CN114875121A

CN114875121A - Device and method for detecting nucleic acid amplification product by adopting lateral chromatography test strip

Info

Publication number: CN114875121A
Application number: CN202210723644.8A
Authority: CN
Inventors: 吴坚; 钱斯雯洁
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-06-23
Filing date: 2022-06-23
Publication date: 2022-08-09

Abstract

The invention discloses a device and a method for detecting a nucleic acid amplification product by using a lateral chromatography test strip. The device comprises three chambers, namely a dilution closed chamber for adding and accommodating a sample solution to be detected, a reaction chamber for nucleic acid amplification reaction and a mixing chamber for chromatography test strip detection; a nucleic acid amplification reagent and a solution are placed in the reaction cavity in advance, and are communicated with the bottom of the dilution closed cavity through a long and thin channel and communicated with the outside atmosphere through the channel; the nucleic acid lateral chromatography test strip is placed in the mixing cavity in advance, and the bottom of the mixing cavity is communicated with the reaction cavity through a long and thin channel and is communicated with the outside atmosphere through the channel. The invention can realize the nucleic acid amplification and the lateral chromatography detection of the product thereof, does not need any manual pipetting step, and the whole process is carried out in an anti-pollution space, thereby having the advantages of simple operation and good performance.

Description

Device and method for detecting nucleic acid amplification product by adopting lateral chromatography test strip

Technical Field

The invention relates to a nucleic acid amplification reaction, a detection device and a method, belonging to the field of nucleic acid analysis, in particular to a device and a method for detecting a nucleic acid amplification product by using a lateral chromatography test strip.

Background

Nucleic acid amplification technologies, including Polymerase Chain Reaction (PCR) and various isothermal amplification technologies, such as loop-mediated isothermal amplification (LAMP), recombinase amplification (RPA), etc., are detection methods with high sensitivity and good specificity, and have very wide applications in various fields such as medicine, agriculture, food safety, environmental detection, etc. Currently, fluorescence detection is often used for detection of amplification products, also referred to as amplicons. Although fluorescence and detection have high sensitivity and can realize real-time detection of amplification products, the fluorescence and detection has the problems of high instrument cost, complex operation and the like.

Lateral flow assay test strips, sometimes referred to as colloidal gold test strips, have been widely used in the field of immunoassays. The colloidal gold pregnancy test paper strip is widely used in families. The labeling substance and the detection substance on the lateral chromatography test strip are modified in a simple way, and the lateral chromatography test strip can also be applied to the detection of a nucleic acid amplification product. Thus, the visual field detection of the amplification product can be realized.

However, the detection of the nucleic acid amplification product by using the lateral chromatography test strip currently has some problems: 1. in most cases, nucleic acid amplification is performed in a separate amplification tube. Thereafter, the amplification tube is opened or cut by various means, and thus the amplification product is applied to the sample pad of the strip for detection. Because the concentration of the amplification product is generally higher, the amplification tube is opened, so that aerosol pollution of the amplification product is easily caused, and false positive of subsequent detection is caused. 2. There are some integrated systems that integrate the test strip detection of nucleic acid amplification and amplification products into a closed system, or open the amplification tube in a closed system, but such systems generally require pumps, valves, blades, etc., which are complicated and costly. 3. For a typical lateral flow assay strip, the solution required to be added to the sample pad is typically 50 microliters or more for a strip 3-4 mm wide in order to achieve detection. Too little solution is not beneficial to the capillary movement of the solution on the chromatographic paper, and enough molecules to be detected cannot be conveyed to the position of the T line to realize detection, so that the detection result is influenced. The volume of the nucleic acid amplification reaction system is generally about 25. mu.l. Due to the high sensitivity of nucleic acid amplification detection, increasing the reaction volume does not bring a significant increase in sensitivity, but rather increases the consumption of biological reagents such as amplification enzymes, thereby increasing the reaction cost. Therefore, in many current test strips for detecting nucleic acid amplification products, certain dilution of amplification solution is required to facilitate subsequent chromatographic detection. How to conveniently mix the amplification solution and the dilution solution without causing aerosol pollution of the amplicon is also a problem which needs to be solved. Aiming at the problems, the invention designs a novel device and a novel method for detecting nucleic acid amplification products by adopting a lateral chromatography test strip, which can simplify the operation steps, reduce the requirements on related instruments and simultaneously effectively improve the detection performance.

Disclosure of Invention

In order to solve the problems in the background art, the invention provides a device and a method for detecting a nucleic acid amplification product by using a lateral chromatography test strip, which can improve the performance of the lateral chromatography test strip in the detection of the nucleic acid amplification product.

The technical scheme of the invention is as follows:

the utility model provides a device for adopting lateral flow chromatography test paper strip to nucleic acid amplification product detects:

the device comprises three chambers which are respectively:

the diluting and sealing cavity is used for adding and containing a sample solution to be measured, and the cavity is vertically arranged;

the reaction cavity is used for nucleic acid amplification reaction, a nucleic acid amplification reagent and a solution are placed in the reaction cavity in advance, one end of the reaction cavity is communicated with the bottom of the dilution closed cavity through a long and thin channel, and is communicated with the outside atmosphere through a vertically arranged channel;

the reaction cavity is pre-solidified with all reagents required for nucleic acid amplification, including amplification enzyme, primers, (dNTP), amplification buffer components and the like.

A mixing chamber for chromatography test paper strip detects, places nucleic acid side direction chromatography test paper strip in advance in the intracavity, communicates through elongated passageway between mixing chamber bottom and the reaction chamber other end, and communicates through the passageway of vertical arrangement and external atmosphere.

The slender means that the ratio of the length to the diameter is more than 3.

A valve for opening and closing is provided in a passage between the reaction chamber and the mixing chamber, and the valve opens or closes the passage as needed.

The top end of the dilution closed cavity can be provided with a pipe cap with a ventilation structure.

The volume of the dilution closed cavity is larger than that of the reaction cavity.

And the reaction cavity and the channel communicated with the external atmosphere, the mixing cavity and the channel communicated with the external atmosphere are all provided with filtering materials, and the filtering materials adopt filter elements, silica particles or silica gel columns and the like.

And a valve for opening and closing is arranged in a channel communicated with the mixing cavity and the outside atmosphere, the valve opens or closes the channel as required, and liquid cannot flow into the mixing cavity from the reaction cavity through the channel through the valve.

The channel for communicating the reaction chamber with the outside atmosphere and the channel for communicating the mixing chamber with the outside atmosphere are horizontally or obliquely or vertically arranged.

The top end of a channel communicated with the outside atmosphere in the reaction cavity is provided with a flexible film without a filter material.

The top end of a channel communicated with the outside atmosphere in the mixing cavity is hermetically provided with a flexible film, a filtering material and a valve are not arranged, a valve is not arranged in the channel between the reaction cavity and the mixing cavity, and a hard material is arranged on the flexible film at the top end of the channel communicated with the outside atmosphere in the mixing cavity.

Secondly, a detection method applied to the device comprises the following steps:

firstly, adding a certain amount of sample solution to be measured into a dilution closed cavity, opening a channel between the dilution closed cavity and a reaction cavity, wherein the liquid level in the dilution closed cavity is higher than that in the reaction cavity, and under the action of static pressure, the sample solution to be measured enters the reaction cavity through the channel between the dilution closed cavity and the reaction cavity; at the moment, a valve is arranged in a channel between the reaction chamber and the mixing chamber and is closed, or a valve is arranged in a channel between the mixing chamber and the external atmosphere and is closed, so that the mixing chamber becomes an airtight chamber and the solution is prevented from flowing into the mixing chamber from the reaction chamber;

after the amplification reaction is finished, a channel between the reaction cavity and the mixing cavity is opened by arranging a valve in the channel between the reaction cavity and the mixing cavity, the channel between the mixing cavity and the external atmosphere is opened by arranging a valve in the channel between the mixing cavity and the external atmosphere, and the mixing cavity is communicated with the external atmosphere through the channel; the liquid level in the diluting closed cavity is higher than the liquid levels in the reaction cavity and the mixing cavity, and the solution in the reaction cavity enters the mixing cavity under the action of the solution static pressure in the diluting closed cavity; due to the hydrostatic pressure of the solution, the sample solution in the dilution closed chamber, which is not amplified, also passes through the reaction chamber and further into the mixing chamber until the water pressure in the dilution closed chamber, the reaction chamber and the mixing chamber is balanced, i.e. the liquid levels in the dilution closed chamber, the reaction chamber and the mixing chamber are at the same level.

After the solution of the sample to be detected enters the reaction cavity, the solution preset in the reaction cavity is dissolved, and before the amplification reaction is finished, the temperature of the reaction cavity is controlled by an external or attached temperature control device to carry out the nucleic acid amplification reaction; at the moment, the solution in the channel is controlled and adjusted through the geometric dimension of the channel, the solution does not flow under the conditions that no external force directly acts and static pressures at two ends of the channel are balanced, namely, the solution of the measured sample in the diluting closed cavity does not enter the reaction cavity through the channel under the action of the static pressure any more, and solute components in the reaction cavity can only enter the channel in a diffusion mode and further enter the diluting closed cavity.

Set up sealed flexible film through the passageway top at mixing chamber and external atmosphere intercommunication, set up hard material on the flexible film, make the mixing chamber unable communicate with each other through passageway and external atmospheric pressure, after the amplification reaction, tear hard material, through set up the valve and open in the passageway between reaction chamber and mixing chamber, make the passageway between reaction chamber and the mixing chamber opened, make solution get into the mixing chamber from the reaction chamber and carry out the chromatography detection, and cause the original gas compression in the mixing chamber simultaneously, flexible film response is outside protruding atmospheric pressure and the outside atmospheric pressure balance of maintaining the mixing chamber, the mixing chamber can be got into to the solution in reaction chamber and the dilution chamber.

The invention has the beneficial effects that:

the device and the method can realize the nucleic acid amplification and the lateral chromatography detection of the product thereof. In the whole detection process, a user only needs two manual operations. Firstly, dripping a certain amount of sample solution to be measured into a dilution closed cavity; secondly, after waiting for a certain nucleic acid amplification time, the whole detection process can be completed by opening one valve. The volume of the nucleic acid amplification reaction can be controlled between several microliters and several tens of microliters by the design of the device, and the volume control does not need to use a high-precision pipetting instrument such as a pipetting gun. After the nucleic acid amplification is finished, the dilution of the amplification solution and the addition of the sample pad to the chromatographic test strip can be finished only by opening a valve without any manual pipetting step, and the whole process is carried out in a pollution-proof space.

Meanwhile, the opening of the valve in the second step can be automatically completed by the instrument under the control of a matched instrument.

Compared with the existing method, the device and the method have the advantages of simple operation and good performance.

Drawings

FIG. 1 is a schematic diagram of the structure of the apparatus of the present invention. 1. Base plate, 2, dilution closed cavity 2, 3, channel, 4,

reaction cavity

4, 5, valve, 6, channel, 7, mixing cavity 7, 8, filter material, 9, channel, 10, valve, 11, filter material, 12, channel.

FIG. 2 is a chromatographic test strip assay for amplification products.

FIG. 3 is a photograph of an amplification gel electrophoresis. The standard nucleic acid fragments of various lengths are shown on the left, the electrophoresis results of the amplification products for amplification in the standard reaction tube are shown in the middle, and the electrophoresis results of the amplification products for amplification in the reaction chamber 4 of the apparatus of the present invention are shown on the right.

FIG. 4 is a ferromagnetic valve in a channel. 51. Ferromagnetic material, 6, channel.

Fig. 5 is a diagram of the use of flexible film material to achieve air-tightness and air pressure balance. 12. Channel, 13, flexible membrane.

Fig. 6 shows the function of air-tightness and valve by using flexible film material and hard material. 9. Channel, 13, flexible film, 14, hard material.

Detailed Description

The invention is further described with reference to the accompanying drawings and the detailed description.

The method and associated apparatus of the present invention are described with reference to fig. 1. Fig. 1 is mainly for convenience of explanation of relevant aspects of the present invention, and for convenience of description, details of construction are highlighted and not drawn to scale.

In the device, three independent cavities, namely a dilution closed cavity 2, a reaction cavity 4 and a mixing cavity 7, are arranged in a substrate 1; the dilution closed cavity 2 is connected with the reaction cavity 4 through a channel 3; the reaction chamber 4 and the mixing chamber 7 are connected by a channel 6.

A valve 5 is provided in the channel 6 between the reaction chamber 4 and the mixing chamber 7, opening or closing the channel as required.

All reagents required for nucleic acid amplification, including amplification enzymes, primers, dNTPs, and amplification buffer components, etc., may be immobilized in the reaction chamber 4 in advance. The mixing chamber 7 is pre-provided with a corresponding nucleic acid lateral chromatography test strip.

The reaction chamber 4 and the mixing chamber 7 are kept at the same and balanced pressure by the

additional channels

12, 9 and the outside. Meanwhile, in the

channels

12, 9, filtering and sealing materials 11, 8 can be arranged, and a filter element, silica particles or silica gel column and the like can be adopted to ensure that only small molecules such as gas and the like can pass through the channels, so that the pollution of the amplicon caused by the leakage of the nucleic acid amplification product to the external environment is avoided under the condition of keeping the air pressure balance inside and outside the cavity.

In the capillary channel 9 of the mixing chamber 7, a valve 10 is arranged to open or close the communication of the channel with the environment. That is, when the valve 10 is closed, the mixing chamber 7 may be set in an airtight mode, preventing liquid from entering the mixing chamber 7 from the reaction chamber 4 via the channel 6.

The following is the engineering process for carrying out the invention

1. In specific application, a certain amount of the sample solution to be measured is added to the dilution closed chamber 2 by using a dropper or the like.

Depending on the nucleic acid amplification reagents, these test sample solutions may be subjected to standard nucleic acid extraction procedures, or may be simply heated or diluted. The amount of sample solution to be measured added can be controlled to be approximately between 50 and 1000 microliters. Under the condition of meeting the requirement of subsequent chromatographic test, the addition amount of the sample solution to be tested can be less.

After the tested sample solution is added, the diluting closed cavity 2 can be closed by a pipe cap with a ventilating structure and the like, so that macromolecules are prevented from entering the surrounding environment from the diluting closed cavity 2 on one hand, and meanwhile, the air pressure balance between the inside of the cavity and the outside can be kept on the other hand. The ventilation structure on the pipe cap can be realized by adopting a filter element, silica particles or silica gel columns and the like.

2. Nucleic acid amplification reaction

After the sample solution to be measured is added into the dilution closed cavity 2, the dilution closed cavity 2 and the reaction cavity 4 are of a communicated structure, and the liquid level of the dilution closed cavity 2 is higher than that of the reaction cavity 4, so that the sample solution to be measured can enter the reaction cavity 4 under the action of static pressure.

At this time, the space between the reaction chamber 4 and the mixing chamber 7 is controlled by the valve 5 or the valve 10: when set, the valve 5 is closed and the solution cannot enter the mixing chamber 7; when the valve 5 is not provided, the valve 10 can be used to close the connection between the passage 9 and the outside air pressure, so that the mixing chamber 7 becomes an airtight chamber to prevent the inflow of liquid.

At the same time, the channel 12 is designed such that it assumes the function of only one gas channel, which is small relative to the volume of the reaction chamber 4. Thus, the volume of liquid flowing into the reaction chamber 4 is mainly determined by the volume of the reaction chamber 4. The volume of the reaction chamber 4 can be controlled between 3 and 80 microliters, so that the corresponding volume of solution can be obtained and reacted in the reaction chamber 4 according to the design of the device as long as the proper sample is added into the dilution closed chamber 2. Volumes of 3 to 80 microliters typically require a high precision pipette for acquisition. The use of pipette guns is complicated for the average user. Through the design of the device, the invention can be realized by a user only by adding a certain amount of sample solution into the diluting closed cavity 2. This is an advantage of the present invention.

In the present invention, the dilution enclosure 2 is larger in volume than the reaction chamber 4, depending on the functional requirements. This allows more solution to be stored in the dilution enclosure 2, facilitating the movement of the liquid in the entire apparatus under the influence of hydrostatic pressure.

After the sample solution to be measured enters the reaction chamber 4, the reaction solution preset in the reaction chamber 4 will be dissolved. The temperature of the reaction chamber 4 can be controlled by an external or attached temperature control device to perform the nucleic acid amplification reaction. Such a reaction may be a PCR reaction, or an isothermal amplification reaction, such as LAMP, or RPA, etc.

During the nucleic acid amplification reaction, the reaction chamber 4 and the dilution chamber 2 are connected together by the channel 3. In the invention, by controlling the geometric dimension of the channel 3, the solution in the channel 3 can not flow under the condition that no external force directly acts and the static pressure at two ends of the solution is balanced. Thus, solute components in the reaction chamber 4 can only enter the channel 3 and further into the dilution enclosure 2 by diffusion. Solute amplification in solution is a slow process, whereas nucleic acid amplification reactions are typically within an hour.

Experiments have shown that at this time scale, when the diameter of the channel 3 is between 50 μm and 1 mm, the exchange of solute components between the reaction chamber 4 and the dilution enclosure 2 due to diffusion is negligible as long as its length is greater than 5 mm.

3. The reaction solution and the reagent also reach the sample pad of the chromatographic strip to carry out chromatographic reaction

After the amplification reaction is finished,

in case a valve 5 is provided, the valve 5 can be opened while the mixing chamber 7 is connected to the outside air pressure via a channel 9. Thus, under the action of the hydrostatic pressure of the solution, the solution in the reaction chamber 4 enters the mixing chamber 7; at the same time, the unamplified sample solution in the diluting enclosed chamber 2 will also pass through the reaction chamber 4 and further into the mixing chamber 7 due to the hydrostatic pressure of the solution until the hydrostatic pressure in the diluting enclosed chamber 2, the reaction chamber 4 and the mixing chamber 7 is balanced. Thus, the valve is opened to dilute the nucleic acid amplification product to a certain volume to meet the detection requirement of the chromatographic test paper.

Instead of the valve 5, the valve 10 may be provided in the passage 9, in which case the valve 5 is not provided. Thus, when the valve 10 is closed, the mixing chamber 7 is an airtight space, and when the solution enters through the elongated passage 6, the gas in the mixing chamber 7 is compressed, thereby causing a rise in gas pressure, which cannot proceed into the mixing chamber 7. When the valve 10 is opened, the solution in the reaction chamber 4 and the dilution sealing chamber can enter the mixing chamber 7 to realize chromatography detection.

In the design of the actual device, the hydrostatic pressure relationship among the diluting sealed cavity, the reaction cavity 4 and the mixing cavity 7 can be controlled by adjusting the relative positions of the diluting sealed cavity and the mixing cavity so as to improve the detection effect.

The specific implementation example is as follows:

1. nucleic acid amplification and test strip detection of vibrio parahaemolyticus

The structure of the device is shown in fig. 1, and the material used is polymethyl methacrylate (PMMA) plate. A figure as shown in fig. 1 is machined on one side of the plate by a mechanical machining method (please note that fig. 1 is not drawn according to actual dimensions for convenience of description, and the dimensions of each structure in the device are based on the following text).

The depth is in the direction perpendicular to the paper surface, the height is in the longitudinal direction of the paper surface, and the width is in the transverse direction of the paper surface.

The depth of the dilution chamber 2, the reaction chamber 4 and the mixing chamber 7 are all 2 mm. The dilution enclosure 2 has a width of 3 mm and a height of 60 mm. The reaction chamber 4 has a width of 4 mm and a height of 3 mm. The mixing chamber 7 has a width of 4 mm and a height of 60 mm.

The channel 3 has a depth of 1 mm, a height of 1 mm and a length of 5 mm. The channel 6 has a depth of 1 mm, a height of 1 mm and a length of 5 mm. The channel 9 has a depth of 0.1 mm, a width of 0.1 mm and a height of 10 mm. The channel 12 has a depth of 0.1 mm, a width of 0.1 mm and a height of 59 mm. Silica gel particles are put in the channel 9 and the channel 12, so that the smooth airflow of the channels can be kept, and macromolecules such as nucleic acid amplification products can be prevented from diffusing to the outside. If desired, capillary valves may be provided in the

channels

9 and 12 to prevent the flow of liquid in the channels. With regard to the specific arrangement of the capillary valve, reference is made to the documents Sensors and Actuators A130-.

After the structure is processed, the structure can be bonded with another PMMA plate through pressure sensitive adhesive and the like to construct a complete cavity. On top of the channel 9 is glued an air-impermeable adhesive tape, acting as a valve 10.

In this example, Vibrio parahaemolyticus is taken as an example, LAMP amplification reaction is carried out with a Thermolabile hemolysin (tlh) gene in Vibrio parahaemolyticus as a target.

The test strip used in the device is a common nucleic acid detection chromatography test strip, streptavidin is fixed on a T line, a FAM antibody is connected on colloidal gold, and the nucleic acid chromatography test strip can be referred to as PLoS ONE8(7), e69355.doi:10.1371/journal. pane.0069355.

Related reaction systems, and curing of the reaction systems in the reaction chamber 4 of the apparatus are described in the references Sensors & initiators B.chemical 305(2020)127440 and Talanta 214(2020) 120818.

The sample Vibrio parahaemolyticus was used at a concentration of about 250 cfu/g. Adding 120 microliters of the nucleic acid extracting solution of the vibrio parahaemolyticus into the dilution closed cavity 2, and heating the reaction cavity 4 in the device by using an additional temperature control electric heating device, wherein the temperature is 65 ℃ and the time is 40 minutes. After heating, the airtight adhesive tape adhered to the top of the channel 9 is torn, and the solution in the reaction chamber 4 and the dilution closed chamber 2 enters the mixing chamber 7, contacts the sample pad of the chromatographic test strip and performs chromatographic detection under the action of capillary force, and after about 5 minutes, a T line appears, as shown in fig. 2. This result indicates that the device and method of the present invention can effectively perform nucleic acid amplification and detection.

Meanwhile, in order to further examine the amplification performance of the device and ensure that the reaction cavity 4 and the dilution closed cavity 2 are communicated through the channel 3 without bringing obvious influence on the overall amplification performance, after the amplification in the device is finished, the device can be opened, and the amplification solution is taken out for gel electrophoresis detection. Meanwhile, the same sample and amplification reagent can be adopted to carry out LAMP amplification reaction in a standard PCR tube and carry out gel electrophoresis detection. The results of detection of both are shown in FIG. 3, which indicates that efficient nucleic acid amplification can be performed even in the apparatus.

The CRISPR system is a very sensitive detection method, can detect target nucleic acid of about 100pM, and the detailed information about CRISPR can be referred to relevant documents (anal. chem.2019,91, 11362-11366). gRNA sequences were designed in the experiment and the CRISPR/Cas12a system was used to detect amplification products that may be present in the dilution chamber 2. Since the dilution enclosed chamber 2 and the reaction chamber 4 are connected by the channel 3, a large amount of amplification products are generated in the reaction chamber 4 during amplification, and these products are likely to move to the dilution enclosed chamber 2 through the channel 3. However, according to the experimental results, even if the concentration of the extract solution of the sample of positive Vibrio parahaemolyticus amplified in the reaction well 4 is 250cfu/g, the amplification product cannot be detected in the dilution-blocked well 2 by the CRISPR technique after the amplification is completed. This result indicates that, under the current conditions of the device, although there is a channel connection between the reaction chamber 4 and the dilution chamber 2, due to the geometrical limitations of the channel, effective solute exchange cannot occur, i.e., the channel arrangement does not have a significant effect on nucleic acid amplification.

Since the amplification product cannot enter the dilution chamber 2 efficiently, it acts as a seal for the reaction chamber 4 as long as sufficient liquid remains in the dilution chamber 2. This closure helps prevent the solution in the reaction chamber 4 from volatilizing into the environment and causing contamination. This is also the reason why this chamber is named dilution enclosure 2.

For the same reaction system, when the other parameters of the apparatus are unchanged, except that the geometry of the channel 3 is changed to the following parameters: 1) the channel 3 has a depth of 0.1 mm, a height of 0.1 mm and a length of 5 mm. 2) The channel 3 has a depth of 0.1 mm, a height of 0.5 mm and a length of 5 mm. 3) The channel 3 has a depth of 0.1 mm, a height of 0.1 mm and a length of 2 mm. Similar results were obtained from the experiments, indicating that the device works properly when the geometry of the channel 3 is within a sub-range.

Theoretically, the longer the length of the channel 3, the more advantageous it is to prevent the exchange of solutes between the reaction chamber 4 and the dilution enclosure 2. However, in practice, too long a channel length causes inconvenience, and the length can be set within 10 cm in general.

In practice, the channel 3 does not have to be arranged horizontally, but may also be arranged obliquely or vertically. In general, the channels are preferably of elongate configuration.

When F3 and B3 of LAMP are used as primers, PCR amplification of Vibrio parahaemolyticus can also be achieved in the reaction chamber 4. In this case, F3 was modified with FAM at the 5 'end, and F3 was modified with biotin at the 5' end. A temperature control device is needed to control the temperature of the reaction chamber 4 to circulate between 95 ℃ and 63 ℃ during the reaction. The rest operations are similar to the LAMP reaction, and similar experiment results can be obtained.

2. Detection of vibrio parahaemolyticus by device with iron wax valve

In this example, the test object was Vibrio parahaemolyticus, and the test reagents used were the same as in example 1, but different in device structure.

In the present embodiment, as shown in fig. 1, a valve 10 is provided so that the mixing chamber 7 is kept in pressure equilibrium with the outside through a passage 9. The provision of a ferromagnetic material 51 as shown in figure 4 in the channel 6 forms a ferrous wax valve.

When the nucleic acid amplification reaction is performed in the reaction chamber 4, the iron wax valve is closed. After the amplification reaction is finished, under the irradiation of laser, the iron wax valve is opened, so that the reaction amplification solution and the sample solution can enter the mixing cavity 7 and contact the sample pad chromatography detection of the chromatography test strip.

The results of the experiment show that, when this structure is employed, nucleic acid detection results similar to those of example 1 can be obtained.

3. Detection of vibrio parahaemolyticus by flexible film enclosed device

In this example, the test object was Vibrio parahaemolyticus, and the test reagents used were the same as in example 1. But differ in device structure.

In this embodiment, as shown in fig. 1, no filter material 11 is provided in the channel 12, but instead, a flexible membrane 13 as shown in fig. 5 is provided only at the top end of the channel 12.

The valve 5 is arranged in the channel 6 and no filter material 8 and no valve 10 are arranged in the channel 9, but instead, just at the top end of the channel 9, a flexible membrane 13 is arranged as shown in fig. 6.

The flexible membrane 13 can, on the one hand, isolate the flow of gas in the channel from the outside, thus achieving an airtight effect and preventing possible contamination due to amplicon contamination, i.e. functioning as the original filter material 8.

Also, since this is a flexible membrane. This membrane can be provided with a great area and flexibility during the design and manufacture of the device, so that when the solution enters the mixing chamber 7 from the reaction chamber 4 after the valve 5 in the channel 6 is opened, causing the original gas in the mixing chamber 7 to be compressed, this flexible membrane will respond quickly, bulging outwards, so as to maintain the balance between the gas pressure in the mixing chamber 7 and the external gas pressure, so that the solution in the reaction chamber 4 and the dilution chamber can enter the mixing chamber 7.

Furthermore, when a flexible membrane 13 is used in the channel 9, the valve 5 may also be disposed in the channel 6, but as shown in fig. 6, a layer of hard material 14 is attached to the top end of the channel 9. Thus, due to the presence of this layer of hard material 14, the mixing chamber 7 is an airtight chamber, into which the solution cannot enter. When the nucleic acid amplification reaction is finished, the hard material 14 layer is torn, and then the solution in the reaction cavity 4 and the dilution closed cavity 2 can enter the mixing cavity 7 for chromatography detection.

Claims

1. The utility model provides an adopt device of side direction chromatography test paper strip to nucleic acid amplification product detection which characterized in that:

the device comprises three chambers which are respectively:

a dilution closed cavity (2) for adding and containing a sample solution to be measured;

the reaction cavity (4) is used for nucleic acid amplification reaction, nucleic acid amplification reagents and solutions are placed in the reaction cavity in advance, one end of the reaction cavity (4) is communicated with the bottom of the dilution closed cavity (2) through a long and thin channel (3), and is communicated with the outside atmosphere through a vertically arranged channel (12);

the mixing cavity (7) is used for detecting the chromatography test strip, the nucleic acid lateral chromatography test strip is placed in the cavity in advance, the bottom of the mixing cavity (7) is communicated with the other end of the reaction cavity (4) through a long and thin channel (6), and the mixing cavity is communicated with the outside atmosphere through a vertically arranged channel (9).

2. The device for detecting the nucleic acid amplification product by using the lateral flow chromatography test strip of claim 1, wherein: a valve (5) for opening and closing is arranged in the channel (6) between the reaction chamber (4) and the mixing chamber (7).

3. The device for detecting the nucleic acid amplification product by using the lateral flow chromatography test strip of claim 1, wherein: the volume of the dilution closed cavity (2) is larger than that of the reaction cavity (4).

4. The device for detecting the nucleic acid amplification product by using the lateral flow chromatography test strip of claim 1, wherein: and filtering materials (11, 8) are arranged in a channel (12) communicated with the outside atmosphere in the reaction cavity (4), a channel (9) communicated with the outside atmosphere in the mixing cavity (7), and the filtering materials (11, 8) adopt a filter element, silica particles or silica gel columns and the like.

5. The device for detecting the nucleic acid amplification product by using the lateral flow chromatography test strip of claim 1, wherein: a valve (10) for opening and closing is provided in a passage (9) communicating the mixing chamber (7) with the outside atmosphere.

6. The device for detecting the nucleic acid amplification product by using the lateral chromatography test strip of claim 1, wherein: a flexible film (13) is arranged at the top end of a channel (12) which communicates the reaction cavity (4) with the outside atmosphere.

7. The device for detecting the nucleic acid amplification product by using the lateral flow chromatography test strip of claim 1, wherein:

the top end of a channel (9) communicated with the outside atmosphere in the mixing cavity (7) is hermetically provided with a flexible film (13), a valve (5) is not arranged in a channel (6) between the reaction cavity (4) and the mixing cavity (7), and a hard material is arranged on the flexible film (13) at the top end of the channel (9) communicated with the outside atmosphere in the mixing cavity (7).

8. A detection method applied to the apparatus according to any one of claims 1 to 7, characterized in that:

firstly, adding a certain amount of sample solution to be measured into the dilution closed cavity (2), and under the action of static pressure, allowing the sample solution to be measured to enter the reaction cavity (4) through a channel (3) between the dilution closed cavity (2) and the reaction cavity (4); at the moment, a valve (5) is arranged in a channel (6) between the reaction chamber (4) and the mixing chamber (7) and is closed, or a valve (10) is arranged in a channel (9) which is communicated with the outside atmosphere and is closed, so that the solution is prevented from flowing into the mixing chamber (7) from the reaction chamber (4);

after the amplification reaction is finished, opening a channel (6) between the reaction cavity (4) and the mixing cavity (7), and communicating the mixing cavity (7) with the external atmospheric pressure through a channel (9); under the action of the hydrostatic pressure of the solution in the dilution closed cavity (2), the solution in the reaction cavity (4) enters the mixing cavity (7); the unamplified sample solution in the dilution enclosed chamber (2) also passes through the reaction chamber (4) and further into the mixing chamber (7) until the water pressure in the dilution enclosed chamber (2), the reaction chamber (4) and the mixing chamber (7) is balanced.

9. The detection method according to claim 8, characterized in that:

after the solution of the sample to be detected enters the reaction cavity (4), the solution preset in the reaction cavity (4) is dissolved, and before the amplification reaction is finished, the temperature of the reaction cavity (4) is controlled by an external or attached temperature control device to carry out the nucleic acid amplification reaction; at the moment, the solution in the channel (3) cannot flow under the conditions that no external force directly acts and the static pressure at the two ends of the channel (3) is balanced through the geometric dimension control and adjustment of the channel (3), and solute components in the reaction cavity (4) can only enter the channel (3) and further enter the dilution closed cavity (2) in a diffusion mode.

10. The detection method according to claim 8, characterized in that:

set up sealed flexible film (13) through passageway (9) top at hybrid chamber (7) and external atmosphere intercommunication, set up hard material (14) on flexible film (13), make hybrid chamber (7) unable communicate with each other through passageway (9) and external atmosphere pressure, after the amplification reaction, tear hard material (14), set up valve (5) and open through passageway (6) between reaction chamber (4) and hybrid chamber (7), make solution get into hybrid chamber (7) from reaction chamber (4) and carry out the chromatography and detect, and cause original gas compression in hybrid chamber (7) simultaneously, flexible film (13) response is outwards protruding to maintain atmospheric pressure and outside atmospheric pressure balance in hybrid chamber (7).