A real-time fluorescence quantitative PCR instrument and detection method thereof

Abstract

The present invention discloses a real-time fluorescence quantitative PCR instrument, comprising a housing, a thermal cycle module, a detection module, and a sample assembly, wherein the thermal cycle module is used to provide the real-time temperature required by the sample assembly during the detection process; the detection module is used to perform real-time detection on the sample assembly; an entrance and an exit are provided on the housing, and the sample assembly enters and exits the housing through the entrance and an exit; the sample assembly comprises a rotating disk and a reagent kit, wherein the reagent kit is placed on the rotating disk and is detachably connected to the rotating disk, the rotating disk is rotatably connected to the housing, and the reagent kit is used to place a reagent tube to be detected. The present invention can effectively improve the efficiency of thermal cycles and ensure that subsequent operations can be seamlessly connected, thereby improving the detection efficiency as a whole. At the same time, the detection structure of the present invention can make the product design more miniaturized.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescence PCR detection, and in particular to a real-time fluorescence quantitative PCR instrument and a detection method using the PCR instrument. Background Art

[0002] The fluorescence quantitative PCR instrument heats the PCR reaction system with the addition of fluorescent groups at a certain temperature for a certain period of time, allowing the reaction system to complete a thermal cycle of high-temperature denaturation, low-temperature renaturation, and appropriate temperature extension, so that the fluorescent element is free in the reaction system. The change in the fluorescence signal is then used to detect the changes in the PCR reaction in real time, and the relationship between the Ct value and the standard curve is used to achieve quantitative analysis of the starting template.

[0003] The qPCR reaction program follows the three basic steps of PCR reaction: denaturation, annealing, and extension. Usually, the denaturation temperature is set at 95°C, the annealing temperature is set at 50-65°C, and the extension temperature is set at 72°C. The annealing temperature and the extension temperature can be set at the same temperature (50-65°C). Fluorescence signal collection is performed during the extension program.

[0004] Conventional PCR instruments, after the cycle is completed, generally place the product in the instrument and "keep" it at about 4 degrees for a period of time to keep the reaction system stable, and then take it out. However, experiments have found that cooling the sample block to this temperature, especially for a long time, will bring unnecessary pressure to the sample block and may shorten its life by more than ten times. In order to avoid this situation, the method adopted in the prior art is to remove the sample immediately after the program ends, or set the instrument to keep the sample at room temperature. This has minimal impact on the PCR product, because DNA is relatively stable at room temperature. In addition, keeping the sample block at low temperature may cause the accumulation of condensed water, which may not only cause corrosion of the block itself, but also damage the electrical components inside the instrument. In the public document "PCR Amplifier, the key to ensuring stable PCR performance, March 23, 2022", the sample processing method after the PCR cycle is completed is clearly mentioned.

[0005] However, in the existing fluorescent quantitative PCR instrument, the reaction temperature is set high during the heating stage, resulting in a high overall temperature of the test tube after the test tube is heated. The experimenter cannot immediately perform subsequent quantitative analysis operations, and a large amount of waiting time is required for cooling, which reduces the work efficiency of the experimenter. Each time the device is placed and taken out, it is necessary to open the device, especially when it is in the detection process. On the one hand, it is necessary to dissipate the heat. On the other hand, after the detection box undergoes high-temperature denaturation and low-temperature renaturation and suitable temperature extension (denaturation annealing and extension process) during the detection process, its own temperature is high, about 72°, and it is easy to get burned during the removal process, making the operation inconvenient. For example, many patents such as patent publication numbers: CN219991591U, CN112172054A, CN116355741A, CN208500928U, etc., all describe technical solutions for preventing burns during manual removal after the test is completed.

[0006] The prior art describes rapid temperature control in PCR testing. For example, the existing public document “Principles, Steps, Precautions, and Frequently Asked Questions of Rapid PCR Reagents, October 27, 2022” discloses an improved method for accelerating temperature changes between temperature stages of PCR thermal cycles, which mentions that the use of a PCR cycler with rapid heat transfer or increasing the heat conduction rate may increase the rate of heating or cooling between different temperature stages, thereby achieving the purpose of FastPCR. The possibility of improving the heat transfer efficiency of traditional PCR cyclers heated by a metal base is small, while PCR instruments using an aerodynamic mode allow the temperature of the heating and cooling air to be adjusted to a great extent, and the PCR sample tubes can also be replaced with materials with higher thermal conductivity, such as capillary glass tubes instead of plastic. In other words, on the basis of traditional temperature control, effective means can be used to increase the rate of temperature increase. However, there is no further elaboration on how to better utilize the heat on the basis of increasing the temperature rate.

[0007] In addition, the existing technology has been committed to the design of product miniaturization, but there has been no proper improvement in the improvement of product detection efficiency brought about by miniaturization design. How to improve the temperature rise and heat utilization efficiency during the detection process while miniaturizing the design of the equipment, and correspondingly improve the detection efficiency is an industry problem that needs to be solved urgently. Summary of the invention

[0008] The present invention aims to solve the problem that the existing detection box needs to be repeatedly placed and taken out of the device manually during the detection process, especially when the detection amount is relatively large, and the subsequent operation cannot be carried out in time due to the high temperature, and the device needs to be opened and closed continuously during the detection process, which leads to the problem of reduced thermal cycle efficiency. A real-time fluorescence quantitative PCR instrument and a detection method thereof are provided, which provides a relatively closed detection environment and a repeated rotation structure that is convenient for continuous operation, effectively improving the thermal cycle efficiency. At the same time, the detection structure of the present invention can make the product design more miniaturized.

[0009] The present invention is achieved through the following technical solutions:

[0010] A real-time fluorescence quantitative PCR instrument comprises a shell, a thermal cycle module, a detection module, and a sample assembly, wherein the thermal cycle module is used to provide the real-time temperature required by the sample assembly during the detection process; the detection module is used to perform real-time detection on the sample assembly; an entrance and an exit are provided on the shell, and the sample assembly enters and exits the shell through the entrance and the exit; the sample assembly comprises a rotating disk and a reagent kit, the reagent kit is placed on the rotating disk and is detachably connected to the rotating disk, the rotating disk is rotatably connected to the shell, the reagent kit is used to place a reagent tube to be detected, the rotating disk is formed by a half disk one and a half disk two connected to each other, the half disk one and the half disk two have the same shape, and are detachably connected to each other to form a rotating disk, and when the reagent tube to be detected is in detection, one of the half disk one and the half disk two is located inside the shell, and the other is located outside the shell.

[0011] In a further embodiment, the reagent kit is placed on a rotating disk, and the reagent kit can be arranged as one or more. When half disk one or half disk two is placed in the shell for detection, the reagent kit arranged thereon is arranged symmetrically relative to the center line.

[0012] In a further solution, a rotating rolling column is provided at the upper end of the shell entrance and exit. When the rotating disk drives the reagent kit and the reagent tubes to be tested thereon to rotate into the shell, the edge of the rotating rolling column contacts the top of some of the reagent tubes to be tested and applies pressure thereto. During this process, the friction force between the reagent tubes to be tested and the rotating rolling column drives the rotating rolling column to rotate; the rotating rolling column is divided into two sections from the midpoint of the entrance and exit, and when the rotating disk rotates, the two sections rotate in opposite directions.

[0013] In a further solution, the rotatable rolling column is movably arranged at the upper end of the shell entrance and exit through an elastic component. The cross-section of the rotatable rolling column is a polygonal structure, and each edge is concave toward its center to form an arc edge. When the rotatable rolling column acts on the reagent tube to be tested and rotates, the arc edge in contact with the reagent tube to be tested always applies pressure to the top of the reagent tube to be tested.

[0014] In a further solution, there is an arc-shaped transition section between two adjacent sides of the polygonal structure. When one of the arc-shaped sides of the rotating rolling column rotates a certain angle driven by a certain reagent tube to be tested, the other adjacent arc-shaped side contacts the top surface of the next reagent tube to be rotated, and the arc-shaped transition section is just placed in the empty space between the two reagent tubes to be tested during this process.

[0015] In a further solution, the elastic force applied by the elastic component causes the rotating rolling column to always apply pressure to the top of the reagent tube to be detected during the process of contacting the top of the reagent tube to be detected.

[0016] In a further solution, the arc-shaped transition section starts from the connection position of the two sections of the rotating rolling column, gradually extends to both ends, and is surrounded by the rotating rolling column.

[0017] In a further scheme, at least one clamping device is included, and openings are provided at both ends of the clamping device. The middle part is bent to form at least one annular structure, and an aisle is formed on the annular structure. The half disk one and the half disk two are provided with buckles that cooperate with the clamping device, and the buckles are connected to the half disk one and the half disk two through a connecting rod. After the buckles enter the clamping device from the opening, they are clamped into the annular structure through the aisle. The aisle is elastic, and the diameter of the contact position between the buckles and the aisle is larger than the width of the aisle opening. When the buckles of the half disk one and the half disk two are respectively clamped into the openings at both ends of the clamping device and placed in the annular structure, the half disk one and the half disk two form a rotating disk as a whole, and the connecting rod is placed at the lower end of the opening.

[0018] In a further solution, a placement cavity and a guide rail are provided on the outer wall of the shell, one end of the guide rail is provided in the placement cavity, and the other end is close to the opening. After the buckle is moved out from the opening, it can run along the guide rail to the placement cavity. At this time, half disk one and / or half disk two where the buckle is located can be placed in the placement cavity, and an elastic guide plate is provided at one end of the guide rail close to the opening. When the buckle applies inward pressure to the elastic guide plate, the elastic guide plate is offset, and the buckle moves from the position of the elastic guide plate to the opening. When the buckle applies outward pressure to the elastic guide plate, the elastic guide plate is offset, and the buckle moves from the position of the elastic guide plate from the opening to the outside.

[0019] A detection method of a real-time fluorescence quantitative PCR instrument, the specific operation mainly includes the following steps:

[0020] Open the entrance and exit of the shell, place the first half disk into the shell through the entrance and exit, connect it to the clamping device, and connect the second half disk to the other end of the clamping device, so that the first half disk and the second half disk form a rotating disk;

[0021] Place the reagent kit equipped with the reagent tubes to be tested at the installation position on the rotating disk;

[0022] Rotate the rotating disk to move the first half disk or the second half disk into the detection position in the housing for detection, and go through the detection processes such as denaturation, annealing and extension;

[0023] After the detection is completed, the rotating disk is rotated to make the detected half disk one or half disk two rotate out from the entrance and exit, and the other half of the half disk two or half disk one rotates in, and the above detection process is repeated;

[0024] While the transferred in reagent tubes to be tested are being tested, the transferred out reagent tubes to be tested that have been tested can be left still or removed, and a new test kit equipped with the reagent tubes to be tested can be added to wait for being transferred to the testing position for the next test;

[0025] After the detection is completed, the buckles of the half disk one and / or half disk two outside the shell are moved out from the opening, slid along the guide rail and placed in the placement cavity, or directly taken out from the elastic guide sheet and placed in the placement cavity.

[0026] The present invention has the following beneficial effects:

[0027] The clamping device and the setting of half disk one and half disk two of the rotating disk can be quickly disassembled and assembled during use. The rotating entry detection method makes the entrance and exit smaller. The detachable and placement method of the rotating disk makes the equipment more miniaturized and can be replaced in time during maintenance.

[0028] After a test is completed, the test kit is rotated out and a new test kit is put into the test, which is preheated by fully utilizing the temperature inside the device and is directly placed in the test position to ensure the sealing of the tube cover of the reagent tube to be tested. No manual clamping is required under high temperature conditions.

[0029] There is no need to open the device, and the temperature of the hot cover is always maintained, and the hot cover is kept pressing the test tube on the reagent kit to prevent the liquid from condensing on the top of the test tube and affecting the test results; the heat in the device can be fully utilized to keep it within a certain temperature range.

[0030] The rotatable and detachable rotating disk, combined with the guide rail and the placement of the cavity, allows the entire detector to be designed to be more compact while having the continuous detection function; during the detection process, it is only necessary to set up a closed structure in a smaller area of ​​the entrance and exit, and use the rotating disk to close the entrance and exit.

[0031] In the prior art, the lid of the reagent tube to be tested is sealed mainly by pressing the hot cover on the top of the lid after entering the detection position, so as to keep it sealed. Because in the process of placing or taking out the test kit in the prior art, the internal temperature is relatively low, even if the lid is not tightly sealed, it will not affect the test reagent. However, the present invention has its particularity, because it is necessary to always maintain a relatively high temperature in the test environment. If the lid of the reagent tube is not well sealed, the test reagent will be volatilized and lost during the process from entering the test environment to pressing the hot cover, thereby having a greater impact on the test result. Therefore, the present invention ensures its sealed state before it rotates to enter the test environment, which effectively cooperates with the purpose of continuous detection of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0033] Figure 1 It is a schematic diagram of the structure of the present invention;

[0034] Figure 2 This is a schematic diagram of the structure when the cavity is placed at the top in an embodiment of the present invention;

[0035] Figure 3 This is a schematic diagram of the structure of a rotating disk in an embodiment of the present invention;

[0036] Figure 4 This is a schematic diagram of the structure of the clamping device in an embodiment of the present invention;

[0037] Figure 5 This is a schematic diagram of the cooperation between the rotating rolling column and the reagent tube to be tested in an embodiment of the present invention;

[0038] Figure 6 It is a schematic diagram of the matching structure between the clamping device and the guide rail in an embodiment of the present invention;

[0039] Figure 7 Schematic diagram of the structure of a rotary rolling column in an embodiment of the present invention.

[0040] Reference numerals shown in the figure: 1-shell, 2-sample assembly, 3-entrance and exit, 4-rotating disk, 5-reagent box, 6-reagent tube to be tested, 7-center line, 8-rotating rolling column, 9-arc edge, 10-arc transition section, 11-clamping device, 12-opening, 13-annular structure, 14-aisle, 15-clip, 16-placing cavity, 17-guide rail, 18-elastic guide plate, 19-connecting rod. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments and drawings. The schematic implementation modes and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention. It should be noted that the present invention is already in the actual development and use stage.

[0042] In this embodiment, Figure 1 and Figure 2 As shown, a real-time fluorescence quantitative PCR instrument of the present invention includes conventional settings such as a thermal cycle module, a detection module, and a heat dissipation module in a conventional quantitative PCR instrument, wherein the thermal cycle module is mainly used to provide the real-time temperature required by the sample component 2 during the detection process; the detection module is mainly used to perform real-time detection on the sample component 2 and obtain relevant detection and analysis data; the present invention mainly improves the heat utilization and continuous detection method of the entire detector. On the basis of the conventional settings, the present invention sets an entrance and exit 3 at the front end of the shell 1. Different from the switch door design in the prior art, the entrance and exit 3 has a smaller open area and is mainly used for the installation and rotation of the rotating disk 4. With a smaller closed structure, it can ensure that the heat inside will not be lost. This setting can provide support for the miniaturized design of the entire device. When it is necessary to perform a test, it is only necessary to open the entrance 3, take out the half disk 1 41 and the half disk 2 42 of the rotating disk 4 placed in the placement cavity 16, and splice them into the rotating disk 4 through the clamping device 11. The specific process can be described as: after taking out the half disk 1 41 from the placement cavity 16, it is rotated from the entrance 3 and placed in the shell 1, and connected with the clamping device 11, and then the half disk 2 42 is connected to the other end of the clamping device 11, so as to form the rotating disk 4. The rotating disk 4 enters and exits the entrance 3 by rotating, and when the half disk 1 41 is completely placed in the shell 1, the half disk 2 42 is completely placed outside the shell 1. The reagent kit 5 is placed on the half disk 1 41 and the half disk 2 42, and the reagent tube 6 to be tested is installed on the reagent kit 5. The half disk 1 41 and the half disk 2 42 rotate alternately to enter the detection position of the shell 1 for detection. The structure of the present invention can effectively provide necessary support for the miniaturization of the entire device, and fully utilize the heat in the device, which is particularly advantageous in the case of a large amount of detection.

[0043] In this embodiment, the clamping device 11 is as follows: Figure 4 and Figure 6The structure described above has openings 12 at both ends, which are used to cooperate with the buckles 15 provided on the half-disc 1 41 and the half-disc 2 42. The middle part is bent to form an annular structure 13. A passage 14 is formed between the opening 12 and the annular structure 13. After the buckle 15 enters from the opening 12, it is inserted into the passage 14 and then placed in the annular structure 13. The passage 14 has a certain elasticity, and its passage diameter is slightly smaller than the size of the opening 12. Therefore, during the process of the buckle 15 being inserted into the passage 14, the operator can clearly know whether it is inserted into the annular structure 13 and is in a suitable position according to the insertion process, thereby ensuring the accuracy of the connection and enabling the reagent tube 6 to be detected in the reagent box 5 to be accurately rotated to the designated detection position. When the buckle 15 is inserted into place, the connecting rod 19 connected to its rear end is placed at the lower end of the opening 12, which can play a role in supporting the entire half-disc 1 41 or half-disc 2 42. The shape of the annular structure 13 matches the shape of the buckle 15. When the buckles 15 of the first half disk 41 and the second half disk 42 are inserted into the annular structure 13, the positions of the two buckles 15 are relatively fixed, so that the entire rotating disk 4 forms a solid integral structure. Figure 1 , Figure 2 and Figure 6 As shown, when the half disk 1 41 and the half disk 2 42 are to be taken out, it is only necessary to pull the half disk 1 41 or the half disk 2 42 so that the buckle 15 can be withdrawn from the passage 14 of the annular structure 13, and then the buckle 15 can be taken out from the position of the opening 12. As another disposal method, the present invention is provided with a guide rail 17 at the opening 12, and an elastic guide piece 18 is provided at the position of the guide rail 17 near the opening 12. When the buckle 15 is to be inserted into the opening 12 from the outside, the elastic guide piece 18 is pushed from the outside to the inside, and the elastic guide piece 18 is deflected inwardly, and the buckle 15 can enter the opening 12 from the position of the elastic guide piece 18. When the buckle 15 needs to be taken out from the inside to the outside, the elastic guide piece 18 is pushed from the inside to the outside, and the elastic guide piece 18 is deflected outwardly, and the buckle 15 can be taken out from the position of the elastic guide piece 18. It is also possible that the buckle 15 does not need to be removed, but the buckle 15 can be directly slid from the opening 12 to the guide rail 17. At this time, the buckle 15 and the guide rail 17 match each other in shape, and the buckle 15 can be slid along the guide rail 17 to the position of the cavity 16 and placed.

[0044] The reagent box 5 is located on the rotating disk 4. Figure 3 The figure shows a reagent kit setting method in this embodiment, wherein the distribution of the reagent tubes 6 to be detected on the reagent kit 5 can be a square arrangement or a circular arrangement, etc. In order to enable the detection module in the detector to obtain accurate data, we set the reagent tubes 6 to be detected on the reagent kit 5 entering the detection position on the half-disc 1 41 or the half-disc 2 42 to be symmetrically arranged, and symmetrically arranged relative to the center line 7, so as to facilitate the detection module to obtain data.

[0045] The present invention is to maintain the detection environment in the shell 1 at a relatively high temperature as much as possible and improve the heat utilization rate by means of a relatively small entrance and exit 3 and a setting method for rotating the reagent box 5 in and out. Therefore, after the reagent tube 6 to be detected on the reagent box 5 enters the detection environment in the shell 1 from the entrance and exit 3, the temperature of the detection reagent therein will rapidly increase after being heated. If the lid of the reagent tube 6 to be detected is not tightly sealed, the detection reagent therein will certainly volatilize due to the rapid increase in temperature, causing a significant impact on the detection result. In addition, after the detection is completed, when the rotating disk 4 is rotated out from the other side of the entrance and exit 3, due to the high temperature in the reagent tube, when the hot cover is no longer pressed on the lid after the detection is completed, the evaporation of the detection reagent therein may push open the lid and volatilize, causing an adverse effect on the detection environment. Based on this, the present invention is provided with a device for detecting and pressing the lid of the reagent tube at the position of the entrance and exit 3, such as Figure 1 , Figure 2 and Figure 7 The rotating rolling column 8 shown in the figure, in this embodiment, we set the rotating rolling column 8 as follows Figure 7 In the structure shown, the rotating rolling column 8 is arranged at the upper end of the entrance and exit 3, and is provided with two sections, which respectively detect and press the lids of the reagent tubes transferred in and out. When the rotating disk 4 drives the reagent box 5 and the reagent tubes 6 to be detected thereon to rotate into the housing 1, the edge of the rotating rolling column 8 contacts the top of part of the reagent tubes 6 to be detected and applies pressure thereto. In this process, the friction between the reagent tubes 6 to be detected and the rotating rolling column 8 drives the rotating rolling column 8 to rotate; when the rotating disk 4 rotates, the two sections of the rotating rolling column 8 rotate in opposite directions driven by the reagent tubes transferred in and out.

[0046] In this embodiment, the cross section of the rotating rolling column 8 is set to a polygonal structure, and each side is concave toward its center to form an arcuate side 9. When the rotating rolling column 8 acts on the reagent tube 6 to be detected and rotates, the arcuate side 9 in contact with the reagent tube 6 to be detected always applies pressure to the top of the reagent tube 6 to be detected. Further, between two adjacent sides of the polygonal structure is an arcuate transition section 10, such as Figure 5 As shown, when one arcuate side 9 of the rotating rolling column 8 rotates a certain angle driven by a certain reagent tube 6 to be detected, another adjacent arcuate side 9 contacts the top surface of the next reagent tube 6 to be detected, and the arcuate transition section 10 is just placed in the empty space between the two reagent tubes 6 to be detected during this process. The rotating rolling column 8 is connected to the housing 1 through an elastic component, and the elastic force exerted by the rotating rolling column 8 causes the rotating rolling column 8 to always apply pressure to the top of the reagent tube 6 to be detected during the process of contacting the top of the reagent tube 6 to be detected.

[0047] During the rotation of the rotary roller column 8, the cooperation between the rotary roller column 8 and the reagent tube 6 to be tested is as follows: Figure 5 As shown, in the present embodiment, the reagent tubes 6 to be detected are arranged in a square in sequence in the reagent box 5, so the arc-shaped transition section 10 starts from the connection position of the two sections of the rotating rolling column 8, gradually extends to both ends and is arranged around the rotating rolling column 8. It can cooperate with the reagent tubes 6 to be detected during its rolling process. Of course, if the arrangement of the reagent tubes 6 to be detected in the reagent box 5 changes, the arrangement of the arc-shaped transition section 10 also needs to be adaptively changed to ensure that the arc-shaped edge 9 can act on the top of each reagent tube 6 to be detected during the rotation. For example, the arrangement of the reagent tubes 6 to be detected in the reagent box 5 is radially arranged relative to the center point of the rotating disk 4 toward the outer edge of the reagent box 5, then the arc-shaped transition section 10 is arranged in a straight strip on the surface of the rotating rolling column 8 without being arranged around.

[0048] In this embodiment, the device housing 1 is provided with a placement cavity 16. Since the rotating disk 4 is rotated from the side of the device into the housing 1, Figure 1 The placement cavity 16 is arranged on the top surface of the housing 1. Of course, the arrangement of the adjustment rail 17 can also be used to arrange the placement cavity 16 on the bottom surface of the housing 1. Combined with the structural design of the rotating disk 4 that can be quickly disassembled and spliced, the entire device can be made flat and miniaturized. Further, the placement cavity 16 can also be arranged on the front surface of the housing 1, such as Figure 2 The conventional display screen and control buttons can be adaptively designed according to the configuration.

[0049] This embodiment also provides a detection method for the above-mentioned PCR instrument, and the specific operation mainly includes the following steps:

[0050] Open the entrance 3 of the housing 1, place the half disk 1 41 into the housing 1 from the entrance 3, connect it to the clamping device 11, connect the half disk 2 42 to the other end of the clamping device 11, so that the half disk 1 41 and the half disk 2 42 form a rotating disk 4; of course, the half disk 1 41 and the half disk 2 42 can also be directly connected through the clamping device 11, and then the half disk 1 41 or the half disk 2 42 is placed in the housing 1 from the entrance 3;

[0051] The reagent kit 5 having the reagent tube 6 to be tested is placed at the installation position on the rotating disk 4; the rotating disk 4 is provided with an installation position for placing the reagent kit 5, so that the reagent kit 5 can be relatively fixed in position during the rotation of the rotating disk 4 and the entire detection process;

[0052] Rotate the rotating disk 4 to move the half disk 1 41 or the half disk 2 42 into the detection position in the housing 1, close the inlet and outlet 3, and then perform the detection, and go through the detection process such as denaturation, annealing and extension;

[0053] After the detection is completed, the entrance 3 is opened again, and the rotating disk 4 is rotated so that the half disk 1 41 or the half disk 2 42 that has been detected is rotated out from the entrance 3, and at the same time, the other half of the half disk 2 42 or the half disk 1 41 is rotated in, and the entrance 3 is closed, and the above detection process is repeated;

[0054] While the transferred in reagent tube 6 to be tested is being tested, the transferred out reagent tube 6 to be tested that has been tested can be left still or removed, and a new reagent kit 5 equipped with the reagent tube 6 to be tested can be added to wait for being transferred to the testing position for the next test;

[0055] After the inspection is completed, the buckle 15 of the half disk 1 41 and / or the half disk 2 42 outside the shell 1 is moved out from the opening 12, slid along the guide rail 17 and placed in the placement cavity 16, or directly taken out from the elastic guide sheet 18 and placed in the placement cavity 16.

[0056] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A real-time fluorescence quantitative PCR instrument, comprising a housing (1), a thermal cycle module, a detection module, and a sample component (2), wherein the thermal cycle module is used to provide the real-time temperature required by the sample component (2) during the detection process; the detection module is used to perform real-time detection on the sample component (2); characterized in that: The housing (1) is provided with an inlet and outlet (3), and the sample assembly (2) enters and exits the housing (1) through the inlet and outlet (3); the sample assembly (2) comprises a rotating disk (4) and a reagent kit (5), the reagent kit (5) being placed on the rotating disk (4) and detachably connected to the rotating disk (4), the rotating disk (4) being rotatably connected to the housing (1), the reagent kit (5) being used to place a reagent tube (6) to be tested, the rotating disk (4) being formed by a half disk (41) and a half disk (42) being connected to each other, the half disk (41) and the half disk (42) being of the same shape, and being detachably connected to each other to form the rotating disk (4). ), when the reagent tube (6) to be tested is under testing, one of the half disk 1 (41) and the half disk 2 (42) is located inside the shell (1), and the other is located outside the shell (1); a rotating rolling column (8) is provided at the upper end of the entrance and exit of the shell (1); when the rotating disk (4) drives the reagent kit (5) and the reagent tube (6) to be tested thereon to rotate into the shell (1), the edge of the rotating rolling column (8) contacts the top of part of the reagent tube (6) to be tested and applies pressure thereto; in this process, the friction between the reagent tube (6) to be tested and the rotating rolling column (8) drives the rotating rolling column (8) to rotate. The column (8) rotates; the rotating rolling column (8) is divided into two sections from the midpoint of the entrance (3); when the rotating disk (4) rotates, the two sections rotate in opposite directions; it comprises at least one clamping device (11); openings (12) are provided at both ends of the clamping device (11); the middle part is bent to form at least one annular structure (13); a passage (14) is formed on the annular structure (13); a buckle (15) cooperating with the clamping device (11) is provided on the half disk (41) and the half disk (42); the buckle (15) is connected to the half disk (41) and the half disk (42) through a connecting rod (19). (42), after the buckle (15) enters the clamping device (11) from the opening (12), it is clamped into the annular structure (13) through the passage (14), the passage (14) is elastic, and the diameter of the contact position between the buckle (15) and the passage (14) is larger than the width of the passage (14) opening. When the buckles (15) of the half disk (41) and the half disk (42) are respectively clamped into the openings (12) at both ends of the clamping device (11) and placed in the annular structure (13), the half disk (41) and the half disk (42) form a rotating disk (4) as a whole, and at this time, the connecting rod (19) is placed at the lower end of the opening (12).

2. A real-time fluorescence quantitative PCR instrument according to claim 1, characterized in that: The reagent kit (5) is placed on the rotating disk (4), and the reagent kit (5) can be arranged as one or more. When the first half disk (41) or the second half disk (42) is placed in the housing (1) for detection, the reagent kits (5) arranged thereon are arranged symmetrically relative to the center line (7).

3. A real-time fluorescence quantitative PCR instrument according to claim 1, characterized in that: The rotating rolling column (8) is movably arranged at the upper end of the shell entrance (3) through an elastic component. The cross section of the rotating rolling column (8) is a polygonal structure, and each side is concave toward its center to form an arcuate side (9). When the rotating rolling column (8) acts on the reagent tube (6) to be detected and rotates, the arcuate side (9) in contact with the reagent tube (6) to be detected always applies pressure to the top of the reagent tube (6) to be detected.

4. A real-time fluorescence quantitative PCR instrument according to claim 3, characterized in that: An arc-shaped transition section (10) is provided between two adjacent sides of the polygonal structure. When one of the arc-shaped sides (9) of the rotating rolling column (8) rotates a certain angle under the drive of a certain reagent tube (6) to be detected, another adjacent arc-shaped side (9) contacts the top surface of the next reagent tube (6) to be detected. During this process, the arc-shaped transition section (10) is just placed in the empty space between the two reagent tubes (6) to be detected.

5. A real-time fluorescence quantitative PCR instrument according to claim 3, characterized in that: The elastic force applied by the elastic component enables the rotating rolling column (8) to always apply pressure to the top of the reagent tube (6) to be tested during the process of contacting the top of the reagent tube (6) to be tested.

6. A real-time fluorescence quantitative PCR instrument according to claim 4, characterized in that: The arc-shaped transition section (10) starts from the connection position of the two sections of the rotating rolling column (8), gradually extends to both ends, and is arranged around the rotating rolling column (8).

7. A real-time fluorescence quantitative PCR instrument according to claim 1, characterized in that: The outer wall of the housing (1) is provided with a placement cavity (16) and a guide rail (17), one end of the guide rail (17) is arranged in the placement cavity (16), and the other end is close to the opening (12). After the buckle (15) is moved out of the opening (12), it can move along the guide rail (17) to the placement cavity (16). At this time, the half disk (41) and / or the half disk (42) where the buckle is located can be placed in the placement cavity (16). When the guide rail (17) is close to the opening (12), the buckle (15) can move along the guide rail (17) to the placement cavity (16). ) is provided with an elastic guide piece (18) at one end; when the buckle (15) applies an inward pressure to the elastic guide piece (18), the elastic guide piece (18) is offset, and the buckle (15) moves from the position of the elastic guide piece (18) to the opening (12); when the buckle (15) applies an outward pressure to the elastic guide piece (18), the elastic guide piece (18) is offset, and the buckle (15) moves from the position of the elastic guide piece (18) to the outside from the opening (12).

8. A detection method of a real-time fluorescence quantitative PCR instrument according to claims 1-7, characterized in that The main steps include: Open the entrance (3) of the housing (1), place the first half disk (41) into the housing (1) through the entrance (3), connect it to the clamping device (11), connect the second half disk (42) to the other end of the clamping device (11), so that the first half disk (41) and the second half disk (42) form a rotating disk (4); Placing a reagent kit (5) equipped with a reagent tube (6) to be tested at a mounting position on the rotating disk (4); Rotating the rotating disk (4) so ​​that the first half disk (41) or the second half disk (42) is rotated to the detection position in the housing (1) for detection, and undergoing the detection processes such as denaturation, annealing and extension; After the detection is completed, the rotating disk (4) is rotated to make the detected half disk one (41) or half disk two (42) rotate out from the entrance (3), and at the same time, the other half of the half disk two (42) or half disk one (41) is rotated in, and the above detection process is repeated; While the transferred in reagent tubes (6) to be tested are being tested, the transferred out reagent tubes (6) to be tested that have been tested can be left to stand or removed, and a new reagent kit (5) equipped with the reagent tubes (6) to be tested can be added to wait for being transferred to the testing position for the next test; After the detection is completed, the buckle (15) of the half disk one (41) and / or the half disk two (42) outside the housing (1) is removed from the opening (12), slid along the guide rail (17) and placed in the placement cavity (16), or directly taken out from the elastic guide sheet (18) and placed in the placement cavity (16).

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