Amplification reagent and target nucleic acid detection method based on limited amplification

Abstract

The invention relates to the field of nucleic acid detection, in particular to an amplification reagent and a target nucleic acid detection method based on limited amplification. The invention provides a non-diagnostic target nucleic acid detection method. The non-diagnostic target nucleic acid detection method comprises the following steps: treating a sample; constructing a system; performing reaction; detecting a result; the hydrogel is obtained by chemical crosslinking of eight-arm polyethylene glycol acrylate and sulfydryl-polyethylene glycol-sulfydryl. The invention provides a rapid detection method based on hydrogel limiting and application. Quantitative rapid detection of target nucleic acid can be realized. The method combines the advantages of short PCR isothermal amplification reaction time, low equipment requirement and hydrogel impurity adsorption. Rapid detection is completed under the condition of crude extraction of a sample, the detection time is greatly shortened, and the kit has sensitivity and specificity which are equal to those of fluorescent PCR. According to the invention, a hydrogel loop-mediated isothermal amplification (LAMP) reagent is subjected to productization conversion, the product form is determined, and meanwhile, a suggested detection device is provided.

Description

[0001] The present application claims priority to the Chinese patent application No. 202410477330.3, filed on April 19, 2024, and entitled "Amplification reagent and target nucleic acid detection method based on limiting amplification", the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of nucleic acid detection, and in particular to an amplification reagent and a target nucleic acid detection method based on limiting amplification. BACKGROUND

[0003] In recent years, digital nucleic acid detection technology has developed rapidly. This technology separates single nucleic acid molecules into individual compartments and performs 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 microfluidic device processing process is too cumbersome and expensive, difficult to use once, and this method also has low bright-dark ratio, is easily affected by inhibitors, and other shortcomings. Therefore, it is urgent to develop a digital quantification detection method with stable technology, simple and flexible operation, and low cost to quickly detect bacteria and viruses in real complex samples. SUMMARY

[0005] Therefore, the present application provides an amplification reagent and a target nucleic acid detection method based on limiting amplification. The present application provides a rapid detection method based on hydrogel limiting and application, which can realize the quantitative rapid detection of target nucleic acids. The present application combines the advantages of short PCR constant temperature amplification reaction time, low equipment requirement, and hydrogel adsorption of impurities. The rapid detection is completed under the condition of sample crude extraction, which greatly reduces the detection time, and has the same sensitivity and specificity as fluorescence PCR. The present application productizes the hydrogel LAMP reagent, determines the product form, and provides a suggested detection device.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0007] The present application provides a detection method of target nucleic acid for non-diagnostic purposes, comprising the following steps:

[0008] S1: sample processing: lysing the sample to be tested;

[0009] S2: System construction: mix the loop-mediated isothermal amplification reagent, hydrogel and the lysed sample to be tested;

[0010] S3: Reaction: cross-linking reaction and loop-mediated isothermal amplification reaction are performed on the mixed components in S2;

[0011] S4: Result detection: whether the target nucleic acid exists in the sample to be tested is determined by detecting whether an amplicon exists in the reacted components in S3. If one or more amplicons exist, the target nucleic acid exists in the sample to be tested. If no amplicon exists, the target nucleic acid does not exist in the sample to be tested.

[0012] The hydrogel is obtained by chemical cross-linking of eight-arm polyethylene glycol acrylate and thiol-polyethylene glycol-thiol.

[0013] The loop-mediated isothermal amplification reagent comprises a primer set, dNTP, magnesium ions, a polymerase, TCEP, a buffer and a freeze-drying protective agent.

[0014] In some embodiments of the present application, the detection in the above detection method S4 comprises quantifying the concentration of the target nucleic acid in the sample based on the number of amplicons.

[0015] In some embodiments of the present application, the hydrogel and the loop-mediated isothermal amplification reagent in the above detection method are divided into two components A and B. Component A comprises a primer set, dNTP, Bst polymerase, TCEP, eight-arm polyethylene glycol acrylate and freeze-drying protective agent A. Component B comprises a buffer, magnesium ions, a dye, TCEP, thiol-polyethylene glycol-thiol and freeze-drying protective agent B.

[0016] The substances contained in component A are bioactive substances and eight-arm polyethylene glycol acrylate, and the freeze-drying protective agent A mainly provides enzyme activity protection. The substances contained in component B are mainly enzyme activity affecting substances and thiol-polyethylene glycol-thiol, and the freeze-drying protective agent B and TCEP mainly provide antioxidant protection for thiol-polyethylene glycol-thiol.

[0017] In some embodiments of the present application, the lysing in the above detection method comprises one or more of a magnetic bead method, a thermal lysing method and / or a sample release method.

[0018] In some embodiments of the present application, the reaction in the above detection method S3 is placed in any one of a glass slide, a polypropylene material container and a polyacrylate material container.

[0019] In some embodiments of the present application, the result detection in the above detection method S4 uses a smartphone camera or a fluorescence microscope to detect whether the amplicon exists.

[0020] In some embodiments of the present application, the molecular weight of the eight-arm polyethylene glycol acrylate in the above detection method is 10-40K; the molecular weight of the mercapto-polyethylene glycol-mercapto is 2-6K.

[0021] In some embodiments of the present application, the molecular weight of the eight-arm polyethylene glycol acrylate in the above detection method is 20K; the molecular weight of the mercapto-polyethylene glycol-mercapto is 4K.

[0022] In some embodiments of the present application, the molar ratio of the eight-arm polyethylene glycol acrylate to the mercapto-polyethylene glycol-mercapto in the above detection method is 1:4.

[0023] In some embodiments of the present application, the concentration of the eight-arm polyethylene glycol acrylate in the above detection method is 1.8-2.75mM; the concentration of the mercapto-polyethylene glycol-mercapto is 7.2-11mM.

[0024] In some embodiments of the present application, the concentration of the eight-arm polyethylene glycol acrylate in the above detection method is 2-2.5mM; the concentration of the mercapto-polyethylene glycol-mercapto is 8-10mM.

[0025] In some embodiments of the present application, the concentration of the eight-arm polyethylene glycol acrylate in the above detection method is 2.5mM; the concentration of the mercapto-polyethylene glycol-mercapto is 10mM.

[0026] In some embodiments of the present application, the concentration of the primer set in the above detection method is 0.1-1×.

[0027] In some embodiments of the present application, the concentration of the primer set in the above detection method is 0.2× or 0.4×.

[0028] In some embodiments of the present application, the concentration of the primer set in the above detection method is 0.2×.

[0029] In some embodiments of the present application, in the 0.2× primer set in the above detection method, a mixture with the concentration of 0.8μM FIP and BIP, 0.1μM F3 and B3, 0.4μM LF and LB is included; without target DNA, no LAMP amplification will produce a fluorescent signal. During the LAMP amplification process, the dye is embedded in the product to release a fluorescent signal to form an amplification point.

[0030] In some embodiments of the present application, the concentration of the dNTP in the above detection method is 0.8-1.4mM.

[0031] In some embodiments of the present application, in the above detection method, the concentration of the dNTP is 1 mM or 1.2 mM.

[0032] In some embodiments of the present application, in the above detection method, the concentration of the dNTP is 1 mM.

[0033] In some embodiments of the present application, in the above detection method, the concentration of the magnesium ion is 3-6.5 mM.

[0034] In some embodiments of the present application, in the above detection method, the concentration of the magnesium ion is 3-4 mM.

[0035] In some embodiments of the present application, in the above detection method, the concentration of the magnesium ion is 4 mM.

[0036] In some embodiments of the present application, in the above detection method, the concentration of the polymerase is 8-12.8 U.

[0037] In some embodiments of the present application, in the above detection method, the concentration of the Bst polymerase is 9.6 U.

[0038] In some embodiments of the present application, in the above detection method, the concentration of the TCEP is 1-4 mM.

[0039] In some embodiments of the present application, in the above detection method, the concentration of the TCEP is 1-2 mM.

[0040] In some embodiments of the present application, in the above detection method, the concentration of the TCEP is 2 mM.

[0041] In some embodiments of the present application, in the above detection method, the freeze-drying protective agent comprises: 12.5 g / L mannitol, 4 g / L BSA, 15 g / L trehalose, 15 mM glycine, 10 g / L cyclodextrin, 20 g / L pullulan.

[0042] In some embodiments of the present application, in the above detection method, the freeze-drying protective agent comprises: freeze-drying protective agent A and freeze-drying protective agent B; the freeze-drying protective agent A comprises: 12.5 g / L mannitol, 4 g / L BSA, 15 g / L trehalose, 15 mM glycine, 10 g / L cyclodextrin;

[0043] The freeze-drying protective agent B comprises: 20 g / L pullulan.

[0044] In the component A of the present application, mannitol and eight-arm polyethylene glycol acrylate have a plasticizing effect in the freeze-drying process, and BSA, trehalose, glycine and cyclodextrin can synergistically stabilize the protein structure and maintain the activity of the enzyme. Glycine can also maintain the stability of the pH value during freeze-drying and reconstitution.

[0045] In the component B of the present application, there is no bioactive ingredient, and the thiol-polyethylene glycol-thiol can not only form a gel network, but also act as a plasticizer in the freeze-drying process. TCEP in component B can effectively reduce and stabilize the thiol (-SH) group, preventing its oxidation into a disulfide bond (-S-S-). The pullulan in the freeze-drying protectant B can form a stable protective film to prevent oxygen and other oxidizing agents from contacting the thiol-polyethylene glycol-thiol, thereby reducing oxidation and degradation.

[0046] In some embodiments of the present application, the freeze-drying protectants A and B are respectively added to components A and B in an amount of 5 μL.

[0047] In some embodiments of the present application, in the above detection method, the time of the loop-mediated isothermal amplification reaction is 15-20 min.

[0048] The present application also provides an amplification reagent, which comprises: a hydrogel, a primer set, dNTP, magnesium ions, a polymerase, TCEP, a buffer and a freeze-drying protectant.

[0049] The hydrogel is obtained after chemical cross-linking of eight-arm polyethylene glycol acrylate and thiol-polyethylene glycol-thiol.

[0050] The molar ratio of the eight-arm polyethylene glycol acrylate to the thiol-polyethylene glycol-thiol is 1:4.

[0051] The present application also provides a kit, which comprises: the above amplification reagent and acceptable adjuvants, carriers and / or devices.

[0052] The present application also provides the use of the above amplification reagent and / or the above kit in the detection of target nucleic acids.

[0053] The present application provides a method for detecting target nucleic acids for non-diagnostic purposes, which comprises the following steps:

[0054] S1: sample processing: lysing the sample to be tested;

[0055] S2: system construction: mixing the loop-mediated isothermal amplification reagent, the hydrogel and the lysed sample to be tested;

[0056] S3: reaction: cross-linking and loop-mediated isothermal amplification of the mixed components in S2;

[0057] S4: result detection: detecting whether the target nucleic acid exists in the component after the reaction in S3, if one or more amplicons appear, the target nucleic acid exists in the sample to be tested; if no amplicon appears, the target nucleic acid does not exist;

[0058] The hydrogel is obtained after chemical cross-linking of eight-arm polyethylene glycol acrylate and thiol-polyethylene glycol-thiol.

[0059] The loop-mediated isothermal amplification reagent comprises a primer set, dNTP, magnesium ions, a polymerase, TCEP, a buffer and a freeze-drying protective agent.

[0060] The beneficial effects of the present application include:

[0061] (1) The sample only needs to be coarsely treated: the preliminary lysis can be thermal lysis (95 DEG C, 5 minutes) and direct lysis of the sample (room temperature, 5 minutes), the sample pretreatment time is short, and the sample treatment effect is close to that of the magnetic bead method.

[0062] (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 simple experimental steps, is fast and efficient, is simple to operate, and is low in cost.

[0063] (3) The hydrogel used in the present application is low in cost, easy to obtain and store, can be directly subjected to LAMP in-situ amplification, and the numerous nanopores in the hydrogel can play a role in isolating inhibitors such as organic matter and heavy metals, so that absolute quantitative analysis of bacteria can be realized, there is no potential risk of sample contamination, and a digital nucleic acid detection technology which is low in price, flexible and simple in operation is provided for bacterial detection in real complex samples.

[0064] (4) The reagent of the present application can be freeze-dried and stored at room temperature, and has the characteristics of high sensitivity and high specificity.

[0065] (5) No gel polymerization step is needed in the detection process, and the gel polymerization step and the LAMP amplification step can be carried out synchronously.

[0066] (6) The detection reaction provided by the present application only needs to be heated at 65 DEG C for 15-20 minutes to observe obvious amplicons. BRIEF DESCRIPTION OF DRAWINGS

[0067] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below.

[0068] Figure 1 Flowchart of an exemplary target nucleic acid;

[0069] Figure 2 Example of sample positive and negative result interpretation;

[0070] Figure 3 Example of gel restriction and fluorescent PCR method on sample; Wherein: The upper fluorescent PCR method amplification curve; The lower water gel restriction loop-mediated isothermal amplification detection gradient dilution;

[0071] Figure 4 Example of different molecular weight water gel monomer on chlamydia trachomatis LAMP test to produce exemplary amplicon image;

[0072] Figure 5 Example of magnesium ion concentration optimization;

[0073] Figure 6 Example of dNTP concentration optimization;

[0074] Figure 7 Example of primer set concentration optimization;

[0075] Figure 8 Example of bst enzyme concentration optimization;

[0076] Figure 9 Example of TCEP concentration optimization;

[0077] Figure 10 Example of water gel concentration optimization;

[0078] Figure 11 Example of the results of the exemplary experiment, showing the effect of reaction time and on the results of the example of the LAMP method in the gel;

[0079] Figure 12 Example of freeze-dried form comparison;

[0080] Figure 13 Example of fluorescence chart after freeze-dried reconstitution;

[0081] Figure 14 Example of the effect of freeze-drying protectants on restriction amplification;

[0082] Figure 15 Example of freeze-drying stability test. DETAILED DESCRIPTION

[0083] The present application discloses amplification reagents and target nucleic acid detection methods based on restriction amplification.

[0084] It should be understood that the expression "one or more of" includes each of the objects recited after the expression and various different combinations of two or more of the recited objects, individually, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0085] The use of the terms "including", "containing", "having" or "comprising" and variations thereof throughout this disclosure is meant to encompass the items listed thereafter and equivalents thereof as well as additional items not specifically listed. Such terms are intended to be open-ended and, in addition to the explicitly recited elements, encompassing those that are alike, equivalent, or similar.

[0086] 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.

[0087] The use of any and all examples, or exemplary language herein, for example, is intended merely to better illuminate 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.

[0088] 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 some example embodiments. 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, comprising the recited starting and ending points. The language "approximately" or "about" preceding a recited numerical value or range thereof is used herein to introduce a range of values that, when measured experimentally, would be expected to include the recited value or range, but which can include values outside of the recited value or range due to the inherent inaccuracy present in all experimental measurements.

[0089] The present application provides a method for target nucleic acid based on hydrogel confinement, the method comprising:

[0090] (1) sample processing: the sample to be tested is subjected to preliminary lysis release;

[0091] (2) system construction: combining reaction components A / B and the sample to form a mixture, the polymer gel is used to fix the nucleic acid in the mixture;

[0092] (3) reaction: placing the hydrogel system under isothermal conditions to simultaneously perform cross-linking reaction and loop-mediated isothermal amplification reaction;

[0093] Result detection: identifying and counting the fluorescence amplification points formed in the hydrogel system, detecting whether the target nucleic acid exists in the mixture, detecting the presence or absence of one or more amplicons produced by the LAMP reaction of the DNA / RNA of the target nucleic acid, wherein the presence of the amplicon indicates the presence of the target nucleic acid in the sample, and the absence of the amplicon indicates the absence of the target nucleic acid in the sample.

[0094] Specifically, 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.

[0095] Specifically, wherein each of the target microorganisms corresponds to one amplicon.

[0096] Specifically, wherein the target nucleic acid is DNA / RNA.

[0097] Specifically, the method further comprises that the preliminary treatment of the sample to be tested can be heat lysis and sample release agent lysis.

[0098] Specifically, the method further comprises that the polymer gel polymerization and LAMP amplification can incubate the mixture synchronously.

[0099] Specifically, the method further comprises using a smartphone camera or a fluorescence microscope to visually detect the presence or absence of amplicons in the reactor.

[0100] Specifically, wherein the cross-linking of the polymer gel is adjusted to form a predetermined mesh size and a predetermined molecular weight between the cross-linking agents in the polymer gel.

[0101] Specifically, the loop-mediated isothermal amplification reagent comprises Bst Buffer, dNTP, magnesium ions, Bst polymerase, dye, TCEP, and primers specific to the target nucleic acid molecule required for amplification.

[0102] Further, the reaction system is divided into components A / B: component A includes primer group 0.2x~0.4x, Bst polymerase 9.6~11.2U, dNTP 1~1.2mM, 2mM~2.75mM eight-arm polyethylene glycol acrylate, TCEP 1mM, freeze-dried protectant A 5μL. Component B includes Bst Buffer 1x, magnesium ions 3~4mM, 8mM~11mM thiol-polyethylene glycol-thiol, dye 2x, TCEP 1mM, freeze-dried protectant B 5μL.

[0103] Further, the 0.2x primer mixture is a mixture with a final concentration of 0.8μM FIP and BIP, 0.1μM F3 and B3, and 0.4μM LF and LB. No fluorescence signal is generated in the absence of target DNA. During the LAMP amplification process, the fluorescence dye is embedded in the amplification product to release the fluorescence signal to form the amplification point.

[0104] Further, the hydrogel monomer can be eight-arm polyethylene glycol acrylate and HS-PEG-SH. Further, the eight-arm polyethylene glycol acrylate has a subweight of 20000, and the HS-PEG-SH has a subweight of 4000.

[0105] Further, the concentration of the eight-arm polyethylene glycol acrylate is 2-2.5 mM; and the concentration of the mercapto-polyethylene glycol-mercapto is 4 times that of the eight-arm polyethylene glycol acrylate.

[0106] In the embodiments 1-9 of the present application, the raw materials and reagents used are commercially available.

[0107] The present application is further described below in conjunction with the embodiments:

[0108] Embodiment 1: Preparation of a Chlamydia trachomatis loop-mediated isothermal amplification detection reagent based on hydrogel limiting

[0109] The dNTP and primers used in the embodiments are from Shanghai Shengong Bioengineering Co., Ltd.; the Bst polymerase in the LAMP reaction system mixture is purchased from Xin Hai Gene, the eight-arm PEG acrylate is purchased from Shanghai Tao Xiang, and the mercapto-PEG-mercapto is purchased from Xi'an Kaixin.

[0110] 1. The primer sequence is as follows:

[0111]

[0112]

[0113] 2. The primer group composition

[0114] The synthesized primers and probes 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 them into 100 μM, and they are fully mixed and dissolved at 2-8℃.

[0115] Take a nuclease-free centrifuge tube, and take the amount of single person's primer probe according to the following configuration table, multiply the preparation person's number, and respectively suck each primer probe into the centrifuge tube, and add nuclease-free water to the specified volume. The prepared primer group is fully mixed.

[0116] Table 2

[0117] 10x Primer Set 1 serving volume CT-5-F3 (100 μΜ) 0.05 CT-5-B3 (100 μΜ) 0.05 CT-5-FIP-1 (100 μΜ) 0.4 CT-5-BIP-2 (100 μΜ) 0.4 CT-5-LF (100 μΜ) 0.2 CT-7-F3 (100 μΜ) 0.05 CT-7-B3 (100 μΜ) 0.05 CT-7-FIP (100 μΜ) 0.4 CT-7-BIP-2 (100 μΜ) 0.4 CT-7-LB (100 μΜ) 0.2 Water Supplemented to 3 μL Total 3

[0118] 3. Reaction component configuration

[0119] The kit contains reagent components A and B, and the concentration of each component is calculated based on a 30 μL reaction system. The reagent component A contains dNTP, primer group, Bst enzyme, eight-arm polyethylene glycol acrylate, TCEP, freeze-drying protective agent A and water. The final concentration of dNTP in A component is 1 mM, the final concentration of primer group is 0.2x, the final concentration of Bst enzyme is 9.6 U / reaction, the final concentration of eight-arm polyethylene glycol acrylate (8Arm-PEG-AC, Mw=20000) is 2.5 mM, the final concentration of TCEP is 1 mM, and the amount of freeze-drying protective agent A added is 5 μL.

[0120] B component contains Bst Buffer, magnesium ion, TCEP, bis-thiol polyethylene glycol, freeze-drying protective agent B and water, in B component, Bst Buffer final concentration is 1x, magnesium ion final concentration is 4mM, SYBR Green final concentration is 2x, TCEP final concentration is 1mM, bis-thiol polyethylene glycol (SH-PEG-SH, Mw=4000) concentration is 10mM, freeze-drying protective agent B is 5μL.

[0121] Table 3

[0122]

[0123]

[0124] 10x Bst Buffer is mixed by betaine, KCl, Tris-HCl, (NH4)2SO4 according to the mass ratio of 50-100:2-5:2-5:1-2:0.8, and the pH value of Tris-HCl is 7.5-8.8.

[0125] 100x SYBR Green dye is obtained by diluting commercially available 10000x SYBR Green I dye by 100 times.

[0126] 0.5mg / μL eight-arm polyethylene glycol acrylate (8Arm-PEG-AC, Mw=20000) and 0.5mg / μL bis-thiol polyethylene glycol (SH-PEG-SH, Mw=4000) are obtained by constant volume in DEPC water from solid powder.

[0127] Freeze-drying protective agent A: 12.5g / L mannitol, 4g / L BSA, 15g / L trehalose, 15mM glycine, 10g / L cyclodextrin.

[0128] Freeze-drying protective agent B: 20g / L pullulan.

[0129] Components A / component B are freeze-dried in light-proof containers, respectively.

[0130] Example 2 A rapid detection of Chlamydia trachomatis based on hydrogel limiting loop-mediated isothermal amplification

[0131] 1. Sample processing

[0132] 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 in a 1.5 mL centrifuge tube (if the secretion is more or the sample is turbid, it is recommended to centrifuge at 3000 rpm for 30 s before taking), centrifuge at 12000 rpm for 5 min, carefully remove the supernatant, add 100 μL of sample release agent, vortex well until there is no obvious precipitate, stand at room temperature for 5 min, centrifuge at 7000 rpm for 30 s, and the supernatant is used for detection.

[0133] 2. Reagent preparation

[0134] Take out components A / B in the kit in Table 3. Reconstitute according to 10 μL per reaction, and add 10 μL of reconstituted component A at the bottom of the tube and 10 μL of component B on the tube cap in each reaction tube.

[0135] 3. Sample addition

[0136] Add 10 μL of the sample to be tested to the prepared reagent reaction tube, mix and centrifuge for a few seconds, and then add the mixture to the reactor and cover it with an evaporation-proof film.

[0137] 4. Incubation

[0138] Place the sample-added reactor on the heating instrument and incubate at 65°C for 15 mins.

[0139] 5. Result detection

[0140] Take out the reactor, invert it and place it under the electron microscope to observe the results under blue light, or directly observe the results using LED ultraviolet light. Choose the appropriate field of view to save the results. (See the detection process diagram in Figure 1 )

[0141] 6. Result interpretation (see the result example diagram in Figure 2 )

[0142] Negative: No green fluorescent amplification point.

[0143] Positive: Contains multiple green fluorescent amplification points (≥1).

[0144] Example 3 Comparison of loop-mediated isothermal amplification and fluorescence PCR detection based on molecular positioning

[0145] CT plasmid construction, 4 μg plasmid dry powder + 1000 μL water dilution to a concentration of 4 ng / μL, by plasmid conversion copy number, gradient plasmid preparation dilution to establish a standard curve CT sample value. The value of the CT sample gradient dilution to 1.00+E6 copies / μL, 1.00+E5 copies / μL, 1.00+E4 copies / μL, 1.00+E3 copies / μL, 1.00+E2 copies / μL, 1.00+E1 copies / μL, 1 copies / μL, 0.4 copies / μL. Respectively, using fluorescent PCR method and water gel limited loop-mediated isothermal amplification detection corresponding to the standard curve, the volume of nucleic acid added is 10 μL, and the consistency of the results of the two methods is compared.

[0146] The amplification curve of the fluorescent PCR method is shown in the upper panel of Figure 3 , which has a good linear relationship (R 2 = 0.99914); as shown in the lower panel of Figure 3 , the water gel limited loop-mediated amplification can observe independent amplification points at 4 copies / reaction ~ 1.00+E6 copies / reaction, and cannot observe independent amplification points at 1.00+E7 copies / reaction; at a concentration of 1000 copies / reaction or less, the water gel limited loop-mediated amplification point count has high consistency with the fluorescent PCR, and at a concentration of 1000 copies / reaction or more, the actual count amplification point is lower than the theoretical amplification point, so this counting method has high accuracy and reliability at 1000 copies / reaction or less.

[0147] The process of chlamydia trachomatis fluorescent detection is as follows:

[0148] 1. Reagent preparation (reagent preparation area)

[0149] (1) Take out each component of the chlamydia trachomatis fluorescent detection kit, place it at room temperature, and mix it thoroughly after complete thawing for standby use.

[0150] (2) Determine the reaction number N, N = the number of samples to be detected (n) + the number of quality control products (2). It is recommended to analyze positive quality control and negative quality control at the same time for each PCR experiment.

[0151] (3) Divide the CT PCR reaction solution into 15 μL / tube into PCR reaction tubes.

[0152] 2. Sample processing and sample addition (sample processing area)

[0153] (1) Sample pretreatment

[0154] Add 1 mL of normal saline to the sample collection tube, shake well, and then pour all the liquid into a 1.5 mL centrifuge tube (discard the swab after squeezing it against the wall of the centrifuge tube), as the sample to be tested.

[0155] (2) Sample processing

[0156] Take out the sample and shake well (frozen samples should be fully dissolved at room temperature before use), take 200 μL of the sample to be tested into a 1.5 mL centrifuge tube (if there is a lot of secretion or the sample is turbid, it is recommended to centrifuge at 3000 rpm for 30 s before taking it up), and use the nucleic acid extraction or purification reagent produced by Aibisheng Technology Co., Ltd. to process the sample. The specific operation is carried out according to the instructions of the kit. The quality control samples in this kit are involved in sample processing.

[0157] (3) Sample addition: Add 10 μL of the sample DNA to be tested, negative quality control, and positive quality control, respectively, to the PCR reaction tube prepared with reagents, cover the tube cap tightly, and centrifuge at a low speed for 5 s.

[0158] 3. PCR amplification (amplification zone) (please refer to the instructions of each instrument for setting)

[0159] (1) Sample setting: Place the PCR reaction tube into the sample slot of the PCR instrument, and set the types and numbers of the samples to be tested, negative quality control, and positive quality control according to the corresponding order.

[0160] (2) Fluorescence channel selection: Select CY5 (internal reference gene), JOE (Chlamydia trachomatis), and 4 channels. Quencher is set to none, and Passive Reference is set to none. Set the sample volume to 25 μL.

[0161] Table 4 Reaction condition setting

[0162]

[0163] (3) Save the file and run the reaction program.

[0164] Example 4

[0165] PEG hydrogel is a kind of gel material insoluble in water formed by cross-linking of hydrophilic polymers. After cross-linking, the polymer aqueous solution will form a hydrogel with a porous structure. By adjusting the molecular weight parameters of the polymer, the pore size and the physicochemical properties of the gel can be controlled.

[0166] The configuration method of the hydrogel system with different molecular weights is as follows: HS-PEG-SH and 8Arm-PEG-AC are variables, and other components are all configured according to Table 3. In 30 μL system, the specific combination of different molecular weights of 8Arm-PEG-AC and HS-PEG-SH is shown in the following table, and the corresponding results after 20 min of reaction are shown in Figure 4

[0167] Table 5 Combination condition setting

[0168]

[0169] As shown in Figure 4 , under the condition that the final concentration of 8Arm-PEG-AC is 2.5 mM and the final concentration of HS-PEG-SH is 10 mM, after 20 min of reaction. The hydrogel combination of HS-PEG-SH-2K and 8Arm-PEG-AC-10K has no amplification point, the hydrogel combination of HS-PEG-SH-4K and 8Arm-PEG-AC-10K has very small amplification point and the number of amplification points is small; the hydrogel combination of HS-PEG-SH-5K / HS-PEG-SH-6K and 8Arm-PEG-AC-10K also has small amplification point.

[0170] In the hydrogel combination of different molecular weights of HS-PEG-SH and 8Arm-PEG-AC-20K, the hydrogel combination of HS-PEG-SH-2K and 8Arm-PEG-AC-20K has small amplification point, the hydrogel combination of HS-PEG-SH-4K and 8Arm-PEG-AC-20K has moderate size, and the hydrogel combination of HS-PEG-SH-5K / 6K and 8Arm-PEG-AC-20K has large amplification point. In the hydrogel combination of different molecular weights of HS-PEG-SH and 8Arm-PEG-AC-20K, the amplification point is large.

[0171] Figure 4 ​The results show that the size of the amplification dots can be controlled by adjusting the molecular weight of HS-PEG-SH and 8Arm-PEG-AC within a certain range, and the effect on the amplification brightness is small. Smaller monomer molecular weight of HS-PEG-SH and 8Arm-PEG-AC forms smaller gel pore size, which may even limit the diffusion of LAMP amplification reagents and affect the sensitivity, resulting in a lower number of amplification dots or no amplification (such as the hydrogel combination of HS-PEG-SH-2K / 4K and 8Arm-PEG-AC-10K). With the increase of the monomer molecular weight of HS-PEG-SH and 8Arm-PEG-AC, the gel pore size becomes larger, and the amplification dots become larger accordingly. Larger gel pore size may lead to the diffusion of amplification dots (such as the hydrogel combination of HS-PEG-SH-4 / 5 / 6K and 8Arm-PEG-AC-40K), which is not conducive to counting for high-concentration samples.

[0172] The combination of 8Arm-PEG-AC-20K with a final concentration of 2.5 mM and HS-PEG-SH with different molecular weights and a final concentration of 10 mM has a moderate amplification dot size, and HS-PEG-SH-4K is the best molecular weight in terms of amplification dot size.

[0173] Example 5 Optimization of the amplification system

[0174] The PEG polymer aqueous solution will form a water-insoluble hydrogel with a porous structure after crosslinking, which will cause the generation of crowding effect. The hydrogel system is adjusted on the basis of the conventional LAMP system, and all the configuration methods refer to Example 1.

[0175] Adjustment of magnesium ion concentration: all other components are configured according to Table 3 except for magnesium ion, and the concentration of magnesium ion is adjusted to 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, and 6.5 mM. The detection results are shown in Figure 5 As shown in the table, non-specific small dots will be generated at a concentration of 4.5 mM or higher, and the higher the concentration, the more non-specific. At a concentration of 3 mM, the amplification dots are small and the number of detection dots is reduced. At a concentration of 3 mM, 3.5 mM, and 4 mM, there are no non-specific small dots, and there is no significant difference in the number of detection dots. The concentration of 4 mM is selected as the best magnesium ion concentration.

[0176] Adjustment of dNTP concentration: all other components are configured according to Table 3 except for dNTP, and the concentration of dNTP is adjusted to 0.8 mM, 1 mM, 1.2 mM, and 1.4 mM. The detection results are shown in Figure 6As shown, non-specific small dots were produced at a concentration of 1.4 mM, the amplification dots were small and the number of detection dots decreased at a concentration of 0.8 mM, and no non-specific small dots were produced at concentrations of 1 mM and 1.2 mM, and there was no significant difference in the number of detection dots. The concentration of 1 mM dNTP was selected as the optimal dNTP concentration.

[0177] Adjustment of primer set concentration: The concentrations of other components except the primer set were all configured according to Table 3, and the concentration of the primer set was adjusted to 0.1x, 0.2x, 0.4x, 0.6x, 0.8x, and 1x. The detection results are shown in Table 4. Figure 7 As shown, non-specific small dots were produced at a concentration of 0.6x or higher, and the higher the concentration of the primer set, the more serious the non-specificity. The amplification dots were small and the number of detection dots decreased at a concentration of 0.1x, and no non-specific small dots were produced at concentrations of 0.2x and 0.4x, and there was no significant difference in the brightness and size of the detection dots. The concentration of 0.2x primer set was selected as the optimal primer set concentration.

[0178] Adjustment of Bst polymerase concentration: The concentrations of other components except Bst polymerase were all configured according to Table 3, and the concentration of the enzyme was adjusted to 8 U, 9.6 U, 11.2 U, and 12.8 U per reaction. The detection results are shown in Table 5. Figure 8 As shown, non-specific small dots were produced at a concentration of 12.8 U per reaction. The amplification dots were small and the number of detection dots decreased at a concentration of 8 U per reaction, and no non-specific small dots were produced at concentrations of 9.6 U per reaction and 11.2 U per reaction, and there was no significant difference in the brightness and size of the detection dots. The concentration of 9.6 U per reaction was selected as the optimal enzyme concentration.

[0179] Adjustment of TCEP concentration: The concentrations of other components except TCEP were all configured according to Table 3, and the concentration of TCEP was adjusted to 0 mM, 1 mM, 2 mM, and 4 mM in components A / B. After mixing components A / B with the sample to be tested, the final concentration of TCEP was 0 mM, 1 mM, 2 mM, and 4 mM. The components A and B were detected at 0, 10, and 20 days after configuration under the condition of 25°C, and the detection results are shown in Table 6. Figure 9 As shown, there was no significant difference in the size of the amplification dots at 0, 1 mM, and 2 mM TCEP concentration on the 0th day after configuration. No amplification dots were observed at a TCEP concentration of 4 mM. On the 10th day after configuration at 25°C, the 0 mM TCEP concentration began to show obvious diffusion, and there was no significant change in the size of the amplification dots at 1 mM and 2 mM TCEP concentration. On the 20th day after configuration at 25°C, there were no amplification dots at 0 mM TCEP concentration, and there was no significant change in the size of the amplification dots at 1 mM and 2 mM TCEP concentration. The results of the size of the amplification dots at different times showed that when the TCEP concentration was 1 mM / 2 mM, the unnecessary oxidation of SH-PEG-SH before polymerization could be prevented, thereby affecting the degree of cross-linking. The concentration of 2 mM was selected as the optimal TCEP concentration.

[0180] Hydrogel concentration optimization: all the reference table 3 configuration except 8Arm-PEG-AC-20K, HS-PEG-SH-4k concentration of other components, according to 8Arm-PEG-AC-20K: HS-PEG-SH-4k is 1:4 ratio of two PEG concentration adjustment, the specific adjustment range as shown in table 6, its detection results as shown in figure 2 Figure 10 As shown in figure 2, with the increase of 8Arm-PEG-AC-20K and HS-PEG-SH-4k concentration, the amplification point gradually becomes smaller. Among them, the concentration 1 appears more obvious diffusion, the concentration 4 amplification point is small, considering the concentration 3 (8Arm-PEG-AC-20K 2.5mM, HS-PEG-SH-4k 10mM) PEG concentration in low power and mobile phone in the effect of taking pictures is better.

[0181] Table 6

[0182] Concentration 1 Concentration 2 Concentration 3 Concentration 4 8 Arm-PEG-AC-20K 1.8 mM 2 mM 2.5 mM 2.75 mM HS-PEG-SH-4k 7.2 mM 8 mM 10 mM 11 mM

[0183] From the above LAMP amplification system of each component concentration optimization, the concentration of each component of molecular limiting amplification is lower than the concentration used in conventional LAMP, especially the concentration of primer set is lower than 0.2 times of the concentration of conventional LAMP (the concentration of primer set commonly used in conventional LAMP is 1 times of the concentration of the present application).

[0184] Example 6 influence of different amplification time

[0185] In order to determine the optimal reaction time of quantitative monitoring of amplification point in hydrogel system. The difference of reaction amplification point was observed at different time points. The configuration method of hydrogel system is shown in table 3.

[0186] Figure 11 As shown in figure 3, the amplification point appeared at 10 min, the count of amplification point reached the maximum value at 15 min, and the number of amplification point did not further increase with the extension of reaction time from 15 min to 30 min, only the size of amplification sub became larger. It may be due to the limited supply of raw materials in the system, and there is no obvious further expansion at 30-40 min. 15-20 min is the optimal reaction time of quantitative monitoring of amplification point in hydrogel system. After the concentration optimization of each component of molecular limiting amplification, the raw materials are limited as much as possible, so that the reaction not only has the characteristics of rapidness, but also has good specificity.

[0187] Example 7 comparison of a kind of hydrogel limiting based on loop mediated isothermal amplification with conventional LAMP and fluorescence PCR detection

[0188] From the optimization of the concentration of each component of the molecular confinement amplification system of Example 5, the concentration of each component of the molecular confinement amplification is lower than that used in the conventional LAMP, especially the concentration of the primer set is lower than 5 times of the conventional LAMP (the concentration of the primer set commonly used in the conventional LAMP is 1 times of the concentration of the present application).

[0189] Conventional LAMP system: CT primer set 1x, Bst polymerase 11.2U, Bst Buffer 1x, dNTP 1.4mM, sybGreen 1x, magnesium ion 6mM.

[0190] Molecular confinement amplification, conventional LAMP and fluorescent PCR were used to detect chlamydia trachomatis, respectively, and the amplification CT values and running times of the three detection methods are shown in the following table. The amplification time of molecular confinement amplification is shorter than that of conventional LAMP and QPCR, and molecular confinement amplification has obvious rapid amplification advantage. For medium concentration samples and low concentration samples, amplification curve can appear within 15 minutes.

[0191] Table 7

[0192]

[0193] Example 8 Comparison of different sample processing methods

[0194] The water gel detection of the present application has high interference resistance to samples, and samples prepared by different sample processing methods can be stably detected. This embodiment compares the effects of heat lysis, magnetic bead method and sample releasing agent on the sample preparation of water gel LAMP

[0195] (1) Sample preparation: 10 CT-positive vaginal swab samples were collected from the clinic and labeled as TQ1-10.

[0196] (2) Extraction of nucleic acid: The nucleic acid extraction process was strictly carried out according to the instructions of each reagent. The final nucleic acid elution / dissolution volume of all extraction methods was set to 100 μL.

[0197] (3) Detection: The extracted nucleic acid was detected by chlamydia trachomatis fluorescent detection reagent, chlamydia trachomatis water gel LAMP reagent in Example 1 and CT LAMP ordinary reagent, respectively.

[0198] 1. Magnetic bead sample processing:

[0199] Take out the sample and shake well (frozen samples should be fully dissolved at room temperature before use), take 1000 μL of sample to be tested, centrifuge at 12000 rpm for 5 minutes. Remove 800 μL of supernatant. The remaining 200 μL is extracted by magnetic bead method, and the elution volume is 100 μL.

[0200] 2. Sample release agent sample processing:

[0201] 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 in a 1.5 mL centrifuge tube (if there is more secretion or the sample is relatively turbid, it is recommended to centrifuge at 3000 rpm for 30 s before sucking), centrifuge at 12000 rpm for 5 min, carefully remove the supernatant, add 100 μL of sample release agent, vortex well until there is no obvious precipitate, and place at room temperature for 5 min, centrifuge at 7000 rpm for 30 s, and the supernatant is used for detection.

[0202] 3. chelex-100 resin pyrolysis:

[0203] 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 in a 1.5 mL centrifuge tube (if there is more secretion or the sample is relatively turbid, it is recommended to centrifuge at 3000 rpm for 30 s before sucking), centrifuge at 12000 rpm for 5 min, carefully remove the supernatant, add 100 μL of sample processing liquid (containing chelex resin, to make the particles evenly distributed, please use the suction head to repeatedly suck and hit each time), vortex well until there is no obvious precipitate, centrifuge at 6000 rpm for 10 sec, 95℃ treatment for 5 min, centrifuge at 7000 rpm for 30 s, and the supernatant is used for detection.

[0204] The detection results are as follows:

[0205] Table 8

[0206]

[0207]

[0208] As shown in the above table, there is no significant difference between the magnetic bead method and the heat lysis method in the fluorescence PCR detection, and the sample release agent is worse than the two methods, and there is also a risk of detection failure for low copy samples. In the hydrogel limiting amplification, the sample release agent, the magnetic bead method, the heat lysis method, and the release agent have no significant difference. The hydrogel method has higher impurity tolerance than the fluorescence quantitative PCR for preliminary sample processing (release agent).

[0209] Example 9 Freeze-drying protectant optimization

[0210] 1. Freeze-drying formula optimization

[0211] In this example, 5 μL of freeze-drying protectant A is added to each person's component A, and 5 μL of freeze-drying protectant B is added to each person's component B. The reagents are freeze-dried with the freeze-drying protectant, and the freeze-drying morphology is observed.

[0212] Table 9 Freeze-drying program

[0213]

[0214] Table 10 Lyophilization protectant formulations

[0215]

[0216] As shown in Figure 12 , lyophilization protectant B1 has a phenomenon of shrinkage, and the other lyophilization protectants are in good condition after lyophilization; part of lyophilization protectant B1 cannot be dissolved, lyophilization protectant B2 dissolves slowly, and lyophilization protectant B3 is good in both lyophilization form and solubility.

[0217] As shown in Figure 13 , after reconstitution of lyophilization protectant B3 and lyophilization protectants A1 / A2 / A3, their amplification efficiencies are tested by mutual matching. According to Figure 13 the fluorescence chart results after reconstitution of lyophilization, the amplification efficiency of lyophilization protectant A3 and lyophilization protectant B3 is the highest, and this combination is selected as the best protectant.

[0218] 2. Effect of lyophilization protectant on reaction

[0219] Table 11

[0220]

[0221] The effects of lyophilization protectants on molecular confinement amplification are tested by respectively configuring no lyophilization protectant, containing one of A / B lyophilization protectants, and containing both A / B lyophilization protectants.

[0222] The results are shown in Figure 14 . In the magnetic bead method for extracting samples, the size of the amplification point in systems 2 and 4 is smaller than that in systems 1 and 3, but the number and brightness of the amplification point do not change significantly. Systems 2 and 4 both contain lyophilization protectant B, indicating that the pullulan in lyophilization protectant B has a certain limiting effect on the size of the amplification product. This limiting effect may be related to the high viscosity of pullulan.

[0223] The results are shown in Figure 14 . In the magnetic bead method for extracting samples, the size of the amplification point in systems 2 and 4 is smaller than that in systems 1 and 3, but the number and brightness of the amplification point do not change significantly. Systems 2 and 4 both contain lyophilization protectant B, indicating that the pullulan in lyophilization protectant B has a certain limiting effect on the size of the amplification product. This limiting effect may be related to the high viscosity of pullulan.

[0224] The numerous nano-holes in the hydrogel can isolate the inhibitors such as organic matter and heavy metal, and the addition of the freeze-drying protective agent further enhances the inhibitor tolerance.

[0225] 3. Freeze-drying stability test

[0226] Freeze-drying protective agent A3 and freeze-drying protective agent B3 were selected, and the components A / B were freeze-dried. After being placed at room temperature for different time, the storage stability of the freeze-dried reagent was detected, and the results are shown in Table 2. Figure 15

[0227] As shown by the results in Table 2, the freeze-dried reagent can still be used to detect CT samples stably after being placed at 45℃ for 15 weeks. Figure 15

[0228] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.​​

Claims

1. A method for detecting a target nucleic acid for non-diagnostic purposes, characterized in that, Comprising the following steps: S1: sample processing: lysing the sample to be tested; S2: system construction: mixing loop-mediated isothermal amplification reagents, hydrogel and the lysed sample to be tested; S3: reaction: cross-linking reaction and loop-mediated isothermal amplification reaction of the mixed components in S2; S4: result detection: detecting whether the target nucleic acid exists in the sample, such as the presence of one or more amplicons, indicating the presence of the target nucleic acid in the sample to be tested; If no amplicon appears, the target nucleic acid does not exist; The hydrogel is obtained by chemical cross-linking of eight-arm polyethylene glycol acrylate and thiol-polyethylene glycol-thiol; The loop-mediated isothermal amplification reagent comprises: primer set, dNTP, magnesium ion, polymerase, TCEP, buffer and lyophilization protectant.

2. The detection method of claim 1, wherein, The result detection in S4 uses a smartphone camera or a fluorescence microscope to detect whether the amplicon exists, and quantifies the concentration of the target nucleic acid in the sample based on the number of amplicons.

3. The detection method of claim 2, wherein, The molecular weight of the eight-arm polyethylene glycol acrylate is 10-40K; the molecular weight of the thiol-polyethylene glycol-thiol is 2-6K.

4. The detection method of claim 3, wherein, The concentration of the eight-arm polyethylene glycol acrylate is 1.8-2.75mM; the concentration of the thiol-polyethylene glycol-thiol is 7.2-11mM; the molar ratio of the eight-arm polyethylene glycol acrylate to the thiol-polyethylene glycol-thiol is 1:4; the concentration of the eight-arm polyethylene glycol acrylate is 1.8-2.75mM; the concentration of the thiol-polyethylene glycol-thiol is 7.2-11mM.

5. The method of claim 4, wherein the detection is performed by a method selected from the group consisting of mass spectrometry, nuclear magnetic resonance, and chromatography. The concentration of the primer set is 0.1-1×.

6. The detection method of claim 5, wherein, The concentration of the dNTP is 0.8-1.4mM; the concentration of the magnesium ion is 3-6.5mM; the concentration of the polymerase is 8-12.8U.

7. The detection method of claim 6, wherein, The concentration of the TCEP is 1-4mM.

8. The detection method of claim 7, wherein, The lyophilization protectant comprises: 12.5g / L mannitol, 4g / L BSA, 15g / L trehalose, 15mM glycine, 10g / L cyclodextrin and 20g / L pullulan.

9. Amplification reagent, characterized in that Comprising: Hydrogel, primer set, dNTP, magnesium ion, polymerase, TCEP, buffer and lyophilization protectant; The hydrogel is obtained by chemical cross-linking of eight-arm polyethylene glycol acrylate and thiol-polyethylene glycol-thiol; The molar ratio of the eight-arm polyethylene glycol acrylate to the thiol-polyethylene glycol-thiol is 1:

4.

10. A kit characterized in that, Comprising: The amplification reagent of claim 9 and acceptable adjuvants, carriers and / or devices.

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