Integrated fluorescence detection kit and testing method thereof

Abstract

The invention discloses an integrated fluorescence detection kit and a testing method thereof.The integrated fluorescence detection kit comprises an upper cover assembly, a lysate tube assembly, a main body assembly, a diluent tube assembly and a decorative cover, and the lower end of the upper cover assembly is connected with the lysate tube assembly and the diluent tube assembly; the side wall of the upper cover assembly is connected with the upper part of the main body assembly, and the main body assembly is nested in the decorative cover; a reagent system in the kit comprises an RT-RAA reaction system and a CRISPR reaction system which are stored in the detection kit at normal temperature in the form of freeze-dried microspheres and closed liquid, and a lysis solution and a diluent are released through the lysis solution tube assembly and the diluent tube assembly and react in the main body assembly. According to the invention, the problems that the existing fluorescence detection operation process is complicated, the instrument is not portable and depends on professional analysis and the like are solved, and the rapid, portable and user-friendly detection on the virus is realized based on the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) detection system.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and particularly relates to an integrated fluorescence detection kit and a testing method thereof. Background Art

[0002] Nucleic acid is the carrier of genetic information of living organisms and an essential component of all known life forms. Nucleic acid is divided into two categories, DNA and RNA, which mainly exist in the cell nucleus and exist in a state combined with proteins. With the rapid development of molecular biology, the research and analysis of nucleic acids have been continuously promoted and applied in the fields of clinical diagnosis, food safety, environmental detection, agriculture, forestry, animal husbandry, etc.

[0003] As a highly sensitive and specific analysis method, CRISPR fluorescence detection technology has been widely applied in many fields such as biomedicine, environmental monitoring, food safety, etc. However, traditional fluorescence detection processes often have problems such as complex operations, expensive equipment, and scattered reagent kits, which limit their popularization and application in practical applications.

[0004] The principle of fluorescence detection technology is based on the fact that certain substances can emit fluorescence of a specific wavelength under the excitation of light of a specific wavelength. By detecting parameters such as the intensity and wavelength of fluorescence, qualitative and quantitative analysis of the target substance can be carried out. This technology has extremely high sensitivity and can detect extremely trace amounts of substances. At the same time, it also has good selectivity and can accurately identify the target substance in complex samples. Therefore, fluorescence detection technology has broad application prospects in biomolecular detection, cell imaging, pathogen diagnosis, etc.

[0005] However, the existing fluorescence detection processes are usually more cumbersome: First, various experimental instruments and reagents need to be prepared, such as pipettes, centrifuge tubes, fluorescent dyes, etc. Then, a series of complex operations such as sample collection, processing, and labeling are carried out. During the detection process, experimental conditions such as temperature and light also need to be precisely controlled to ensure the accuracy of the detection results. It is difficult for existing nucleic acid detection kits to combine the entire processes of nucleic acid extraction and nucleic acid detection into the same closed space. As a result, during the nucleic acid detection process, aerosol cross - contamination of nucleic acid samples easily causes false positives in the subsequent nucleic acid detection of samples due to the need to separate the entire processes of nucleic acid extraction and nucleic acid detection, and the detection efficiency is relatively low.

[0006] In addition, traditional fluorescence detection equipment is usually large in volume and expensive, and requires professional operators for maintenance and use. This not only increases the detection cost but also limits its application in grass - roots laboratories and on - site detections. Summary of the Invention

[0007] Based on the technical problems existing in the prior art, the present invention provides an integrated fluorescence detection kit and its testing method, which solves the problems of complex operation process, inconvenient portability of equipment and non-user-friendly design in the existing nucleic acid detection technology.

[0008] According to the first aspect of the technical solution of the present invention, an integrated fluorescence detection kit is provided, which includes an upper cover assembly, a lysis solution tube assembly, a main body assembly, a dilution solution tube assembly and a decorative cover; The lower end of the upper cover assembly is connected to the lysis solution tube assembly and the dilution solution tube assembly, the side wall of the upper cover assembly is connected to the upper part of the main body assembly, and the main body assembly is nested in the decorative cover.

[0009] Preferably, the upper surface of the upper cover assembly is provided with a placement channel for the lysis solution tube assembly and a placement channel for the dilution solution tube assembly, and an annular gasket is designed on the contact surface of the upper cover assembly with the reaction tube, and the annular gasket contacts the contact surfaces of the lysis solution tube assembly and the dilution solution tube assembly.

[0010] Preferably, the lysis solution tube assembly includes a lysis solution tube push rod assembly and a lysis solution tube body.

[0011] More preferably, the lysis solution tube as a whole adopts a cylindrical structure, and the inner surface of the lysis solution tube is smoothed.

[0012] Preferably, the inside of the lysis solution tube adopts a tapered transition design, gradually narrowing from the inner diameter of the lysis solution tube to the inner diameter of the liquid outlet.

[0013] Preferably, the dilution solution tube assembly includes a dilution solution tube push rod assembly, a dilution solution tube and a sealing ring. The dilution solution tube is a slender cylinder. One end of the dilution solution tube is connected to the dilution solution tube push rod assembly to be closed, and the other end is open and provided with a sealing ring and a sealing film.

[0014] Preferably, the dilution solution push rod of the dilution solution tube push rod assembly is composed of a plastic rod body and a silicone push head. The rod body is a slender cylinder, and the push head is in interference fit with the inner wall of the dilution solution tube; the biological shape of the push head is adapted to the cross section of the dilution solution tube.

[0015] Furthermore, a pointed cone cavity is provided in the sealing ring, and CRISPR freeze-dried microspheres are pre-placed in the cavity. The CRISPR freeze-dried microspheres are separated from the dilution solution to ensure the stability of the CRISPR freeze-dried microspheres.

[0016] Even further, the upper part of the reaction tube main body is in a flat cylindrical shape, and the lower part is a triangular reaction area, which is integrally injection-molded.

[0017] Based on the second aspect of the present invention, a testing method for an integrated fluorescence detection kit is characterized by including the following steps: Step S1, processing of the sample to be tested: Open the sealed aluminum film at the liquid addition port at the top of the lysis tube, immerse the sample to be tested in the lysis solution, install the push rod assembly of the lysis tube at the liquid addition port at the top of the lysis tube, form a constant-pressure sealed structure inside the lysis tube, and let it stand for 5 min; Step S2, RT-RAA amplification and incubation: Remove the sealing piece below the lysis tube, apply downward pressure to push the push rod of the lysis tube, and push the push rod of the lysis tube to the bottom, so that a certain amount of fluid flows into the reaction area of the reaction tube body, and quickly dissolve the RT-RAA lyophilized microspheres placed in the triangular reaction area; Place the triangular reaction area in a constant-temperature heating environment to complete the RT-RAA nucleic acid amplification reaction; Step S3, CRISPR reaction: After incubation, apply pressure to push the dilution push rod to the bottom, and the tapered structure pierces the sealing film. The diluent dissolves the CRISPR lyophilized microspheres and flows to the reaction area of the reaction tube body, and reacts with the RT-RAA nucleic acid amplification product obtained in Step S2. According to the set reaction temperature, heat the triangular reaction area of the reaction tube body at a constant temperature to complete the nucleic acid cleavage in the triangular reaction area of the reaction tube body; Step S4, judging the test result according to the fluorescence intensity in the reaction area: Use blue light to irradiate the sheared nucleic acid in the triangular reaction area of the reaction tube body obtained in Step S3, and use a fluorescence image analysis system for analysis, and judge the test result according to the fluorescence intensity in the reaction area.

[0018] Compared with the prior art, an integrated fluorescence detection kit and its test method of the present invention have the following beneficial technical effects: The present invention has a unique self-appearance design, highly integrating the whole processes of nucleic acid extraction, nucleic acid amplification and fluorescence detection in a kit with dimensions of 5.5 * 3.0 * 8.5 cm in length, width and height. The kit includes a lysis tube assembly, an upper cover assembly, a diluent tube assembly, a main body assembly and a decorative cover. Through a unique design, the whole process of nucleic acid detection is completed in the same closed space in different time periods. During the nucleic acid detection process, aerosol cross-contamination is avoided, and the detection efficiency is high. 2. The present invention has a special reagent storage form, including pre-isolated placement of RT-RAA and CRISPR lyophilized microspheres. The lyophilized microspheres are stored in a sealed and isolated manner to ensure stability and activity, and can be quickly dissolved and reacted when encountering fluid; The lysis solution and diluent are pre-injected and sealed; The lysis tube adopts a separate storage strategy, and is inserted into the kit through the placement channel of the lysis tube assembly during use to avoid nucleic acid sample contamination and ensure the accuracy of the results.

[0019] 3. The present invention has a unique operation process, including injecting the RT-RAA and CRISPR reagent systems into the reaction zone through two channels (the channel for placing the lysis tube and the channel for placing the dilution tube) respectively; the method of releasing the fluid is through a rigorous sealing design, pressurized by a push rod, so that the tapered structure pierces the sealing film and flows to the reaction zone; the quantitative control of the liquid is achieved by applying pressure to the push rod from the outside, changing the pressure in the closed space inside the reaction tube, thereby realizing the quantitative outflow of the liquid; nucleic acid amplification, cleavage, and detection reactions are all carried out in the same reaction zone.

[0020] 4. The present invention integrates the RAA isothermal amplification technology, CRISPR gene cleavage technology, and fluorescence detection technology, realizing an integrated process from nucleic acid sample processing to the output of detection results, greatly simplifying the fluorescence detection process, and is a user-friendly design. Brief Description of the Drawings

[0021] Figure 1 is a schematic diagram of the overall structure of the integrated fluorescence detection kit according to the present invention; Figure 2 is a schematic diagram of the structure of the lysis tube assembly of the integrated fluorescence detection kit according to the present invention; Figure 3 is a schematic diagram of the structural position relationship among the main body assembly, the upper cover assembly, and the dilution tube assembly of the integrated fluorescence detection kit according to the present invention; Figure 4 is a schematic diagram of the main body assembly structure according to the present invention; Figure 5 is a schematic diagram of the upper cover assembly and the dilution tube structure according to the present invention; Figure 6 is a schematic diagram of the structural position relationship between the dilution tube and the push rod according to the present invention; Figure 7 is a visual presentation diagram of the results of the detection example according to the present invention; Figure 8 is a diagram presenting the data of three repeated experiments of the detection example according to the present invention.

[0022] Explanation of the reference numerals in the drawings: 1. Lysis tube push rod; 2. Lysis tube assembly; 3. RT-RAA freeze-dried microspheres; 4. Upper cover assembly; 5. Dilution push rod; 6. Main body assembly; 7. Dilution tube assembly; 8. CRISPR freeze-dried microspheres; 9. Decorative cover; 10. Lysis solution; 11. Lysis tube push rod assembly; 13. Lysis tube; 14. Dilution solution; 15. Reaction tube main body; 16. Sealing ring. 17. Dilution tube. Detailed Embodiments

[0023] In order to make the technical problems to be solved, the technical solutions adopted and the beneficial effects obtained by the present invention more clearly understood, the present invention will be further described in detail below with reference to specific embodiments. The specific embodiments described herein are only used to explain the present invention and do not constitute any limitation to the present invention. Unless otherwise defined, all terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs.

[0024] Specific embodiments are provided below to help understand the present invention. It should be understood that the embodiments and test examples listed in the present invention are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims.

[0025] The present invention discloses an integrated fluorescence detection kit and its testing method. The integrated fluorescence detection kit includes an upper cover assembly, a lysis solution tube assembly, a main body assembly, a dilution solution tube assembly and a decorative cover. The lower end of the upper cover assembly is connected to the lysis solution tube assembly and the dilution solution tube assembly, and the side wall of the upper cover assembly is connected to the upper part of the main body assembly. The main body assembly is nested in the decorative cover. The reagent system in the kit includes an RT-RAA reaction system and a CRISPR reaction system, which are stored at room temperature in the detection kit in the form of freeze-dried microspheres and a sealing liquid, and mainly release the lysis solution and the dilution solution through the lysis solution tube and the dilution solution tube, and react in the main body assembly. The present invention solves the problems of complex operation process of existing fluorescence detection, inconvenient portability of instruments, and dependence on professional analysis, etc. Based on the CRISPR detection system, it realizes rapid, portable and user-friendly detection of viruses. The integrated fluorescence detection kit can be a point-of-care nucleic acid detection kit.

[0026] The integrated fluorescence detection kit of the present invention, as Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 and Figure 6 shown, it includes an upper cover assembly 4, a lysis solution tube assembly 2, a main body assembly 6, a dilution solution tube assembly 7 and a decorative cover 9; the lower end of the upper cover assembly 4 is connected to the lysis solution tube 2 and the dilution solution tube 7, and the side wall of the upper cover assembly 4 is connected to the upper part of the main body assembly 6. The main body assembly 6 is nested in the decorative cover 9.

[0027] The upper cover assembly 4 is used to assist in the precise installation of the lysis solution tube and the diluent tube, and at the same time achieve a good sealing effect. Its overall shape is a flat cylinder, and there are two circular grooves in the middle. The upper surface of the upper cover assembly 4 is provided with a placement channel for the lysis solution tube assembly 2 and a placement channel for the diluent tube assembly 7. On one side of the upper cover assembly 4, there is a circular groove specifically for assembling the diluent tube assembly 7. The diameter of the circular groove is slightly larger than the outer diameter of the diluent tube assembly 7. In a preferred embodiment, the depth of the circular groove is about 49 mm to ensure that the diluent tube assembly 7 can be stably embedded therein. Inside the circular groove, there are positioning protrusions and tenon structures that cooperate with the corresponding features on the diluent tube assembly 7 to prevent it from rotating or falling off during use. On the other side opposite to the assembly position of the diluent tube assembly 7, an insertion channel for the lysis solution tube assembly 2 is provided. The insertion channel for the lysis solution tube assembly 2 is cylindrical, and the diameter of the insertion channel for the lysis solution tube assembly 2 is slightly larger than the outer diameter of the lysis solution tube 13. In a preferred embodiment, the diameter length of the insertion channel for the lysis solution tube assembly 2 is about 41 mm. Inside the insertion channel for the lysis solution tube assembly 2, there are also guiding structures and sealing ring installation grooves to guide the smooth insertion of the lysis solution tube assembly 2 and achieve a good sealing effect. An annular sealing gasket is designed on the contact surface of one end of the upper cover assembly 4 with the lysis solution tube assembly 2 and the diluent tube assembly 7. The thickness of the sealing gasket is slightly larger than the distance between the upper cover assembly 4 and the lysis solution tube assembly 2 and the diluent tube assembly 7 to generate appropriate compressive deformation during assembly to form a reliable seal. In a preferred embodiment, the material of the sealing gasket is silicone rubber with high temperature resistance and chemical corrosion resistance, and its hardness is about 45 degrees on the Shore scale, which can not only provide good sealing performance but also maintain elasticity after multiple uses.

[0028] The diluent tube assembly 7 is used to hold and release the diluent 14, and at the same time dissolve the CRISPR freeze-dried microspheres 8 to trigger the nucleic acid detection reaction. The diluent tube assembly 7 includes a diluent push rod assembly 5 and a diluent tube 17. The diluent push rod 5 is composed of a plastic rod body and a silicone push head. The rod body is a slender cylinder, with a length of about 19 mm and a diameter of about 6.5 mm, and is made of high-strength plastic to provide sufficient thrust. The push head has an interference fit with the inner wall of the diluent tube 17, and the biological shape of the push head is adapted to the cross-section of the diluent tube 17, usually being circular or oval. In this embodiment, the push head is circular. In a preferred embodiment, the material of the push head is selected as silicone with good sealing performance and wear resistance. The diluent tube 17 below the diluent tube assembly 7 is a slender cylinder, with a length of about 26 mm, an outer diameter of about 9.5 mm, and an inner diameter of about 7.5 mm. The diluent tube 17 is made of transparent high-molecular PP plastic, which has good chemical stability and low liquid adsorption. The thickness of the thin wall at the bottom of the diluent tube 17 is preferably 0.1 mm - 0.9 mm. In a preferred embodiment, the thickness of the thin wall at the bottom of the diluent tube 17 is about 0.1 mm. The material of the thin wall at the bottom of the diluent tube 17 is selected as PP plastic that is easy to be punctured but can maintain sealing under normal storage conditions. One end of the diluent tube assembly 7 is closed, and the other end is open and provided with a sealing ring 16 and a sealing film to keep the internal diluent 14 sterile and stable before leaving the factory. The surface of the diluent tube 17 is marked with scales to accurately indicate the volume of the diluent 14. When initially installed, 350 ul of the diluent 14 is accurately filled into the diluent tube 17 through a high-precision filling device in a sterile environment. The filling process is carried out in a sterile environment to ensure the purity and sterility of the diluent 14. After filling, the diluent 14 is sealed to ensure the reliability and stability of the seal.

[0029] There is a cavity with a pointed cone below the sealing ring 16. The CRISPR freeze-dried microspheres 8 are placed in the cavity, separated from the diluent 14 to ensure the stability of the CRISPR freeze-dried microspheres 8. There is a sharp hard plastic cone in the cavity, and the cone is directly below the sealing ring 16. In a specific embodiment, the height of the cone is about 2.5 mm, and the apex angle is about 60 degrees, so that when the diluent tube is inserted in place and a certain pressure is applied, it can accurately puncture the sealing film on the sealing ring 16 at the bottom of the diluent tube. A triangular funnel is arranged below the cavity. After the diluent dissolves the CRISPR freeze-dried microspheres, it flows through the triangular funnel to the reaction area of the reaction tube main body to react with the obtained RT-RAA nucleic acid amplification product.

[0030] The lysis solution tube assembly 2 is used to process nucleic acid samples and hold and release the lysis solution 10. It is integrally cylindrical and injection-molded from PP plastic. The lysis solution tube assembly 2 includes a lysis solution tube push rod 11 and a lysis solution tube 13. The inner diameter of the lysis solution tube 13 is 5.8 mm, the outer diameter is 7.6 mm, and the height is 49 mm. The wall thickness of the tube body of the lysis solution tube 13 is uniform, with a thickness of 0.9 mm, to provide sufficient structural strength and stability, while avoiding excessive material use and reducing costs. The inner surface of the lysis solution tube 13 is smoothed. In this embodiment, the roughness is controlled below Ra1.0. More preferably, in this preferred embodiment, the roughness is controlled below Ra0.8, to reduce liquid wall adhesion and residue, and ensure that the lysis solution 10 can be completely released. The length of the lysis solution tube push rod 1 of the lysis solution tube push rod assembly 11 in the lysis solution tube assembly 2 should be the same as the height of the lysis solution tube push rod 1. The diameter of the lysis solution tube push rod 1 is slightly larger than the inner diameter of the lysis solution tube 13, generally 7.9 mm, to ensure that the push rod can move smoothly within the lysis solution tube body and can push out a certain amount of lysis solution 10 during use. The part of the lysis solution tube push rod 1 in contact with the lysis solution tube 2 is provided with a sealing gasket or sealing ring to prevent the lysis solution 10 from leaking from the top during the movement of the lysis solution tube push rod 1.

[0031] The liquid filling port is located at the top of the lysis solution tube 13, with an inner diameter of 7.5 mm, an outer diameter of 9 mm, and a height of 5 mm. The edge of the liquid filling port is designed to be arc-shaped, facilitating the pouring of liquid and avoiding liquid splashing during the liquid filling process. An annular structure with a raised ring is provided inside the liquid filling port to provide better support and sealing effect during heat sealing. Preferably, the sealing of the liquid filling port at the top of the lysis solution tube 13 adopts the aluminum film heat sealing method. The thickness of the aluminum film is 100 um, which has good flexibility and sealing performance. During heat sealing, the aluminum film is fused with the plastic at the edge of the liquid filling port through a heating device to form a tight seal. The width of the heat sealing area is 0.8 mm to ensure the reliability of the seal. A raised tear tab is provided at one corner of the aluminum film, facilitating the operator to quickly open the aluminum film during use. In the preferred embodiment, the edge of the liquid filling port of the lysis solution tube 13 is designed to be arc-shaped, facilitating the pouring of liquid and avoiding liquid splashing during the liquid filling process. The liquid outlet is located at the bottom of the lysis solution tube 13 and is integrally formed with the lysis solution tube 13. The inner diameter of the liquid outlet is 0.5 mm, and the outer diameter is 1 mm. The inside of the lysis solution tube 13 adopts a tapered transition design, gradually reducing from the inner diameter of the lysis solution tube 13 to the inner diameter of the liquid outlet, to reduce the resistance when the liquid flows out and ensure the smooth outflow of the lysis solution 10. The end of the liquid outlet of the lysis solution tube 13 is rounded to avoid sharp edges from damaging subsequent operations and connecting components. A relatively shallow pre-scratch is engraved at the liquid outlet to ensure a stable liquid outflow volume.

[0032] The main body component 6 is used to provide a reaction area for nucleic acid amplification and nucleic acid detection, and is also the observation area for the detection results. Its shape is an overall flat cylinder; the main body component 6 further includes a reaction tube body 15 and RT-RAA freeze-dried microspheres 3. The reaction tube body 15 is designed in a conical shape. The upper part of the reaction tube body 15 is injection-molded into a flat column shape with high-transparency PC plastic, and the height is about 78 mm. The special shape provides sufficient internal space to accommodate the reagents and nucleic acid samples required for the reaction, and at the same time can be adapted to common detection devices; the high-transparency PC plastic not only has excellent optical properties, with a transmittance of over 95% in the visible light and the fluorescence range of specific wavelengths, but also has good mechanical strength and chemical stability; the PC plastic is specially treated to further reduce the fluorescence background of the material and reduce the interference with the detection signal. The lower part of the reaction tube body 15 is a triangular reaction area, and the wall thickness is strictly controlled to not exceed 0.8 mm, which helps the fluid flow and heat transfer during the reaction process. The surface of this area is mirror-treated, and the surface roughness Ra value does not exceed 0.8, so as to minimize the scattering and reflection of light on the surface and improve the accuracy and sensitivity of fluorescence detection. Before leaving the factory, the triangular reaction area at the lower part of the reaction tube body 15 is precisely pre-loaded with RT-RAA freeze-dried microspheres 3. The RT-RAA freeze-dried microspheres 3 are evenly distributed and have a moderate density to ensure that the nucleic acid sample can quickly and fully contact and react with them after being added.

[0033] The decorative cover 9 is used to facilitate the batch storage and transportation of the integrated fluorescence detection kit, and its shape is a rounded rectangular cuboid.

[0034] The reagent system in the kit includes an RT-RAA reaction system and a CRISPR reaction system. The RAA isothermal amplification technology is a new type of nucleic acid amplification method that gets rid of the dependence on thermal cycling equipment in traditional PCR technology. The core of the RAA technology lies in the synergistic action of recombinase, single-stranded binding protein and DNA polymerase. The recombinase can recognize and bind to the homologous region of the primer and the target nucleic acid sequence to form a complex, and accurately guide the primer to the target DNA position. The single-stranded binding protein then stabilizes the displaced single-stranded DNA to prevent its accidental annealing. The DNA polymerase then rapidly extends along the single-stranded DNA starting from the primer to complete the nucleic acid amplification. This unique mechanism enables RAA to rapidly amplify nucleic acids under relatively constant temperature conditions (usually 37 - 42 °C), greatly shortening the detection time. At the same time, the RAA technology has high sensitivity and specificity, and can detect trace amounts of nucleic acids, providing strong support for the early diagnosis of diseases.

[0035] The CRISPR system relies on the functions of Cas proteins and guide RNAs (gRNAs). The gRNA can specifically recognize the target nucleic acid sequence and guide the Cas protein to bind to it. The Cas protein has endonuclease activity and can cleave the target sequence at specific sites. In the integrated fluorescence detection consumables, the application of CRISPR technology further improves the detection specificity, can accurately distinguish the target nucleic acid from similar sequences, and reduces the occurrence of false positive and false negative results.

[0036] The working principle of the present invention: Open the sealed aluminum film at the liquid addition port at the top of the lysate tube body, immerse the sample to be tested in the lysate, and the cells or pathogens are lysed to release nucleic acids. Cover the pusher assembly of the lysate tube to form a constant pressure sealing structure inside the lysate tube. Break off the sealing piece at the bottom of the lysate tube, and by applying pressure to the pusher of the lysate tube, a certain amount of fluid flows into the triangular reaction area, quickly dissolving the RT-RAA freeze-dried microspheres. Place the reaction area in a constant temperature heating environment to complete the RT-RAA nucleic acid amplification reaction. Then apply pressure to the diluent pusher, and the tapered structure pierces the sealing film, quickly dissolving the CRISPR freeze-dried microspheres and flowing into the reaction area to react with the existing RT-RAA products, and heat the triangular reaction area at a constant temperature according to the reagent reaction temperature to complete the nucleic acid cleavage and detection process.

[0037] The sealing performance of the lysate tube is crucial for the accuracy and reliability of the integrated fluorescence detection kit. To ensure that the lysate tube can maintain good sealing performance under various conditions, the present invention adopts the following strict test methods: Prepare a batch of lysate tubes to be tested. In a clean environment, use a high-precision pipette to accurately add 500 microliters of lysate to each lysate tube. Carefully place the lysate tubes with added lysate in an oven that has been pre-set and calibrated to a stable 60 °C. During the entire 192-hour (8-day) test process, continuously monitor and record the temperature of the oven to ensure that the temperature always remains within the specified range of 60 °C, with the fluctuation amplitude controlled within a very small allowable range to simulate relatively harsh usage environmental conditions. Observe and record the lysate tubes every 48 hours. Use precise measuring tools, such as a magnifying glass with scale or a high-precision liquid level measuring instrument, to accurately measure the liquid level height and record the measurement results in detail to check whether there are obvious signs of liquid decline. To more precisely determine whether the liquid has decreased, mark and measure the initial liquid level position of each lysate tube before the test and record the initial liquid level height data. Compare the current liquid level height with the initial liquid level height during each observation. If after the 192-hour test, the liquid in the lysate tube has not significantly decreased and the change in liquid level height is within the acceptable error range (the liquid level drops by no more than 1% of the total liquid level), then the sealing performance of the lysate tube is considered qualified. For those lysate tubes that show obvious liquid reduction during the test, mark them as unqualified products and conduct a detailed inspection and analysis of their sealing structure to find possible sealing defects, such as loose sealing at the liquid outlet, gaps at the tube body connection, poor heat sealing of the aluminum film at the liquid addition port, etc. Based on the analysis results, improve and optimize the production process and sealing design of the lysate tube to improve the sealing quality and stability of subsequent products.

[0038] Test the sealing performance of the main component of the integrated fluorescence detection kit: By using the method of visual inspection, a preliminary assessment of the appearance of the main component was carried out. No obvious cracks, notches or assembly incompleteness and other defects that may affect the sealing performance were found. Subsequently, the pressure test method was used. Connect the main component to a dedicated pressure test device, gradually increase the internal pressure to the set value and maintain it for a period of time. During this period, use a high-precision pressure sensor to continuously monitor the pressure change. The results show that the pressure remains stable within the specified time and there is no obvious drop, indicating that the main component can withstand the predetermined pressure without leakage.

[0039] In another embodiment, a detection method using an integrated fluorescence detection kit is provided, which includes the following steps: Step S1, processing the sample to be tested: Open the sealed aluminum film at the liquid addition port at the top of the lysate tube, immerse the sample to be tested in the lysate, install the pusher assembly of the lysate tube at the liquid addition port at the top of the lysate tube, form a constant-pressure sealing structure inside the lysate tube, and let it stand for 5 minutes; Step S2, RT-RAA amplification and incubation: Remove the sealing piece under the lysis tube, push down the lysis tube pusher with pressure, and push the lysis tube pusher to the bottom. A certain amount of fluid flows into the triangular reaction zone, quickly dissolving the RT-RAA freeze-dried microspheres. Place the reaction zone in a constant-temperature heating environment to complete the RT-RAA nucleic acid amplification reaction.

[0040] Step S3, CRISPR reaction: After incubation, push the diluent pusher to the bottom with pressure. The tapered structure pierces the sealing film, and the diluent dissolves the CRISPR freeze-dried microspheres and flows into the reaction zone of the reaction tube body, reacting with the RT-RAA nucleic acid amplification product obtained in Step S2. Heat the triangular reaction zone of the reaction tube body at a constant temperature according to the set reaction temperature to complete the nucleic acid cleavage in the triangular reaction zone of the reaction tube body.

[0041] Step S4, Interpret the test result according to the fluorescence intensity in the reaction zone: Use blue light to irradiate the cleaved nucleic acid in the triangular reaction zone of the reaction tube body obtained in Step S3, and use a fluorescence image analysis system for analysis. Interpret the test result according to the fluorescence intensity in the reaction zone.

[0042] Example 1, using an integrated fluorescence detection kit to detect the nucleic acid of SARS-CoV-2, a novel coronavirus, which includes the following steps: Step T1: Nucleic acid sample treatment: Open the sealed aluminum film at the liquid addition port on the top of the lysis tube, immerse the quantified nucleic acid sample in the lysis solution, and detect the full genome sequence of SARS-CoV-2 at 10 3 / 10 2 / 10 1 / 10 0 / 0.5 / 0.05 copy / µL respectively. Cover the lysis tube pusher assembly, and the lysis tube forms a constant-pressure sealed structure. Let it stand for 5 min.

[0043] Step T2: RT-RAA amplification and incubation: Remove the sealing piece under the lysis tube, push down the lysis tube pusher with pressure, and push the lysis tube pusher to the bottom. A certain amount of fluid flows into the triangular reaction zone, quickly dissolving the RT-RAA freeze-dried microspheres. Place the reaction zone in a metal bath, set the metal bath temperature to 43°C, and the time to 25 min, and start incubation.

[0044] Step T3: CRISPR reaction: After incubation, push the diluent pusher to the bottom with pressure. The tapered structure pierces the sealing film, and the diluent dissolves the CRISPR freeze-dried microspheres and flows into the reaction zone. Heat at a constant temperature of 37°C and incubate for 25 min to complete the cleavage and detection process of the nucleic acid of the novel coronavirus SARS-CoV-2.

[0045] Step T4: Interpret the test result based on the fluorescence intensity in the reaction zone: Irradiate the sheared nucleic acid in the triangular reaction zone of the reaction tube main body obtained in Step S3 with blue light, and analyze it using a fluorescence image analysis system. Interpret the test result based on the fluorescence intensity in the reaction zone. Specifically, within the same test time, if the fluorescence intensity value of the experimental group is more than 3 times higher than that of the negative control (ddH2O), it is determined as a positive result.

[0046] The experimental results are as Figure 7 and Figure 8 shown. The results of three repeated experiments show that the fluorescence results of positive and negative samples can be visually distinguished in the reaction zone of the kit. Positive fluorescence results as low as 0.5 copy / µL can be observed. The background interference of the kit is less and the results are clear.

[0047] In the present invention, the CRISPR gene shearing technology plays a key role; the CRISPR system consists of a Cas protein and a guide RNA (gRNA). The gRNA can specifically recognize the target nucleic acid sequence and guide the Cas protein to bind to it. The Cas protein has endonuclease activity. After forming a complex with the gRNA, when the gRNA recognizes the target nucleic acid, it can activate the cleavage activity of the Cas protein and cleave the target nucleic acid at a specific site. In the integrated fluorescence detection of the present invention, the application of the CRISPR technology further improves the specificity of the detection, can accurately distinguish the target nucleic acid from similar sequences, and reduces the occurrence of false positive and false negative results. Further, the fluorescence detection technology is based on the property that substances produce fluorescence when excited by light of a specific wavelength; when the Cas protein is activated, it will simultaneously activate the collateral cleavage effect. After the reporter probe is cleaved to release a specific fluorescent label group, a detectable fluorescent signal will be generated under the irradiation of the excitation light; by detecting parameters such as the intensity and wavelength of the fluorescent signal, the presence and concentration of the target nucleic acid can be analyzed quantitatively or qualitatively. The fluorescence detection technology adopted in the present invention has high sensitivity and high selectivity, can detect extremely weak changes in fluorescent signals, and thus realizes the accurate detection of trace nucleic acids. At the same time, through the careful design of the fluorescently labeled probe, the simultaneous detection of multiple target nucleic acids can be achieved, improving the detection throughput and efficiency.

[0048] The design of the integrated fluorescence detection kit of the present invention fully considers the requirements of multiple links such as nucleic acid sample processing, nucleic acid amplification, gene cleavage, and fluorescence detection. The separation design of the lysis solution tube and the integrated fluorescence detection consumable body component not only facilitates the collection and processing of nucleic acid samples but also ensures the safety and accuracy of the detection process. The lysis solution in the lysis solution tube can effectively destroy the cell structure in the sample to be tested and release nucleic acids. The RAA freeze-dried microspheres and CRISPR freeze-dried microspheres are pre-isolated and placed in the integrated fluorescence detection kit, ensuring the stability and long-term preservation of the reagents. During the detection process, these freeze-dried microspheres can quickly dissolve and participate in the reaction to achieve nucleic acid amplification and cleavage.

[0049] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications all fall within the protection scope of the present invention.

[0050] In addition, it should be noted that, in the various specific technical features described in the above specific embodiments, they can be combined in any way without conflict. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods. In addition, any combination can be made between various different embodiments of the present invention, as long as it does not violate the idea of the present invention, it should also be regarded as the content disclosed by the present invention.

Claims

1. An integrated fluorescence detection kit, characterized in that, It includes an upper cover assembly (4), a lysate tube assembly (2), a main body assembly (6), a diluent tube assembly (7) and a decorative cover (9); The lower end of the upper cover assembly (4) is connected to the lysate tube assembly (2) and the diluent tube assembly (7), the side wall of the upper cover assembly (4) is connected to the upper part of the main body assembly (6), and the main body assembly (6) is nested in the decorative cover (9).

2. The integrated fluorescence detection kit according to claim 1, wherein The upper surface of the upper cover assembly (4) is provided with a placement channel for the lysate tube assembly (2) and a placement channel for the diluent tube assembly (7). An annular gasket is designed on the contact surface of the upper cover assembly (4) with the reaction tube, and the contact surface of the annular gasket contacts the lysate tube assembly (2) and the diluent tube assembly (7).

3. The integrated fluorescence detection kit according to claim 2, wherein, The lysate tube assembly (2) includes a lysate tube push rod assembly (11) and a lysate tube body (13).

4. The integrated fluorescence detection kit according to claim 3, wherein The whole lysate tube (13) adopts a cylindrical structure, and the inner surface of the lysate tube (13) is smoothed.

5. The integrated fluorescence detection kit according to claim 4, wherein The inside of the lysate tube (13) adopts a tapered transition design, and the inner diameter of the lysate tube (13) gradually decreases to the inner diameter of the liquid outlet.

6. The integrated fluorescence detection kit according to claim 1, wherein The diluent tube assembly (7) includes a diluent tube push rod assembly (5), a diluent tube (17) and a sealing ring (16). The diluent tube (17) is a slender cylinder. One end of the diluent tube (7) is connected to the diluent tube push rod assembly (5) to be sealed, and the other end is open and provided with a sealing ring (16) and a sealing film.

7. The integrated fluorescence detection kit according to claim 5, wherein The diluent push rod of the diluent tube push rod assembly (5) is composed of a plastic rod body and a silicone push head. The rod body is a slender cylinder, and the push head is in interference fit with the inner wall of the diluent tube; the biological shape of the push head is adapted to the cross-section of the diluent tube.

8. The integrated fluorescence detection kit according to claim 6, wherein A cavity with a pointed cone is provided below the sealing ring (16). The CRISPR freeze-dried microspheres (8) are pre-placed in the cavity, and the CRISPR freeze-dried microspheres (8) are separated from the diluent (14) to ensure the stability of the CRISPR freeze-dried microspheres (8).

9. The integrated fluorescence detection kit according to claim 5, wherein The upper part of the reaction tube main body (15) is in a flat cylindrical shape, and the lower part is a triangular reaction area, which is integrally injection-molded.

10. A test method using the integrated fluorescence detection kit according to any one of claims 1-9, characterized in that, It includes the following steps: Step S1, treatment of the sample to be tested: Open the sealed aluminum film at the liquid addition port at the top of the lysate tube, immerse the sample to be tested in the lysate, install the lysate tube push rod assembly at the liquid addition port at the top of the lysate tube, form a constant pressure sealing structure in the lysate tube, and stand for 5 min; Step S2, RT-RAA amplification and incubation: Remove the sealing piece below the lysate tube, push down the lysate tube push rod under pressure, and push the lysate tube push rod to the bottom, so that a certain amount of fluid flows into the reaction area of the reaction tube main body, and quickly dissolve the RT-RAA freeze-dried microspheres placed in the triangular reaction area; Place the triangular reaction area in a constant temperature heating environment to complete the RT-RAA nucleic acid amplification reaction; Step S3, CRISPR reaction: After the incubation ends, apply pressure to the diluent pusher until it reaches the bottom, and the tapered structure pierces the sealing film. The diluent dissolves the CRISPR freeze-dried microspheres and flows into the reaction zone of the reaction tube body, reacting with the RT-RAA nucleic acid amplification product obtained in Step S2. Heat the triangular reaction zone of the reaction tube body at a constant temperature according to the set reaction temperature to complete nucleic acid cleavage in the triangular reaction zone of the reaction tube body. Step S4, Interpret the detection result according to the fluorescence intensity in the reaction zone: Irradiate the cleaved nucleic acid in the triangular reaction zone of the reaction tube body obtained in Step S3 with blue light, and analyze it using a fluorescence image analysis system. Interpret the detection result according to the fluorescence intensity in the reaction zone.

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