Plug-in type reaction device suitable for containing LAMP (loop-mediated isothermal amplification) detection substance to detect cummerus fumoniliforme

Abstract

The invention relates to a plug-in type reaction device suitable for containing LAMP (loop-mediated isothermal amplification) detection substances so as to detect carufuronia, which belongs to the technical field of biology, consists of a liquid storage tube (25) and a reaction tube (26), and is characterized in that a liquid storage tube plug (13) is arranged at the lower part of the liquid storage tube (25), four liquid flowing tubes (9) are arranged, and each liquid flowing tube (9) is a flat tube with an inclined angle of 45 degrees; after the plugging plate (6) is positioned at the outer opening of the liquid flowing pipe (9), candle liquid or vaseline is coated on the periphery of the outer opening of the liquid flowing pipe (9) and the inner surface of the through hole; a reaction system required by the LAMP reaction is contained in the reaction tube (26); lAMP reaction is carried out by utilizing the reaction tube (26), and the color change of the solution is directly observed by naked eyes after the reaction is finished, so that whether the cupofuronia exists in the sample or not is judged according to the color change; the method is easy and convenient to operate, low in cost and visual in result.

Description

Technical Field

[0001] The invention relates to a plug-in reaction device suitable for containing LAMP detection objects to detect Cupiophora carunculata, belonging to the technical field of biology. Background Art

[0002] Ciboria carunculoides belongs to the phylum Ascomycota, class Leotiomycetes, order Helotiales, family Sclerotiniaceae, and genus Ciboria. It can cause the fruitlets of mulberry to swell and protrude, making the infected mulberry look like popcorn and easy to fall off, leaving the fruit axis. It is currently the dominant pathogen of mulberry sclerotinia in my country. At present, most fruit mulberry varieties in my country are susceptible varieties, and spraying chemical fungicides is the main method for preventing and controlling mulberry sclerotinia. Therefore, establishing a method for rapid detection of mulberry sclerotinia bacteria to monitor the occurrence of the disease is conducive to reducing the use of chemical pesticides and the development of pathogen resistance, and reducing the losses caused by mulberry sclerotinia to the mulberry industry. At present, there are few detection methods for mulberry sclerotinia: one is to identify the pathogen through traditional methods, namely morphology. Since it is extremely difficult for Caruncle-shaped Cup-disc Fungus to grow on artificial culture media, it is now mostly identified by observing the symptoms of diseased fruits and the morphology of the fruiting bodies of the pathogen. However, this method relies on the experience of the tester, and it is difficult for ordinary personnel to make accurate judgments in the field, and it is time-consuming and laborious. The second is to identify pathogens through molecular biological methods. The existing methods include the high sensitivity of real-time fluorescence quantitative PCR and the sequence polymorphism based on enzyme cutting to detect Caruncle-shaped Cup-disc Fungus and other mulberry sclerotinia pathogens. These methods have high detection accuracy and strong specificity, and can be well used for the identification and disease detection of mulberry sclerotinia pathogens, but the detection time is long, and thermal cycling equipment is required, which is costly. The test results also need to be presented by agarose gel electrophoresis, which cannot meet the needs of rapid field detection. Loop-mediated isothermal amplification (LAMP) is a new type of nucleic acid amplification technology. It is widely used in food safety testing, plant and aquatic quarantine, human disease detection and other fields because of its advantages such as simple operation, low cost, strong specificity, and visualization of results. This technology designs 4 specific primers for 6 regions of the target sequence. Under the action of Bst DNA polymerase, the target sequence is amplified in vitro in a specific exponential manner within the range of 60-65°C, and the amplification reaction can be completed in about 60 minutes. The results of LAMP detection can be judged not only by agarose gel electrophoresis, but also by directly observing the test results with the naked eye using indicator reactions such as fluorescent dyes and indicators. Since the reaction conditions required for this technology are constant temperature conditions, a water bath or equipment that can provide stable temperature conditions can meet the conditions of the amplification reaction. The detection operation steps are simple, the cost is low, and the test results are visible, which can greatly improve the efficiency of field detection. At present, there is no LAMP detection method for Caruncle-shaped Cup Disk in the detection methods of mulberry sclerotinia. The methods for judging the results of LAMP reactions include agarose gel electrophoresis, fluorescent dye method, turbidity method, metal ion indicator method, etc.Hydroxynaphthol blue (HNB) is a metal ion indicator. At the beginning of the reaction, hydroxynaphthol blue combines with magnesium ions in the reaction solution to make the solution appear purple. As the amplification reaction proceeds, magnesium ions react with the precipitated pyrophosphate ions to form magnesium pyrophosphate precipitates. After hydroxynaphthol blue loses magnesium ions, the reaction solution appears blue (positive), while the solution without amplification reaction remains purple (negative). Regarding the relevant technologies for storing reagents for faster use, the main ones are currently iodine-related cotton swabs, such as a traditional Chinese medicine portable double-headed iodine cotton swab that can disinfect and care for wounds (application number CN202221479358.3); cotton swabs (application number CN202320676649.X); integrated venous blood collector (application number CN202323060000.4); a dry-wet separation cotton swab jar (application number CN2024204949 30.6); a dressing for circumcision surgery (application number CN202221085379.7); an iodine cotton swab (application number CN202122529459.9); a new type of iodine cotton swab (application number CN202121214359.0); a splash-proof disposable disinfected iodine cotton swab (application number CN202120469581.9); a medical iodine cotton swab (application number CN20202093 8083.X); a sanitary iodine cotton swab (application number CN201720862012.4); an improved iodine cotton swab (application number CN201720866451.2); an improved easy-to-use iodine cotton swab (application number CN201720403594.X); a negative pressure iodine cotton swab and its use method (application number CN201710250620.4); an improved cosmetic iodine cotton swab (application number CN 201710613931.2); a cosmetic iodine cotton swab (application number CN201611186200.6); a medical iodine cotton swab (application number CN201520435537.0); a medical iodine cotton swab (application number CN201520336328.0); a variety of iodine cotton swabs were announced, which use a variety of means to store iodine liquid, supplemented by a special structure, so that people can use it in different occasions. The present invention draws on the principle of the above-mentioned iodine cotton swab storage to design a tubular plug-in reaction device suitable for holding LAMP test objects to detect caruncle-shaped cup disc fungus, which is very convenient for holding LAMP test objects. At the same time, it is also convenient to place the entire device in a water bath to maintain a constant LAMP reaction. Summary of the invention

[0003] In view of the technical shortcomings of the existing methods for detecting Cupidiscus carnosus, the present invention aims to provide a rapid detection method for Cupidiscus carnosus based on LAMP technology, so as to solve the problems of high cost and complicated operation of Cupidiscus carnosus detection in the prior art.

[0004] The technical solution adopted by the present invention to solve its technical problem is:

[0005] The present invention is suitable for containing LAMP detection objects to detect caruncle-shaped cup disc fungus plug-in reaction device, which is composed of a liquid storage tube 25 and a reaction tube 26, and is characterized in that the liquid storage tube 25 is glass, the upper end of the liquid storage tube 25 is a pipe mouth 27 that can be inserted into another liquid storage tube plug 13, and the length of the entire liquid storage tube 25 below the pipe mouth is 1 / 5 of the upper part of the liquid storage tube 25, the thickness of the upper part of the liquid storage tube wall 1 is 1-5 mm, the height is 5-10 mm, and it is cylindrical, the outer diameter of the cylinder is 5-20 mm, and the inner diameter is 3-10 mm ; Four anti-slip sheets 5 are arranged in the middle of the inner surface of the upper part of the liquid storage tube 25, and the line between the central points of two adjacent anti-slip sheets 5 and the center of the circle where the cross section is located forms an angle of 90°. The middle part of the liquid storage tube 25 is the main part of the liquid storage tube 25, and the thickness of the middle part of the liquid storage tube wall 1 is 0.5-2 mm, the height is 15-30 mm, and it is cylindrical. The outer diameter of the cylinder is 5-20 mm and the inner diameter is 4-16 mm; the lower part of the liquid storage tube 25 is the liquid storage tube plug 13; the liquid storage tube plug 13 is barrel-shaped, and the liquid storage tube plug 13 can be divided from top to bottom The plug 13 is divided into three parts: a connection part, a central part and a bottom part; the connection part of the liquid storage tube plug 13 starts from the top edge of the inclined surface with an angle of 45° in the middle of the liquid storage tube 25, and ends at the bottom end of the liquid storage tube wall 1 with an outer diameter of 5-20 mm, and the thickness of the connection part of the liquid storage tube plug 13 is 1.5-7 mm; the central part of the liquid storage tube plug 13 refers to the part from the bottom end of the liquid storage tube wall 1 with an outer diameter of 5-20 mm, and ends at the lower edge of the opening of the outer end of the liquid flow tube 9. Four spring-fixing cavities 7 are arranged on the central outer surface of the central part of the liquid storage tube plug 13, and at this point On the cross section of the central part of the liquid storage tube plug 13, the two connecting lines from the center of the two adjacent fixed spring chambers 7 to the center of the cross section circle form an angle of 90 degrees; the bottom of the liquid storage tube plug 13 refers to the bottom edge of the opening of the outer end of the liquid flow tube 9, all the way to the bottom edge of the entire liquid storage tube 25. The bottom of the liquid storage tube plug 13 consists of three parts: the liquid storage tube bottom 10, the liquid storage tube bottom convex 11, and the liquid storage tube bottom concave 12. The upper part of the bottom of the liquid storage tube plug 13 is the liquid storage tube bottom convex 11, which is conical. The lower edge of the liquid storage tube bottom convex 11 and the lower edge of the opening of the inner end of the liquid flow tube 9 are 0.5-1 mm, there are 4 liquid flow tubes 9, and the two connecting lines between the central points of the inner openings of two adjacent liquid flow tubes 9 and the center of the bottom circle of the convex 11 of the liquid storage tube bottom form an angle of 90°; each liquid flow tube 9 is a flat tube inclined at 45°; the solid part below the convex 11 of the liquid storage tube bottom is the liquid storage tube bottom 10; the concave 12 of the liquid storage tube bottom is the part of the lower surface of the liquid storage tube bottom 10 that is concave upward; a convex 3 is set on the upper outer surface of the liquid storage tube wall 1 at a distance of 3-6 mm from the top of the liquid storage tube wall 1, and the convex 3 is in a circular ring shape, and the inner wall of the circular convex 3 is fused with the liquid storage tube wall 1, and the cross-section of the convex 3 ring body is rectangular, and the length of the rectangular convex 3 ring body is 2-5 mm, width of 1-3 mm; float 4 is the storage tube 25 into the water bath reaction when the uppermost storage tube 25 is set outside the upper part of the liquid storage tube 25, for the storage tube 25 and the reaction tube 26 inserted together to provide buoyancy, the float 4 consists of a float body 21 and a float cavity 22, the float body 21 is a foam plastic material, annular, the outer diameter of the float body 21 is 10-50 mm, the inner diameter is 5-20 mm, the thickness of 5-10 mm; the float cavity 22 is a cavity in the center of the float 4, cylindrical, the diameter of the float cavity 22 is 5-20 mm, the height of 5-10 mm; the anti-slip sheet 5 is arranged in the anti-slip sheet groove 14, a total of 4 , each anti-slip sheet 5 is composed of an anti-slip sheet body 15 and a connecting rubber 16, the anti-slip sheet body 15 is made of manganese steel, the outer surface of the upper end of the anti-slip sheet body 15 forms an inclined surface, and the lower end of the anti-slip sheet body 15 is flush; the connecting rubber 16 is made of silica gel; the blocking plate 6 is made of glass, a total of 4, the blocking plate 6 is divided into an upper and lower part, the upper part is the head, and the lower part is the handle. The width of the head of the blocking plate 6 is 2-3 times the width of the handle, the height is 1-2 mm, the thickness is 0.2-0.5 mm, and the width direction is arc-shaped; the cross-section of the blocking plate 6 handle coincides with the arc-shaped through-hole inner cavity in the connecting part of the liquid storage tube plug 13, and there is a shallow groove on the inner surface of the middle part, which is shaped The sealing plate 6 is located at the outer opening of the liquid flow tube 9, and the outer opening periphery and the inner surface of the through hole of the liquid flow tube 9 are coated with candle liquid or vaseline; each spring cavity 7 is in the shape of a rectangular parallelepiped, the height of the spring cavity 7 is 2.1-5.2 mm, the length is 0.7-1.5 mm, and the width is 0.6-1.2 mm. The central axis of the spring cavity 7 in the height direction is in a straight line with the center of the outer opening of the liquid flow tube 9. A spring 8 is arranged in each spring cavity 7; the length of the spring 8 is 2-5 mm, the diameter is 0.5-1 mm, the spiral pitch is 0.2-0.5 mm, and the diameter of the cross section of the spring steel wire is 0.1-0.2 mm, an arc-shaped through hole is provided in the connection part of the liquid storage tube plug 13 corresponding to the position of the fixed spring chamber 7; the liquid storage tube wall concave 17 is a semicircular groove located on the inner surface of the upper part of the liquid storage tube 25, and there are 2 liquid storage tube wall concave 17, and the 2 liquid storage tube wall concave 17 are located on a diameter; the liquid storage tube wall convex 18 is a semicircular protrusion located on the inner surface of the upper part of the liquid storage tube 25, and there are 2, and the 2 liquid storage tube wall convex 18 is located on another diameter perpendicular to the diameter of the 2 liquid storage tube wall concave 17; each liquid storage tube wall concave 17 or liquid storage tube wall convex 18 is arranged between two adjacent anti-slip sheets 5; the liquid storage tube plug concave 19 is a semicircular groove located on the central part and the bottom outer surface of the liquid storage tube plug 13, and there are 2 liquid storage tube plug concave 19, and the 2 liquid storage tube plug concave 19 are located on a diameter; the liquid storage tube plug convex 20 is a semicircular protrusion located on the central part and the bottom outer surface of the liquid storage tube plug 13 , there are two, the two storage tube plug convex 20 is located on another diameter perpendicular to the diameter where the two storage tube plug concave 19 is located; the lower end of the storage tube 25 is the storage tube plug 13, and the through hole of the storage tube plug 13 is inserted with a blocking plate 6, the wall around the storage tube 25 is the storage tube wall 1, and the cavity surrounded by the storage tube wall 1 is the storage tube cavity 2; the reaction tube 26 is glass, the wall thickness at the upper opening is 1-5 mm, the height is 5-10 mm, and it is cylindrical, the outer diameter of the cylinder is 5-20 mm, and the inner diameter is 3-10 mm; 4 anti-slipping sheets 5 are set in the middle of the upper inner surface of the reaction tube 26, and the line between the central points of two adjacent anti-slipping sheets 5 and the center of the circle where the cross section is located forms an angle of 90°; the lower part of the reaction tube 26 is a gradually tapering structure, and finally blocked at the bottom, forming a small tube-like shape with a lower end size of 1 / 5-1 / 4 of the upper end. .

[0006] The present invention uses the calmodulin (CaM) gene sequence of Caruncle-shaped Cup disc fungus as a target sequence (as shown in Seq ID No:5), and designs 4 LAMP primers. The LAMP detection primer composition for detecting the calmodulin gene sequence of Caruncle-shaped Cup disc fungus comprises: a forward inner and outer primer FIP-GC142, a reverse inner primer BIP-GC142, a forward outer primer F3-GC142 and a reverse outer primer B3-GC142, wherein the F3 sequence is shown in Seq ID No:1; the B3 sequence is shown in Seq ID No:2; the FIP sequence is shown in Seq ID No:3; and the BIP sequence is shown in Seq ID No:4.

[0007] The present invention also provides application of the primer combination in detecting C. carunculata.

[0008] The present invention also provides a liquid storage tube (25) and a reaction tube (26) for detecting C. carunculata using the primer combination. The liquid storage tube (25) and the reaction tube (26) contain a reaction system required for a LAMP reaction.

[0009] The reaction system contained: 0.1 μM forward outer primer F3-GC142, 0.1 μM reverse outer primer B3-GC142, 0.8 μM forward inner primer FIP-GC142 and 0.8 μM reverse inner primer BIP-GC142, 12.5 μL 2×HNB lsoAmpMix, 8 U / μL Bst DNA polymerase, ddH2O and 1.0 μL template DNA.

[0010] The present invention also provides a LAMP detection method for Caruncle-shaped Cupiucophyllum, which uses a reaction tube (26) to carry out a LAMP reaction, and after the reaction is completed, directly observe the color change of the solution with the naked eye to determine whether Caruncle-shaped Cupiucophyllum is present in the sample.

[0011] The LAMP detection method can refer to the following steps: extract the DNA of the sample to be detected, take 1.0 μL of DNA solution as a template, add it to 24 μL of the kit detection solution for LAMP reaction, the reaction procedure is 65°C reaction amplification for 45-60min, and after the reaction, 85°C heat inactivation can be selected for 5 min. The result of the LAMP reaction can be directly observed by naked eye to observe the color change of the solution or by agarose gel electrophoresis. If the color of the solution changes from purple to blue, or a ladder-like electrophoresis band is observed under ultraviolet light, it indicates that the sample to be detected contains caruncle-shaped cup disc fungus; if the color of the solution does not change and is still purple, or a ladder-like electrophoresis band is not observed under ultraviolet light, it indicates that the sample to be detected does not contain caruncle-shaped cup disc fungus.

[0012] The beneficial effects of the present invention are:

[0013] (1) Simple operation and low cost: The detection method of Caruncle-shaped Cupidella caruncle based on LAMP technology provided by the present invention overcomes the problems of complicated operation steps, the need for thermal cycle equipment, and high cost in the prior art. The detection method of the present invention can quickly detect Caruncle-shaped Cupidella caruncle within 60 minutes under a constant temperature of 65°C. It has simple requirements for detection equipment and does not require expensive thermal cycle equipment, and is suitable for promotion and use at the grassroots level.

[0014] (2) Result visualization: The detection method of the present invention can be directly observed by the naked eye through color changes, which improves the detection efficiency to a certain extent and facilitates the rapid on-site detection of C. carunculata. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention is further described below in conjunction with the accompanying drawings.

[0016] Figure 1 The present invention is a schematic diagram of the overall longitudinal section structure of a liquid storage tube of a plug-in reaction device suitable for containing LAMP test objects to detect Carunculaceae.

[0017] Figure 2 The present invention is a schematic diagram of the cross-sectional structure of the anti-detachment sheet of the liquid storage tube of the plug-in reaction device suitable for containing LAMP test objects to detect Carunculaceae.

[0018] Figure 3 The present invention is a schematic diagram of the cross-sectional structure of a liquid storage tube plug of a plug-in reaction device suitable for containing LAMP test objects to detect Carunculum carunculum.

[0019] Figure 4 The figure is a schematic diagram of the overall structure of a floating ring of a plug-in reaction device suitable for containing LAMP test objects to detect Carunculaceae.

[0020] Figure 5 The schematic diagram of the longitudinal section structure of the reaction tube of the plug-in reaction device suitable for containing LAMP test objects to detect Carunculaceae.

[0021] Figure 1-5 In the embodiment, 1. liquid storage tube wall, 2. liquid storage tube cavity, 3. convex block, 4. floating ring, 5. anti-slip sheet, 6. blocking plate, 7. fixed spring cavity, 8. fixed spring, 9. liquid flow tube, 10. liquid storage tube bottom, 11. convex bottom of liquid storage tube, 12. concave bottom of liquid storage tube, 13. liquid storage tube plug, 14. anti-slip sheet groove, 15. anti-slip sheet body, 16. connecting rubber, 17. concave wall of liquid storage tube, 18. convex wall of liquid storage tube, 19. concave plug of liquid storage tube, 20. convex plug of liquid storage tube, 21. floating ring body, 22. floating ring cavity, 23. reaction tube wall, 24. reaction tube cavity, 25. liquid storage tube, 26. reaction tube, 27. tube mouth.

[0022] Figure 6 A diagram of the binding sites of the LAMP primers of the caruncle-shaped Cupiucophylla in the target sequence of a plug-in reaction device suitable for containing LAMP test materials to detect the caruncle-shaped Cupiucophylla.

[0023] Figure 7The LAMP detection method of the plug-in reaction device suitable for holding LAMP test objects to detect Ciboria carunculoides can only detect Ciboria carunculoides among 8 pathogens, and other pathogens cannot be detected. In the figure, A is the result of the color change of the reaction solution, B is the result of 1% agarose gel electrophoresis to detect the LAMP reaction product, and M is DL 2000 marker. Among them: 1. Blank control; 2. Ciboria carunculoides; 3. Sclerotiniasclerotiorum; 4. Fusarium fujikuroi; 5. Rhizoctonia solani; 6. Leptosphaerulina arachidicola; 7. Penicillium italicum; 8. Colletotrichum orbiculare; 9. Aspergillus japonicus.

[0024] Figure 8 The sensitivity verification result of the LAMP detection method for Caruncle-shaped Cup disc fungus is a plug-in reaction device suitable for containing LAMP detection objects to detect Caruncle-shaped Cup disc fungus. In the figure, A is the result of the color change of the reaction solution, B is the result of 1% agarose gel electrophoresis to detect the LAMP reaction product, and M is DL 2000 marker. Among them: 1. 10 ng / μL; 2. 1 ng / μL; 3. 100 pg / μL; 4. 10 pg / μL; 5. 1 pg / μL; 6. 100 fg / μL; 7. 10 fg / μL; 8. 1 fg / μL; 9. Blank control. DETAILED DESCRIPTION

[0025] Embodiment 1

[0026] The plug-in reaction device suitable for containing LAMP test objects to detect caruncle-shaped cup disc fungus is composed of a liquid storage tube wall 1, a liquid storage tube cavity 2, a convex block 3, a floating ring 4, an anti-slip sheet 5, a blocking plate 6, a fixed spring cavity 7, a fixed spring 8, a liquid flow tube 9, a liquid storage tube bottom 10, a liquid storage tube bottom convex 11, a liquid storage tube bottom concave 12, a liquid storage tube plug 13, an anti-slip sheet groove 14, an anti-slip sheet body 15, a connecting rubber 16, a liquid storage tube wall concave 17, a liquid storage tube wall convex 18, a liquid storage tube plug concave 19, a liquid storage tube plug convex 20, a floating ring body 21, a floating ring cavity 22, a reaction tube wall 23, a reaction tube cavity 24, a liquid storage tube 25, a reaction tube 26, and a tube mouth 27. The liquid storage tube 25 is used to contain a primer combination, HNB lsoAmp Mix, Bst for detecting the mulberry sclerotinia pathogen caruncle-shaped cup disc fungus. The tube for the detection solution prepared by DNA polymerase and ddH2O is made of glass. The upper end of the liquid storage tube 25 is a tube mouth 27 into which another liquid storage tube plug 13 can be inserted. The lower part of the tube mouth occupies 1 / 5 of the length of the entire liquid storage tube 25, which is the upper part of the liquid storage tube 25. The thickness of the upper part of the liquid storage tube wall 1 is 1-5 mm, the height is 5-10 mm, and it is cylindrical. The outer diameter of the cylinder is 5-20 mm, and the inner diameter is 3-10 mm. The middle of the inner surface of the upper part of the liquid storage tube 25 is provided with Four anti-slip sheets 5 are arranged, and the line connecting the central points of two adjacent anti-slip sheets 5 and the center of the circle where the cross section is located forms an angle of 90°. The middle of the liquid storage tube 25 is the main part of the liquid storage tube 25. The thickness of the middle part of the liquid storage tube wall 1 is 0.5-2 mm, the height is 15-30 mm, and it is cylindrical. The outer diameter of the cylinder is 5-20 mm and the inner diameter is 4-16 mm; the lower part of the liquid storage tube 25 is the liquid storage tube plug 13; the liquid storage tube plug 13 is the liquid storage tube 25 from which The nozzle 27 of the other storage tube 25 is inserted into the upper part of the other storage tube 25 or the nozzle 27 of the reaction tube 26 is inserted into the upper part of the other storage tube 25 of the reaction tube 26. The entire storage tube plug 13 is barrel-shaped. The storage tube plug 13 can be divided into three parts from top to bottom: a connecting part, a central part and a bottom part. The connecting part of the storage tube plug 13 starts from the top edge of the inclined surface with an inclination of 45° in the middle of the storage tube 25 and extends to the storage tube wall 1 with an outer diameter of 5-20 mm. The thickness of the connection part of the liquid storage tube plug 13 is 1.5-7 mm; the central part of the liquid storage tube plug 13 is from the bottom end of the liquid storage tube wall 1 with an outer diameter of 5-20 mm to the lower edge of the opening of the outer end of the liquid flow tube 9. Four spring chambers 7 are arranged on the outer surface of the central part of the liquid storage tube plug 13. On the cross section of the central part of the liquid storage tube plug 13, the two connecting lines from the center of two adjacent spring chambers 7 to the center of the cross section circle form an angle of 90°.The bottom of the liquid storage tube plug 13 refers to the bottom edge of the opening at the outer end of the liquid flow tube 9, all the way to the bottom edge of the entire liquid storage tube 25. The bottom of the liquid storage tube plug 13 consists of three parts: the liquid storage tube bottom 10, the liquid storage tube bottom convex 11, and the liquid storage tube bottom concave 12. The upper part of the bottom of the liquid storage tube plug 13 is the liquid storage tube bottom convex 11, which is conical. The lower edge of the liquid storage tube bottom convex 11 and the lower edge of the opening at the inner end of the liquid flow tube 9 are 0.5-1 mm apart. There are 4 liquid flow tubes 9, and the two connecting lines between the central points of the inner openings of two adjacent liquid flow tubes 9 and the center of the bottom circle of the liquid storage tube bottom convex 11 are at an angle of 90°; each liquid flow tube 9 is a flat tube inclined at 45°. After the sealing plate 6 outside the outer opening is pushed open to expose the opening at the outer end of the liquid flow tube 9, the liquid in the liquid storage tube cavity 2 can flow along the liquid flow tube 9 flows out; the solid part below the convex 11 of the liquid storage tube bottom is the liquid storage tube bottom 10, which is used to block the lower end of the liquid storage tube cavity 2; the concave 12 of the liquid storage tube bottom is the part that is concave upward in the center of the lower surface of the liquid storage tube bottom 10. On the one hand, the concave 12 of the liquid storage tube bottom can reduce the weight of the entire liquid storage tube 25. On the other hand, when the liquid storage tube plug 13 is inserted into the liquid storage tube 25 or the reaction tube 26 from the pipe mouth 27, the change range of the inner cavity volume of the liquid storage tube 25 or the reaction tube 26 can be minimized, so that the resistance of the liquid in the liquid storage tube cavity 2 to flow downward is reduced to a minimum, thereby ensuring the smooth flow of the liquid; a convex step 3 is set on the upper outer surface of the liquid storage tube wall 1 at a distance of 3-6 mm from the top of the liquid storage tube wall 1, that is, the upper surface of the convex step 3 is 3-6 mm away from the top of the liquid storage tube wall 1, and the convex step 3 is The inner wall of the annular convex 3 is fused with the liquid storage tube wall 1, and the cross section of the convex 3 ring body is rectangular. The length of the rectangular convex 3 ring body is 2-5 mm and the width is 1-3 mm. When the lower end of the liquid storage tube 25 is inserted into the float ring 4, the float ring 4 can be sleeved on the outside of the liquid storage tube 25 below the convex 3. At this time, the convex 3 can block the float ring 4 to prevent the float ring 4 from slipping out from the upper end of the liquid storage tube 25; the float ring 4 is sleeved on the outside of the uppermost liquid storage tube 25 when the liquid storage tube 25 is placed in a water bath for reaction, and provides buoyancy for the liquid storage tube 25 and the reaction tube 26 inserted together. The float ring 4 is composed of a float ring body 21 and a float ring cavity 22. The float ring body 21 is made of foam plastic and is annular. The outer diameter of the float ring body 21 is 10-50 mm and the inner diameter is 5-20 mm, thickness of 5-10 mm; the float ring cavity 22 is a cavity in the center of the float ring 4, which is cylindrical, and the diameter of the float ring cavity 22 is 5-20 mm and the height is 5-10 mm; the liquid storage tube 25 is inserted downward from the top of the float ring cavity 22 and finally stops below the convex gear 3; the anti-slip sheet 5 is arranged in the anti-slip sheet groove 14, a total of 4, each anti-slip sheet 5 is composed of an anti-slip sheet body 15 and a connecting rubber 16, the anti-slip sheet body 15 is made of manganese steel, and the upper outer surface of the anti-slip sheet body 15 forms an inclined surface to facilitate the insertion of the liquid storage tube plug 13, and the lower end of the anti-slip sheet body 15 is flush, which can resist the anti-slip sheet groove 14 or the inner surface of the bottom wall of the spring cavity 7; the connecting rubber 16 is made of silica gel, which can adhere the anti-slip sheet 5 to the anti-slip sheet groove 14 under the action of the adhesive;When the liquid storage tube plug 13 is inserted into the liquid storage tube 25 or the reaction tube 26 from the tube opening 27, the anti-slip sheet 5 first presses the anti-slip sheet body 15 into the anti-slip sheet groove 14. Since the connecting rubber 16 is made of silicone and has elasticity, it will not block the pressing of the anti-slip sheet body 15. Once the anti-slip sheet 5 reaches the position of the fixed spring cavity 7, under the elastic action of the elastic connecting rubber 16, a part of the anti-slip sheet body 15 can be pushed into the fixed spring cavity 7. However, at this time, the fixed spring cavity 7 also has a spring steel fixed spring 8, and the anti-slip sheet body 15 can only be located at the gap between the anti-slip sheet groove 14 and the fixed spring cavity 7, so that the two liquid storage tubes 25 or the liquid storage tube 25 and the reaction tube 26 that are inserted into each other are pressed against each other. 6 are tightly connected together and are not easy to fall off, so they play a role of preventing them from falling off; the blocking plates 6 are made of glass, and there are 4 of them in total. The blocking plates 6 can block the outer opening of the liquid flow tube 9. When the liquid in the liquid storage tube 25 needs to flow out of the liquid flow tube 9, it is pushed away from the outer opening of the liquid flow tube 9 by the liquid storage tube wall 1 around the tube opening 27, so that the liquid can flow out. In order to ensure that the blocking plates 6 function normally, the blocking plates 6 are designed to be divided into two parts, the upper part is the head, and the lower part is the handle. The width of the head of the blocking plate 6 is 2-3 times the width of the handle, the height is 1-2 mm, the thickness is 0.2-0.5 mm, and the width direction is arc-shaped. The head of the blocking plate 6 can prevent the blocking plate 6 from flowing out of the through hole The cross section of the handle of the blocking plate 6 coincides with the inner cavity of the arc-shaped through hole in the connecting part of the liquid storage tube plug 13, and a shallow groove is provided on the inner surface of the middle part, forming a part of the fixed spring cavity 7. When the fixed spring 8 presses against the upper and lower bottom walls of the fixed spring cavity 7, the fixed spring 8 is equivalent to locking the blocking plate 6 and the liquid storage tube plug 13 together, which can ensure that the blocking plate 6 will not move up and down and can better block the outer opening of the liquid flow tube 9; once the external force acting on the handle of the blocking plate 6 is slightly larger, the deformation of the fixed spring 8 can be pushed, thereby pushing the blocking plate 6 away from the outer opening of the liquid flow tube 9 and losing the blocking ability; but when the two liquid storage tubes 25 or the liquid storage tube 25 and the reaction tube 26 are tightly connected together, the fixed spring 8 has a recovery function. The tendency of complex deformation, and then under the action of the force generated by the elasticity of the connecting rubber 16, the two push the anti-slip sheet 15 to the gap between the anti-slip sheet groove 14 and the fixed spring cavity 7; after the sealing plate 6 is located at the outer opening of the liquid flow tube 9, candle liquid or vaseline is applied to the outer opening periphery of the liquid flow tube 9 and the inner surface of the through hole, so as to seal the gap that may appear at the connection between the sealing plate 6 and other structures; each fixed spring cavity 7 is rectangular, the height of the fixed spring cavity 7 is 2.1-5.2 mm, the length is 0.7-1.5 mm, and the width is 0.6-1.2 mm. The central axis of the fixed spring cavity 7 in the height direction is in a straight line with the center of the outer opening of the liquid flow tube 9, and a fixed spring 8 is arranged in each fixed spring cavity 7;The fixing spring 8 is made of spring steel wire wound in a spiral shape. The fixing spring 8 has a length of 2-5 mm, a diameter of 0.5-1 mm, a spiral pitch of 0.2-0.5 mm, and a cross-sectional diameter of the spring steel wire of 0.1-0.2 mm. An arc-shaped through hole is provided at each position corresponding to the fixing spring cavity 7 in the connecting portion of the liquid storage tube plug 13, for the blocking plate 6 to pass through the hole. At the same time, when the liquid storage tube plug 13 is inserted from the pipe opening 27 of another liquid storage tube 25 into the upper part of another liquid storage tube 25 or from the pipe opening 27 of the reaction tube 26 into the upper part of another liquid storage tube 25 of the reaction tube 26, the lower bottom surface of the central part of the liquid storage tube plug 13 extending outward from the bottom end of the liquid storage tube wall 1 with an outer diameter of 5-20 mm and the top surface of the tube wall around the pipe opening 27 are close to each other. After the contact, on the one hand, it can prevent the liquid storage tube plug 13 from continuing to be inserted, and on the other hand, it can also use the tube wall around the tube mouth 27 to push the sealing plate 6 into the arc-shaped through hole. In addition, the total length of the central part and the bottom of the liquid storage tube plug 13 is greater than the height of the upper part of the liquid storage tube wall 1. After the sealing plate 6 is pushed into the arc-shaped through hole, the opening of the outer end of the liquid flow tube 9 is just below the upper part of the liquid storage tube wall 1. In addition, since the inner surface of the upper part of the liquid storage tube wall 1 and the outer surface of the central part and the bottom of the liquid storage tube plug 13 are respectively provided with liquid storage tube wall recesses 17, liquid storage tube wall protrusions 18, liquid storage tube plug recesses 19, and liquid storage tube plug protrusions 20 that can be embedded together, the inner diameter of the upper part of the liquid storage tube wall 1 and the outer diameter of the central part and the bottom of the liquid storage tube plug 13 are the same, and there is almost no The gap connects the two tubes relatively tightly together, ensuring that the liquid in the liquid storage tube cavity 2 can only flow down from the liquid flow tube 9 smoothly and fall into the tube cavity below, and will not flow out from the arc-shaped through-hole of the connecting part of the liquid storage tube plug 13; the liquid storage tube wall concave 17 is a semicircular groove located on the inner surface of the upper part of the liquid storage tube 25, and there are 2 liquid storage tube wall concave 17, and the 2 liquid storage tube wall concave 17 are located on one diameter; the liquid storage tube wall convex 18 is a semicircular convexity located on the inner surface of the upper part of the liquid storage tube 25, and there are 2, and the 2 liquid storage tube wall convex 18 are located on another diameter perpendicular to the diameter of the 2 liquid storage tube wall concave 17; each liquid storage tube wall concave 17 or liquid storage tube wall convex 18 is arranged between two adjacent anti-slip sheets 5; the liquid storage tube plug concave 19 is located in the liquid storage tube The semicircular grooves at the center and the bottom outer surface of the plug 13, the two liquid storage tube plug recesses 19, are located on one diameter; the liquid storage tube plug protrusions 20 are semicircular protrusions located at the center and the bottom outer surface of the liquid storage tube plug 13, there are two, the two liquid storage tube plug protrusions 20 are located on another diameter perpendicular to the diameter where the two liquid storage tube plug recesses 19 are located; when the liquid storage tube plug 13 is inserted into the upper part of the liquid storage tube 25 or the reaction tube 26, the liquid storage tube wall recess 17 and the liquid storage tube plug protrusion 20 or the liquid storage tube wall protrusion 18 and the liquid storage tube plug recess 19 are mutually embedded, so that the liquid storage tube plug 13 can be tightly and accurately inserted into the upper part of the liquid storage tube 25 or the reaction tube 26, and the structures that need to correspond to each other will also accurately correspond to each other;The reaction tube 26 is used to hold the sample DNA solution, and the detection solution that falls into it undergoes LMAP reaction in the reaction tube 26 under the temperature condition provided by the water bath. The reaction tube 26 is made of glass, and the wall thickness at the upper opening is 1-5 mm, the height is 5-10 mm, and it is cylindrical. The outer diameter of the cylinder is 5-20 mm, and the inner diameter is 3-10 mm. Four anti-slipping sheets 5 are arranged in the middle of the upper inner surface of the reaction tube 26, and the line connecting the central points of two adjacent anti-slipping sheets 5 and the center of the circle where the cross section is located forms an angle of 90°. The lower part of the reaction tube 26 is a gradually tapering structure, and finally it is blocked at the bottom to form a small tube-like shape that is approximately conical and the size of the lower end is 1 / 5-1 / 4 of the upper end. Since the amount of liquid used in the reaction process is relatively small, there are inclined surfaces with an inclination of 45 degrees between the upper and lower ends of the middle part of the liquid storage tube 25 and the upper and lower tube walls, so that the liquid can flow in or out smoothly without the liquid residue that may be caused by the bend at the connection due to surface adsorption; the lower end of the liquid storage tube 25 is the liquid storage tube plug 13, and the plugging plate 6 is inserted into the through hole of the liquid storage tube plug 13. The walls around the liquid storage tube 25 are the liquid storage tube wall 1, and the cavity surrounded by the liquid storage tube wall 1 is the liquid storage tube cavity 2; let the detection solution in the liquid storage tube 25 fall into the reaction tube 26 inserted together below, and the detection solution and the sample DNA solution in the reaction tube 26 undergo LAMP reaction. Since the detection solution contains hydroxynaphthol blue, the detection result can be observed by naked eyes through the hydroxynaphthol blue indication after the reaction. If the reaction solution is blue, it means that there is caruncle-shaped cup disc fungus in the sample to be detected, and if the reaction solution is purple, it means that there is no caruncle-shaped cup disc fungus in the sample to be detected. ;

[0027] The present invention is further described below in conjunction with specific examples. The attached drawings are only for reference and description purposes and are not intended to limit the present invention.

[0028] Example 1: Detection of C. carunculata by LAMP method.

[0029] A LAMP primer composition designed according to the calmodulin gene sequence of the caruncle-shaped cup disc fungus comprises a forward inner primer FIP-GC142, a reverse inner primer BIP-GC142, a forward outer primer F3-GC142 and a reverse outer primer B3-GC142, and the specific sequence of each primer is as follows. The binding site of each primer in the target sequence is as follows Figure 6 shown.

[0030] F3-GC142: 5'-GGTCGATGCCGATAACAAC-3'

[0031] B3-GC142:5'-ATTTGCCATGGTAAACAAGA-3'

[0032] FIP-GC142: 5'-GTCACCTTCCGAGTCATGAAGGTACCATTGATTTCCCAGG-3'

[0033] BIP-GC142: 5'-GCTAAGAATTTCTCACCATGATGGCGCTAACCTTGAATGCCTCTC-3'

[0034] The LAMP detection reagent tube for detecting Carunculaceae comprises the above primer combination, 2×HNB lsoAmpMix, Bst DNA polymerase and ddH2O, wherein the concentrations of each reagent are: 0.1 μM forward outer primer F3-GC142, 0.1 μM reverse outer primer B3-GC142, 0.8 μM forward inner primer FIP-GC142, 0.8 μM reverse inner primer BIP-GC142 and 8 U / μL Bst DNA polymerase.

[0035] The detection method of the above-mentioned LAMP kit for detecting caruncle-shaped cup disc fungus is as follows: extract the test sample DNA, take 1.0 μL of the test sample DNA solution and add it to 24 μL of the kit solution to create a total of 25 μL reaction system, and perform LMAP amplification reaction after preparing the system. The reaction procedure is a constant temperature amplification reaction of 65°C for 45-60 min, and optional heat inactivation at 85°C for 5 min. After the amplification reaction is completed, the color of the reaction solution can be directly observed by naked eyes or detected by agarose gel electrophoresis. If the reaction solution is blue, or there are ladder bands in the electrophoresis detection, it means that caruncle-shaped cup disc fungus exists in the sample to be detected. If the reaction solution is purple, or there are no ladder bands in the electrophoresis detection, it means that caruncle-shaped cup disc fungus does not exist in the sample to be detected.

[0036] Example 2: Specificity verification of LAMP detection of C. carunculata.

[0037] In order to verify the specificity of LAMP method for detecting Ciboria carunculoides, Ciboria carunculoides, Sclerotinia sclerotiorum, Fusarium fujikuroi, Rhizoctonia solani, Leptosphaerulina arachidicola, Penicillium italicum, Colletotrichumorbiculare and Aspergillus japonicus were selected as test strains. DNA of the test strains was extracted, and 1.0 μL DNA solution was taken to prepare LAMP reaction system for amplification. The amplification results showed that the solution in the amplification reaction tube using only the DNA of Carunculum carunculum as the template was blue, and ladder-like bands appeared in 1% agarose gel electrophoresis, indicating a positive result; the solution in the blank control and other amplification reaction tubes using the DNA of the test strain other than Carunculum carunculum as the template was purple, and no ladder-like bands appeared in 1% agarose gel electrophoresis, indicating a negative result ( Figure 7 ).

[0038] Example 3: Sensitivity verification of LAMP detection of C. carunculata.

[0039] The DNA of Chrysosporium carnosus was extracted and the concentration was determined. After 10-fold gradient dilution, the concentrations of Chrysosporium carnosus DNA solutions were 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, and 1 fg / μL, respectively.

[0040] The above DNA solution was used as a template to perform LAMP reaction. The reaction procedure was a constant temperature amplification reaction at 65°C for 45-60 minutes. The amplification reaction results showed that the solution in the amplification reaction tube with a concentration of 100 pg / μL-10 ng / μL was blue in color, and ladder-like bands appeared in the electrophoresis detection, which was a positive result; the solution in the blank control and other concentrations of the amplification reaction tube was purple in color, and no ladder-like bands appeared in the electrophoresis detection, which was a negative result ( Figure 8 ). This indicates that the sensitivity of the LAMP reaction is 100 pg / μL.

[0041] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected, and the scope of the present invention to be protected is defined by the attached claims and their equivalents.

Claims

1. A plug-in reaction device suitable for containing LAMP test materials to detect Carunculaceae, comprising a liquid storage tube (25) and a reaction tube (26), characterized in that: The liquid storage tube (25) is made of glass. The upper end of the liquid storage tube (25) is a tube mouth (27) into which another liquid storage tube plug (13) can be inserted. The lower part of the tube mouth occupies 1 / 5 of the length of the entire liquid storage tube (25) as the upper part of the liquid storage tube (25). The thickness of the upper part of the liquid storage tube wall (1) is 1-5 mm, the height is 5-10 mm, and it is cylindrical. The outer diameter of the cylinder is 5-20 mm and the inner diameter is 3-10 mm. Four anti-slipping sheets (5) are arranged in the middle of the inner surface of the upper part of the liquid storage tube (25). The central points of two adjacent anti-slipping sheets (5) are at the same cross section as the central points of the two adjacent anti-slipping sheets (5). The connecting line between the centers of the circles forms an angle of 90°. The middle part of the liquid storage tube (25) is the main part of the liquid storage tube (25). The thickness of the middle part of the liquid storage tube wall (1) is 0.5-2 mm, the height is 15-30 mm, and it is cylindrical. The outer diameter of the cylinder is 5-20 mm and the inner diameter is 4-16 mm. The lower part of the liquid storage tube (25) is the liquid storage tube plug (13). The liquid storage tube plug (13) is in the shape of a cylinder. The liquid storage tube plug (13) can be divided into three parts from top to bottom: a connecting part, a central part and a bottom part. The connecting part of the liquid storage tube plug (13) is composed of a storage tube and a central part. The middle part of the liquid tube (25) is inclined at a 45° angle from the top edge of the inclined surface to the bottom end of the liquid storage tube wall (1) with an outer diameter of 5-20 mm, and the thickness of the connecting part of the liquid storage tube plug (13) is 1.5-7 mm; the central part of the liquid storage tube plug (13) refers to the part from the bottom end of the liquid storage tube wall (1) with an outer diameter of 5-20 mm to the lower edge of the opening at the outer end of the liquid flow tube (9). The central outer surface of the central part of the liquid storage tube plug (13) is provided with 4 spring chambers (7). At this point, on the cross section of the central part of the liquid storage tube plug (13), adjacent spring chambers (7) are arranged. The two connecting lines from the center of the two spring chambers (7) to the center of the cross-section circle form an angle of 90°; the bottom of the liquid storage tube plug (13) refers to the bottom edge of the opening of the outer end of the liquid flow tube (9) starting from the bottom edge to the bottom edge of the entire liquid storage tube (25). The bottom of the liquid storage tube plug (13) consists of three parts: the liquid storage tube bottom (10), the liquid storage tube bottom convex (11), and the liquid storage tube bottom concave (12). The upper part of the bottom of the liquid storage tube plug (13) is the liquid storage tube bottom convex (11), which is conical. The distance between the lower edge of the liquid storage tube bottom convex (11) and the lower edge of the opening of the inner end of the liquid flow tube (9) is 0.5-1 mm, there are 4 liquid flow tubes (9), and the two connecting lines between the central points of the inner openings of two adjacent liquid flow tubes (9) and the center of the bottom circle of the liquid storage tube bottom convex (11) form an angle of 90°; each liquid flow tube (9) is a flat tube inclined at 45°; the solid part below the liquid storage tube bottom convex (11) is the liquid storage tube bottom (10); the liquid storage tube bottom concave (12) is the part of the lower surface of the liquid storage tube bottom (10) that is concave upward; a convex step (3) is set on the upper outer surface of the liquid storage tube wall (1) at a distance of 3-6 mm from the top of the liquid storage tube wall (1), the convex step (3) is in a circular ring shape, the inner wall of the circular ring convex step (3) is fused with the liquid storage tube wall (1), the cross section of the convex step (3) is rectangular, and the rectangular convex step (3) The length of the ring body is 2-5 mm and the width is 1-3 mm; the float ring (4) is placed on the outer surface of the uppermost liquid storage tube (25) when the liquid storage tube (25) is placed in the water bath for reaction, and provides buoyancy for the liquid storage tube (25) and the reaction tube (26) inserted together. The float ring (4) is composed of a float ring body (21) and a float ring cavity (22). The float ring body (21) is made of foam plastic and is in a circular ring shape. The outer diameter of the float ring body (21) is 10-50 mm, the inner diameter is 5-20 mm, and the thickness is 5-10 mm; the float ring cavity (22) is a hollow cavity in the center of the float ring (4) and is cylindrical. The diameter of the float ring cavity (22) is 5-20 mm and the height is 5-10 mm; The anti-slip sheet (5) is arranged in the anti-slip sheet groove (14), and there are 4 anti-slip sheets (5). Each anti-slip sheet (5) is composed of an anti-slip sheet body (15) and a connecting rubber (16). The anti-slip sheet body (15) is made of manganese steel, and the outer surface of the upper end of the anti-slip sheet body (15) forms an inclined surface, and the lower end of the anti-slip sheet body (15) is flush; the connecting rubber (16) is made of silica gel; the blocking plate (6) is made of glass, and there are 4 blocking plates (6) in total. The blocking plate (6) is divided into an upper and lower part, the upper part is the head, and the lower part is the handle. The width of the head of the blocking plate (6) is 2-3 times the width of the handle, the height is 1-2 mm, the thickness is 0.2-0.5 mm, and the width direction is arc-shaped; the cross section of the blocking plate (6) handle coincides with the arc-shaped through-hole inner cavity in the connecting part of the liquid storage tube plug (13), and the middle The inner surface of the part has a shallow groove, which forms a part of the spring cavity (7); the plugging plate (6) is located at the outer opening of the liquid flow tube (9), and candle liquid or vaseline is applied to the outer opening periphery of the liquid flow tube (9) and the inner surface of the through hole; each spring cavity (7) is rectangular, the height of the spring cavity (7) is 2.1-5.2 mm, the length is 0.7-1.5 mm, and the width is 0.6-1.2 mm. The central axis of the spring cavity (7) in the height direction is on a straight line with the center of the outer opening of the liquid flow tube (9), and a spring (8) is arranged in each spring cavity (7); the length of the spring (8) is 2-5 mm, the diameter is 0.5-1 mm, the spiral pitch is 0.2-0.5 mm, and the diameter of the cross section of the spring steel wire is 0.1-0.2 mm, the liquid storage tube plug (13) corresponding to the position of the spring cavity (7) in the connecting portion is provided with an arc-shaped through hole; the liquid storage tube wall concave (17) is a semicircular groove located on the inner surface of the upper portion of the liquid storage tube (25), the liquid storage tube wall concave (17) has 2, and the 2 liquid storage tube wall concave (17) is located on a diameter; the liquid storage tube wall convex (18) is a semicircular convexity located on the inner surface of the upper portion of the liquid storage tube (25), there are 2, the 2 liquid storage tube wall convex (18) is located with the 2 liquid storage tube wall concave ( 17) is located on another diameter perpendicular to the diameter thereof; each liquid storage tube wall concave (17) or liquid storage tube wall convex (18) is arranged between two adjacent anti-dropping sheets (5); the liquid storage tube plug concave (19) is a semicircular groove located on the central part and the outer surface of the bottom of the liquid storage tube plug (13), and there are two liquid storage tube plug concave (19), and the two liquid storage tube plug concave (19) are located on one diameter; the liquid storage tube plug convex (20) is a semicircular groove located on the central part and the outer surface of the bottom of the liquid storage tube plug (13). There are two circular protrusions, and the two liquid storage tube plug protrusions (20) are located on another diameter perpendicular to the diameter where the two liquid storage tube plug recesses (19) are located; the lower end of the liquid storage tube (25) is the liquid storage tube plug (13), and a blocking plate (6) is inserted into the through hole of the liquid storage tube plug (13); the wall around the liquid storage tube (25) is the liquid storage tube wall (1), and the cavity surrounded by the liquid storage tube wall (1) is the liquid storage tube cavity (2); the reaction tube (26) is glass, and the wall thickness at the upper opening is 1-5 mm, 5-10 mm in height, cylindrical, with an outer diameter of 5-20 mm and an inner diameter of 3-10 mm; 4 anti-slipping sheets (5) are arranged in the middle of the inner surface of the upper part of the reaction tube (26), and the line connecting the central points of two adjacent anti-slipping sheets (5) and the center of the circle where the cross section is located forms an angle of 90°; the lower part of the reaction tube (26) is a gradually tapering structure, and finally blocked at the bottom, forming a small tube-like shape with a lower end size of 1 / 5-1 / 4 of the upper end. .

2. The plug-in reaction device suitable for containing LAMP test objects to detect C. carunculata according to claim 1, characterized in that: The reaction tube (26) contains a reaction system required for LAMP reaction; the primer composition in the reaction system comprises: 0.1 μM forward outer primer F3-GC142, 0.1 μM reverse outer primer B3-GC142, 0.8 μM forward inner primer FIP-GC142 and 0.8 μM reverse inner primer BIP-GC142, and the reaction system also contains 12.5 μL 2×HNB lsoAmp Mix, 8 U / μL Bst DNA polymerase, ddH2O and 1.0 μL template DNA; the LAMP detection method of caruncle-shaped cup disc fungus comprises using the reaction tube (26) to carry out LAMP reaction, and directly observing the color change of the solution after the reaction is completed to judge whether caruncle-shaped cup disc fungus exists in the sample; the F3 sequence is shown in Seq ID No: 1; the B3 sequence is shown in Seq ID No: 2; the FIP sequence is shown in Seq ID No: 3; the BIP sequence is shown in Seq ID No: 4; the LAMP detection method can refer to the following steps: extract the DNA of the sample to be detected, take 1.0 μL of DNA solution as a template, add it to 24 μL of the kit detection solution for LAMP reaction, the reaction procedure is 65°C reaction amplification for 45-60 min, and after the reaction, 85°C heat inactivation can be selected for 5 min; the judgment of the LAMP reaction result can be directly observed by naked eye The color change of the solution or detected by agarose gel electrophoresis. If the color of the solution changes from purple to blue, or a ladder-like electrophoresis band is observed under ultraviolet light, it indicates that Carunculaceous Cup disc fungus exists in the sample to be detected; If the solution color does not change and remains purple, or no ladder-like electrophoresis bands are observed under ultraviolet light, it means that there is no Carunculaceae in the sample to be tested.

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