A high-throughput isothermal amplification device

Abstract

The utility model discloses a high flux constant temperature amplification device belongs to constant temperature amplification device technical field, the utility model solves the problem of tedious operation and easy to be contaminated when carrying out a variety of virus parallel detection to the nucleic acid sample of same source in prior art. The utility model includes the substrate, the primer storage area of substrate includes several primer storage cavities, and the amplification primer is stored in primer storage cavity all; nucleic acid sample adding area includes several nucleic acid temporary storage cavities, and adjacent nucleic acid temporary storage cavity is communicated, and the even hole and second sample adding hole are arranged on the substrate, and every nucleic acid temporary storage cavity all communicates with a primer storage cavity, and the reaction area includes several reaction cavities, and every reaction cavity all communicates with a nucleic acid temporary storage cavity, and the colorless transparent structure that can observe the situation in reaction cavity is arranged on the substrate. The utility model realizes high flux constant temperature amplification, and effectively improves virus detection efficiency, is applicable to the scene such as entry -exit port quarantine that needs to realize virus detection quickly.

Description

Technical Field

[0001] This utility model belongs to the technical field of isothermal nucleic acid amplification devices, specifically relating to a high-throughput isothermal amplification device. Background Technology

[0002] Against the backdrop of my country's continued expansion of foreign agricultural trade, rapid cross-customs zone virus screening for exported crops, live animals, and their products is a core and crucial link for customs to strengthen the national biosecurity defense line, ensure the efficiency of export cargo clearance, and circumvent overseas quarantine technical barriers. In the current export quarantine scenario, a single batch of samples to be inspected often simultaneously covers multiple viruses that pose a risk of cross-border transmission. Traditional laboratory testing procedures are time-consuming and involve many steps, which can easily lead to fresh agricultural products and live animals being held in ports beyond their permitted time limit.

[0003] In the current customs-standardized viral nucleic acid testing system, while the traditional quantitative PCR method is supported by mature national standards and has stable accuracy, it requires multiple rounds of temperature-controlled amplification using a precision thermal cycler in the laboratory. A single batch of tests typically takes more than 1.5 hours, and the entire process must be completed in a certified molecular biology laboratory, making it unsuitable for direct deployment to frontline locations such as ports, animal quarantine facilities, and fresh agricultural product inspection points. Even with the expansion of target numbers through multiple fluorescence channels, issues such as primer dimer interference, non-specific amplification, and imbalances in amplification efficiency for different targets frequently arise due to differences in the annealing temperatures of different viral primers.

[0004] In recent years, isothermal nucleic acid amplification technology has become the mainstream technology for rapid on-site testing at customs due to its core advantages of not requiring sophisticated thermal cycling equipment, adapting to a wide temperature range, and having a fast amplification speed. Currently, several mature technical routes have been formed, including loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), multi-enzyme isothermal rapid amplification (MIRA), and nucleic acid sequence-dependent amplification (NASBA). Different technologies are adapted to different quarantine scenarios: LAMP can achieve efficient amplification of low-copy seed-borne viruses at a constant temperature of 65℃, suitable for screening for virus-carrying crops; NASBA can directly complete one-step amplification of RNA viruses at a constant temperature of 41℃, suitable for rapid detection of animal RNA viruses such as foot-and-mouth disease and African swine fever; MIRA can complete amplification in 5-30 minutes in a wide temperature range of 20-41℃, and can be deployed at outdoor inspection points without a professional laboratory environment. The entire process only requires a simple temperature control device or even vehicle-mounted power supply, completely breaking the dependence of traditional PCR on laboratory conditions.

[0005] However, the application of all mainstream isothermal amplification technologies in customs quarantine scenarios still faces a common technical bottleneck across the industry: most existing commercial testing products and publicly available research solutions follow a single-tube, single-target detection logic. To conduct parallel screening for multiple cross-border viruses on the same exported animal throat swab or crop leaf / seed nucleic acid sample, the only method is to manually pre-split multiple reaction solutions and repeatedly open and add samples, distributing the sample to dozens of independent reaction tubes for separate amplification. This method is not only cumbersome, but more importantly, the repeated opening and adding of samples easily leads to cross-contamination between different samples and nucleic acid aerosol contamination from the diffusion of amplification products, ultimately resulting in a significantly increased false-positive rate in on-site testing, directly affecting the validity of quarantine results. Utility Model Content

[0006] To address the problems of cumbersome operation and easy cross-contamination of samples and nucleic acid aerosol contamination caused by repeated opening and adding of samples in existing technologies for parallel detection of multiple viruses from the same source, this invention provides a high-throughput isothermal amplification device.

[0007] The technical solution adopted in this utility model is as follows:

[0008] A high-throughput isothermal amplification device includes a substrate, on which a primer storage area, a nucleic acid sample loading area, and a reaction area are sequentially disposed;

[0009] The primer storage area includes several primer storage cavities arranged in a row. Each primer storage cavity is not connected to the others, and each primer storage cavity stores amplification primers. A first sample application hole is provided on the side edge of the substrate near the primer storage area. The first sample application hole is connected to each primer storage cavity.

[0010] The nucleic acid loading area includes several nucleic acid storage cavities arranged in a row. Each pair of adjacent nucleic acid storage cavities is connected. A uniform hole and a second loading hole are respectively provided on two edges of the substrate adjacent to the first loading hole. The second loading hole is connected to the nucleic acid storage cavity closest to it. The uniform hole is connected to the nucleic acid storage cavity closest to it. Each nucleic acid storage cavity is connected to a primer storage cavity.

[0011] The reaction zone includes several reaction chambers arranged in a row, each of which is connected to a nucleic acid storage chamber. A colorless and transparent structure that allows observation of the contents of the reaction chamber is provided on the substrate at the position corresponding to each reaction chamber.

[0012] Both the first and second sample feeding holes are equipped with sealing structures, and the uniform feeding hole is equipped with an openable and closable structure.

[0013] This technical solution involves pre-storing primers for detecting various viruses in primer storage chambers. Each primer storage chamber is relatively independent, preventing primer confusion. During use, the substrate is first tilted so that the second sample well faces upwards. The nucleic acid sample of the target virus is added to the substrate through the second sample well. Under gravity, the nucleic acid sample gradually flows downwards from the first nucleic acid storage chamber until all nucleic acid storage chambers are filled. Then, the homogenization well and the second sample well are closed to create a sealed environment within the substrate, preventing the nucleic acid sample in each storage chamber from moving randomly. Next, the substrate is flipped so that the first sample well faces upwards, and the homogenization well is opened. Other auxiliary agents required for the amplification reaction (all auxiliary agents are pre-mixed before addition), such as RNA transcriptase, are added to each primer storage chamber through the first sample well. The incoming auxiliary agents are mixed with the lyophilized primer powder in each primer storage chamber to obtain a mixed system. Under the influence of gravity, the mixed system continues to flow downwards into each nucleic acid storage chamber and then into each reaction chamber. The nucleic acid samples in each nucleic acid storage chamber also flow into each reaction chamber under the influence of gravity. In this way, the construction of multiple virus detection amplification systems is completed simultaneously. In order to improve the uniformity of the mixing of various raw materials, the system can be thoroughly mixed by shaking the substrate. Then, the reaction temperature is provided to achieve isothermal nucleic acid amplification. With the colorless and transparent structure and a handheld fluorescent flashlight, the fluorescence signal can be directly observed with the naked eye without professional operation. High-throughput isothermal amplification is achieved, which effectively improves the efficiency of virus detection and is suitable for scenarios such as entry and exit port quarantine that require rapid virus detection.

[0014] Preferably, the heating chamber is also included and is adapted to the substrate. The heating chamber is provided with a heating groove. At least the reaction zone on the substrate can enter the heating groove. The shape and size of the heating chamber are such that the inner wall of the heating chamber is in contact with the substrate. The heating groove is provided with a heating mechanism. The upper side of the heating groove on the heating chamber is a brown glass plate, and the other sides are opaque.

[0015] With this technical solution, in extreme cases where the preferred amplification temperature for the primers in the corresponding isothermal amplification technology is around 37°C, the body temperature can be used to provide the required reaction temperature (e.g., MIRA technology), achieving good detection results. To accommodate situations where the preferred amplification temperature differs significantly from 37°C, this invention further provides a heating chamber adapted to the substrate size. This chamber allows at least the entire reaction area of ​​the substrate to be placed within the heating chamber, and the heating mechanism within the chamber ensures the reaction system reaches the preferred amplification temperature, guaranteeing isothermal amplification effectiveness.

[0016] Preferably, the heating chamber is fixedly mounted on the base, the base is provided with a sliding mechanism, the sliding mechanism is provided with a flipping mechanism, the flipping mechanism is provided with a support plate for supporting the substrate, and the support plate and / or the substrate is provided with a limiting mechanism for restricting the substrate from shifting during the flipping process.

[0017] After adopting this technical solution, the substrate on the support plate can be inserted into the heating tank by the sliding mechanism on the base. The state of the support plate can be adjusted by the flipping mechanism, so that the first sample feeding hole or the second sample feeding hole on the substrate on the support plate can face upwards, making it easier for the operator to complete the sample feeding.

[0018] Preferably, the flipping mechanism includes four damping buffer telescopic rods arranged in pairs in a row. One end of each damping buffer telescopic rod is fixedly connected to the sliding mechanism, and the other end is hinged to the support plate ball.

[0019] After adopting this technical solution, the operator can manually drive the damping buffer telescopic rod to extend and retract, so that the adjacent support plate is in the required state. Since the damping buffer telescopic rod itself has a large damping and the support plate and base plate are lightweight, it is sufficient to support the base plate and maintain this shape after adjustment.

[0020] Preferably, the sliding mechanism includes a groove disposed on the base plate, the groove extending from the distance from the heating chamber to the distance from the heating chamber, and a slider slidably disposed in the groove, the slider being connected to the flipping mechanism.

[0021] With this technical solution, the substrate can be inserted into the heating tank to complete the amplification by sliding the slider in the groove.

[0022] Preferably, the substrate has a protrusion protruding from the edge of the substrate at both the uniform hole and the second sample feeding hole, and the support plate has two limiting grooves that respectively cooperate with the two protrusions.

[0023] With this technical solution, when the substrate is placed on the support plate from top to bottom, the protrusion engages in the limiting groove to achieve positioning with the substrate.

[0024] Preferably, the sealing structure consists of a first rubber plug and a second rubber plug fixedly disposed in the first sample feeding hole and the second sample feeding hole, respectively, and the first rubber plug and the second rubber plug are puncture-resistant self-sealing structures.

[0025] The uniform pore includes a first pore and a second pore that are interconnected. The diameter of the first pore is larger than the diameter of the second pore. The first pore is connected to the outside, and the second pore is connected to the nucleic acid storage chamber. The openable structure includes a third rubber soft plug disposed in the first pore. The third rubber soft plug is provided with a third pore. The third rubber soft plug can be rotated within the first pore until the third pore is aligned with or staggered with the first pore.

[0026] Preferably, the first sample loading well is connected to several non-interconnected first channels, and each of the first channels is connected to a primer storage cavity.

[0027] Each pair of adjacent nucleic acid temporary storage chambers is connected by a second channel;

[0028] Each of the aforementioned nucleic acid temporary storage chambers is connected to a primer storage chamber via a third channel disposed on the substrate;

[0029] Each of the reaction chambers is connected to a nucleic acid storage chamber via a fourth channel disposed on the substrate.

[0030] Preferably, the diameters of the first channel, the second channel, the third channel, and the fourth channel are all in the micrometer range.

[0031] Preferably, the size and shape of each primer storage chamber are completely consistent, the size and shape of each nucleic acid temporary storage chamber are completely consistent, and the size and shape of each reaction chamber are completely consistent.

[0032] Preferably, there are six primer storage chambers, six nucleic acid temporary storage chambers, and six reaction chambers, which together form a rectangular array of six rows and three columns.

[0033] Preferably, each of the fourth channels is tilted, and when the second sample port is facing upward, the position where the third channel is connected to the primer storage chamber is higher than the position where the third channel is connected to the nucleic acid temporary storage chamber; each of the fifth channels is tilted, and when the second sample port is facing upward, the position where the fourth channel is connected to the reaction chamber is higher than the position where the fourth channel is connected to the nucleic acid temporary storage chamber.

[0034] In summary, due to the adoption of the above technical solution, the beneficial effects of this utility model are:

[0035] This invention pre-stores primers for detecting various viruses in primer storage chambers, with each chamber being relatively independent to avoid primer confusion. Multiple virus detection amplification systems can be constructed simultaneously with only two sample additions, achieving high-throughput isothermal amplification and effectively improving virus detection efficiency. It is suitable for scenarios requiring rapid virus detection, such as border crossing quarantine. Attached Figure Description

[0036] This utility model will be described by way of example and with reference to the accompanying drawings, wherein:

[0037] Figure 1 This is a schematic diagram of the external structure of the substrate in Example 1;

[0038] Figure 2 This is a schematic diagram of the internal structure of the substrate in Example 1;

[0039] Figure 3 This is a schematic diagram of the external structure of the substrate in Example 2;

[0040] Figure 4 This is a schematic diagram of the heating chamber structure in Example 3 when the temperature is controlled by zones;

[0041] Figure 5 This is a schematic diagram of the substrate structure in Example 3 when the temperature is controlled in zones;

[0042] Figure 6 This is a schematic diagram of the structure when the substrate is placed horizontally in Example 4;

[0043] Figure 7 This is a schematic diagram of the structure when the first sample feeding hole is facing upwards in Example 4;

[0044] Figure 8 This is a schematic diagram of the structure when the second sample feeding hole is facing upwards in Example 4;

[0045] Figure 9 This is a schematic diagram of the substrate structure in Example 6.

[0046] Figure label:

[0047] Wherein: 1-substrate, 2-first sample loading hole, 3-uniform hole, 4-second sample loading hole, 5-reaction chamber, 6-first channel, 7-primer storage chamber, 8-third channel, 9-nucleic acid temporary storage chamber, 10-fourth channel, 11-second channel, 12-support plate, 13-flipping mechanism, 14-base, 15-slider, 16-heating tank, 17-heating chamber, 18-brown glass plate, 19-slide groove, 20-limiting groove, 21-heat insulation silicone strip, 22-slot, 23-cover plate, 24-card slot, 25-pressure relief port, 26-transparent soft silicone baffle. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. The components of the embodiments of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely represents selected embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0049] In the description of the embodiments of this application, it should be noted that the terms "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship commonly used when the utility model product is in use. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application. In addition, the terms "first," "second," and "third," etc., are only used to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0050] The following is combined Figures 1-8 This utility model will be described in detail.

[0051] It should be noted that this device is adaptable to all scenarios and is compatible with the reaction systems and detection processes of various mainstream isothermal nucleic acid amplification technologies, such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and multi-enzyme isothermal rapid amplification (MIRA). In order to clearly present the core functions and operating logic of the device, the specific embodiments below will only use multi-enzyme isothermal rapid amplification (MIRA) as a typical application scenario for explanation, and will not limit the technical coverage of this device. The amplification system targeted by this device is the same as that of the existing RNA isothermal rapid amplification kit (such as the Amp Future RNA Isothermal Rapid Amplification Kit (Fluorescent)-II), and its total reaction volume is 50 μL. Therefore, the volume of each reaction chamber 5, nucleic acid temporary storage chamber 9, and primer storage chamber 7 in this device is set to be greater than 50 μL. The amount of each raw material in the amplification system is allowed to have a certain deviation. Specifically, the deviation of the amount of core components such as RNA sample and upstream and downstream primers in the 50 μL system is controlled within ±0.5 μL, and the deviation of other auxiliary agents is relaxed to ±2 μL to avoid affecting the amplification efficiency due to excessive deviation of the system volume.

[0052] Example 1

[0053] like Figure 1 and Figure 2 As shown, a high-throughput isothermal amplification device is described. The substrate 1 is integrally injection molded from scratch-resistant rigid transparent PC plastic with a thickness of 2mm. The substrate 1 has a length and width of 100mm × 60mm. Three functional areas are arranged sequentially along the length of the substrate 1: a primer storage area, a nucleic acid loading area, and a reaction area. The primer storage area includes five primer storage cavities 7 arranged in a row. The nucleic acid loading area includes five nucleic acid temporary storage cavities 9 arranged in a row. The reaction area includes several reaction cavities 5 arranged in a row. The five primer storage cavities 7, the five nucleic acid temporary storage cavities 9, and the five reaction cavities 5 together form a rectangular array of five rows and three columns. The rectangular array is centrally located within the effective working area of ​​the substrate 1.

[0054] like Figure 2 As shown, five primer storage chambers 7 are arranged equidistantly along the width of the substrate 1, with a center-to-center distance of 8 mm between adjacent chambers 7. Each primer storage chamber 7 has a uniform volume of 50 μL. All primer storage chambers 7 are completely isolated from each other, and each chamber 7 is pre-encapsulated with MIRA-specific amplification primers (lyophilized powder) targeting different viruses. In this embodiment, the five primer storage chambers 7 respectively correspond to Tomato Brown Curly Virus (preferred amplification temperature is 37°C), Tomato Spotted Wilt Virus (preferred amplification temperature is 38°C), and Citrus Huanglongbing Fungicide (preferred amplification temperature is 37°C). MIRA-specific amplification primers for five quarantine plant viruses that are prevalent in agricultural production: Tomato mosaic virus (preferred amplification temperature is 36.5℃), Cucumber green mottle mosaic virus (preferred amplification temperature is 37.5℃), and Tomato mosaic virus (preferred amplification temperature is 36.5℃). (Primer lyophilized powder is a conventional commercial product in this field and can be ordered directly from biotechnology companies such as Mike Biotechnology and Qitian Biotechnology.) The amount of primer in each primer storage chamber 7 is set according to the amount required to detect the virus, which can directly match the primer concentration requirements of the subsequent amplification system. The independent chamber structure completely avoids cross-contamination between different primers.

[0055] The substrate 1 has a first sample loading hole 2 with a diameter of 3 mm at the top edge near the primer storage area. The bottom of the first sample loading hole 2 is connected to 5 completely unconnected first channels 6. The end of each first channel 6 is connected to the inlet of a primer storage cavity 7. The inner diameter of all first channels 6 is within the micron-level microfluidic channel range, allowing liquid to flow in a directional manner only under gravity, and preventing reagent cross-flow between different primer storage cavities 7.

[0056] To ensure that the amplification aid injected through the first sample loading well 2 is distributed as evenly as possible into each primer storage chamber 7 (the principle is the same as that of microfluidic technology), the volumetric flow rate of each first channel 6 must be similar. However, the distances from the primer storage chamber 7 to the first sample loading well 2 are unequal. Therefore, to ensure that the volumetric flow rate of each first channel 6 is similar, the diameter and length of each first channel 6 must satisfy the following formula:

[0057] Under viscosity-dominated laminar flow conditions (following the Hagen-Poiseuille law), the volumetric flow rate... Q With the inner diameter of the first channel D It is proportional to the fourth power and is related to the channel length. L Inversely proportional, that is, Therefore, the diameter of each first channel 6 must satisfy:

[0058] .

[0059] like Figure 3 As shown, five nucleic acid storage chambers 9 are arranged equidistantly along the width of the substrate 1, with a center-to-center distance of 8 mm between adjacent nucleic acid storage chambers 9. Each nucleic acid storage chamber 9 has a volume of 5 μL (matching the 5 μL sample loading volume of nucleic acid). Each pair of adjacent nucleic acid storage chambers 9 is connected by a second channel 11 with an inner diameter of 70 μm. On the right and left edges of the substrate 1 (adjacent to the top edge where the first loading well 2 is located), there are uniform holes 3 with a diameter of 3 mm and second loading wells 4 with a diameter of 3 mm, respectively. The second loading well 4 is directly connected to the nearest first nucleic acid storage chamber 9, and the uniform hole 3 is directly connected to the nearest last nucleic acid storage chamber 9. Each nucleic acid storage chamber 9 is individually connected to a corresponding primer storage chamber 7 through a third channel 8 with an inner diameter of 70 μm. The five third channels 8 are completely non-intersecting, realizing a one-to-one correspondence between the primer storage chamber 7 and the nucleic acid storage chamber 9.

[0060] like Figure 3 As shown, five reaction chambers 5 are arranged equidistantly along the width of the substrate 1, with a center-to-center distance of 8 mm between two adjacent reaction chambers 5. Each reaction chamber 5 has a volume of 200 μL and is connected to a corresponding nucleic acid storage chamber 9 via a fourth channel 10 with an inner diameter of 70 μm. The area on the substrate 1 corresponding to each of the five reaction chambers 5 is integrally formed using a colorless, transparent, high-transmittance PMMA plate, forming an observation window that can be directly viewed or used with a handheld fluorescent flashlight to collect signals. The fluorescence color development inside the reaction chamber 5 can be directly observed without opening the cover, which is suitable for observation needs under strong field light.

[0061] It should be noted that each of the first channel 6, the second channel 11, the third channel 8, and the fourth channel 10 is a vertical channel, and its length is as short as possible to reduce the amount of amplification reagent remaining in it, thereby reducing the deviation in the amount of amplification reagent used. The amount of each amplification reagent added can also take into account the amount lost in this part, and the amount used is increased accordingly to make the amount of each component in the amplification system relatively accurate.

[0062] The first sample loading port 2 is internally fixed with a 3mm diameter first rubber soft stopper, and the second sample loading port 4 is internally fixed with a 3mm diameter second rubber soft stopper. Both the first and second rubber soft stoppers have a cross-shaped through-hole injection port in their central areas. The edges of the cross-shaped cut of the injection port remain tightly fitted in their natural state, forming an interference seal structure based on the elasticity of the rubber itself. This allows for a long-term maintenance of a sealed environment inside the tube without external puncture, effectively preventing the intrusion of external aerosols and leakage of nucleic acid samples from the tube. Figure 2 As shown, there is a relatively large buffer space at the connection between the first sample loading hole 2 and each of the first channels 6. The first rubber soft stopper is only set at the end of the first sample loading hole 2, occupying less than 10% of the volume of the first sample loading hole 2. The auxiliary agent injected from the first sample loading hole 2 is temporarily stored in the buffer space, so that the auxiliary agent is roughly evenly distributed into each primer storage cavity 7 under the action of gravity and capillary action of the first channel 6.

[0063] The size of the first sample loading well 2 matches the outer diameter of the pipette tip (the outer diameter of the tip of a 1000μL pipette is approximately 1.2-2.0mm), allowing the pipette tip to be inserted into the injection port at the first sample loading well 2 for sample loading. Both rubber stoppers are made of low-temperature resistant medical-grade butyl rubber, maintaining good sealing performance in field environments of 0~40℃. They automatically spring back to seal after puncture, preventing chloroplast impurities and field dust from entering the cavity and causing contamination. The uniform pore 3 consists of two coaxially connected sections: the upper section is a first pore with a diameter of 3mm, and the lower section is a second pore with a diameter of 1mm. The first pore is directly connected to the outside atmosphere, and the second pore is connected to the nearest last nucleic acid storage chamber 9. The closable structure of the uniform pore 3 is a third rubber soft plug embedded in the first pore. A limiting structure is required in the first pore to prevent the third rubber soft plug from falling out, but the third rubber soft plug can rotate. For example, an annular baffle is set at the edge of the first pore, and the outer diameter of the third rubber soft plug is smaller than the inner diameter of the annular baffle, thereby preventing the third rubber soft plug from falling out. The third rubber soft stopper detaches; the third rubber soft stopper is a weather-resistant silicone rotary stopper. A 1mm diameter third hole is formed on the side wall of the third rubber soft stopper. Operators can rotate the third rubber soft stopper (a rod of similar size to the second hole can be inserted into the second hole and rotated, thus rotating the third rubber soft stopper) to align the third hole with the second hole in the lower section, achieving air pressure communication between the inside and outside of the cavity. Alternatively, the third hole can be rotated until it completely overlaps with the second hole, achieving complete sealing of the entire nucleic acid sample loading area and preventing accidental reagent leakage in field wind conditions. To ensure precise alignment between the third and second holes, marking points can be set around the first hole on the substrate 1. When the third rubber soft stopper is rotated to align with the marking points, the third hole and second hole are completely aligned. The rotatable third rubber soft stopper allows for the opening and closing of the uniform hole 3, which, compared to a removable plug, is less prone to contamination caused by placing the plug in other locations after removal.

[0064] It should be noted that this device is for single use only. Before use, it should be packaged in a sterile sealed bag. To maintain the effectiveness of the primers, the entire device needs to be stored in a low-temperature, light-protected environment at -20℃±5℃ for long-term storage, which can ensure the stable activity of the pre-loaded lyophilized primer powder for more than 12 months. For short-term temporary storage, it can be stored in a normal refrigerated environment at 2~8℃, with a shelf life of about 3 months, which is fully compatible with the conventional refrigerator storage conditions of grassroots plant protection stations. The storage environment should be kept dry, with the relative humidity controlled at 30%~60%. At the same time, it should be kept away from direct sunlight and strong acid and alkali corrosion environments to prevent the PC / PMMA substrate 1 from aging and cracking, which would affect the sealing performance.

[0065] Example 2

[0066] like Figure 3As shown, the main structure of this embodiment is completely identical to that of Embodiment 1, with the only difference being that the areas on the substrate 1 corresponding to the five nucleic acid storage chambers 9 and the areas corresponding to the five primer storage chambers 7 are all integrally molded from colorless and transparent PMMA material, forming a full-area visualization window. After completing reagent loading, operators can directly visually observe whether the auxiliary agent in each primer storage chamber 7 is completely dissolved and whether the crude nucleic acid sample extracted from plant leaves in each nucleic acid storage chamber 9 is completely and evenly distributed, promptly checking for abnormalities such as sample fiber residue and reagent unevenness, and avoiding amplification failure caused by impurities in the plant sample.

[0067] Example 3

[0068] The main structure of this embodiment is completely the same as that of Embodiment 1 or Embodiment 2, with the addition of a temperature control component adapted to the substrate 1:

[0069] The component includes a lightweight aluminum heating chamber 17, weighing only approximately 280-300g. It can be powered by a standard power bank. Inside the heating chamber 17 is a heating groove 16 that perfectly matches the shape of the substrate 1. The internal dimensions of the heating groove 16 match the dimensions of the substrate 1, allowing the reaction zone of the substrate 1 to be completely embedded within the heating groove 16. After embedding, the inner wall of the heating groove 16 is completely flush with the upper and lower surfaces of the substrate 1, achieving seamless heat conduction. For MIRA amplification scenarios at approximately 37°C, this device does not require activating the heating mechanism. Operators can place the sealed substrate 1 close to their body in a clothing pocket, relying on body heat to provide a stable 37°C incubation environment for the reaction system. Amplification can be completed in 15-20 minutes, making it perfectly suitable for remote farmland scenarios without any power supply.

[0070] When the preferred amplification temperature of the target differs significantly from 37°C, the low-power PTC ceramic heating element embedded at the bottom of the heating bath 16 is activated, which can rapidly raise the temperature inside the heating bath 16 to any set temperature within the range of 30°C to 45°C within 2 minutes. The temperature control accuracy is stable at ±0.5°C, which perfectly matches the optimal reaction temperature for MIRA amplification of different plant viruses. No complex thermal cycling procedure is required, and the total power consumption of a single detection does not exceed 5Wh.

[0071] The top opening of the heating chamber 17 is sealed with a 3mm thick anti-reflective brown glass plate 18, which can completely block direct sunlight interference in the field environment and prevent the fluorescence signal during the amplification process from being obscured by strong background light. In this embodiment, the top of the heating chamber 17 is provided with an observation window that communicates with the heating tank 16, and the side of the heating chamber 17 is provided with a connecting groove that communicates with the observation window. The side wall of the observation window is provided with an insertion groove that matches the connecting groove. The brown glass plate 18 can be inserted into the insertion groove through the connecting groove and cover the observation window, thereby realizing the detachable connection between the brown glass plate 18 and the heating chamber 17. When interpreting the results, the brown glass plate 18 can be removed to observe the amplification results. Except for the top brown glass plate 18, the other five outer surfaces of the heating chamber 17 are all made of aluminum alloy to form an opaque structure, further isolating external stray light. With the help of a regular handheld fluorescent flashlight, the results can be directly interpreted by the naked eye under strong midday light without the need for professional fluorescence detection instruments.

[0072] In another embodiment, such as Figure 7 and Figure 8 As shown, the heating tank 16 is divided into five relatively independent heating chambers by heat-insulating silicone strips 21. The reaction area on the substrate 1 also has slots 22 that fit the heat-insulating silicone strips 21, allowing each reaction chamber to be inserted into one of the five heating chambers. The original single PTC heating element in the heating tank 16 is replaced with five sets of independent micro-film heating elements. One micro-film heating element is placed in each heating chamber, and the size of each set of heating elements perfectly matches the size of a single reaction chamber, correspondingly attaching to the bottom of each of the five reaction chambers. Each set of micro-heating elements is equipped with an NTC thermistor with an accuracy of ±0.1℃. The five sets of temperature measurement signals are connected to independent micro-PID temperature control chips, allowing for individual setting of the target temperature for each heating chamber, achieving independent temperature control within the range of 35℃ to 42℃, fully covering the optimal amplification temperature for each of the five targets. This zoned temperature control does not require altering the original heating chamber's dimensions and remains compatible with existing power supplies. The heating power of a single chamber is only 0.3W, and the total power consumption does not exceed 2W, without significantly increasing the battery life of portable devices. This approach can simultaneously verify amplification efficiency at different temperatures on a single substrate, and can also set exclusive amplification parameters for some temperature-sensitive mutant strains, further expanding the adaptability of the device.

[0073] Example 4

[0074] The main structure of this embodiment is completely the same as that of embodiment 3, with the addition of a sliding mechanism and a flipping component:

[0075] like Figure 6-8As shown, the lightweight base 14, made of ABS material, weighs only 1-1.2 kg. The heating chamber 17 is fixedly installed on the rear side of the base 14 by bolts. The front side of the base 14 is provided with a sliding mechanism extending in the front-back direction. The sliding mechanism is equipped with a flipping mechanism 13 that can move in the front-back direction. An ABS material support plate 12 for supporting the substrate 1 is fixedly installed on the top of the flipping mechanism 13. The surface of the support plate 12 is provided with a limiting structure that adapts to the shape of the substrate 1, which can limit the horizontal displacement of the substrate 1 during the flipping process.

[0076] The flipping mechanism 13 in this embodiment adopts a four-group damping buffer telescopic rod structure (its model can be GC-05-15, made of Q235 / Q355 high-strength steel, rated damping force: 8N, the maximum load of a single rod can reach 1.2kg, and the total load of the four parallel rods can cover the total weight of the support plate 12 + base plate 1 (the total weight does not exceed 0.3kg), with sufficient load margin): the four groups of damping buffer telescopic rods are arranged in pairs along the left and right directions, the bottom of each damping buffer telescopic rod is fixedly connected to the top of the slider 15 of the sliding mechanism, and the top is ball-jointed to the bottom of the support plate 12 through a universal ball joint. Relying on the different extension lengths of the four groups of telescopic rods, the support plate 12 can be tilted at any angle within the range of 0°~120°. By adjusting the extension stroke of different telescopic rods, for example, the support plate 12 can be precisely adjusted to two fixed states: the first state supports the support plate 12 in a fixed position. The side of plate 12 with the second sample well 4 is tilted upwards at approximately 60°, so that the second sample well 4 of the substrate 1 faces upwards, facilitating the operator to inject nucleic acid samples into the nucleic acid temporary storage chamber 9; in the second state, the side of the support plate 12 with the first sample well 2 is tilted upwards at approximately 60°, so that the first sample well 2 of the substrate 1 faces upwards, facilitating the operator to inject MIRA amplification aids into the primer storage chamber 7 (the relevant MIRA amplification aids are all commercially available products in this field and can be directly purchased. MIRA-related reagents can be obtained from suppliers such as AMP Future. MIRA amplification aids can be: 1. Core enzyme system: recombinase UvsX, single-strand binding protein SSB, strand displacement DNA polymerase Bsu, some RNA targets require the additional addition of MMLV reverse transcriptase, adapted to the nucleic acid type of plant viruses. 2. Reaction buffer system: containing 20mM A mixture of Tris-HCl, 100mM KAc, 8mM MgAc2, and dNTPs provides the necessary ionic environment and substrate for amplification. 3. Fluorescence detection components: SYBR Green I fluorescent dye or specific probes are added, allowing for direct visual observation of the fluorescence signal using a handheld fluorescent flashlight. 3. Auxiliary stabilizers: Trehalose, glycerol, and other lyophilization protectants can be combined with primers to form a lyophilized powder suitable for the long-term low-temperature storage requirements of the device. The entire process eliminates the need to manually lift the substrate 1 to adjust the angle, preventing sample spillage and contamination due to hand tremors during field operations.

[0077] As an alternative implementation of the equivalent replacement, the flipping mechanism 13 can also adopt a structure of four sets of low-power stepper electric telescopic rods (the model can be a miniature DC electric push rod YNT-02 with a rated thrust of 50N. Four rods connected in parallel can easily bear the total load of the support plate 12 + substrate 1 with sufficient load margin). The bottom of each electric telescopic rod is fixedly connected to the top of the slider 15 of the sliding mechanism, and the top is ball-jointed to the bottom of the support plate 12 through a universal ball joint. The extension and retraction stroke of the four sets of telescopic rods can be automatically controlled by a preset program, and the two sample loading postures can be switched with one button to realize the fully automated operation without the need for manual adjustment of the tilt angle.

[0078] The sliding mechanism includes a groove 19 formed on the upper surface of the base 14. The groove 19 extends in the front direction from the area away from the heating chamber 17 to the area near the rear of the heating chamber 17. A steel slider 15 that can slide freely along the groove is embedded inside the groove 19. The top of the slider 15 is fixedly connected to the bottom of the flipping mechanism 13 by bolts. After all sample addition and reagent mixing operations are completed, the slider 15 is pushed backward along the groove 19, which can drive the substrate 1 on the support plate 12 to be smoothly inserted into the heating groove 16 of the heating chamber 17. There is no need to manually touch the substrate throughout the process, avoiding the influence of hand temperature on the initial temperature of the reaction system and ensuring the stability of the amplification results. It should be noted that in this embodiment, the lowest position of the flipping mechanism 13 can be set to be flush with the heating groove 16, so that the substrate 1 can be inserted into the heating groove 16 when the flipping mechanism 13 is adjusted to the lowest position.

[0079] On the substrate 1, at the edge of the uniform hole 3 and the second sample feeding hole 4, there are two protrusions that extend outward by 1 cm. On the surface of the support plate 12, corresponding to the positions of the two protrusions, there are two limiting grooves 20 with perfectly matched sizes. When the substrate 1 is placed on the support plate 12, the two protrusions can be completely inserted into the corresponding limiting grooves 20, which completely prevents the substrate 1 from shifting and falling off during outdoor bumps, flips, and sliding.

[0080] The complete operating procedure of this device is fully adapted to the scenario of virus detection in crop fields:

[0081] Sample loading steps: The sliding mechanism moves the support plate 12, causing the substrate 1 to slide to a forward operating position away from the heating chamber 17. The flipping mechanism is then used to adjust the support plate 12 to a 60° tilt, so that the second sample loading hole 4 faces upwards. The third rubber stopper of the uniform hole 3 is rotated to align the third hole with the second hole, connecting the internal and external air pressure. A pipette is inserted into the injection port of the second rubber stopper (a gap exists between the pipette and the second rubber stopper, which also serves to release pressure), allowing the nucleus extracted from field crop leaves to be added to the substrate through the second sample loading hole 4. The acid sample (a nucleic acid sample that has been processed and can be directly amplified) flows down the first nucleic acid storage chamber 9 closest to the second sample well 4 under the action of gravity, along the connected second channel 11, until all 5 nucleic acid storage chambers 9 are filled, completing the entire sampling. When it is observed that the nucleic acid storage chamber 9 directly connected to the uniform well 3 is nearly full, the third rubber soft stopper is rotated to completely seal the uniform well 3. At this time, a sealed space is formed inside the substrate, and the nucleic acid samples in each nucleic acid storage chamber 9 will not flow or shift randomly under the capillary resistance of the microchannel.

[0082] Reagent mixing steps: Adjust the tilt of the support plate from 12° to 60° using the flipping mechanism, so that the first sample loading port 2 of the substrate 1 is in a higher position; then open the uniformity hole 3 to maintain pressure balance between the inside and outside of the substrate 1. Add the premixed isothermal amplification aid system through the first sample loading port 2. Specifically, insert a pipette into the injection port of the first rubber soft stopper (there is a gap between the pipette and the first rubber soft stopper, which also serves to relieve pressure), thus adding the premixed MIRA amplification aid into the substrate 1 through the first sample loading port 2. Under the same flow resistance and capillary action conditions, the M... IRA amplification aids are approximately evenly distributed into five primer storage chambers 7 through five first channels 6 with equal flow rates, and mixed with lyophilized plant virus primers pre-stored in each primer storage chamber 7. Simultaneously, nucleic acid samples in each nucleic acid temporary storage chamber 9 flow into the reaction chamber 5 under gravity. Once the aids have completely entered each primer storage chamber 7, the uniform distribution hole 3 is closed, the substrate 1 is removed, and the substrate 1 is placed horizontally. The substrate 1 is then gently shaken along its width to mix the primers and aids, obtaining a pre-reaction system. The substrate 1 is then placed back onto the support plate 12, keeping the first sample loading hole 2 facing upwards.

[0083] Under the influence of gravity, the pre-reaction system flows downward through the third channel 8 into the corresponding nucleic acid temporary storage chamber 9, and continues to move downward into the corresponding reaction chambers 5. Then, the uniformity hole 3 is closed, the substrate 1 is removed, and the substrate 1 is shaken for about 3 to 5 seconds to further improve the mixing uniformity (when shaking, make sure that the first sample loading hole 2 is facing upward). Then, it is placed back on the support plate 12 (when experimental conditions are available, a portable oscillation mixing device (such as a multi-functional vortex mixer) can also be used to assist in mixing the substrate 1 after sample loading to further improve the mixing effect).

[0084] Amplification and interpretation steps: Push the slider 15 backward along the slide groove 19 to smoothly insert the substrate 1 into the heating chamber 16 of the heating chamber 17. It should be noted that if the preferred amplification temperature of the target is 37℃, there is no need to activate the heating mechanism of the heating chamber. Simply remove the substrate 1 and place it close to your body in a pocket. Incubation for 15 minutes using body temperature will complete the amplification. If the preferred amplification temperature of the target deviates significantly from 37℃, activate the heating mechanism of the heating chamber 17 to adjust the temperature to the corresponding optimal value. Incubation at a constant temperature for 15 minutes will complete the MIRA isothermal amplification of all 5 crop viruses. Remove the substrate 1, remove the brown glass plate 18, and use a regular handheld blue fluorescent flashlight to illuminate the reaction area. The fluorescence signal of each reaction chamber can be directly observed with the naked eye. The detection results of the 5 plant viruses can be interpreted within 10 seconds. No professional PCR equipment is required throughout the process. The operation can be completed even in remote mountainous farmlands without external power, fully meeting the actual needs of crop origin quarantine and rapid screening of virus-free seedlings.

[0085] Example 5

[0086] This embodiment is basically the same as embodiment 4, except that: five relatively independent injection holes are set at the first sample loading hole 2, each injection hole is connected to a first channel 6, and a second rubber soft stopper is set at each injection hole. Each second rubber soft stopper has a cross-shaped cut. The premixed MIRA amplification agent can be injected into the first channel 6 through the second rubber soft stopper by a pipette, thereby realizing the function of injecting MIRA amplification agent into each primer storage cavity 7 separately, which can further ensure the accurate dosage of each component in the amplification system.

[0087] Example 6

[0088] This embodiment is basically the same as embodiment 4, except that: Figure 9As shown, a pressure relief port 25 communicating with the outside is provided on the high-transmittance PMMA plate corresponding to each reaction chamber 5 on the substrate 1. The pressure relief port 25 is located on the side of the reaction chamber 5 near the nucleic acid temporary storage chamber 9. A transparent soft silicone baffle 26 is provided at each pressure relief port 25. The upper surface of the transparent soft silicone baffle 26 is flush with the surface of the substrate 1. A cross-shaped cut is provided on each transparent soft silicone baffle 26. A cover plate 23 (transmittance PMMA material) is slidably connected to the substrate 1. A pressure relief port 25 is provided at each end of the cover plate 23. Each substrate 1 has a locking block and a locking slot 24 on each side. The locking block and the locking slot 24 cooperate to slide the cover plate 23 and prevent the cover plate 23 from detaching from the substrate 1. The friction between the locking block and the locking slot 24 is greater than the weight of the cover plate 23, so that the cover plate 23 cannot move freely on the substrate 1 due to its own weight. The bottom surface of the cover plate 23 abuts against the upper surface of the transparent soft silicone baffle 26. When the cover plate 23 is closed, the cross-shaped cut on the transparent soft silicone baffle 26 cannot be opened outward, thus keeping the substrate 1 in a sealed state.

[0089] To ensure the smooth entry of liquid from each channel into the reaction chamber 5, when the cover plate 23 is not closed on the transparent soft silicone baffle 26, the tip of a syringe bulb (30ml small syringe bulb or 60ml medium syringe bulb with an outer diameter of approximately 2.2-2.5mm, matching the size of the first sample loading port 2) can be inserted through the first sample loading port 2. This injects air into the substrate 1, driving the liquid to flow into the reaction chamber 5, ensuring the smooth entry of liquid into each reaction chamber 5. During this process, pressure is released at the transparent soft silicone baffle 26. Since the volume of the reaction chamber 5 (200μL) is much larger than the total volume of the reaction system (approximately 50μL), the liquid level in the reaction chamber 5 cannot reach the pressure relief port 25 when the first sample loading port 2 is tilted upwards, thus preventing leakage. After all the liquid has entered the reaction chamber 5, the cover plate 23 is slid down and closed on the transparent soft silicone baffle 26, thereby sealing the pressure relief port. Then, the substrate 1 is adjusted to be horizontal and inserted into the heating chamber 17 for subsequent operations.

[0090] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. A high-throughput isothermal amplification device, characterized in that: Includes a substrate (1), on which a primer storage area, a nucleic acid sample loading area, and a reaction area are sequentially disposed; The primer storage area includes several primer storage cavities (7) arranged in a row. Each primer storage cavity (7) is not connected to the others, and each primer storage cavity (7) stores amplification primers. A first sample loading hole (2) is provided on one side edge of the substrate (1) near the primer storage area. The first sample loading hole (2) is connected to each primer storage cavity (7). The nucleic acid loading area includes several nucleic acid storage chambers (9) arranged in a row. Each pair of adjacent nucleic acid storage chambers (9) are connected. On the substrate (1), uniform holes (3) and second loading holes (4) are respectively provided at two edges adjacent to the first loading hole (2). The second loading hole (4) is connected to the nearest nucleic acid storage chamber (9). The uniform hole (3) is connected to the nearest nucleic acid storage chamber (9). Each nucleic acid storage chamber (9) is connected to a primer storage chamber (7). The reaction zone includes several reaction chambers (5) arranged in a row. Each reaction chamber (5) is connected to a nucleic acid storage chamber (9). A colorless and transparent structure is provided on the substrate (1) at the position corresponding to each reaction chamber (5) so that the situation inside the reaction chamber (5) can be observed. The first sample feeding hole (2) and the second sample feeding hole (4) are both provided with a sealing structure, and the uniform hole (3) is provided with an openable and closable structure.

2. The high-throughput isothermal amplification device according to claim 1, characterized in that: It also includes a heating chamber (17) adapted to the substrate (1), wherein a heating groove (16) is provided in the heating chamber (17), at least the reaction zone on the substrate (1) can enter the heating groove (16), and the shape and size of the heating chamber (17) make the inner wall of the heating chamber (17) contact the substrate (1), a heating mechanism is provided in the heating groove (16), the upper side of the heating groove (16) on the heating chamber (17) is a brown glass plate (18), and the other sides are opaque structures.

3. The high-throughput isothermal amplification device according to claim 2, characterized in that: Includes a base (14), the heating chamber (17) is fixedly disposed on the base (14), the base (14) is provided with a sliding mechanism, the sliding mechanism is provided with a flipping mechanism (13), the flipping mechanism (13) is provided with a support plate (12) for supporting the substrate (1), and the support plate (12) and / or the substrate (1) are provided with a limiting mechanism for limiting the displacement of the substrate (1) during the flipping process.

4. The high-throughput isothermal amplification device according to claim 3, characterized in that: The flipping mechanism (13) includes four damping buffer telescopic rods, which are arranged in pairs in a row. One end of each damping buffer telescopic rod is fixedly connected to the sliding mechanism, and the other end is ball-jointed to the support plate (12).

5. A high-throughput isothermal amplification device according to claim 3, characterized in that: The flipping mechanism (13) includes four electric telescopic rods, one end of which is fixedly connected to the sliding mechanism, and the other end is ball-jointed to the support plate (12).

6. A high-throughput isothermal amplification device according to claim 3, characterized in that: The sliding mechanism includes a groove (19) disposed on the base plate. The groove (19) extends from away from the heating chamber (17) to near the heating chamber (17). A slider (15) is slidably disposed in the groove (19). The slider (15) is connected to the flipping mechanism (13).

7. A high-throughput isothermal amplification device according to claim 3, characterized in that: The substrate (1) is provided with uniform holes (3) and a second sample feeding hole (4), both of which are provided with protrusions protruding from the edge of the substrate (1). The support plate (12) is provided with two limiting grooves (20) that respectively cooperate with the two protrusions.

8. A high-throughput isothermal amplification device according to any one of claims 1-7, characterized in that: The sealing structure consists of a first rubber soft plug and a second rubber soft plug, which are respectively fixedly installed in the first sample feeding hole (2) and the second sample feeding hole (4). The first rubber soft plug and the second rubber soft plug are puncture-resistant self-sealing structures. The uniform pore (3) includes a first pore and a second pore that are interconnected. The diameter of the first pore is larger than the diameter of the second pore. The first pore is connected to the outside, and the second pore is connected to the nucleic acid storage chamber (9). The openable structure includes a third rubber soft plug disposed in the first pore. The third rubber soft plug is provided with a third pore. The third rubber soft plug can be rotated in the first pore until the third pore is aligned with or staggered with the first pore.

9. A high-throughput isothermal amplification device according to any one of claims 1-7, characterized in that: The first sample well (2) is connected to several non-interconnected first channels (6), and each first channel (6) is connected to each primer storage cavity (7); Each pair of adjacent nucleic acid storage chambers (9) are connected by a second channel (11); Each of the nucleic acid temporary storage cavities (9) is connected to a primer storage cavity (7) through a third channel (8) disposed on the substrate (1); Each of the reaction chambers (5) is connected to a nucleic acid storage chamber (9) via a fourth channel (10) disposed on the substrate (1).

10. A high-throughput isothermal amplification device according to claim 9, characterized in that: The diameters of the first channel (6), the second channel (11), the third channel (8), and the fourth channel (10) are all in the micrometer range.

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