Molecular detection instrument, heating bag and use method of diagnostic instrument

Abstract

An obliquely arranged excitation light source channel is formed in one side of a reaction chamber, an excitation light source is provided for a sample, the heating bag is placed in the reaction chamber, an internal heating mode is adopted, additional heating equipment is not needed, and the operation is simple and convenient. An image after reaction is collected at the upper end of the reaction chamber, an excitation light source channel is obliquely arranged, so that a certain angle is formed between an excitation light path of an oblique light path and a fluorescence receiving light path, the fluorescence can fully enter the bottom of a reagent tube with a certain depth, the reaction sufficiency is ensured, a heating bag is matched with a CRISPR freeze-drying ball, and preparation is performed according to the temperature required by the molecular detection reaction; according to the device, the required appropriate temperature is provided for the amplification stage and the detection reaction stage of the molecular reaction, the device can complete detection on the premise of not increasing additional steps in the amplification and detection process, aerosol pollution is avoided, the reaction time is shortened, the instrument structure is simple, and complex hardware equipment is not needed.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular detection, and relates to a molecular detection instrument, a heating pack and a method for using the diagnostic instrument. Background Art

[0002] The emerging CRISPR molecular diagnostic technology has made great progress in the field of nucleic acid detection in recent years. It guides Cas proteins to recognize and cut nucleic acid targets through CRISPR RNA. Researchers have combined it with isothermal amplification technology to effectively make up for the lack of specificity caused by isothermal amplification alone. Among them, RT-RPA is considered to be the best amplification reaction for CRISPR molecular diagnosis because its reaction temperature range (36-42°C) overlaps with the optimal reaction temperature (37-42°C) of the commonly used CRISPR Cas13a and Cas12a enzymes. However, the introduction of amplification also brings new problems, that is, the reaction processes of the two will affect each other. CRISPR reagents contain Cas enzymes, and the activated Cas enzymes will shear primers and templates, resulting in decreased sensitivity or even complete loss of detection ability. Due to its trans-cutting and cis-cutting activities, the activated Cas enzymes will shear primers and templates, inhibiting the amplification reaction, thereby reducing the concentration of the amplified product and resulting in decreased sensitivity (as shown in the HOLMESv2 method, the sensitivity drops from 10-18M to 10-14M, or even completely lose the detection ability). Therefore, most molecular diagnostic technologies that combine amplification with CRISPR require amplification first, then opening the lid and adding the CRISPR detection system, and finally mixing and centrifuging. They cannot be performed in a "one-pot" system, which easily leads to aerosol contamination and increases the number of operation steps. However, if the amplification and detection systems are placed together in a one-pot reaction system, the sol contamination caused by opening the lid and centrifugation can be avoided, and the number of operation steps can be reduced. However, the problems of low sensitivity and long reaction time need to be solved urgently.

[0003] To address the incompatibility of RPA amplification and CRISPR detection reactions in one-pot nucleic acid detection, the researchers introduced the following devices for isolation:

[0004] In 2019, Wang Yongming and others placed the Cas12 enzyme on the wall of the Ep tube, and the other components (RPA reagent, ssDNA and crRNA) at the bottom of the centrifuge tube. Although the two were in the same tube, they were not in contact in space. After the RPA reaction at the bottom of the Ep tube was completed, the Cas12 enzyme on the tube wall was mixed into the bottom reaction liquid by instantaneous centrifugation for detection reaction. Its sensitivity reached 10 copies / μL within 30 minutes (a total of 1μL nucleic acid template was added), achieving rapid and highly sensitive detection. However, the centrifugation operation introduced requires additional instruments and reaction steps, which limits the use scenarios and instrument integration.

[0005] In 2023, Liu Changchun and others combined the RPA reaction solution with CRISPR gel to construct a simple CRISPR gel biosensor platform for detecting human immunodeficiency virus (HIV) RNA. In this CRISPR gel biosensor platform, the RPA reaction solution is in the upper layer of the Ep tube, and the CRISPR-cas12a reaction solution in the lower layer is embedded in the agarose gel, providing a spatially separated but connected reaction interface with the RPA reaction solution. During isothermal incubation, the RPA reaction initially occurs in the upper layer of the CRISPR gel. When the RPA product is fully amplified and reaches the CRISPR gel, the CRISPR reaction occurs throughout the test tube. Its sensitivity reached 30 copies / T within 30 minutes. However, its system is not stable and only supports placing the Ep tube containing the reaction solution vertically, otherwise the spatial stratification effect cannot be fully exerted.

[0006] In 2022, Zhou Xiaoming and others added glycerol to the RPA amplification and CRISPR detection reaction solutions. Since glycerol has a certain viscosity, the CRISPR reagent and the RPA reagent are separated in the initial stage of the PRA reaction, delaying the fusion of the two, thereby improving the detection efficiency. Its detection sensitivity reached 10 copies / T within 50 minutes, which is 100 times higher than the traditional one-pot method without the addition of glycerol. However, its reaction time is slightly longer. If the nucleic acid extraction step cannot be avoided (the extraction process requires expensive instruments and well-trained operators, and the extraction time is more than one hour), it will take two hours to obtain the test results. Of course, its system is not stable. It only supports the vertical placement of the Ep tube containing the reaction solution. If it is placed horizontally, the phase separation characteristics brought by the viscosity of glycerol will be greatly reduced, limiting its application in various scenarios.

[0007] In 2022, Peng Niancai and others designed a "tube in a tube" structure, in which the RPA reaction is carried out in an inner tube with two hydrophobic holes at the bottom. After the amplification reaction is completed, the reaction solution enters the outer tube containing the pre-stored Cas13a reagent under the action of centrifugation or vibration. The inner and outer tubes are combined into an independent reaction chamber, and nucleic acid detection can be completed without opening the lid. No specialized equipment is required, and small automated instruments can be used to meet the needs of rapid on-site nucleic acid detection. Its detection sensitivity reached 3 copies / μL within 25 minutes. However, its inevitable nucleic acid extraction step limits the application of portable self-testing.

[0008] In 2023, Zhou Xiaoming and others developed a new light-activated CRISPR-Cas12 system using photochemically activated NPOM caged crRNA. The caged crRNA prevents Watson-Crick base pairing between crRNA and the target, thereby silencing the activity of CRISPR-Cas12. Rapid activity recovery can be achieved by inducing crRNA decaging with UV light. After the RPA amplification reaction is completed, ultraviolet light is introduced to irradiate the reaction solution to activate CRISPR-Cas12 and start the detection reaction. Its detection sensitivity reached 1copy / μL within 30-40 minutes (a total of 15μL nucleic acid template was added), achieving fast and efficient detection. However, the introduction of UV lamps limits the usage scenarios and instrument integration, and adds additional instruments and equipment; the inevitable nucleic acid extraction step further limits the usage scenarios.

[0009] In order to reduce the complexity of the equipment, portable heating methods such as self-heating packs are used for on-site rapid nucleic acid testing. In 2022, Dami Kim and others used commercially available heat packs as heat sources to develop a LAMP-based nucleic acid amplification and nanogold test paper detection device, which successfully achieved a femtomolar (<6.8×102 copies / rxn) detection limit within 40 minutes (excluding the RNA extraction step). The use of self-heating packs avoids the use of fixed power supplies, reduces the complexity and cost of the equipment, and has a wider range of application scenarios. However, this type of self-heating pack purchased directly has problems such as long preheating time and difficulty in controlling the temperature on demand.

[0010] Therefore, the current one-pot nucleic acid detection device has the following four shortcomings: first, the system is unstable and only supports vertical placement of sample tubes (horizontal placement will result in poor effects such as phase separation); second, during the reaction, additional equipment (centrifuge, UV lamp, etc.) or operating steps (centrifugation, mixing, etc.) are required, which can easily lead to aerosol contamination and prolong the reaction time; at the same time, the instrument structure is complex, such as the need to introduce a centrifuge or even UV lamps that are more dangerous to users; most instruments require a fixed power supply to use, and cannot achieve instant detection; for some instruments that use heating packs to provide heat sources, the commercial heating packs used are all purchased, and there are problems such as long preheating time and difficulty in controlling temperature as needed. Summary of the invention

[0011] The purpose of the present invention is to solve the problems in the prior art that the reaction process requires additional equipment for assistance, which easily leads to aerosol pollution and prolongs the reaction time, and the instrument has a complex structure and is inconvenient to operate. The heating pack in the instrument has a long preheating time and the temperature is difficult to control and cannot match the temperature of the detection reaction, resulting in a long detection time and low detection accuracy. A molecular detection instrument, a heating pack and a method for using a diagnostic instrument are provided.

[0012] In order to achieve the above object, the present invention adopts the following technical solutions:

[0013] A molecular detection instrument comprises a main housing, a reaction chamber is arranged inside the main housing, a test tube to be detected is placed on the upper end of the reaction chamber, a heating pack is arranged inside the reaction chamber, an image acquisition port is opened on the upper end of the main housing, an emission filter is arranged on the image acquisition port, and an LED light source lamp is also arranged inside the main housing;

[0014] An LED light source integrated chamber is arranged on one side of the main shell, an excitation light source channel is provided in the LED light source integrated chamber, an excitation filter is arranged near the port of the test tube to be detected in the excitation light source channel, and the excitation light source channel is inclined in the horizontal direction.

[0015] A further improvement of the present invention is:

[0016] The inclination angle of the end of the excitation light source channel close to the reaction chamber along the vertical direction is 66.57-76.57°.

[0017] A placement groove is provided at the upper end of the reaction chamber, and a hinge and a buckle are arranged on the outer side of the placement groove. The hinge and the buckle are used to cooperate and fix the test tube to be tested.

[0018] The wavelength of the excitation filter is 470nm±5nm; the wavelength of the emission filter is 532nm±5nm.

[0019] A heating pack for a molecular detection instrument comprises, by weight, 5-7 parts of iron powder, 5-6 parts of vermiculite, 5-6 parts of carbon powder, 10-12 parts of sodium chloride solution and 13-16 parts of resin.

[0020] The mass fraction of the sodium chloride solution is 7-13%.

[0021] A method for using a molecular detection instrument comprises the following steps:

[0022] The test tube to be tested is placed at the upper end of the reaction chamber, and the heating pack is placed inside the reaction chamber. The RPA reagent and the CRISPR freeze-dried ball are placed in advance in the test tube to be tested, and the CRISPR freeze-dried ball is located at the upper end of the liquid surface of the RPA reagent, and the outside of the CRISPR freeze-dried ball is wrapped with paraffin;

[0023] The heating pack starts to heat up, entering the amplification reaction stage. After the paraffin melts, the CRISPR freeze-dried ball enters the lower layer of the RPA reagent and enters the detection reaction stage.

[0024] After the reaction phase to be detected is completed, the LED light source lamp and the LED light source integrated chamber are started, the image of the test tube to be detected is collected through the image acquisition port, and the detection result is determined based on the image.

[0025] It also includes a heating pack that enters an amplification reaction phase after heating for 3-7 minutes.

[0026] The amplification reaction phase is 4-6 minutes, the detection reaction phase is 13-18 minutes, and at the end of the amplification reaction, the heating temperature of the heating pack reaches the melting point of paraffin.

[0027] When the heating pack is heated, the temperature rises to 36°C within 0-3 minutes, rises to 40°C within 3-7 minutes, and gradually rises to 42°C after 7 minutes. The temperature of the heating pack can be maintained between 40-42°C for 23-60 minutes.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention discloses a molecular detection instrument. An excitation light source channel is arranged at an angle on one side of a reaction chamber to provide an excitation light source for a sample. A heating pack is placed inside the reaction chamber to adopt an internal heating method without the need for additional heating equipment. An image after the reaction is collected at the upper end of the reaction chamber. The excitation light source channel is arranged at an angle so that an excitation light path of the oblique light path and a fluorescence receiving light path have a certain angle, and the excitation light path can be fully incident on the bottom of a reagent tube with a certain depth to ensure the adequacy of the reaction. The device can complete the detection without opening the shell, thereby avoiding aerosol pollution and shortening the reaction time. The instrument has a simple structure and does not require complex hardware equipment.

[0030] The present invention discloses a heating pack for a molecular detection instrument. The heating pack is prepared according to the temperature required for the molecular detection reaction, provides the required suitable temperature for the amplification stage and the detection reaction stage of the molecular reaction, and can control the pre-amplification reaction to be performed below 40°C to meet the shortest pre-amplification time. The heating pack can control the reaction time and the required temperature of each stage, shortens the reaction time, and improves the accuracy and detection speed of the detection result.

[0031] The present invention discloses a method for using a molecular detection instrument. Paraffin is wrapped around the outside of a CRISPR freeze-dried ball. In the initial stage, the CRISPR freeze-dried ball is located at the upper end of the liquid surface of an RPA reagent, satisfying the requirement of physical isolation between an amplification system and a CRISPR detection system in a pre-amplification stage. The temperature of a heating package is controlled so that the paraffin melts between a pre-amplification reaction and a shearing reaction. After the shortest pre-amplification time is satisfied, the reaction temperature is increased to 40°C, and the melted paraffin slowly releases the CRISPR detection reagent to enter a detection reaction stage. The method provides the temperature conditions required for pre-amplification and CRISPR detection reactions by combining a heating package and paraffin, and has a short detection time and high detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0033] Figure 1 The overall structure of the present invention Figure 1 ;

[0034] Figure 2 The overall structure of the present invention Figure 2 ;

[0035] Figure 3 It is the internal structure diagram of the present invention;

[0036] Figure 4 is a structural diagram of a reaction chamber of the present invention;

[0037] Figure 5 is a flow chart of the method of the present invention;

[0038] Figure 6 This is a graph of the endpoint fluorescence results of samples of different concentrations after 17 minutes in an embodiment of the present invention;

[0039] Figure 7 This is a real-time fluorescence intensity graph of samples with different concentrations after 17 minutes in an embodiment of the present invention (wherein the fluorescence intensity is replaced by grayscale values, which are identified using imagJ);

[0040] Figure 8 It is a graph of thermal parameters of the heating pack of the present invention (wherein a is a graph of temperature characteristics of self-heating packs with different iron powder contents; b is a graph of constant temperature performance of the heating pack; and c is a schematic diagram of preheating time corresponding to different iron powder contents).

[0041] Among them: 1-excitation filter; 2-clasp; 3-test tube; 4-hinge; 5-reaction chamber; 6-LED light source lamp; 7-main shell; 8-emission filter; 9-fixing gasket; 10-LED light source integrated chamber; 11-excitation light source channel. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0043] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0045] In the description of the embodiments of the present invention, it should be noted that if the terms "upper", "lower", "horizontal", "inner", etc. indicate an orientation or positional relationship based on the orientation or positional relationship shown in the drawings, or the orientation or positional relationship in which the product of the invention is usually placed when in use, it is only for the convenience of describing the present invention and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention. In addition, the terms "first", "second", etc. are only used to distinguish the description, and cannot be understood as indicating or implying relative importance.

[0046] In addition, if the term "horizontal" appears, it does not mean that the component must be absolutely horizontal, but can be slightly tilted. For example, "horizontal" only means that its direction is more horizontal than "vertical", which does not mean that the structure must be completely horizontal, but can be slightly tilted.

[0047] In the description of the embodiments of the present invention, it is also necessary to explain that, unless otherwise clearly specified and limited, the terms "set", "install", "connect", and "connect" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal connection of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0048] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0049] See also Figure 1The present invention discloses a molecular detection instrument, including a main shell 7, a reaction chamber 5 is arranged inside the main shell 7, a test tube 3 to be detected is placed on the upper end of the reaction chamber 5, a heating pack is arranged inside the reaction chamber 5, an image acquisition port is opened at the upper end of the main shell 7, an emission filter 8 is arranged on the image acquisition port, and an LED light source lamp 6 is also arranged inside the main shell 7; an LED light source integrated chamber 10 is arranged on one side of the main shell 7, an excitation light source channel 11 is opened in the LED light source integrated chamber 10, an excitation filter 1 is arranged near the port of the test tube 3 to be detected in the excitation light source channel 11, and the excitation light source channel 11 is inclined in the horizontal direction.

[0050] Furthermore, openings are provided on both sides of the reaction chamber 5, one side of which is a rectangular parallelepiped structure with an opening, and the other side is an arched opening, so that the user can hold the temperature-controlled reaction chamber 5 and load the instrument from the lower end of the main shell 7. Rectangular through holes are provided on the other two sides of the reaction chamber 5 that are perpendicular to the side with the opening to ensure that oxygen can enter and fully contact with the reactants in the heating package.

[0051] Furthermore, a placement slot is provided at the upper end of the reaction chamber 5, and four placement slots are provided. The test tubes 3 to be tested are placed in the placement slots. A hinge 4 is provided on one side of the four placement slots, and a buckle 2 is provided on the other side. The hinge 4 is hinged to the upper end of the reaction chamber 5. The hinge 4 can be buckled above the placement slot and cooperate with the buckle 2 to fix the test tubes 3 to be tested inside.

[0052] Furthermore, in this embodiment, the test tube 3 to be tested is placed in parallel. If the test tube 3 to be tested is placed vertically and fluorescence is collected from the top, the water vapor generated will condense into water droplets on the top cover of the Ep tube, which will greatly interfere with the fluorescence collection. Since the area of ​​the Ep tube cover is small, the density of the water droplets is large, so the sample should be placed horizontally.

[0053] Furthermore, the inclination angle of the end of the excitation light source channel 11 close to the reaction chamber 5 along the vertical direction is 66.57-76.57°, specifically 71.57°. In this embodiment, since the sample is placed horizontally, both the orthogonal and confocal optical paths will interfere with each other and fail to fully illuminate the sample. The excitation optical path of the oblique optical path and the fluorescence receiving optical path have a certain angle, which can fully incident on the bottom of the reagent tube with a certain depth. With the oblique optical path structure as a reference, a certain angle adjustment is made to make it more suitable for the detection performance requirements and structural requirements of the instrument in this article.

[0054] Furthermore, in this embodiment, a battery box is configured in the LED light source lamp 6 to provide power.

[0055] Furthermore, a fixing gasket 9 is provided at the image acquisition port, and the emission filter 8 is fixed by the fixing gasket 9 .

[0056] Furthermore, in this embodiment, the excitation light source channel 11 is a blue light source with an excitation wavelength band between 400nm-480nm, which is integrated in the LED light source integrated chamber 10.

[0057] Furthermore, in this embodiment, the wavelengths of light passed by the excitation filter 1 and the emission filter 8 are 470 nm±5 nm and 532 nm±5 nm, respectively.

[0058] Furthermore, in this embodiment, the main housing 7 and the LED light source integrated chamber 10 are matched with a user-operable buckle through a hinge, and the instrument as a whole is opaque, with a size of 105.6mm×90mm×106mm. When selecting materials, the reaction temperature and the thermoplasticity of the material are taken into consideration, and polylactic acid (PLA) is used for printing.

[0059] Furthermore, in this embodiment, a mobile phone is placed on the image acquisition port for image acquisition, and the shooting distance in this device is 10 cm.

[0060] The invention discloses a heating pack for a molecular detection instrument, which comprises 5-7 parts of iron powder, 5-6 parts of vermiculite, 5-6 parts of carbon powder, 10-12 parts of sodium chloride solution and 13-16 parts of resin in terms of mass.

[0061] Furthermore, during preparation, the reactants in the heating bag are placed in a microporous breathable membrane. The pores of the microporous breathable membrane can control the slow entry of oxygen to avoid excessively fast oxygen entry rate resulting in a violent reaction and inability to continuously release heat, and finally the reaction is vacuum packaged.

[0062] The vacuum outer packaging bag in this embodiment is a layer of vacuum transparent plastic film that isolates oxygen. After the user tears it open, the self-heating bag comes into contact with oxygen in the air, and the iron powder inside reacts with oxygen and releases heat.

[0063] Furthermore, the non-woven bag is used as a microporous breathable membrane. The size of the microporous breathable membrane is 10×12 cm, and the powder loading is 35-50 g. Putting the temperature control reaction raw materials into the non-woven bag helps to control the speed of oxygen entry, prolong the heat release time and maintain a constant temperature effect.

[0064] Furthermore, the heat pack provides heat for the reaction based on the exothermic principle of the reaction between iron powder and oxygen, maintaining the reaction temperature at 37°C-42°C, which is the optimal temperature range for RPA reaction and CRIPSR shear reaction, and is placed into the cavity of the temperature-controlled reaction chamber from the front.

[0065] The present invention also discloses a method for using the molecular detection instrument, comprising the following steps:

[0066] The test tube 3 to be tested is placed at the upper end of the reaction chamber 5, and the heating pack is placed inside the reaction chamber 5. The RPA reagent and the CRISPR freeze-dried ball are pre-placed in the test tube 3 to be tested, and the CRISPR freeze-dried ball is located at the upper end of the liquid surface of the RPA reagent, and the outside of the CRISPR freeze-dried ball is wrapped with paraffin;

[0067] The heating pack starts to heat up, entering the amplification reaction stage. After the paraffin melts, the CRISPR freeze-dried ball enters the lower layer of the RPA reagent and enters the detection reaction stage.

[0068] After the reaction phase to be detected is completed, the LED light source lamp 6 and the LED light source integrated chamber 10 are started, the image of the test tube 3 to be detected is collected through the image acquisition port, and the detection result is determined based on the image.

[0069] In this method, when the heating pack is placed in, the vacuum packaging bag can be opened, and after sampling, the throat swab is placed in the sample release agent to fully lyse for 5 minutes, and then two drops are squeezed into the Ep tube containing the RPA reagent.

[0070] When collecting the final image, use the smartphone camera to take a photo through the emission filter to obtain the fluorescence image of the reaction endpoint. If green fluorescence appears, the sample is positive, and if no fluorescence is produced, the sample is negative.

[0071] The amplification reaction reagents in this method include enzymes, primers, and templates required for the RPA reaction. The enzymes are directly purchased from commercial kits. The primers are designed on the PrimerBLAST website based on the Orf1ab gene sequence of the COVID-19 virus.

[0072] The extraction-free reagent is a nucleic acid sample release agent, the main components of which are surfactants and proteases. Surfactants can destroy the cell membrane structure and release nucleic acids from the membrane. Proteases can degrade proteins on the membrane and further promote the release of nucleic acids.

[0073] The CRISPR freeze-dried pellet in this method includes CRISPR RNA (crRNA), LbaCas12a, Cas12a buffer and single-stranded DNA probe labeled with a fluorescent quencher; it is freeze-dried by Tolo Biotechnology Company.

[0074] Heat solid paraffin to form liquid paraffin, add an appropriate amount of xylene to reduce the viscosity of the paraffin, and then evenly wrap it on the surface of the CRISPR freeze-dried ball. Place it in a 4-degree Celsius refrigerator. The paraffin will quickly solidify on its surface to form a paraffin film. Figure 5 Then place the packaged freeze-dried balls in a fume hood to allow xylene to evaporate completely and avoid entering the solution.

[0075] refer to Figure 5 , the use process of the present invention:

[0076] The user takes a throat swab sample with a cotton swab and puts it into the sampling reagent, squeezes and mixes it, and then drops it into the nucleic acid detection reagent. The reagent tube 12 containing the sample and the nucleic acid detection reagent is placed in the open slot of the temperature-controlled reaction tank 2, and the vacuum packaging bag of the heating bag is torn open. The heating bag is placed in the temperature-controlled reaction tank 2, and the amplification reaction begins. Turn on the LED light source 6, and observe the change in the fluorescence of the sample in the reagent tube through the emission filter 8. If fluorescence appears, it is positive, and if no fluorescence is negative, it is negative. Positive samples with higher concentrations will show fluorescence first.

[0077] refer to Figure 8 b. The self-heating package specially made for the reaction completes the heating process within 0-2.8 minutes and completes the RPA amplification reaction within 2.8-6.8 minutes. At 6.8 minutes, the paraffin melting point (40 degrees Celsius) is reached and the paraffin melts to release the CRISR lyophilized reagent wrapped therein. At this time, the concentration of the amplified product has reached a level that is not affected by Cas12 enzyme cutting. Thereafter, the amplification reaction and the detection reaction are carried out simultaneously.

[0078] refer to Figure 6 to Figure 7 For samples of different concentrations, the present invention can detect them within 17 minutes. Including the sampling and lysis process, it takes a total of 22 minutes to obtain the test results.

[0079] The present invention designs primers based on the novel coronavirus. The minimum detection limit of the 30μL system is 1 copies / μL, the final concentration is 0.17 copies / μL, and the detection rate of 12 clinical positive samples is 100%. While the sensitivity is high, no false positives or false negatives are generated.

[0080] In this method, the nucleic acid extraction step is avoided. After the user places the sample into the nucleic acid lysis solution for 5 minutes, the test can be carried out directly. There is no need to go to the hospital to use a bulky and expensive nucleic acid extractor for extraction, which realizes true portability; the system is stable, and accidental shaking or horizontal placement will not affect the reaction; the method of paraffin-wrapped freeze-dried reagents is introduced, which prolongs the shelf life of the reaction reagents and realizes a rapid and highly sensitive one-pot reaction without the need for additional equipment (centrifuge, ultraviolet lamp, etc.) or operating steps (centrifugation, mixing, etc.), avoiding aerosol contamination and shortening the reaction time; the instrument has a simple structure and does not require complex hardware equipment. The purposefully designed self-heating bag not only reduces the complexity of the equipment, but also can release heat on demand according to the temperature requirements of the reaction process.

[0081] In the detection, the method makes the heat release of the self-heating bag and the melting of paraffin cleverly cooperate to control the entire reaction process. Combined with the RT-RPA reaction temperature range (25-48°C) and the optimal temperature range of the CRISPR / Cas12a detection reaction (37-42°C), the paraffin melts between the pre-amplification reaction and the shearing reaction, so its melting point should be between the suitable temperatures of these two reactions. The present invention customizes paraffin-wrapped CRISPR lyophilized reagents with a melting point of about 40°C to meet the requirements of physical isolation of the amplification system and the CRISPR detection system in the pre-amplification stage. The pre-amplification reaction is carried out below 40°C. After at least meeting the shortest pre-amplification time, the reaction temperature is raised to 40°C, the melted paraffin slowly releases the CRISPR detection reagent, and then the temperature is maintained below 42°C to complete the entire reaction. In order to reduce the complexity of the equipment and meet the needs of low-cost on-site rapid detection, a self-heating bag is used to provide the temperature conditions required for the above-mentioned pre-amplification and CRISPR detection reactions.

[0082] The present invention determines the melting point of paraffin and the content of iron powder that need to be customized according to the overall reaction process. It is further determined that the iron powder content in the designed self-heating bag meets the reaction temperature requirements, and heat can be released as needed for one-pot nucleic acid detection in the whole process. The self-heating bag rises from room temperature to 36°C (preheating) within 0-3min; rises from 36°C to 40°C within 3-7min (pre-amplification reaction is carried out at 36-40°C, two-step reaction stage; paraffin melts at 40°C); the time maintained at 40°C-42°C is 23min (temperature range suitable for both amplification and detection reactions, one-step reaction stage). Next, the self-heating bag is used as a temperature-controlled heat source to complete the entire reaction. In the strategy of using the self-heating bag as a temperature-controlled reaction heat source, the RT-RPA amplification time is 4min, the CRISPR detection time is 13min, and the total reaction requires a total of 17min.

[0083] See Table 1 for the composition of all materials in the reaction process:

[0084] Table 1 Thermodynamic parameters of each component in the temperature-controlled reaction chamber

[0085]

[0086] Referring to the thermodynamic parameters of each component in Table 1, the total heat required for the reaction is calculated to be 4503 J according to formula (1):

[0087] Q i =C i m i △T i (1)

[0088] According to Carnot's theorem, the maximum efficiency of a heat transfer system between reservoirs of different temperatures depends only on the temperature and has nothing to do with the specific implementation of the heat transfer system. The efficiency of a heat transfer system between two reservoirs of different temperatures can only reach its maximum value in a reversible cycle, which is the Carnot efficiency (ηC):

[0089]

[0090] Where Th and Tc are the temperatures of the hot reservoir and the cold reservoir, respectively.

[0091] According to formula (2), select the initial time (T C1 =22℃,T h1 =23.5℃) and the time when the temperature difference is the largest (T C2 =42℃,T h2 =52℃) to calculate, η C1 and η C2 They are 0.0638 and 0.1923 respectively.

[0092] Real heat transfer systems develop along irreversible paths under the inevitable constraints of finite time and finite size. In actual production, the efficiency should be between ηC / 2 and ηC / 2+ηC2 / 8. Therefore, 0.0319<η1<0.0324, 0.0962<η2<0.1008. We take 3% and 10% as the heat engine efficiency of this system. Combined with the total heat of reaction (4503J) obtained in the previous article, the heating pack should provide 45300J to 150100J of heat.

[0093] The temperature performance of the self-heating package (such as heating rate, maximum temperature, duration of optimal temperature, etc.) is mainly determined by the iron powder content. According to the above, the heat required for the reaction is determined to be between 45300J and 150100J. According to the enthalpy change equation of the reaction heat, the amount of iron powder required for the reaction is calculated to be 0.055mol to 0.182mol, and the mass of the iron powder is calculated to be 3.08g to 10.19g. Therefore, iron powder in the range of 3g to 10g is selected for the test.

[0094] 4Fe+3O2=2Fe2O3 (3)

[0095] The reaction enthalpy of formula (3) becomes:

[0096] △H=-3296.8kJ<0 (4)

[0097] Eight groups of tests were set up in the range of 3g-10g iron powder content. According to the mass fraction, the vermiculite and activated carbon contents were set to 5g, 10% NaCl solution was 12g, and hydrogel was 13g. Then different masses of iron powder were added respectively and loaded into a microporous breathable membrane to prepare a self-heating bag. The self-heating bag was placed in the cavity of the temperature-controlled reaction chamber, and the Ep tube filled with double distilled water was placed in the top reaction tank of the temperature-controlled reaction chamber. The temperature of the double distilled water in the Ep tube was measured using a Uni-T UT320D thermocouple thermometer. The temperature of the double distilled water was recorded every 15s to evaluate the temperature characteristics of the self-heating bags with different iron powder contents, such as Figure 8 As shown in a.

[0098] In Turu Figure 8 In a, when the iron powder content is 5, 5.5 or 6 g, the temperature conditions required for the aforementioned amplification and detection reaction combination are met. When the iron powder content is 6 g, the temperature rise interval is 4 min, which is just suitable for the shortest pre-amplification time. Figure 8 b. It is preliminarily estimated that the optimal iron powder content is 6g.

[0099] For the three self-heating packs with iron powder contents of 5, 5.5 or 6 g that meet the temperature conditions, the maintenance time in the specific temperature zone is different, that is, the preheating time, RT-RPA reaction time and CRISPR reaction time are different. Figure 8 As shown in Figure c, when the iron powder content is 6 g, the preheating time is the shortest (~3 min), the time maintained in the RT-RPA amplification reaction is the longest (~4 min), and the performance is the best.

[0100] The special heating pack Figure 8 Point B in b melts the paraffin and cooperates with the paraffin to achieve one-pot reaction temperature control, so that the whole process can be completed automatically without centrifugation. While reducing the number of operation steps and costs, it has a high detection sensitivity (1copy / μL), reaching the level of clinical application.

[0101] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A molecular detection instrument, characterized in that: The invention comprises a main shell (7), a reaction chamber (5) is arranged inside the main shell (7), a test tube (3) to be detected is placed at the upper end of the reaction chamber (5), a heating pack is arranged inside the reaction chamber (5), an image acquisition port is opened at the upper end of the main shell (7), an emission filter (8) is arranged at the image acquisition port, and an LED light source lamp (6) is also arranged inside the main shell (7); An LED light source integrated chamber (10) is arranged on one side of the main housing (7), an excitation light source channel (11) is provided in the LED light source integrated chamber (10), an excitation filter (1) is arranged near the port of the test tube (3) to be detected in the excitation light source channel (11), and the excitation light source channel (11) is inclined in the horizontal direction.

2. A molecular detection instrument according to claim 1, characterized in that: The inclination angle of the end of the excitation light source channel (11) close to the reaction chamber (5) along the vertical direction is 66.57-76.57°.

3. A molecular detection instrument according to claim 1, characterized in that: A placement groove is provided at the upper end of the reaction chamber (5), and a hinge (4) and a buckle (2) are arranged outside the placement groove. The hinge (4) and the buckle (2) are used to cooperate and fix the test tube (3) to be tested.

4. A molecular detection instrument according to claim 1, characterized in that: The wavelength of the excitation filter (1) is 470nm±5nm; the wavelength of the emission filter (8) is 532nm±5nm.

5. A heating pack for a molecular detection instrument, characterized in that: Calculated by mass, the composition comprises 5-7 parts of iron powder, 5-6 parts of vermiculite, 5-6 parts of carbon powder, 10-12 parts of sodium chloride solution and 13-16 parts of resin.

6. A heating pack for a molecular detection instrument according to claim 5, characterized in that: The mass fraction of the sodium chloride solution is 7-13%.

7. A method for using a molecular detection instrument, characterized in that: The following steps are involved: The test tube (3) to be tested is placed at the upper end of the reaction chamber (5), and the heating pack is placed inside the reaction chamber (5). The RPA reagent and the CRISPR freeze-dried ball are placed in advance in the test tube (3), and the CRISPR freeze-dried ball is located at the upper end of the liquid surface of the RPA reagent. The outside of the CRISPR freeze-dried ball is wrapped with paraffin; The heating pack starts to heat up, entering the amplification reaction stage. After the paraffin melts, the CRISPR freeze-dried ball enters the lower layer of the RPA reagent and enters the detection reaction stage. After the reaction phase to be detected is completed, the LED light source lamp (6) and the LED light source integrated chamber (10) are started, an image of the test tube (3) to be detected is collected through the image collection port, and the detection result is determined based on the image.

8. The method for using a molecular detection instrument according to claim 7, characterized in that: It also includes a heating pack that enters an amplification reaction phase after heating for 3-7 minutes.

9. The method for using a molecular detection instrument according to claim 8, characterized in that: The amplification reaction phase is 4-6 minutes, the detection reaction phase is 13-18 minutes, and at the end of the amplification reaction, the heating temperature of the heating pack reaches the melting point of paraffin.

10. The method for using a molecular detection instrument according to claim 9, characterized in that: When the heating pack is heated, the temperature rises to 36°C within 0-3 minutes, rises to 40°C within 3-7 minutes, and gradually rises to 42°C after 7 minutes. The temperature of the heating pack can be maintained between 40-42°C for 23-60 minutes.

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