Kit for detecting aeromonas in aquatic products

Abstract

The invention belongs to the technical field of monad detection, and particularly relates to a kit for detecting aeromonas in aquatic products, which comprises an operation plate for uniformly and quantitatively adding samples to a plurality of PCR tubes in a PCR plate; the operation plate comprises a plurality of quantitative latex containers made of elastic materials, injection ports of the quantitative latex containers are fixed relative to the upper plate, the upper plate does not seal the injection ports of the quantitative latex containers, the sealed ends of the quantitative latex containers are fixed relative to the lower frame, and the upper plate and the lower frame move relatively linearly to be away from or close to each other. The volume of the quantitative latex container can be changed after the quantitative latex container is stretched, the distance between the upper plate and the lower frame corresponds to the fixed volume of the quantitative latex container, and when the quantitative latex container is used, the volume of the quantitative latex container is adjusted according to the volume of a solution needing to be filled into each PCR tube, so that the maximum volume of the quantitative latex container at the moment is the volume of a target solution filled into the PCR tube.

Description

Technical Field

[0001] The invention belongs to the technical field of Aeromonas detection, and particularly relates to a kit for detecting Aeromonas in aquatic products. Background Art

[0002] When testing for Aeromonas in aquatic samples, the multiplex PCR method is currently commonly used. The multiplex PCR method involves placing the same sample into multiple PCR tubes for testing. Specific primers targeting different Aeromonas genes are added to different PCR tubes, allowing for the simultaneous detection of multiple Aeromonas or their different typing and virulence genes. For example, when testing for Aeromonas hydrophila in aquatic products, primers targeting its 16S rDNA conserved sequence gene, hemolysin gene, and quorum sensing signal molecule gene can be designed. Multiplex PCR can be performed in multiple PCR tubes, enabling the detection of multiple targets in a single experiment, thereby improving experimental efficiency.

[0003] The current multiplex PCR detection method places a sample into multiple PCR tubes, and uses a handheld pipette to transfer the quantitative samples one by one into multiple PCR tubes. However, the number of PCR tubes is usually dozens or even hundreds, and the tube openings of the PCR tubes are small, making the operation cumbersome, inefficient, and prone to causing hand soreness. Summary of the Invention

[0004] To solve the problems raised in the above background technology, the present invention provides a kit for detecting Aeromonas in aquatic products, which has the characteristics of being able to simultaneously load a quantitative sample solution into multiple PCR tubes at one time, being easy to operate and saving time.

[0005] To achieve the above-mentioned object, the present invention provides the following technical solutions: a kit for detecting Aeromonas in aquatic products, comprising an operation plate for uniformly and quantitatively adding samples to multiple PCR tubes in a PCR plate;

[0006] The operating panel includes several quantitative latex containers made of elastic material. The injection port of the quantitative latex container is fixed relative to the upper plate, and the upper plate does not close the injection port of the quantitative latex container. The closed end of the quantitative latex container is fixed relative to the lower frame, and the upper plate and the lower frame move linearly away or closer.

[0007] As a preferred Aeromonas detection kit in aquatic products of the present invention, the quantitative latex container includes a conical container wall, a hard ring portion is fixedly connected to the injection port of the quantitative latex container, and a spherical end is provided at the closed end of the quantitative latex container.

[0008] As a preferred embodiment of the Aeromonas detection kit in aquatic products of the present invention, the inner side of the lower frame is fixedly connected to a plurality of auxiliary supports through a plurality of main supports, and an open groove is formed at one end of the auxiliary support.

[0009] As a preferred embodiment of the Aeromonas detection kit in aquatic products of the present invention, a control rod is slidably connected to the outer side of one end of the auxiliary bracket, and a plurality of push rods are provided on the control rod.

[0010] As a preferred Aeromonas detection kit in aquatic products of the present invention, one end of the main bracket is fixedly connected to a threaded sleeve, the inner side of the threaded sleeve is threadedly connected to a threaded rod, and one end of the threaded rod is used to contact the surface of the upper plate.

[0011] As a preferred embodiment of the Aeromonas detection kit in aquatic products of the present invention, the upper plate is fixedly connected with a fence around it, and a diversion nozzle is provided at one end of the fence.

[0012] As a preferred embodiment of the Aeromonas detection kit in aquatic products of the present invention, a hard ruler is fixedly connected to the side surface of one end of the upper plate, and the surface of the ruler is in contact with the surface of the lower frame.

[0013] As a preferred embodiment of the Aeromonas detection kit in aquatic products of the present invention, the operation panel is further equipped with a hard water-absorbing plate for removing water stains on the surface of the upper panel.

[0014] As a preferred method of the Aeromonas detection kit in aquatic products of the present invention, the PCR plate includes a plate frame and a PCR tube, and a flexible layer providing static friction is fixedly connected to the position where the plate frame and the PCR tube cooperate.

[0015] As a preferred embodiment of the Aeromonas detection kit in aquatic products of the present invention, the control rod is fixedly connected to the buckle on the auxiliary bracket to limit the sliding movement.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: by moving the upper plate and the lower frame in opposite directions, the quantitative latex container can be stretched and its volume can be changed. The distance between the upper plate and the lower frame will correspond to the fixed volume of the quantitative latex container. When in use, the volume of the quantitative latex container is adjusted according to the volume of solution required to be loaded into each PCR tube, so that the maximum volume of the quantitative latex container at this time is the target solution volume loaded into the PCR tube. The sample solution is poured onto the upper plate so that each quantitative latex container is filled with the sample solution. Then, the upper plate is slightly tilted to pour out the excess solution on the upper plate, or a scraper is used. Scrape off the excess solution on the upper plate, turn the PCR plate upside down on the operation plate, and align the tube mouth of each PCR tube with the injection port of the corresponding quantitative latex container. Since the quantitative latex container is made of flexible material, it has a good sealing effect and can achieve a temporary sealed connection between the quantitative latex container and the PCR tube. Finally, flip the PCR plate and the operation plate as a whole to avoid misalignment between the two. After flipping, the sample solution in the quantitative latex container will enter the PCR tube, so that multiple PCR tubes can be loaded with quantitative sample solution at the same time, which is simple to operate and saves time. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0018] Figure 1 It is a schematic diagram of the overall structure of the present invention;

[0019] Figure 2 This is a cross-sectional view of the structure of the PCR plate of the present invention;

[0020] Figure 3 For the present invention Figure 2 Schematic diagram of the enlarged structure at A;

[0021] Figure 4 This is a front view of the overall structure of the operating panel of the present invention;

[0022] Figure 5 This is a schematic diagram of the back side of the overall structure of the operating panel of the present invention;

[0023] Figure 6 This is a schematic structural diagram of the quantitative latex container of the present invention;

[0024] Figure 7 Schematic diagram of the connection structure of the threaded sleeve in the present invention;

[0025] Figure 8 Schematic diagram of the opening position of the opening slot in the present invention;

[0026] In the picture:

[0027] 1. PCR plate; 2. Operation plate; 3. Water absorption plate; 4. Ruler; 5. Buckle;

[0028] 11. Plate rack; 12. Flexible layer; 13. PCR tube;

[0029] 21. Upper plate; 22. Lower frame; 23. Quantitative latex container; 231. Container wall; 232. Hard ring; 233. Spherical end;

[0030] 211, enclosure; 212, diversion nozzle;

[0031] 24. Main bracket; 25. Auxiliary bracket; 26. Open slot; 241. Threaded sleeve; 242. Threaded rod;

[0032] 27. Control lever; 28. Push rod; 29. ​​Hand push part. DETAILED DESCRIPTION

[0033] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] like Figures 1-8 As shown:

[0035] A kit for detecting Aeromonas in aquatic products, comprising an operation plate 2 for uniformly and quantitatively adding samples to a plurality of PCR tubes 13 in a PCR plate 1;

[0036] The operating panel 2 includes several quantitative latex containers 23 made of elastic material. The injection port of the quantitative latex container 23 is fixed relative to the upper plate 21, and the upper plate 21 does not close the injection port of the quantitative latex container 23. The closed end of the quantitative latex container 23 is fixed relative to the lower frame 22, and the upper plate 21 and the lower frame 22 move linearly relative to each other away from or closer to each other.

[0037] Aeromonas is a common species of bacteria found in aquatic products. Aeromonas also includes common Aeromonas hydrophila, Aeromonas sobria, Aeromonas vernix, Aeromonas dakar and Aeromonas sobria. It can infect crucian carp, bream, dace, common carp, silver carp, bighead carp, grass carp, sweetfish, wolf seabass, rainbow trout, Nile tilapia, channel catfish, yellow eel, eel and shrimp, causing disease or death.

[0038] When detecting Aeromonas in aquatic samples, the multiplex PCR method is currently commonly used to detect Aeromonas. The multiplex PCR method is to place the same sample into multiple PCR tubes 13 for detection. Specific primers for different Aeromonas genes are added to different PCR tubes 13, which can simultaneously detect multiple Aeromonas or their different types and virulence genes. For example, when detecting Aeromonas hydrophila in aquatic products, primers can be designed for its 16SrDNA conserved sequence gene, hemolysin gene, and quorum sensing signal molecule gene. Multiplex PCR can be performed in multiple PCR tubes 13, which can complete the detection of multiple targets in one experiment, thereby improving experimental efficiency.

[0039] If a single PCR amplifies only one gene, it cannot accurately determine the characteristics of a pathogen. Using multiple PCR to detect target bacteria from different angles can increase the accuracy and reliability of the test, help to more comprehensively understand the situation of microorganisms in the sample, and avoid the false positive or false negative results that may occur in single primer detection.

[0040] At the same time, when detecting monocytosine, positive and negative controls will be set up; samples known to contain the target monocytosine DNA are added to the positive control tube to verify whether the PCR reaction system and experimental operation are normal. If there is no expected amplification product in the positive control, it indicates that there is a problem with the experiment; samples that do not contain the target monocytosine, such as sterile water or samples known to not contain the bacteria, are added to the negative control tube to detect whether there is contamination during the experiment. If amplification bands appear in the negative control, it indicates that the experiment is contaminated and the results are unreliable.

[0041] When optimizing the reaction conditions, the optimal conditions for PCR amplification of different gene fragments of Bacillus may be different, such as annealing temperature, Mg 2+ concentration, primer concentration, etc.; the sample is placed in multiple PCR tubes 13, and different reaction conditions can be set for each tube to perform a gradient experiment to find the PCR reaction conditions that are most suitable for the detection of the target bacteria, thereby improving the sensitivity and specificity of the detection, and providing a basis for optimizing the reaction system and for subsequent more accurate and efficient detection of bacteria;

[0042] In the current multiplex PCR method, when a sample is placed into multiple PCR tubes 13, a handheld pipette is used to transfer the quantitative sample one by one into the multiple PCR tubes 13. However, the number of PCR tubes 13 is usually dozens or even hundreds, and the tube openings of the PCR tubes 13 are small, which makes the operation cumbersome, inefficient, and easily causes hand fatigue.

[0043] In this embodiment, the volume of the quantitative latex container 23 can be changed after being stretched by moving the upper plate 21 and the lower frame 22 in opposite directions. The distance between the upper plate 21 and the lower frame 22 will correspond to the fixed volume of the quantitative latex container 23. When in use, the volume of the quantitative latex container 23 is adjusted according to the volume of solution required to be loaded into each PCR tube 13, so that the maximum volume of the quantitative latex container 23 at this time is the target solution volume loaded into the PCR tube 13, and the sample solution is poured onto the upper plate 21 so that each quantitative latex container 23 is filled with the sample solution. Then, the upper plate 21 is slightly tilted to pour out the excess solution on the upper plate 21, or a scraper is used to scrape the upper plate 21. Scrape off the excess solution on the plate 21, turn the PCR plate 1 upside down on the operating plate 2, and align the tube mouth of each PCR tube 13 with the injection port of the corresponding quantitative latex container 23. Since the quantitative latex container 23 is made of flexible material, it has a good sealing effect and can achieve a temporary sealed connection between the quantitative latex container 23 and the PCR tube 13. Finally, flip the PCR plate 1 and the operating plate 2 as a whole to avoid misalignment between the two. After flipping, the sample solution in the quantitative latex container 23 will enter the PCR tube 13, so that multiple PCR tubes 13 can be filled with quantitative sample solution at the same time, which is simple to operate and saves time.

[0044] In an optional embodiment, the quantitative latex container 23 includes a conical container wall 231, a hard ring portion 232 is fixedly connected to the injection port of the quantitative latex container 23, and a spherical end cap 233 is provided at the closed end of the quantitative latex container 23.

[0045] In this embodiment, the hard ring portion 232 is provided to facilitate the injection port of the quantitative latex container 23 to be fixed on the upper plate 21, such as Figure 7 As shown, when the quantitative latex container 23 passes through the hole opened on the upper plate 21 from top to bottom, the diameter of the hard ring portion 232 is larger than the hole opened on the upper plate 21, so that the injection port of the quantitative latex container 23 is limited (that is, the part including the hard ring portion 232 is limited). The setting of the hard ring portion 232 not only makes it more convenient to fix the injection port of the quantitative latex container 23, but also facilitates disassembly and replacement. The forming of the hard ring portion 232 can be to fix a hard plastic ring at the opening of the one-piece quantitative latex container 23 by gluing, thereby forming the hard ring portion 232; the purpose of setting the spherical end 233 is to facilitate the closed end of the quantitative latex container 23 to be clamped and fixed.

[0046] In an optional embodiment, a plurality of auxiliary supports 25 are fixedly connected to the inner side of the lower frame 22 via a plurality of main supports 24 , and an open slot 26 is formed at one end of the auxiliary support 25 .

[0047] In this embodiment, the main bracket 24 serves as the main bearing structure and is arranged perpendicular to the auxiliary bracket 25, which is beneficial to reducing the overall manufacturing cost. The open groove 26 on the auxiliary bracket 25 can allow the connecting part between the spherical end 233 and the container wall 231 to pass through. Under the elastic force of the container wall 231 itself, the spherical end 233 can be engaged by the open groove 26, thereby limiting the effect of the closed end position of the quantitative latex container 23.

[0048] In an optional embodiment, a control rod 27 is slidably connected to the outer side of one end of the auxiliary bracket 25 , and a plurality of push rods 28 are provided on the control rod 27 .

[0049] In this embodiment, as described in the above embodiments, the PCR plate 1 and the operation plate 2 need to be flipped as a whole during operation. The sample solution enters the PCR tube 13 from the quantitative latex container 23 through its own fluidity. The flow of the solution requires a certain amount of time, and due to the interaction force between molecules, part of the sample solution will remain on the inner surface of the quantitative latex container 23. Therefore, by suddenly separating the spherical end 233 from the opening groove 26, the spherical end 233 will be ejected toward the hard ring portion 232 under the influence of the elasticity of the quantitative latex container 23 itself, thereby quickly allowing the sample solution in the quantitative latex container 23 to quickly enter the PCR tube 13 and reduce the liquid residue on the surface of the quantitative latex container 23. In order to facilitate the operation of this step, by moving the control rod 27, the control rod 27 will drive several push rods 28 to move synchronously, so that the push rod 28 pushes the spherical end 233 away from the opening groove 26. The hand push portion 29 can be fixed at the end of the control rod 27 to facilitate operation.

[0050] In an optional embodiment, one end of the main bracket 24 is fixedly connected to a threaded sleeve 241 , the inner side of the threaded sleeve 241 is threadedly connected to a threaded rod 242 , and one end of the threaded rod 242 is used to contact the surface of the upper plate 21 .

[0051] In this embodiment, Figure 5 As shown, a hexagonal groove can be opened inward at the end face of the threaded rod 242 to facilitate the use of an Allen wrench to drive the threaded rod 242 to rotate. The rotation of the threaded rod 242 can push the upper plate 21 and the lower frame 22 to separate, so as to facilitate precise control of the separation distance between the two. Due to the elastic properties of the quantitative latex container 23 itself, there is always an elastic force between the upper plate 21 and the lower frame 22 to move them closer to each other.

[0052] In an optional embodiment, a baffle 211 is fixedly connected to the four sides of the upper plate 21 , and a guide nozzle 212 is provided at one end of the baffle 211 .

[0053] In this embodiment, the baffle 211 is provided to prevent the sample solution guided on the upper plate 21 from flowing to other parts. The diversion nozzle 212 is provided, and when the operating plate 2 is tilted or a scraper is used to scrape off excess sample solution, the diversion nozzle 212 has a good diversion effect, which facilitates the centralized collection of excess sample solution.

[0054] In an optional embodiment, a hard ruler 4 is fixedly connected to the side surface of one end of the upper plate 21 , and the surface of the ruler 4 is in contact with the surface of the lower frame 22 .

[0055] In this embodiment, the distance between the upper plate 21 and the lower frame 22 can be visually observed by setting the ruler 4. At the same time, the ruler 4 stabilizes the relative position of the two and reduces the offset in other directions. The ruler 4 can be set around the operating panel 2.

[0056] In an optional embodiment, the operating panel 2 is further equipped with a hard water-absorbing plate 3 for removing water stains on the surface of the upper panel 21 .

[0057] In this embodiment, even after tilting or scraping, some sample solution may still remain on the upper plate 21. When the operation plate 2 is turned over, the workbench may be contaminated. There are many spaces on the upper plate 21, and the traditional method of wiping with a paper towel is troublesome. By covering the upper plate 21 with a hard absorbent plate 3 that matches the shape of the upper plate 21, the excess sample solution on the upper plate 21 can be absorbed, thereby preventing the sample solution from falling when the operation plate 2 is turned over. The hard absorbent plate 3 is more convenient to cooperate with the upper plate 21, and is easy to operate.

[0058] In an optional embodiment, the PCR plate 1 includes a plate frame 11 and PCR tubes 13 , and a flexible layer 12 providing static friction is fixedly connected at the position where the plate frame 11 and the PCR tubes 13 cooperate.

[0059] In this embodiment, when the PCR plate 1 is turned upside down on the operating plate 2, in order to prevent the PCR tube 13 from being separated from the plate rack 11 or the relative position of the two from being unstable, resulting in the PCR tube 13 being unable to fit mouth to mouth with the quantitative latex container 23, a flexible layer 12 is added to provide static friction force, so that when the PCR plate 1 is flipped over, the PCR tube 13 will not shake or fall off, and the operation of removing the PCR tube 13 from the plate rack 11 is not complicated.

[0060] In an optional embodiment, the control rod 27 is fixedly connected to the buckle 5 on the auxiliary bracket 25 to limit sliding.

[0061] In this embodiment, the buckle 5 is fixed to the auxiliary bracket 25 by screws, which facilitates the installation of the control rod 27 and reduces production costs.

[0062] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A kit for detecting Aeromonas in aquatic products, characterized by: It comprises an operation plate (2) for uniformly and quantitatively adding samples to a plurality of PCR tubes (13) in a PCR plate (1); The operating panel (2) includes a plurality of quantitative latex containers (23) made of elastic material. The injection ports of the quantitative latex containers (23) are fixed relative to the upper panel (21), and the upper panel (21) does not close the injection ports of the quantitative latex containers (23). The closed ends of the quantitative latex containers (23) are fixed relative to the lower frame (22), and the upper panel (21) and the lower frame (22) can move linearly relative to each other to move away from or closer to each other.

2. The kit for detecting Aeromonas in aquatic products according to claim 1, characterized in that: The quantitative latex container (23) comprises a conical container wall (231), a hard ring portion (232) is fixedly connected to the injection port of the quantitative latex container (23), and a spherical end cap (233) is provided at the closed end of the quantitative latex container (23).

3. The kit for detecting Aeromonas in aquatic products according to claim 1 or 2, characterized in that: The inner side of the lower frame (22) is fixedly connected with a plurality of auxiliary supports (25) through a plurality of main supports (24), and an opening groove (26) is provided at one end of the auxiliary support (25).

4. The kit for detecting Aeromonas in aquatic products according to claim 3, characterized in that: One end of the auxiliary bracket (25) is slidably connected to the outside of a control rod (27), and a plurality of push rods (28) are provided on the control rod (27).

5. The kit for detecting Aeromonas in aquatic products according to claim 3, characterized in that: One end of the main bracket (24) is fixedly connected to a threaded sleeve (241), the inner side of the threaded sleeve (241) is threadedly connected to a threaded rod (242), and one end of the threaded rod (242) is used to contact the surface of the upper plate (21).

6. The kit for detecting Aeromonas in aquatic products according to claim 1, characterized in that: The upper plate (21) is fixedly connected with a fence (211) on all sides, and a diversion nozzle (212) is provided at one end of the fence (211).

7. The kit for detecting Aeromonas in aquatic products according to claim 1 or 5, characterized in that: A hard ruler (4) is fixedly connected to the side surface of one end of the upper plate (21), and the surface of the ruler (4) is in contact with the surface of the lower frame (22).

8. The kit for detecting Aeromonas in aquatic products according to claim 1, characterized in that: The operating panel (2) is also equipped with a hard water-absorbing plate (3) for removing water stains on the surface of the upper panel (21).

9. The kit for detecting Aeromonas in aquatic products according to claim 1, wherein: The PCR plate (1) comprises a plate frame (11) and a PCR tube (13); a flexible layer (12) providing static friction is fixedly connected at a position where the plate frame (11) and the PCR tube (13) cooperate.

10. The kit for detecting Aeromonas in aquatic products according to claim 4, characterized in that: The control rod (27) is fixedly connected to the buckle (5) on the auxiliary bracket (25) to limit the sliding movement.

Images