Agarose and target nucleic acid detection method based on limited amplification

Abstract

The invention relates to the field of biological detection, in particular to agarose and a target nucleic acid detection method based on limited amplification. And the hydrogel comprises 3-5% (w / v) of agarose. The invention provides a nucleic acid limiting rapid detection method based on high-resolution agarose gel, which can realize rapid detection of naked eye identification and quantification of target nucleic acid without the help of a complex detection instrument. Quicker detection (10-15 minutes) of a sample and lower-cost detection are realized, and meanwhile, the kit has sensitivity and specificity which are equal to those of fluorescent PCR (Polymerase Chain Reaction). Meanwhile, the invention provides a corresponding simple detection device.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection, in particular to agarose and target nucleic acid detection method based on limiting amplification. BACKGROUND

[0002] The common detection methods of microorganisms include culture method, immunization method and nucleic acid detection method. The culture method is the "gold standard" for the detection of many pathogens, but it has the limitations of long operation time and culture and identification of certain species. The antigen detection method such as immunization method has low sensitivity and specificity. Nucleic acid (DNA) detection has high sensitivity and specificity, short sample-to-result time (3 to 5 hours), and is considered as a new gold standard test, becoming the main detection means of microorganisms. The loop-mediated isothermal amplification (LAMP) is widely used in the rapid diagnosis of various pathogens due to its short detection time, high accuracy and low equipment requirement. However, due to the complex primer design and the significant disadvantage of background (non-specific) amplification, the clinical application of LAMP, especially the LAMP based on six primers, is limited by many factors. In addition, the performance of primers is unpredictable.

[0003] In recent years, digital nucleic acid detection technology has developed rapidly. This technology separates single bacterial nucleic acid molecules in individual compartments and carries out independent nucleic acid amplification reactions to identify the absolute accurate concentration of target nucleic acids. Higher specificity and accuracy, better variant analysis capability, and end-point detection and absolute quantification make digital amplification technology expand in various detections.

[0004] The existing digital loop-mediated isothermal amplification method (digital LAMP) based on microfluidic chip array reaction chamber has relatively high design and use cost, limited scalability, and more importantly, the processing process of microfluidic device is too complicated and expensive, and it is difficult to be used once. In addition, this method also has the disadvantages of low bright-dark ratio and easy to be affected by inhibitors. Therefore, it is urgent to develop a digital quantification detection method with stable technology, simple and flexible operation, and low cost to rapidly detect bacteria in real complex samples. SUMMARY

[0005] Therefore, the present application provides agarose and target nucleic acid detection method based on limiting amplification. The present application provides a high-resolution agarose gel-based nucleic acid limiting rapid detection method, which can realize the naked-eye identification and quantitative rapid detection of target nucleic acid without the aid of complex detection instruments. The sample can be detected more quickly (10-15 min), the detection cost is lower, and the sensitivity and specificity are equivalent to those of fluorescent PCR. A simple detection device is also provided.

[0006] To achieve the above object, the present application provides the following technical solutions.

[0007] The present application provides a hydrogel, comprising: 3-5% (w / v) agarose.

[0008] In some embodiments of the present application, the above hydrogel is 4% (w / v) agarose.

[0009] The present application also provides an application of the above hydrogel in detecting target nucleic acid by loop-mediated isothermal amplification.

[0010] The present application also provides a method for detecting target nucleic acid by loop-mediated isothermal amplification, comprising the following steps:

[0011] S1: sample processing: pretreating the sample to be tested;

[0012] S2: component mixing and gel preparation: mixing component B with the sample to be tested, centrifuging, heating, cooling, and mixing with component A;

[0013] S3: reaction: performing loop-mediated isothermal amplification reaction on the mixed components after S2;

[0014] S4: result detection: counting the fluorescence amplification point of the mixture after reaction in S3, and when there is more than one amplicon, the sample to be tested has target nucleic acid; and when there is no amplicon, the sample to be tested has no target nucleic acid;

[0015] The component A comprises: primer group, dNTP and BST enzyme.

[0016] The component B comprises: the above hydrogel, magnesium ion and buffer.

[0017] In some embodiments of the present application, in the above method, the concentration of the hydrogel is 3-5% (w / v).

[0018] In some embodiments of the present application, in the above method, the concentration of the hydrogel is 4% (w / v).

[0019] In some embodiments of the present application, in the above method, the temperature of heating in S2 is 95℃, and the time is 5 min.

[0020] In some embodiments of the present application, in the above method, the temperature of cooling in S2 is 40℃, and the time is 2 min.

[0021] In some embodiments of the present application, in the above method, the time of loop-mediated isothermal amplification reaction in S3 is 5-20 min.

[0022] In some embodiments of the present application, the method described above, the time of the loop-mediated isothermal amplification reaction described in S3 is 15 min.

[0023] In some embodiments of the present application, the method described above, the temperature of the loop-mediated isothermal amplification reaction described in S3 is 65℃.

[0024] In some embodiments of the present application, the method described above, the pretreatment described in S1 adopts a magnetic bead method or a sample release method.

[0025] In some embodiments of the present application, the method described above, the reaction described in S3 is placed in a polypropylene material.

[0026] In some embodiments of the present application, the polypropylene material described in the method described above contains low-melting paraffin.

[0027] In some embodiments of the present application, the method described above, the result detection described in S4 adopts one or more of an ultraviolet LED flashlight, a fluorescence microscope, and a camera.

[0028] In some embodiments of the present application, the method described above, the component A comprises:

[0029] Primer group Final concentration 0.3x

[0030] dNTP Final concentration 1.2mM BST enzyme 8U;

[0031] The component B comprises:

[0032] Hydrogel Final concentration 3-5% (w / v)

[0033] Magnesium ion Final concentration 4.8mM Buffer Final concentration 1x.

[0034] The present application provides a hydrogel, comprising: 3-5% (w / v) of agarose.

[0035] The beneficial effects of the present application include:

[0036] (1) The sample only needs to be treated at 95℃ for 5 minutes, which reduces the sample pretreatment time as the step is combined with the gel preparation, and the treatment effect is close to the magnetic bead method in the agarose limiting loop-mediated isothermal amplification.

[0037] (2) Compared with the microfluidic chip digital PCR, the method of the present application can complete the digital analysis of nucleic acids without complex microfluidic devices and chips, temperature control devices and other high-cost equipment, has the characteristics of simple experimental steps, rapid and efficient, simple operation and low cost.

[0038] (3), the agarose gel used in the application has low cost, common and easy-to-obtain raw materials, and is easy to store, and can directly carry out LAMP in-situ amplification, and the numerous nano-pores in the hydrogel can play a role in isolating organic matter, heavy metals and other inhibitors, so as to realize absolute quantitative analysis of bacteria, and there is no potential risk of sample pollution, and a digital nucleic acid detection technology with low price, flexible and simple operation is provided for bacterial detection in real complex samples.

[0039] (4), has the characteristics of high sensitivity and high specificity.

[0040] (5), the detection reaction only needs to be heated at 65 DEG C for 15 min, and the obvious amplicon can be observed. BRIEF DESCRIPTION OF DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the application or the prior art, the drawings needed to be used in the embodiment or the prior art description will be briefly introduced below.

[0042] Figure 1 Sample positive and negative result interpretation example;

[0043] Figure 2 Agarose limited loop-mediated isothermal amplification detection and fluorescence PCR comparison schematic diagram; wherein: the upper part shows the fluorescence PCR amplification curve; the lower part shows the agarose limited loop-mediated isothermal amplification detection gradient dilution;

[0044] Figure 3 Different agarose gel concentration comparison results;

[0045] Figure 4 Agarose gel results under different amplification times (65 DEG C, 5 min, 10 min, 15 min, 20 min);

[0046] Figure 5 Comparison of agarose gel and PEG hydrogel; wherein: a) shows the PEG hydrogel 15 min incubation result; b) shows the PEG hydrogel 20 min incubation result; c) shows the agarose gel 15 min incubation result; d) shows the agarose gel 20 min incubation result; e shows the PEG hydrogel eight-tube in-tube amplification 25 min product result; f shows the agarose gel eight-tube in-tube amplification 25 min product result;

[0047] Figure 6 Slide incubation chamber;

[0048] Figure 7 Gel reactor device schematic diagram;

[0049] Figure 8 Comparison of different reactor reaction results (camera shooting results). DETAILED DESCRIPTION

[0050] The present application discloses agarose and target nucleic acid detection method based on limiting amplification.

[0051] It should be understood that the expression "one or more of something" includes each of the objects of the recited expression individually as well as various combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in connection with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0052] The use of the terms "including", "has", "have" or "contains", including their grammatical conjugations, should generally be understood to be open-ended, e.g., not excluding additional, unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0053] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the application remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0054] The use of any and all examples, or exemplary language herein, e.g., "such as" or "including", is intended merely to better illustrate the application and does not pose a limitation on the scope of the application unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0055] Further, the numerical ranges and parameters setting forth the broadest scope of the application are approximations, and are only used to convey generally understood precision. Numerical parameters are only approximations of numerical values in the specific examples described herein. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Therefore, unless otherwise specifically indicated, all ranges disclosed herein are approximations, and are understood to be open-ended ranges, encompassing the plain lower and upper values and sub-ranges thereof. In this context, "about" is understood to encompass the plain recited numerical value plus or minus 10%, 5%, 1%, or 0.5% of the numerical value.

[0056] The present application provides a rapid detection method based on agarose limiting loop-mediated isothermal amplification, comprising the following steps:

[0057] (1) Sample processing: the sample to be tested is preliminarily processed.

[0058] (2) Component mixing and gel preparation: combining component B and the sample to form a mixture, heating at 95℃ for 5 minutes for nucleic acid lysis and agarose melting. Adding component A to the mixture after cooling, and fixing the nucleic acid in the mixture at room temperature.

[0059] (3) reaction: the hydrogel system is heated under isothermal conditions to perform the loop-mediated isothermal amplification reaction.

[0060] (4) result detection: the fluorescence amplification points formed in the hydrogel system are counted to detect whether the target nucleic acid exists in the mixture, and the presence or absence of one or more amplicons produced by the LAMP reaction of the DNA / RNA of the target nucleic acid is detected, wherein the presence of the amplicon indicates that the target nucleic acid exists in the sample, and the absence of the amplicon indicates that the target nucleic acid does not exist in the sample.

[0061] Further, the agarose is high-resolution agarose. The high-resolution agarose has a lower melting temperature and gel temperature than conventional agarose, and the gel can be converted into a gel when the temperature reaches above 75℃. Specifically, the high-resolution agarose suspension is agarose powder that swells by absorbing water at room temperature, but does not have enough heat to promote cross-linking between agarose molecules.

[0062] Further, the sol can be quickly dissolved by heating at 95℃ for 5 minutes, and the melting state can be maintained at 40℃.

[0063] Further, the method further comprises quantifying the concentration of the target nucleic acid in the sample based on the number of amplicons detected after the LAMP reaction. Wherein each target microorganism corresponds to one amplicon.

[0064] Further, the method further comprises that the preliminary treatment of the sample to be tested can be thermal lysis and other forms of lysis.

[0065] Further, the concentration of the agarose gel is adjusted to form a predetermined mesh size and a predetermined molecular weight.

[0066] Further, the loop-mediated isothermal amplification reagent includes buffer, dNTP, magnesium ions, Bst polymerase, primers for specific amplification of target nucleic acid molecules, and high-resolution agarose.

[0067] Specifically, the Bst polymerase is a hot-start polymerase, which is activated above 45℃.

[0068] Further, the concentration of the high-resolution agarose is 3-5% high-resolution agarose.

[0069] Further, in step (2), the hydrogel system is placed in a 10mm diameter round bottom, 3mm height flat bottom polypropylene container for isothermal amplification reaction. The polypropylene container contains low melting point paraffin (40℃) in advance. The low melting point paraffin is used to form a liquid sealing film in the constant temperature amplification to prevent water evaporation in the hydrogel, ensuring the accuracy of the detection results. The polypropylene reaction container also contains a sealing film or cover to further prevent evaporation or contamination.

[0070] Further, after the reaction is completed, the paraffin forms a solid and cannot flow at room temperature. The reaction tube is inverted for observation of the fluorescence amplification point. Direct counting analysis is performed using an ultraviolet LED flashlight or a fluorescence microscope.

[0071] In Examples 1 to 8 of the present application, the primers are from Shanghai Shengong Bioengineering Co., Ltd.; the bst enzyme and dNTP in the LAMP reaction system mixture are purchased from Xin Hai Gene Co., Ltd.; and the high-resolution synthetic agarose is purchased from Yuan Ye Biological Co., Ltd. The raw materials and reagents used can be purchased from the market.

[0072] The present application will be further described below in conjunction with examples:

[0073] Example 1 Preparation of a Chlamydia trachomatis nucleic acid rapid detection kit based on agarose limiting

[0074] 1. The primer sequence is as follows:

[0075] Table 1

[0076]

[0077] 2. Primer mix composition

[0078] The synthesized primers are centrifuged at 10000 rpm for 1 min, and an appropriate amount of nuclease-free water (calculated according to the synthesis amount) is added to dissolve to 50 μM, and fully mixed and dissolved at 2-8℃.

[0079] Take a nuclease-free centrifuge tube, and according to the following configuration table, take the amount of single person's primer multiplied by the number of prepared people, and respectively suck each primer into the centrifuge tube, and add nuclease-free water to the specified volume. The prepared 5×primer mix is fully mixed.

[0080] Table 2

[0081]

[0082]

[0083] 3. Reaction component configuration

[0084] The reaction solution is divided into two components, A and B. Component A, which needs to be frozen, contains the primers MIX, dNTP, and Bst enzyme. Component B contains Bst Buffer, magnesium ions, agarose, and SYBR Green. The single-person reagent ratio is shown in Table 3. The reaction system mixture (25 μL) is composed of the following components (final concentration): 1 × Bst Buffer, 4.8 mM magnesium ions, 1.2 mM dNTP, 8 U Bst enzyme, 0.3 × primer MIX, 2 × SybGreen, 1 mg agarose, and 5 μL template DNA.

[0085] Table 3

[0086]

[0087] Example 2: Rapid detection of Chlamydia trachomatis based on agarose restriction loop-mediated isothermal amplification

[0088] 1. Sample processing

[0089] Take out the sample and shake well (frozen samples should be fully dissolved at room temperature before use). Take 500 μL of the sample to be tested, centrifuge at 12000 rpm for 5 minutes. Discard the supernatant and add 50-100 μL of nucleic acid release agent. Vortex until there is no obvious sediment, centrifuge at 7000 rpm for 30 seconds, and use the supernatant for detection.

[0090] 2. Reagent preparation

[0091] Take out components A and B from the kit and place them at room temperature. Mix component A thoroughly after it is completely thawed and set aside. Add 16.3 μL of component B to each reaction tube.

[0092] 3. Component mixing and gel preparation

[0093] Add 5 μL of the sample to be tested to the reaction tube containing component B. Mix well and centrifuge for a few seconds. Heat at 95°C for 5 minutes. Place the mixture on a 40°C heater for 2 minutes or more. Add 3.7 μL of component A and mix well. Add the mixture to the reactor and cover it with an evaporation-proof film at room temperature.

[0094] 4. Reaction

[0095] Place the sample-loaded reactor on the heating instrument and incubate at 65°C for 15 minutes.

[0096] 5. Result detection

[0097] Remove the reactor and cool it to room temperature. Observe the results under a blue light microscope or directly using LED ultraviolet light. Choose the appropriate field of view to save the results.

[0098] 6. Result interpretation (see the example of the result in Figure 1 the figure).

[0099] Negative: no green fluorescence amplification point.

[0100] Positive: contains multiple green fluorescence amplification points (≥1).

[0101] Example 3 Chlamydia trachomatis agarose-based LAMP detection and fluorescence PCR comparison

[0102] The CT plasmid was constructed, 4 μg of plasmid dry powder was diluted with 1000 μL of water to a concentration of 4 ng / μL, and the copy number was calculated by plasmid conversion. The CT sample was valued by establishing a standard curve of gradient plasmid preparation dilution. The valued CT sample was gradient diluted to 2.00+E6 copies / μL, 2.00+E5 copies / μL, 2.00+E4 copies / μL, 2.00+E3 copies / μL, 2.00+E2 copies / μL, 2.00+E1 copies / μL, 2 copies / μL, 1 copies / μL. The corresponding standard curves were made by fluorescence PCR and agarose-based LAMP detection, respectively, and the nucleic acid was added in a volume of 5 μL. The consistency of the results of the two methods was compared.

[0103] The amplification curve of fluorescence PCR is shown in Figure 2 , which has good linearity (R 2 = 0.99914); as Figure 2 shown below, independent amplification points can be observed by agarose-based LAMP at 1 copies / μL to 2.00+E5 copies / μL, and independent amplification points cannot be observed at a concentration of 2.00+E6 copies / μL; at a concentration below 1000 copies / reaction, the consistency of the agarose-based LAMP point count and the fluorescence PCR is higher, and at a concentration above 1000 copies / reaction, the actual count of the amplification point is lower than the theoretical amplification point, so this counting method has high accuracy and reliability below 1000 copies / reaction. There is no difference in the detection limit between the two methods (due to the limitation of the microscope field of view, the amplification points in the figure below are listed within one field of view).

[0104] Example 4 Comparison of different agarose concentrations

[0105] The gel used in this example is high-resolution agarose, which has the following significant improvements compared to traditional agarose:

[0106] (1) Close to the resolution of polyacrylamide gel: the 2%-4% gel resolution can approach the resolution of 4%-8% polyacrylamide gel. Polyacrylamide gel is known for its high resolution, but the operation process is complex and has certain toxicity. High-resolution agarose provides a safer and easier alternative.

[0107] (2) The ability to make higher concentration gels: up to 5% gels can be made, and higher gel concentrations help to improve the resolution of these small fragments. The gels are highly transparent with low background: gels with greater than 85% transparency are easy to view and photograph, and the low background makes the results much clearer.

[0108] The agarose concentration will affect the gel pore size and the permeability of the LAMP amplification components. Different gel concentrations have different resolutions for fragment ranges, and to ensure the ideal cross-linking effect, the agarose concentration needs to be optimized. From the following table, it can be seen that agarose 3% to 5% of agarose may play a better limiting role for LAMP reaction stem loop products, while not hindering the free movement of segment fragments primers. 3%, 4%, 5% agarose concentration were selected for comparison.

[0109] Table 4

[0110] Linear DNA fragment size (bp) 20-250 50-500 100-1200 500-2000 Agarose concentration (%) 5.0 4.0 3.0 2.0

[0111] Agarose concentration screening of agarose limiting: the reagent components in this example use the proportion in Example 1.

[0112] Table 5

[0113]

[0114]

[0115] The amplification results of different agarose concentration systems are shown in Figure 3 . The 3% gel amplification point is larger and appears a certain degree of diffusion, the 4% gel amplification point size is appropriate, and the 5% amplification point is smaller and not easy to distinguish. Select 4% agarose as the best limiting concentration of the system.

[0116] Example 5 Optimal reaction time of loop-mediated isothermal amplification based on agarose limiting

[0117] This example explores the effect of different amplification times on the amplification results, according to Figure 4 It can be seen that the tiny amplification point appears at 5 min, the count of amplification point reaches the maximum at 15 min, and after 15 min, the fluorescence point number does not further increase, only the size of the amplicon becomes larger. 15 min is the optimal reaction time for quantitative monitoring of amplification points in hydrogel system.

[0118] Example 6 Comparison of the effect of different hydrogel limiting systems on the formation of amplification points

[0119] It has been reported that four-arm polyethylene glycol acrylate with a molecular weight of 10000 MW and thiol-polyethylene glycol-thiol with a molecular weight of 3400 MW, at a molar ratio of 1:2, 1.6 mg of four-arm polyethylene glycol acrylate and 1.1 mg of SH-PEG-SH are added to 25 μL of a reaction system, and a PEG hydrogel can be formed by Michael addition at room temperature. This embodiment compares the detection performance difference of PEG hydrogel and agarose gel.

[0120] 1. PEG hydrogel system

[0121] The reaction system mixture (25 μL) is composed of the following components: 1 x Bst Buffer, 4.8 mM magnesium ions, 1.2 mM dNTP, 8 U of Bst enzyme, 0.3 x primer MIX, 2 x Syb Green, 1.6 mg of four-arm PEG acrylate and 1.1 mg of SH-PEG-SH, and 5 μL of template DNA. Nuclease-free water is added to a final volume of 25 μL. The mixture is injected into the incubation chamber on the glass slide, and after uniform distribution, a sealing sheet is covered. The prepared simple gel chip is placed into a small heater and heated at 65°C for 20 min.

[0122] 2. Agarose gel system

[0123] The reaction system mixture (25 μL) is composed of the following components: 1 x Bst Buffer, 4.8 mM magnesium ions, 1.2 mM dNTP, 8 U of Bst enzyme, 0.3 x primer MIX, 2 x Syb Green, 1 mg of agarose, and 5 μL of template DNA. Nuclease-free water is added to a final volume of 25 μL. The mixture is injected into the incubation chamber on the glass slide, and after uniform distribution, a sealing sheet is covered. The prepared simple gel chip is placed into a small heater and heated at 65°C for 20 min.

[0124] The results are shown in Figure 5 (a-d), a) PEG hydrogel 15 min incubation result; b) PEG hydrogel 20 min incubation result; c) agarose gel 15 min incubation result; d) agarose gel 20 min incubation result; e) PEG hydrogel eight-tube in-tube amplification product after 25 min; f) agarose gel eight-tube in-tube amplification product after 25 min.

[0125] Figure 5 The results show that under the same amplification conditions, the agarose gel amplification point is larger. Specifically, there is no significant difference between the agarose amplification point at 15 min and the PEG hydrogel system amplification point at 20 min.

[0126] The two hydrogels were tested on a fluorescent PCR instrument, as shown in the following table. The agarose gel has a faster reaction speed, and the same template can appear 3-5 min earlier. For example Figure 5 (e, f) The PEG hydrogel and agarose gel were amplified in the eight-tube for 20 min. Although the product cannot be accurately counted due to the lack of flat spreading, it can still be observed that the brightness and size of the amplification point of the agarose gel are better than those of the PEG hydrogel.

[0127] Table 6

[0128] Sample name PEG hydrogel (Ct value) Agarose gel (Ct value) Fluorescent PCR (Ct value) Sample 1 8.24 5.18 28.44 Sample 2 13.91 9.24 32.51 Sample 3 16.66 10.44 34.52 Sample 4 17.54 11.65 35.29

[0129] Example 7 Comparison of different sample processing methods

[0130] The sample releasing agent of the present application contains 4% chelex-100 resin and 1% Tween 20, which can remove part of the interfering substances and assist in releasing nucleic acids. By performing 95℃, 5 minutes of thermal lysis to release nucleic acids and melt agarose. This embodiment compares the effects of sample releasing agent and magnetic bead method on sample preparation of agarose confined loop-mediated isothermal amplification.

[0131] a, Sample preparation: 10 CT-positive vaginal swab samples were collected from the clinic and labeled as TQ1-10.

[0132] b, Nucleic acid extraction: The nucleic acid extraction process was strictly in accordance with the instructions of each reagent. The final nucleic acid elution / dissolution volume of all extraction methods was set to 50 μL.

[0133] c, Detection: The extracted nucleic acid was detected by chlamydia trachomatis fluorescent detection reagent, chlamydia trachomatis agarose confined loop-mediated isothermal amplification reagent in example 1 and QPCR respectively.

[0134] 1, Magnetic bead sample processing:

[0135] Take out the sample and shake well (frozen samples should be fully dissolved at room temperature before use), take 1000 μL of the sample to be tested, centrifuge at 12000 rpm for 5 minutes. Remove 800 μL of supernatant. The remaining 200 μL is extracted by magnetic beads.

[0136] 2, Sample releasing agent sample processing: refer to example 2:

[0137] The detection results are as follows:

[0138] Table 7

[0139]

[0140]

[0141] The fluorescent PCR can detect all samples (40 holes in total), and the sample processing method of magnetic beads is better than the release agent. Both the magnetic beads method and the sample release agent can detect all samples in the agarose limiting LAMP, and there is no significant difference in the amplification CT value, indicating that the agarose limiting LAMP system has certain anti-interference ability and can stably detect the preliminary processed samples.

[0142] Example 8: Comparison of detection effects of different reactors

[0143] The reaction system mixture (25 μL) is composed of the following components: 1 × Bst Buffer, 4.8 mM magnesium ion, 1.2 mM dNTP, 8 U Bst enzyme, 0.3 × primer MIX, 2 × SybGreen, 1 mg agarose, and 5 μL template DNA. On this basis, add nuclease-free water to a final volume of 25 μL.

[0144] 1. Slide incubation chamber Figure 6

[0145] Purchase 3M VHB or PET double-sided tape with a thickness of 0.25 mm, and use a punch to make a 10 mm diameter circular incubation chamber. Pre-bond the glass slide with the double-sided tape to make a simple chip. Inject the mixture into the incubation chamber on the glass slide, and then cover it with a sealing sheet. The prepared simple gel chip is placed in a heater at 65°C for 15 min.

[0146] 2. Gel reactor

[0147] Use the gel reactor in Figure 7 , which contains low-melting-point paraffin (40°C) in advance. Inject the mixture into the gel reactor, and then cover it with a PCR pressure-sensitive film. Place the prepared gel reactor in a heater at 65°C for 15 min.

[0148] As shown in Figure 8 , there is no significant difference between the slide incubation chamber and the gel reactor. When the film is covered, air bubbles are easily formed, which can affect visual observation. In the gel reactor, low-melting-point paraffin is used to prevent evaporation, and the PCR pressure-sensitive film does not directly contact the LAMP reaction solution, so air bubbles are not easily formed. Due to the difference in material between the two reactors, the brightness of the amplification point in the gel reactor is more obvious, which is convenient for observation in practical application.

[0149] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered as the protection scope of the present application.​

Claims

1. A hydrogel, characterized in that, The application relates to a water gel and a preparation method thereof. 3-5% (w / v) agarose.

2. The application of the water gel in claim 1 in loop-mediated isothermal amplification detection of target nucleic acid.

3. A method for detecting a target nucleic acid by loop-mediated isothermal amplification, characterized by, The method comprises the following steps: S1: sample processing: pretreatment of the sample to be tested; S2: component mixing and gel preparation: mixing of component B with the sample to be tested, centrifugation, heating, cooling, and mixing with component A; S3: reaction: loop-mediated isothermal amplification reaction of the mixed components after S2; S4: result detection: fluorescence amplification point recognition counting of the mixture after the reaction in S3, if more than one amplicon appears, the sample to be tested contains target nucleic acid; if no amplicon appears, the sample to be tested does not contain target nucleic acid; The component A comprises: a primer group, dNTP and BST enzyme; The component B comprises: the water gel in claim 1, magnesium ions and buffer.

4. The method of claim 3, wherein, The concentration of the water gel is 3-5% (w / v).

5. The method of claim 3 or 4, wherein, The heating temperature in S2 is 95 DEG C, and the time is 5 min.

6. The method according to any one of claims 3 to 5, characterized in that, The cooling temperature in S2 is 40 DEG C, and the time is 2 min.

7. The method according to any one of claims 3 to 6, characterized in that, The loop-mediated isothermal amplification reaction time in S3 is 5-20 min.

8. The method according to any one of claims 3 to 7, characterized in that, The pretreatment in S1 adopts a magnetic bead method or a sample release method.

9. The method according to any one of claims 4 to 8, characterized in that, The reaction in S3 is placed in a polypropylene material.

10. The method of claim 9, wherein, The polypropylene material contains low-melting-point paraffin.

11. The method according to any one of claims 4 to 10, characterized in that, The result detection in S4 adopts one or more of an ultraviolet LED flashlight, a fluorescence microscope and a camera.

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