阅读说明：本技术 一种基于核酸恒温扩增反应阻塞流动的微流控目视现场快速检验装置及其检测方法 (Micro-fluidic visual field rapid inspection device based on nucleic acid isothermal amplification reaction blocking flow and detection method thereof ) 是由 李彪 陈以铖 张馨予 李雯曦 李禹克 马晟利 聂桓 葛文玉 李慧 于 2020-07-29 设计创作，主要内容包括：本发明公开了一种基于核酸恒温扩增反应阻塞流动的微流控目视现场快速检验装置及其检测方法。步骤1：对待测样本进行扩增；步骤2：将步骤1中扩增后的反应体系与含乙醇溶液混合,扩增的阳性样本与扩增的阴性样本呈现出不同的核酸沉淀效果；步骤3：对步骤2中的核酸沉淀通过阻塞微珠堆积出的多孔介质微通道的效果,分辨核酸沉淀的差异；步骤4：通过微通道显色针对步骤3中沉淀的差异,供目视辨识反应的阴性和阳性结果。本发明针对病毒核酸检测,可在感染初期得出准确结果,无需复杂的核酸检验设备。(The invention discloses a micro-fluidic visual field rapid inspection device based on nucleic acid isothermal amplification reaction blocking flow and a detection method thereof. Step 1: amplifying a sample to be detected; step 2: mixing the reaction system amplified in the step 1 with an ethanol-containing solution, wherein the amplified positive sample and the amplified negative sample show different nucleic acid precipitation effects; and step 3: distinguishing the difference of the nucleic acid precipitates by the effect of blocking the micro-channels of the porous medium accumulated by the microbeads on the nucleic acid precipitates in the step 2; and 4, step 4: the difference in the precipitation in step 3 was visualized by microchannel visualization for visual identification of negative and positive results of the reaction. The invention aims at virus nucleic acid detection, can obtain accurate results at the initial stage of infection, and does not need complex nucleic acid detection equipment.)

1. The microfluidic visual field rapid inspection device based on nucleic acid isothermal amplification reaction blocking flow is characterized by comprising an inlet section, a mixing section, at least one blocking channel (9) and a color development cavity (7), wherein the blocking channel (9) is communicated with the color development cavity (7).

2. The micro-fluidic visual on-site rapid inspection device for nucleic acid isothermal amplification reaction based flow blockage according to claim 1, wherein the inlet section comprises 2 inlets (1) and branch pipes (2), each inlet (1) is communicated with one branch pipe (2), 2 branch pipes (2) are communicated with the mixed bent channel (3), and the mixed bent channel (3) is communicated with the blockage channel (9).

3. The microfluidic visual field rapid inspection device based on nucleic acid isothermal amplification reaction blocked flow according to claim 1, wherein the blocking channel (9) comprises a backflow prevention mechanism (4), a pre-assembly inlet (5) and a blocking starting structure (6), each blocking channel (9) is provided with one backflow prevention mechanism (4) inside a starting end channel connected with the mixed bending channel (3), each blocking channel (9) is provided with one blocking starting structure (6) inside a channel connected with the chromogenic cavity (7), the blocking channel (9) between the backflow prevention mechanism (4) and the blocking starting structure (6) is provided with the pre-assembly inlet (5), and each blocking channel (9) is communicated with the chromogenic cavity (7).

4. The microfluidic visual field rapid inspection device based on nucleic acid isothermal amplification reaction blocking flow according to claim 1, wherein a pneumatic outlet (8) is arranged on the color development cavity (7), and the pneumatic outlet (8) is connected with a negative pressure device.

5. The micro-fluidic visual on-site rapid inspection device based on nucleic acid isothermal amplification reaction blocking flow is characterized in that the blocking channel (9) is a porous medium channel.

6. The device for the on-site rapid detection of nucleic acid based on microchannel blockage of claim 3, wherein the diameter of the particle material filled in the blockage channel (9) is 5-30 μm.

7. The microfluidic visual field rapid inspection device based on nucleic acid isothermal amplification reaction blocking flow according to claim 4, wherein the negative pressure device is an injector and a laboratory micro-injection pump.

8. The method for testing the microfluidic visual field rapid test device based on the nucleic acid isothermal amplification reaction blocking flow according to claim 1, wherein the test method is implemented by using the principle that nucleic acid amplified by alcohol precipitation blocks a porous medium in a microchannel, and comprises the following steps:

step 1: amplifying the positive or negative sample and injecting it into the test device through an inlet (1);

step 2: the ethanol-containing solution is injected into the testing device through another inlet (1);

and step 3: mixing the amplified sample with an ethanol-containing solution in a mixing section to enable the amplified positive sample and the amplified negative sample to present different nucleic acid precipitation effects;

and 4, step 4: injecting particles into the blocking channel (9) through the pre-assembly inlet (5) to form a porous medium micro-channel between the blocking starting structure (6) and the pre-assembly inlet (5), and distinguishing the difference of nucleic acid precipitates by the porous medium micro-channel formed by accumulation of the blocking microbeads on the nucleic acid precipitates in the step 3;

and 5: the difference in precipitation in step 4 was visualized by porous media microchannel for visual identification of negative and positive results of the reaction.

9. The inspection method of the micro-fluidic visual on-site rapid inspection device based on nucleic acid isothermal amplification reaction blocking flow according to claim 1, wherein the isothermal amplification reaction system is a nucleic acid sequence dependent amplification NASBA system, a loop-mediated isothermal amplification reaction LAMP system, a rolling circle amplification RCA system, a transcription-mediated amplification TMA system or a strand displacement amplification SDA system.

10. The method for testing the microfluidic visual on-site rapid test device based on the nucleic acid isothermal amplification reaction blocking flow according to claim 1, wherein the ethanol volume concentration of the alcohol solution is not less than 50%.

Technical Field

The invention belongs to the field of nucleic acid detection, and particularly relates to a micro-fluidic visual field rapid inspection device based on nucleic acid isothermal amplification reaction blocking flow and a detection method thereof.

Background

At present, nucleic acid detection is widely applied to the field of clinical medicine, and compared with a colloidal gold chromogenic detection method for detecting an antibody of a patient, the nucleic acid detection has the capability of detecting in the early stage of infection, and can obtain precious time lead when being applied to epidemic prevention detection. Since the amount of nucleic acid in the sample to be detected is very limited, amplification of a trace amount of nucleic acid is required. The nucleic acid amplification method proposed at the earliest is Polymerase Chain Reaction (PCR), which is also the most widely used nucleic acid amplification method at present. However, PCR requires highly trained personnel to operate sophisticated temperature cycling equipment to effect denaturation, annealing and extension of nucleic acids, which limits the possibilities for convenient applications. With the generation of a series of nucleic acid isothermal amplification methods, the complexity of the instrument is reduced, the reaction process is simple and efficient, and the miniaturization and field arrangement of detection equipment are facilitated. However, there is still a great challenge to Point-of-Care Testing (POCT) for nucleic acids in the field.

How to popularize nucleic acid detection in the global scope, especially in developing areas and less developed areas, faces a series of problems of how to overcome pollution, reduce cost, remove complex instruments, simplify operation and the like. The micro-fluidic chip provides a solution, integrates a plurality of reaction steps into a micro-fluidic chip with the square centimeter, realizes the miniaturization of a detection instrument, the simplification of sample pretreatment, the high efficiency and the accuracy of experimental operation and the economy of detection cost.

The nucleic acid detection system used in the current market mainly has detection technology based on PCR and isothermal amplification method, most of the nucleic acid detection systems use fluorescence detection, and need professional detection equipment to interpret results, and due to background fluorescence, the interpretation time of negative samples is long; the other uncapping operation after amplification in a non-laboratory environment is easy to cause aerosol pollution; some detection results are not high in visual identification degree, and some detection reagents are easy to interfere and interrupt amplification reaction. These all pose challenges to POCT's requirements of being efficient, accurate, simple and fast.

Disclosure of Invention

The invention provides a micro-fluidic visual field rapid inspection device based on nucleic acid isothermal amplification reaction blocking flow and a detection method thereof, aiming at virus nucleic acid detection, an accurate result can be obtained at the initial stage of infection, a complex temperature control system and nucleic acid inspection equipment are not needed, the safety is ensured by negative pressure and alcohol inactivation in the whole process, a sample reagent is heated in a closed manner at the working temperature of the used isothermal amplification reagent, and the detection result can be visually judged by the color development effect of a chip by giving negative pressure drive at room temperature.

The invention is realized by the following technical scheme:

a micro-fluidic visual field rapid inspection device based on nucleic acid isothermal amplification reaction blocking flow comprises an inlet section, a mixing section, at least one blocking channel 9 and a color development cavity 7, wherein the blocking channel 9 is communicated with the color development cavity 7.

Further, the inlet section comprises 2 inlets 1 and branch pipes 2, each inlet 1 is communicated with one branch pipe 2, 2 branch pipes 2 are communicated with the mixed bent channel 3, and the mixed bent channel 3 is communicated with the blocking channel 9.

Further, the blocking channel 9 comprises a backflow prevention mechanism 4, a pre-assembly inlet 5 and a blocking starting structure 6, wherein each backflow prevention mechanism 4 is arranged inside a starting end channel of a connecting end of the blocking channel 9 and the mixed bending channel 3, a blocking starting structure 6 is arranged inside a channel of a connecting end of the blocking channel 9 and the color developing cavity 7, the pre-assembly inlet 5 is arranged on the blocking channel 9 between the backflow prevention mechanism 4 and the blocking starting structure 6, and each blocking channel 9 is communicated with the color developing cavity 7.

Further, an air pressure outlet 8 is arranged on the color development cavity 7, and the air pressure outlet 8 is connected with a negative pressure device.

Further, the blocking channel (9) is a porous medium channel.

Furthermore, the diameter of the particle substances filled in the precipitation blocking section 6 is 5-30 μm.

Further, the negative pressure device is an injector and a laboratory micro-injection pump.

A method for testing a microfluidic visual field rapid testing device based on nucleic acid isothermal amplification reaction blocking flow comprises the following steps:

step 1: amplifying the positive or negative sample and injecting it into the test device through an inlet (1);

step 2: the ethanol-containing solution is injected into the testing device through another inlet (1);

and step 3: mixing the amplified sample with an ethanol-containing solution in a mixing section to enable the amplified positive sample and the amplified negative sample to present different nucleic acid precipitation effects;

and 4, step 4: injecting particles into the blocking channel (9) through the pre-assembly inlet (5) to form a porous medium micro-channel between the blocking starting structure (6) and the pre-assembly inlet (5), and distinguishing the difference of nucleic acid precipitates by the porous medium micro-channel formed by accumulation of the blocking microbeads on the nucleic acid precipitates in the step 3;

and 5: the difference in precipitation in step 4 was visualized by porous media microchannel for visual identification of negative and positive results of the reaction.

The invention has the beneficial effects that:

1. aiming at virus nucleic acid detection, the invention can obtain an accurate result at the initial stage of infection without a complex temperature control system and nucleic acid detection equipment, and the detection result can be visually judged by the color development effect of the chip only by hermetically heating a sample reagent at the working temperature of the used constant-temperature amplification reagent and driving the sample reagent at the room temperature under negative pressure.

2. In a certain time, the device can complete visual discrimination on-site real-time qualitative nucleic acid detection in a whole closed cover state. Particularly, compared with other existing methods which additionally need to use a high-temperature or ultraviolet irradiation method for inactivation, the method uses a high-concentration ethanol solution to directly inactivate the reagent in the detection process, thereby ensuring better safety in the field test process of the amplification reaction and subsequent pollution-free treatment.

3. The invention has simple additional using devices, mainly comprises a 200 mu L standard test tube for detection and a common medical injector for providing negative pressure.

Drawings

FIG. 1 is a schematic structural diagram of the present invention.

FIG. 2 is a schematic diagram of the structure of two blocking channels of the present invention.

FIG. 3 is a schematic illustration of a blocked channel structure according to the present invention.

FIG. 4 is a schematic diagram of an embodiment of the present invention.

FIG. 5 is a diagram of an experimental test for testing the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Chip of PDMS substrate: the manufacturing method is characterized in that a soft photoetching processing manufacturing mode is adopted, and PDMS is used for bonding with glass finally through glue evening, exposure, development, pouring, stripping and punching.

Chip of PMMA substrate: the manufacturing method of the hot-pressing method is characterized in that the method comprises the steps of exposure, development, fixation, water rinsing, postbaking, chromium corrosion, glass corrosion, photoresist removal and chromium removal of chromium plate glass to manufacture a template, placing the processed PMMA substrate and the template on a workbench of a tablet press for mould pressing, and finally bonding with a PMMA cover plate to complete the manufacturing.

The blocking channel is designed into a slender structure to facilitate blocking, the sum of the cross sections of the channel is not more than 500 μm, and the height is not less than 30 μm. The rear half part of the blocking channel is a blocking section (starting from a blocking initial structure 6), tiny particles with the diameter of 5-30 mu m are filled from an assembly opening in the blocking section, the pre-filling of the blocking section of the channel is realized, the assembly opening is closed after the microspheres are compactly arranged, and the assembly of the detection channel is completed. In this case, the passage allows water and the solution after the amplification reaction of the negative sample to pass through, whereas the solution after the amplification reaction of the positive sample is difficult to pass through due to clogging by the nucleic acid precipitate of the large structure.

As shown in FIGS. 1 to 3, the microfluidic visual field rapid inspection device for blocking flow based on nucleic acid isothermal amplification reaction comprises an inlet section, a mixing section, at least one blocking channel 9 and a developing cavity 7, wherein the blocking channel 9 is communicated with the developing cavity 7.

As shown in fig. 1-3, further, the inlet section includes 2 inlets 1 and branch pipes 2, each of the inlets 1 is communicated with one branch pipe 2, 2 branch pipes 2 are communicated with the mixing curved channel 3, and the mixing curved channel 3 is communicated with the blocking channel 9. The blocking channel 9 comprises an anti-backflow mechanism 4, a pre-assembly inlet 5 and a blocking starting structure 6, wherein each blocking channel 9 is internally provided with the anti-backflow mechanism 4 at the starting end channel of the connecting end of the mixed bending channel 3, each blocking channel 9 is internally provided with the blocking starting structure 6 at the channel connecting end of the blocking channel 9 and the developing cavity 7, the blocking channel 9 between the anti-backflow mechanism 4 and the blocking starting structure 6 is provided with the pre-assembly inlet 5, and each blocking channel 9 is communicated with the developing cavity 7.

As shown in fig. 1-3, further, after the particles are injected from the pre-assembly inlet 5, the particles are blocked by the blocking starting structure 6, and are assembled between the pre-assembly inlet 5 and the blocking starting structure 6 at a position close to the blocking starting structure 6, the gap size of the blocking starting structure 6 is larger than the diameter of the particles, but the liquid can pass through when the nucleic acid precipitation is not enough to form the blocking, so that in the negative case, the indicator can normally pass through the particle assembly section to make the color of the color developing chamber 7 visible to the naked eye.

Further, the blocking channel 9 can be a blocking section based on microchannel blocking, and can be a porous medium channel prepared directly or formed by assembling particles into the channel.

Further, the total width of the cross section of all the blocking channels 9 is not more than 500 μm, and the height is not less than 30 μm.

As shown in fig. 1-3, further, an air pressure outlet 8 is provided on the color development chamber 7, and the air pressure outlet 8 is connected to a negative pressure device.

Further, as shown in fig. 4, the negative pressure device is a syringe or a mechanical micro syringe pump.

A method for testing a microfluidic visual field rapid testing device based on nucleic acid isothermal amplification reaction blocking flow comprises the following steps:

step 1: amplifying a sample to be detected, and connecting the solution after reaction to the chip device through one of the inlet 1 and the catheter;

step 2: the solution containing ethanol is connected to the chip device through a conduit and the other port of the inlet 1;

and step 3: adding equal positive pressure to two ports of the inlet 1, and simultaneously adding negative pressure to the air pressure outlet 8 to drive the amplified sample and the solution containing ethanol to enter a channel, wherein the two solutions can be passively (or actively under the action of vibration) mixed in a mixing section to generate nucleic acid precipitation;

and 4, step 4: nucleic acid precipitation in step 3 is performed by passing the porous medium microchannel between the pre-assembled inlet 5 and the blocking starting structure 6, and the nucleic acid precipitation with different total amount and structure size can block the porous medium microchannel, thereby distinguishing the difference of nucleic acid precipitation. The negative or positive result of the sample detection is visually identified by the color development of the color development chamber 7.

Specifically, 20 μ L of the sample to be tested and the amplification reaction solution are put into a 200 μ L test tube, mixed for 30s by swirling, a 100 μ L syringe is connected to the front end of the liquid supply tube A (as shown in FIG. 4), 20 μ L of oily colored indicator solution is first sucked, 10 μ L of air is then sucked, and finally 20 μ L of reaction system solution is sucked, the oily indicator solution plays a role of sealing the reaction system besides indication, and then the tail end of the liquid supply tube is connected to the chip, and the front-end syringe is disconnected. Immersing the chip and the liquid supply tube into a constant-temperature water bath for heating, ensuring that the chip and the liquid supply tube are completely immersed under the water surface, starting amplification, and heating in the constant-temperature water bath at 63 ℃ for 20 min. After completion, the mixture was cooled at room temperature for 1min, and a 100. mu.L syringe was connected to the tip of the liquid supply tube B (see FIG. 4), and 20. mu.L of the oily colored indicator solution + 10. mu.L of air + 30. mu.L of alcohol were sequentially aspirated, after which the tip of the liquid supply tube was connected to the chip, and the tip syringe was disconnected. And a common medical syringe is used at the outlet of the color development cavity, 8mL of negative pressure is added to drive the liquid in the chip to flow. As a large amount of magnesium pyrophosphate precipitates are generated in the LAMP nucleic acid amplification reaction process, the diameters of the precipitates are within the range of 2-7 mu m. Meanwhile, nucleic acid precipitates can be formed by mixing ethanol with the solution after the amplification reaction, and the precipitates have larger diameters, so that the blockage of positive detection results can be obviously enhanced. Particularly, after the ethanol is introduced, the liquid in the mixing section area is subjected to ultrasonic vibration or vibration mixing, so that the rapid mixing of the ethanol and the reaction system is accelerated, and the nucleic acid precipitation is enhanced. When the reaction system mixed with ethanol flows through the blocked channel section, a plurality of nucleic acid precipitates are generated in the reaction liquid of the positive sample under the action of the ethanol and are accumulated on particles in the channel, so that the flow rate in the channel is remarkably reduced, and the colored oily fluid of the negative sample can rapidly pass through the blocked section and enter the color development cavity. And finally, judging the detection result by depending on whether the upstream oily colored indicating liquid reaches the color development cavity or not, and making an accurate judgment by visual observation. According to the design result, the negative sample can be controlled to flow through the channel to develop color within 5 minutes, and the positive sample does not pass through and does not develop color for not less than 10 minutes, so that the nucleic acid detection result can be visually judged within 30 minutes.

The detection device and the operation are simple, and the field implementation is easy. Amplification can be carried out in a liquid supply tube or a detection tube, and the driving force for the flow in the chip can be either negative pressure operation as described above or positive pressure driving flow at the tip of the liquid supply tube. In addition, in the whole detection process, the reagent is directly inactivated by using a high-concentration ethanol solution under a negative pressure environment, so that the safety of the field test process of the amplification reaction is fully ensured, and the subsequent field harmless treatment is facilitated.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.