the moving bar 417 moves, it drives the rectangular block 419 to move through the cylinder 418. When the rectangular block 419 moves, it drives the rotating column 420 to fit the outer side of the mixing cylinder, which is convenient for clamping and fixing the cylinder, making the installation between the cylinder and the concave disk 44 more stable, preventing the cylinder and the concave disk 44 from separating, and further improving the safety of the cylinder mixing. Then the personnel pulls the rotating plate 42, which rotates outside the positioning column 41 and drives the vertical plate 410 and the limiting plate 411 to fit together, and then they are loosened. At this time, the rotating plate 42 swings back and forth under the action of force, and the cylinder swings back and forth when the rotating plate 42 swings back and forth, so that the preservation liquid and the sample inside the cylinder are mixed. At this time, since the limiting ring 43 swings due to the swinging of the rotating plate 42, the limiting ring 43 drives the positioning block 47 to move when it swings, and the positioning block 47 drives the worm 46 to move when it moves. At this time, the tooth grooves of the gear 48 and the inner wall of the arc ring 49 are meshed, so that the gear 48 is generated during the movement of the gear 48. The gear 48 rotates by itself, and the worm 46 rotates by itself. The worm 46 rotates by itself, and the worm wheel 45 rotates by itself. The worm wheel 45 rotates by itself, and the concave plate 44 rotates by itself. The concave plate 44 rotates by itself, and the cylinder rotates outside the rotating column 420. The swing of the cylinder can be controlled by simple manual operation, which changes the difficulty of swing control and the manual swinging method in the prior art. This method reduces the labor intensity of personnel, and the cylinder swings back and forth to better achieve uniform mixing between the preservation solution and the sample, which helps to ensure the accuracy of each test result and reduce false positive or false negative results caused by uneven mixing. Then the personnel will extract nucleic acid from the mixed preservation liquid, mix the extracted nucleic acid with the fluorescent quantitative PCR premix, and put the mixing tube into the swing component 4. At this time, the oscillation of the swing component 4 can effectively promote the full mixing of the two liquids to ensure that the concentration and ratio of the reactants are accurate. The mixed liquid forms a qPCR reaction liquid, which is then transferred to the qPCR reaction tube and tested using a qPCR instrument to complete the overall detection method.

[0029] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them.

Claims

1. A fungal pathogen qPCR detection kit, characterized in that: The invention comprises a packaging box (1), wherein a placement box (2) is arranged inside the packaging box (1), and placement grooves (3) are equidistantly provided at the top of the placement box (2); a swing assembly (4) is fixedly installed at one end of the placement box (2) at a position inside the placement groove (3), and the swing assembly (4) comprises a positioning column (41) fixedly installed inside the placement box (2), and a rotating plate (42) is rotatably connected to the outer side of the positioning column (41), and two limiting rings (43) are fixedly installed on one end surface of the rotating plate (42), and one of the limiting rings (43) is fixedly installed. A concave disc (44) is rotatably connected to the inside of the concave disc (43), a worm wheel (45) is fixedly installed at the bottom end of the concave disc (44), a worm gear (45) is meshingly connected to the outside of the worm gear (45), a positioning block (47) is symmetrically rotatably connected to the outside of the worm gear (46), and the positioning block (47) is fixedly installed at the bottom end of the limiting ring (43), a gear (48) is fixedly installed at one end of the worm gear (46), an arc ring (49) is sleeved on the outside of the gear (48), and the arc ring (49) is fixedly installed inside the placement groove (3) of the placement box (2).

2. The fungal pathogen qPCR detection kit according to claim 1, characterized in that: A tooth groove is provided at the bottom end of the inner wall of the arc-shaped ring (49), and the outer ring of the gear (48) is meshedly connected to the inside of the tooth groove.

3. The fungal pathogen qPCR detection kit according to claim 2, characterized in that: The back of the positioning column (41) is symmetrically mounted with a limiting plate (411), one end of one of the limiting plates (411) being bonded with a rubber pad (412), and a vertical plate (410) is fixedly mounted inside the rotating plate (42).

4. The fungal pathogen qPCR detection kit according to claim 2, characterized in that: A clamping plate (413) is integrally formed at one end of the positioning column (41), a manual telescopic rod (414) is embedded and installed at the top end of the inner wall of the rotating plate (42), and a clamping block (415) is clamped and installed at the bottom end of the manual telescopic rod (414).

5. The fungal pathogen qPCR detection kit according to claim 4, characterized in that: A triangular groove is formed at the top of the clamping plate (413), and the inner side of the triangular groove and the outer surface of the clamping block (415) fit together.

6. The fungal pathogen qPCR detection kit according to claim 2, characterized in that: A spring rod (416) is symmetrically embedded and installed on one end surface of the rotating plate (42); a same moving bar (417) is fixedly installed between one end surfaces of the two spring rods (416); a cylinder (418) is fixedly installed on one end of the moving bar (417); and a rectangular block (419) is clamped and installed on the outer side of the cylinder (418).

7. The fungal pathogen qPCR detection kit according to claim 6, characterized in that: One end surface of the rectangular block (419) is rotatably connected to a rotating column (420), and a center line of the rotating column (420) and a center line of the limiting ring (43) are located in the same vertical plane.

8. The fungal pathogen qPCR detection kit according to claim 2, characterized in that: A groove is formed on one end surface of the rotating plate (42), and the spring rod (416), the moving bar (417), the cylinder (418) and the rectangular block (419) are all located inside the groove.

9. A detection method applicable to the fungal pathogen qPCR detection kit according to claim 8, characterized in that: The following steps are involved: Step 1: Sample preparation: Collect samples according to the test object, such as blood, saliva, swab samples, etc.; Step 2: Nucleic acid extraction: Use a special nucleic acid extraction kit to extract the nucleic acid in the sample; Step 3: Preparation of qPCR reaction system: According to the instructions, the extracted nucleic acid and the fluorescent quantitative PCR premix and other reagents are mixed in a certain proportion, and stirred and mixed by a rocking component (4) to prepare a qPCR reaction solution; Step 4: qPCR reaction: inject the prepared reaction solution into the qPCR reaction tube, use the qPCR instrument to react and collect the fluorescence signal; Step 5: Result analysis: Analyze the fluorescence signal obtained from the qPCR reaction; Step 6: Waste disposal: After completing the test, properly dispose of used test kits, consumables and other items in accordance with the laboratory's safety management requirements to ensure the hygiene of the laboratory environment.

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