Reagent system for absolute quantitative detection of nucleic acid and application thereof

Abstract

The invention relates to a reagent system for absolute quantitative detection of nucleic acid and application of the reagent system. A reagent system for absolute quantitative detection of nucleic acid is designed, a specific solid-phase carrier reagent is designed, a specific hydrogel system can be formed, a specific porous structure can effectively provide support for nucleic acid amplification reaction, a to-be-detected sample and an amplification reaction system are uniformly limited in micropores for reaction, and the detection accuracy is improved. The method can effectively realize high-flux rapid independent reaction, facilitates fluorescence imaging, calculates the specific initial copy number of the target nucleic acid molecule in the sample according to the number of fluorescent bright spots in the hydrogel, realizes absolute quantification, does not need an additional physical structure and does not need to make a standard curve, obviously reduces the cost, and simplifies the process.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and relates to a reagent system for absolute quantitative detection of nucleic acids and its application. Background Art

[0002] Nucleic acids are the general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which are biological macromolecular compounds polymerized from nucleotide monomers and are one of the most basic substances of life. In vitro nucleic acid amplification and quantification are one of the core technologies in the field of molecular biology and are applied in research fields such as molecular sequencing, gene expression analysis, gene mutation research, early molecular diagnosis of diseases, single nucleotide polymorphism, and drug screening.

[0003] Currently, nucleic acid quantitative detection technologies include fluorescence detection technology: nucleic acid quantitative detection uses specially matched fluorescence detection technology, and fluorescence dyes are used to detect the concentration of specific target molecules, enabling accurate quantification of DNA and RNA; multiplex nucleic acid detection technology: this technology is mainly divided into multiplex polymerase chain reaction (PCR), multiplex isothermal amplification technology (ITA), multiplex nucleic acid detection technology based on clustered regularly interspaced short palindromic repeats / associated proteins (CRISPR / Cas), and metagenomic sequencing (mNGS); real-time fluorescence PCR technology (qPCR): nucleic acid amplification is monitored by real-time collection of fluorescence signals. During the exponential phase of PCR amplification, there is a linear relationship between the Ct value of the template and its initial copy number, enabling quantitative detection; digital PCR technology (Droplet digtial PCR, ddPCR): through microfluidic technology, the target nucleic acid and PCR reaction solution are distributed in multiple reaction units for PCR amplification. By detecting the fluorescence signal intensity of each reaction unit after amplification, the number of reaction units containing the target nucleic acid is judged, and the number of target nucleic acid molecules in the starting sample is calculated based on the Poisson distribution, achieving absolute quantitative detection of nucleic acid concentration.

[0004] Currently, faster, more sensitive, more accurate, and high-throughput have become the mainstream trends in detection, posing new challenges to current nucleic acid quantitative detection technologies, such as limited sensitivity and accuracy of qPCR technology, limited scalability of dPCR technology, and low detection throughput. Therefore, how to provide a nucleic acid absolute quantitative method with high throughput, technical stability, and simple operation is one of the problems that urgently need to be solved in the current field of molecular biology. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art and actual needs, the present invention provides a reagent system for absolute quantitative detection of nucleic acids and its application to achieve rapid, accurate, and high-throughput absolute quantitative detection of nucleic acids.

[0006] To achieve this purpose, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a reagent system for absolute quantification detection of nucleic acids. The reagent system includes a solid-phase carrier reagent, and the solid-phase carrier reagent includes at least one of the following reagent combinations:

[0008] (1) including poly(ethylene glycol) acrylate compounds and poly(ethylene glycol) thiol compounds;

[0009] (2) including poly(ethylene glycol) maleimide compounds and poly(ethylene glycol) thiol compounds;

[0010] (3) including N-isopropylacrylamide (NIPAM), a first cross-linking agent, and a first initiator;

[0011] (4) including 2-hydroxyethyl methacrylate (HEMA), a second cross-linking agent, a second initiator, and a co-initiator;

[0012] (5) including acrylamide (AM), a third cross-linking agent, and a third initiator;

[0013] (6) including acrylamide (AM), N,N'-methylenebisacrylamide (MBAA), a fourth cross-linking agent, and a fourth initiator;

[0014] (7) including 2-acrylamido-2-methylpropanesulfonic acid (AMPS) and acrylamide.

[0015] The reagent system designed for absolute quantification detection of nucleic acids in the present invention includes designing a specific solid-phase carrier reagent, which can form a specific hydrogel system. The porous structure therein can effectively provide support for the nucleic acid amplification reaction, uniformly confine the sample to be tested and the amplification reaction system in the micropores for reaction, effectively realize high-throughput rapid independent reactions, and facilitate fluorescence imaging. The specific initial copy number of the target nucleic acid molecules in the sample is calculated according to the number of fluorescent bright spots in the hydrogel. The calculation methods can include direct counting method, Poisson distribution counting method, and hemocytometer counting method, etc. (wherein, in the direct counting method, the number of fluorescent bright spots is equivalent to the copy number of the target nucleic acid, and the initial copy number of the target nucleic acid is calculated according to the loading volume; the Poisson distribution counting method is the same as the ddPCR calculation method; the hemocytometer counting method is to calculate the number of fluorescent bright spots in the whole system according to the number of fluorescent bright spots in a part of the volume of the hydrogel system and the proportion of this volume, and calculate the initial copy number of the target nucleic acid according to the loading volume.), achieving absolute quantification, without the need for additional physical structures and making standard curves, significantly reducing costs and simplifying the process.

[0016] Preferably, the reagent system further includes primers for target nucleic acid molecules and reagents for nucleic acid amplification reactions.

[0017] It can be understood that the primers for target nucleic acid molecules and the reagents for nucleic acid amplification reactions can be selected according to actual needs.

[0018] Preferably, the polyethylene glycol acrylate compound is selected from at least one of polyethylene glycol diacrylate (AC-PEG-AC), three-arm polyethylene glycol acrylate (3Arm-PEG-AC), four-arm polyethylene glycol acrylate (4Arm-PEG-AC), eight-arm polyethylene glycol acrylate (8Arm-PEG-AC) and other polyethylene glycol acrylate compounds having two or more acrylate groups.

[0019] Preferably, the polyethylene glycol maleimide compound is selected from at least one of bismaleimide polyethylene glycol (MAL-PEG-MAL), three-arm polyethylene glycol maleimide (3Arm-PEG-MAL), four-arm polyethylene glycol maleimide (4Arm-PEG-MAL), six-arm polyethylene glycol maleimide (6Arm-PEG-MAL), eight-arm polyethylene glycol maleimide (8Arm-PEG-MAL) and other polyethylene glycol maleimide compounds having two or more maleimide groups.

[0020] Preferably, the polyethylene glycol mercapto compound is selected from at least one of bis-mercapto polyethylene glycol (SH-PEG-SH), four-arm polyethylene glycol mercapto (4Arm-PEG-SH) and other polyethylene glycol mercapto compounds having two or more mercapto groups.

[0021] The mercapto group can undergo a Michael addition reaction with maleimide groups or carbon-carbon double bonds such as acrylate groups, causing two monomers carrying corresponding groups to polymerize to form a hydrogel.

[0022] Preferably, the mass ratio of the polyethylene glycol acrylate compound to the bis-mercapto polyethylene glycol is (1-30):(10-1), for example, it can be 1:10, 3:7, 1:1, 7:3, 10:1, 25:1 or 29:1, etc.

[0023] Preferably, the mass ratio of the polyethylene glycol diacrylate to the bis-mercapto polyethylene glycol is (5-60):(2-10), for example, it can be 5.8:10, 11:5, 29:10, 39:10 or 58:2, etc.

[0024] Preferably, the mass ratio of the three-arm polyethylene glycol acrylate to the bis-mercapto polyethylene glycol is (5-60):(3-15), for example, it can be 5.7:15, 23.4:12, 29:10, 22:4 or 58.2:3, etc.

[0025] Preferably, the mass ratio of the eight-arm polyethylene glycol acrylate to the bis-mercapto polyethylene glycol is (3-31):(4-20), for example, it can be 3:20, 14.5:20, 29:10, 24:5 or 29.2:4, etc.

[0026] Preferably, the first crosslinking agent includes N,N-methylenebisacrylamide (MBAA).

[0027] Preferably, the first initiator includes potassium persulfate (KPS).

[0028] Preferably, the mass ratio of the N-isopropylacrylamide, the first crosslinking agent, and the first initiator is (80-98):(0.5-5):(0.1-1), such as 81:1:0.5, 82:4:0.8, 89:2:0.2, 97:3:0.3, 90:0.6:0.9, 84:0.8:0.4, or 94:4.5:0.5, etc.

[0029] Preferably, the second crosslinking agent includes ethylene glycol dimethacrylate (EGDMA).

[0030] Preferably, the second initiator includes ammonium persulfate (APS).

[0031] Preferably, the co-initiator includes tetramethylethylenediamine (TMEDA).

[0032] Preferably, the mass ratio of the 2-hydroxyethyl methacrylate, the second crosslinking agent, the second initiator, and the co-initiator is (70-95):(0.5-5):(0.1-1):(0.1-1).

[0033] Preferably, the third crosslinking agent includes polyethylene glycol diacrylate (PEGDA).

[0034] Preferably, the third initiator includes 2-hydroxy-2-methylpropiophenone (HMPP).

[0035] Preferably, the mass ratio of the acrylamide, the third crosslinking agent, and the third initiator is (70-90):(5-20):(0.1-1), such as 71:6:0.2, 80:12:0.5, 80:8:0.7, 80:16:0.3, 80:14:0.4, 80:10:0.6, or 80:7:0.9, etc.

[0036] Preferably, the mass ratio of the 2-acrylamido-2-methylpropanesulfonic acid and the acrylamide is 1:(2-10), including but not limited to 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, or 1:9, etc.

[0037] Preferably, the fourth crosslinking agent includes tetramethylethylenediamine (TMEDA), and the fourth initiator includes ammonium persulfate.

[0038] Preferably, the mass ratio of the acrylamide, N,N-methylenebisacrylamide, fourth crosslinking agent and fourth initiator is (152-228):(8-12):(0.1-2):(0.1-2).

[0039] Preferably, the weight-average molecular weight of the polyethylene glycol acrylate compound is 5000-40000; the weight-average molecular weight of the polyethylene glycol maleimide compound is 5000-40000, the weight-average molecular weight of the bis-mercapto polyethylene glycol is 1000-20000, and the weight-average molecular weight of the four-arm polyethylene glycol mercapto is 1000-20000.

[0040] In a second aspect, the present invention provides the use of the nucleic acid absolute quantification detection system described in the first aspect in the preparation of a nucleic acid absolute quantification kit and / or nucleic acid absolute quantification detection.

[0041] In the present invention, a specific hydrogel solid-phase system capable of providing a solid-phase support for nucleic acid amplification reactions is designed, which is convenient for realizing high-throughput and rapid nucleic acid quantification detection and has broad application prospects.

[0042] In a third aspect, the present invention provides a nucleic acid absolute quantification detection kit, and the kit includes the nucleic acid absolute quantification detection system described in the first aspect.

[0043] In a fourth aspect, the present invention provides a nucleic acid absolute quantification method, and the nucleic acid absolute quantification method includes:

[0044] Preparing the nucleic acid absolute quantification detection system described in the first aspect, performing a nucleic acid amplification reaction, performing fluorescence imaging on the reaction product, calculating the number of fluorescent bright spots, and calculating the copy number of the target nucleic acid molecule according to the number of fluorescent bright spots.

[0045] It can be understood that the nucleic acid absolute quantification detection system designed by the present invention is applicable to any nucleic acid amplification reaction.

[0046] Preferably, the nucleic acid amplification reaction may include any one of loop-mediated isothermal amplification reaction (LAMP), recombinase polymerase amplification reaction (RPA), polymerase chain amplification reaction (PCR), rolling circle amplification reaction (RCA) or other common nucleic acid amplification reactions in the art.

[0047] It can be understood that common fluorescence analysis methods in the art are all applicable to the present invention. For example, the software hAmplicon_count-master can be selected to count the amplification points and quantify the fluorescence signal. The target nucleic acid can be quantified through the Poisson distribution formula, and further the concentration of the target nucleic acid in the original sample can be calculated to achieve nucleic acid absolute quantification without making a standard curve.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] The reagent system designed for absolute quantification detection of nucleic acids in the present invention includes a designed specific solid-phase carrier reagent, which can form a specific hydrogel system. Among them, the specific porous structure can effectively provide support for nucleic acid amplification reactions, uniformly confine the sample to be tested and the amplification reaction system in micropores for reaction, effectively realizing high-throughput rapid independent reactions, and being convenient for fluorescence imaging. According to the number of fluorescent bright spots in the hydrogel, the specific initial copy number of target nucleic acid molecules in the sample is calculated to achieve absolute quantification, without additional physical structures and making standard curves, significantly reducing costs and simplifying the process. Brief Description of the Drawings

[0050] Figure 1 It is a result diagram for the consistency analysis of the hydrogel LAMP and ddPCR results in Example 1. In the figure, A-F are the quantification effects of hydrogel systems with different monomer mass ratios (4Arm-PEG-AC:SH-PEG-SH = 1:10, 3:7, 1:1, 7:3, 10:1, 16:11).

[0051] Figure 2 It is a bar chart showing the relationship between different monomer mass ratios of the hydrogel (4Arm-PEG-AC:SH-PEG-SH = 1:10, 3:7, 1:1, 7:3, 10:1, 16:11) and the radius of the fluorescent spots.

[0052] Figure 3 It is a result diagram for the consistency analysis of the hydrogel LAMP and ddPCR results in Example 2.

[0053] Figure 4 It is a result diagram for the consistency analysis of the hydrogel LAMP and ddPCR results in Example 3.

[0054] Figure 5 It is a result diagram for the consistency analysis of the hydrogel LAMP and ddPCR results in Example 4.

[0055] Figure 6 It is a result diagram for the consistency analysis of the hydrogel LAMP and ddPCR results in Example 5.

[0056] Figure 7 It is a result diagram for the consistency analysis of the hydrogel LAMP and ddPCR results in Example 6.

[0057] Figure 8 It is a result diagram for the consistency analysis of the hydrogel LAMP and ddPCR results in Example 7.

[0058] Figure 9 It is a result diagram for the consistency analysis of the hydrogel LAMP and ddPCR results in Example 8.

[0059] Figure 10 This is the result chart for the consistency analysis of the hydrogel LAMP and ddPCR results in Example 9.

[0060] Figure 11 This is the ROC curve graph for comparing the hydrogel LAMP experimental results and the clinical tuberculosis diagnosis results in Example 10.

[0061] Figure 12 This is the result chart of the area under the ROC curve for comparing the hydrogel LAMP experimental results and the clinical tuberculosis diagnosis results in Example 11. Detailed implementation manners

[0062] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation manners. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of the patent protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0063] For those not specifying specific technologies or conditions in the examples, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0064] Example 1

[0065] In this example, the hydrogel LAMP system prepared with different mass ratios of hydrogel monomers was used for quantitative analysis of the Escherichia coli 23S ribosomal gene.

[0066] Primers were designed for the 23S ribosomal gene of Escherichia coli. 10 μL of Escherichia coli bacterial solution (ATCC: 25922) was mixed evenly with 90 μL of DNA Extraction Solution 1.0 (Biosearch Technologies). After being placed at 65 °C for 1 min, it was placed at 95 °C for 1 min to extract the sample DNA. The 25 μL reaction system for hydrogel LAMP was as follows: 12.5 μL of WarmStart LAMP 2X Master Mix (NEB), 2.5 μL of 10× primer mixture, 0.5 μL of LAMP Fluorescent Dye (NEB), 4Arm-PEG-AC (Mw: 10000) with different mass ratios (1:10, 3:7, 1:1, 7:3, 10:1, 16:11) (0.12 mg, 0.97 mg, 1.62 mg, 2.27 mg, 3.12 mg, 1.92 mg respectively) and SH-PEG-SH (Mw: 3400) (3.12 mg, 2.27 mg, 1.62 mg, 0.97 mg, 0.12 mg, 1.32 mg respectively), 2.5 μL of the DNA of the sample to be tested, 2 μL of sterile water. Systems containing the DNA of the sample to be tested with different copy numbers were prepared, and the system without adding the DNA of the sample to be tested was used as a control (NTC). After the prepared systems were mixed evenly, they were sealed in 200 μL centrifuge tubes and allowed to gel at room temperature for 2 min. After gelling, the hydrogels were placed in a PCR amplifier for amplification (the LAMP reaction conditions were amplification at 65 °C for 10 min). After amplification, the samples were installed on an electric translation stage through tube clamps, and fluorescence signal detection was carried out using a light sheet fluorescence microscope. The software hAmplicon_count-master was used to count the fluorescence amplification points. The quantitative results of the hydrogel systems with monomer mass ratios of 1:10, 3:7, 1:1, 7:3, 10:1, 16:11 are shown in Figure 1 . Figure 1The quantitative effects of hydrogel systems with different monomer mass ratios (4Arm-PEG-AC:SH-PEG-SH = 1:10, 3:7, 1:1, 7:3, 10:1, 16:11) are shown as A - F. The experimental results were compared with those analyzed by the ddPCR system for the same samples. The experiments with 4Arm-PEG-AC:SH-PEG-SH = 1:10, 3:7, 1:1, 7:3, 10:1 had excellent consistency, obtaining an intraclass correlation coefficient of 0.998, while the experiment with 4Arm-PEG-AC:SH-PEG-SH = 16:11 was 0.996. At the same time, compared with 4Arm-PEG-AC:SH-PEG-SH = 16:11, the fluorescence amplification point diffusion range in the hydrogel systems formed by cross-linking monomers in the experiments with 4Arm-PEG-AC:SH-PEG-SH = 1:10, 3:7, 1:1, 7:3, 10:1 was smaller. The results are shown in Figure 2 , the gelation time and nucleic acid amplification time were significantly shortened, and the anti-interference ability of the system was also stronger. The specific data are shown in Table 1, indicating that designing a specific proportion of solid-phase carrier reagents in the present invention can significantly improve the detection effect.

[0067] Table 1

[0068]

[0069]

[0070] Example 2

[0071] In this example, the hydrogel LAMP system prepared with different hydrogel monomer mass ratios was used for quantitative analysis of the 23S ribosomal gene of Escherichia coli.

[0072] Primers were designed for the Escherichia coli 23S ribosomal gene. 10 μL of Escherichia coli bacterial solution (ATCC: 25922) was mixed evenly with 90 μL of DNA Extraction Solution 1.0 (Biosearch Technologies). After placing it at 65 °C for 1 min, it was placed at 95 °C for 1 min to extract the sample DNA. The 25 μL reaction system of hydrogel LAMP was as follows: 12.5 μL of WarmStart LAMP 2X Master Mix (NEB), 2.5 μL of 10× primer mixture, 0.5 μL of LAMP Fluorescent Dye (NEB), AC-PEG-AC (Mw: 10000) with different mass ratios (29:50, 11:5, 29:10, 39:10, 29:1) (1.192 mg, 2.222 mg, 2.411 mg, 2.576 mg, 3.132 mg respectively) and SH-PEG-SH (Mw: 3400) (2.048 mg, 1.018 mg, 0.829 mg, 0.664 mg, 0.108 mg respectively), 2.5 μL of the DNA of the sample to be tested, 2 μL of sterile water. Systems containing the DNA of the sample to be tested with different copy numbers were prepared, and the system without adding the DNA of the sample to be tested was used as a control (NTC). After the prepared systems were mixed evenly, they were sealed in 200 μL centrifuge tubes and allowed to gel at room temperature for 2 min. After gelling, the hydrogels were placed in a PCR amplifier for amplification (the LAMP reaction conditions were amplification at 65 °C for 10 min). After amplification, the samples were installed on an electric translation stage through tube clamps, and a light sheet fluorescence microscope was used to detect the fluorescence signal, and the software hAmplicon_count-master was used to count the fluorescence amplification points. This example had the same quantitative effect as Example 1. As Figure 3 shown, when the experimental results were compared with the results of analyzing the same samples using a ddPCR system, the intraclass correlation coefficient was between 0.997 and 0.999, showing excellent consistency.

[0073] Example 3

[0074] In this example, a hydrogel LAMP system prepared with different mass ratios of hydrogel monomers was used for quantitative analysis of the Escherichia coli 23S ribosomal gene.

[0075] Primers were designed for the Escherichia coli 23S ribosomal gene. 10 μL of Escherichia coli bacterial solution (ATCC: 25922) was mixed evenly with 90 μL of DNA Extraction Solution 1.0 (Biosearch Technologies), placed at 65 °C for 1 min, and then placed at 95 °C for 1 min to extract the sample DNA. The 25 μL reaction system for hydrogel LAMP was: 12.5 μL of WarmStart LAMP 2X Master Mix (NEB), 2.5 μL of 10× primer mixture, 0.5 μL of LAMP Fluorescent Dye (NEB), 3Arm-PEG-AC (Mw: 10000) with different mass ratios (19:50, 39:20, 29:10, 11:2, 97:5) (0.905 mg, 2.138 mg, 2.411 mg, 2.636 mg, 3.081 mg respectively) and SH-PEG-SH (Mw: 3400) (2.048 mg, 1.018 mg, 0.829 mg, 0.604 mg, 0.159 mg respectively), 2.5 μL of the DNA of the sample to be tested, 2 μL of sterile water, to prepare systems containing the DNA of the sample to be tested with different copy numbers, and the system without adding the DNA of the sample to be tested was used as a control (NTC). After the prepared system was mixed evenly, it was sealed in a 200 μL centrifuge tube and placed at room temperature for 2 min to form a gel. After the gel was formed, the hydrogel was placed in a PCR amplifier for amplification (the LAMP reaction conditions were amplification at 65 °C for 10 min). After the amplification was completed, the sample was installed on an electric translation stage through a tube clamp, and a light sheet fluorescence microscope was used to detect the fluorescence signal, and the software hAmplicon_count-master was used to count the fluorescence amplification points. This example had the same quantitative effect as Example 1, as Figure 4 shown. When the experimental results were compared with the results of analyzing the same sample using a ddPCR system, the intra-group correlation coefficient was between 0.997 and 0.999, showing excellent consistency. This example had the same quantitative effect as Example 1, as Figure 4 shown. When the experimental results were compared with the results of analyzing the same sample using a ddPCR system, the intra-group correlation coefficient was between 0.997 and 0.999, showing excellent consistency.

[0076] Example 4

[0077] In this example, a hydrogel LAMP system prepared with different mass ratios of hydrogel monomers was used for quantitative analysis of the Escherichia coli 23S ribosomal gene.

[0078] Primers were designed for the 23S ribosomal gene of Escherichia coli. 10 μL of Escherichia coli bacterial liquid (ATCC: 25922) was mixed evenly with 90 μL of DNA Extraction Solution 1.0 (Biosearch Technologies). After being placed at 65 °C for 1 min, it was placed at 95 °C for 1 min to extract the sample DNA. The 25 μL reaction system of hydrogel LAMP was as follows: 12.5 μL of WarmStart LAMP 2X Master Mix (NEB), 2.5 μL of 10× primer mixture, 0.5 μL of LAMP Fluorescent Dye (NEB), 8Arm-PEG-AC (Mw: 10000) with different mass ratios (3:20, 29:40, 29:10, 24:5, 73:10) (0.411 mg, 1.364 mg, 2.411 mg, 2.686 mg, 2.848 mg respectively) and SH-PEG-SH (Mw: 3400) (2.829 mg, 1.876 mg, 0.829 mg, 0.554 mg, 0.392 mg respectively), 2.5 μL of the DNA of the sample to be tested, 2 μL of sterile water. Systems containing the DNA of the sample to be tested with different copy numbers were prepared, and the system without adding the DNA of the sample to be tested was used as a control (NTC). After the prepared systems were mixed evenly, they were sealed in 200 μL centrifuge tubes and allowed to gel at room temperature for 2 min. After gelling, the hydrogels were placed in a PCR amplifier for amplification (the LAMP reaction conditions were amplification at 65 °C for 10 min). After amplification, the samples were installed on an electric translation stage through a tube clamp, and a light sheet fluorescence microscope was used to detect the fluorescence signal, and the software hAmplicon_count-master was used to count the fluorescence amplification points. This example had the same quantitative effect as Example 1. As Figure 5 shown, when the experimental results were compared with the results of analyzing the same samples using the ddPCR system, the intraclass correlation coefficient was between 0.997 and 0.999, showing excellent consistency.

[0079] Example 5

[0080] In this example, a hydrogel LAMP system prepared from hydrogel monomers with different weight average molecular weights was used for quantitative analysis of the 23S ribosomal gene of Escherichia coli.

[0081] Primers were designed for the 23S ribosomal gene of Escherichia coli. 10 μL of Escherichia coli bacterial solution (ATCC: 25922) was mixed evenly with 90 μL of DNA Extraction Solution 1.0 (Biosearch Technologies), placed at 65 °C for 1 min, and then placed at 95 °C for 1 min to extract the sample DNA. The 25 μL reaction system of hydrogel LAMP was: 12.5 μL of WarmStart LAMP 2X Master Mix (NEB), 2.5 μL of 10× primer mixture, 0.5 μL of LAMP Fluorescent Dye (NEB), 1.92 mg of 4Arm-PEG-AC with different weight-average molecular weights (5000, 8000, 12000, 16000, 20000 respectively) and 1.32 mg of SH-PEG-SH with different weight-average molecular weights (1000, 5000, 10000, 15000, 20000 respectively), 2.5 μL of the DNA of the sample to be tested, 2 μL of sterile water. Systems containing the DNA of the sample to be tested with different copy numbers were prepared, and the system without adding the DNA of the sample to be tested was used as a control (NTC). After the prepared systems were mixed evenly, they were sealed in 200 μL centrifuge tubes and allowed to gel at room temperature for 5 min. After gelling, the hydrogels were placed in an amplifier for amplification (the LAMP reaction conditions were amplification at 65 °C for 25 min). After amplification, the samples were installed on an electric translation stage through a tube clamp, and a light sheet fluorescence microscope was used to detect the fluorescence signal, and the software hAmplicon_count-master was used to count the fluorescence amplification points. This example had the same quantitative effect as Example 1. As Figure 6 shown, when the experimental results were compared with the results of analyzing the same samples using a ddPCR system, the intra-class correlation coefficient was between 0.997 and 0.999, showing excellent consistency.

[0082] Example 6

[0083] In this example, a hydrogel LAMP system prepared using different polyethylene glycol acrylate monomers was used for quantitative analysis of the 23S ribosomal gene of Escherichia coli.

[0084] Primers were designed for the 23S ribosomal gene of Escherichia coli. 10 μL of Escherichia coli bacterial solution (ATCC: 25922) was mixed evenly with 90 μL of DNA Extraction Solution 1.0 (Biosearch Technologies). After being placed at 65 °C for 1 min, it was placed at 95 °C for 1 min to extract the sample DNA. The 25 μL reaction system for hydrogel LAMP was as follows: 12.5 μL of WarmStart LAMP 2X Master Mix (NEB), 2.5 μL of 10× primer mixture, 0.5 μL of LAMP Fluorescent Dye (NEB), different polyethylene glycol acrylates (Mw: 10000) (3.84 mg of polyethylene glycol diacrylate (AC-PEG-AC), 2.56 mg of three-arm polyethylene glycol acrylate (3Arm-PEG-AC), 1.92 mg of four-arm polyethylene glycol acrylate (4Arm-PEG-AC), 0.96 mg of eight-arm polyethylene glycol acrylate (8Arm-PEG-AC) respectively), 1.32 mg of SH-PEG-SH, 2.5 μL of the DNA of the sample to be tested, 2 μL of sterile water. Systems containing the DNA of the sample to be tested with different copy numbers were prepared, and the system without adding the DNA of the sample to be tested was used as a control (NTC). After the prepared systems were mixed evenly, they were sealed in 200 μL centrifuge tubes and placed at room temperature for 5 min to form gels. After gel formation, the hydrogels were placed in a PCR amplifier for amplification (the LAMP reaction conditions were amplification at 65 °C for 25 min).

[0085] After the amplification was completed, the samples were installed on the electric translation stage through tube clamps, and fluorescence signal detection was carried out using a light sheet fluorescence microscope. The software hAmplicon_count-master was used to count the fluorescence amplification points. This example had the same quantitative effect as Example 1, as Figure 7 shown. Among them, the polyethylene glycol acrylates were respectively selected from AC-PEG-AC, 3Arm-PEG-AC (3-arm-PEG-AC), 4Arm-PEG-AC (4-arm-PEG-AC), 8Arm-PEG-AC (8-arm-PEG-AC). The experimental results were compared with the results of analyzing the same samples using a ddPCR system. The intra-class correlation coefficients were 0.996, 0.998, 0.998, and 0.997 respectively.

[0086] Example 7

[0087] In this example, a hydrogel LAMP system prepared using different polyethylene glycol maleimide monomers was used for quantitative analysis of the 23S ribosomal gene of Escherichia coli.

[0088] Primers (SEQ ID NO:1~SEQ ID NO:6) were designed for the 23S ribosomal gene of Escherichia coli. 10 μL of Escherichia coli bacterial solution (ATCC: 25922) was mixed evenly with 90 μL of DNA Extraction Solution 1.0 (Biosearch Technologies). After being placed at 65 °C for 1 min, it was placed at 95 °C for 1 min to extract the sample DNA. The 25 μL reaction system of hydrogel LAMP was as follows: 12.5 μL of WarmStart LAMP 2X Master Mix (NEB), 2.5 μL of 10× primer mixture, 0.5 μL of LAMP Fluorescent Dye (NEB), different polyethylene glycol maleimides (Mw: 10000) (3.84 mg of polyethylene glycol dimaleimide (MAL-PEG-MAL), 2.56 mg of three-arm polyethylene glycol maleimide (3Arm-PEG-MAL), 1.92 mg of four-arm polyethylene glycol maleimide (4Arm-PEG-MAL), 0.96 mg of eight-arm polyethylene glycol maleimide (8Arm-PEG-MAL) respectively), 1.32 mg of SH-PEG-SH (Mw: 3400), 2.5 μL of the DNA of the sample to be tested, and 2 μL of sterile water were used to prepare systems containing the DNA of the sample to be tested with different copy numbers, and the system without adding the DNA of the sample to be tested was used as a control (NTC). After the prepared systems were mixed evenly, they were sealed in 200 μL centrifuge tubes and placed at room temperature for 5 min to form gels. After gel formation, the hydrogels were placed in a PCR amplifier for amplification (the LAMP reaction condition was amplification at 65 °C for 25 min).

[0089] After the amplification was completed, the samples were installed on the electric translation stage through tube clamps, and the fluorescence signals were detected using a light sheet fluorescence microscope. The software hAmplicon_count-master was used to count the fluorescence amplification points. This example had the same quantitative effect as Example 1, as Figure 8 shown. Among them, the polyethylene glycol maleimides were respectively selected from MAL-PEG-MAL, 3Arm-PEG-MAL, 4Arm-PEG-MAL, and 8Arm-PEG-MAL. The experimental results were compared with the results of analyzing the same samples using a ddPCR system. The intra-class correlation coefficients were 0.997, 0.997, 0.997, and 0.998 respectively.

[0090] Example 8

[0091] In this example, a hydrogel LAMP system prepared using different polyethylene glycol thiol monomers was used for quantitative analysis of the 23S ribosomal gene of Escherichia coli.

[0092] Primers were designed for the Escherichia coli 23S ribosomal gene (SEQ ID NO:1 to SEQ ID NO:6). 10 μL of Escherichia coli bacterial solution (ATCC: 25922) was mixed evenly with 90 μL of DNA Extraction Solution 1.0 (Biosearch Technologies). After placing at 65 °C for 1 min, it was placed at 95 °C for 1 min to extract the sample DNA. The 25 μL reaction system of hydrogel LAMP was as follows: 12.5 μL of WarmStart LAMP 2X Master Mix (NEB), 2.5 μL of 10× primer mixture, 0.5 μL of LAMP Fluorescent Dye (NEB), 1.92 mg of 4Arm-PEG-AC (Mw: 10000), different polyethylene glycol thiols (Mw: 3400) (1.32 mg of bis-thiol polyethylene glycol (SH-PEG-SH) and 0.65 mg of tetra-arm polyethylene glycol thiol (4Arm-PEG-SH) respectively), 2.5 μL of the DNA of the sample to be tested, 2 μL of sterile water. Systems containing DNA of the sample to be tested with different copy numbers were prepared, and the system without adding the DNA of the sample to be tested was used as a control (NTC). After the prepared systems were mixed evenly, they were sealed in 200 μL centrifuge tubes and placed at room temperature for 5 min to form gels. After gel formation, the hydrogels were placed in a PCR amplifier for amplification (the LAMP reaction conditions were amplification at 65 °C for 25 min).

[0093] After amplification, the samples were installed on an electric translation stage through tube clamps, and fluorescence signal detection was performed using a light sheet fluorescence microscope. The fluorescence amplification points were counted using the software hAmplicon_count-master. This example had the same quantitative effect as Example 1, as Figure 9 shown. Among them, the polyethylene glycol thiols were selected from SH-PEG-SH and 4Arm-PEG-SH respectively. The experimental results were compared with the results of analyzing the same samples using a ddPCR system, and the intra-class correlation coefficients were 0.996 and 0.998 respectively.

[0094] Example 9

[0095] In this example, different hydrogel LAMP systems were used for quantitative analysis of the Escherichia coli 23S ribosomal gene.

[0096] The components in the hydrogel include: 4Arm-PEG-AC (Mw: 10000), SH-PEG-SH (Mw: 3400), HEMA (Mw: 130.14), EGDMA (Mw: 198.22), APS (Mw: 228.2), NIPAM (Mw: 113.16), K2S2O8 (Mw: 270.32), MBAA (Mw: 154.17), AM (Mw: 71.08), PEGDA (Mw: 700), HMPP (Mw: 164.21), AMPS (Mw: 207.2).

[0097] Primers were designed for the 23S ribosomal gene of Escherichia coli. 10 μL of Escherichia coli bacterial liquid (ATCC: 25922) was mixed evenly with 90 μL of DNA Extraction Solution 1.0 (Biosearch Technologies), placed at 65 °C for 1 min, and then placed at 95 °C for 1 min to extract the sample DNA. The 25 μL reaction system for hydrogel LAMP was: 12.5 μL of WarmStart LAMP 2X Master Mix (NEB), 2.5 μL of 10× primer mixture, 0.5 μL of LAMP Fluorescent Dye (NEB), PEG hydrogel system (1.92 mg 4Arm-PEG-AC, 1.32 mg SH-PEG-SH) or pHEMA hydrogel system (17.2 mmol HEMA, 0.5 mmol EGDMA, 0.105 mmol APS) or PNIPAM hydrogel system (1.04 mg NIPAM, 0.05 mg K2S2O8, 0.01 mg MBAA) or PAM hydrogel system (2.5 mg AM, 0.31 mg PEGDA and 0.03 mg HMPP, or, 2.38 mg AM, 0.13 mg MBAA, 12.5 μg APS and 12.5 μg TMEDA) or P(AMPS-AM) hydrogel system (0.88 mg AMPS, 1.76 mg AM, 0.08 mg MBAA, 0.22 mg HMPP), 2.5 μL of the DNA of the sample to be tested, and finally made up to 25 μL with sterile water to prepare a system containing the DNA of the sample to be tested with different copy numbers, and the system without adding the DNA of the sample to be tested was used as a control (NTC). After the prepared system was mixed evenly, it was sealed in a 200 μL centrifuge tube and placed at room temperature for 5 min to form a gel. After the gel was formed, the hydrogel was placed in an amplifier for amplification (the LAMP reaction condition was amplification at 65 °C for 25 min). After the amplification was completed, the sample was installed on an electric translation stage through a tube clamp, and the fluorescence signal was detected using a light sheet fluorescence microscope, and the fluorescence amplification points were counted using the software hAmplicon_count-master. This example had the same quantitative effect as Example 1, as Figure 10 shown. Among them, for the PEG hydrogel system, pHEMA hydrogel system, PNIPAM hydrogel system, PAM hydrogel system, and P(AMPS-AM) hydrogel system, the experimental results were compared with the results of analyzing the same sample using the ddPCR system, and the intraclass correlation coefficients were 0.998, 0.997, 0.998, 0.998, and 0.997 respectively.

[0098] Example 10

[0099] This example uses a hydrogel LAMP system for quantitative analysis of CXCL10 mRNA in clinical whole blood samples.

[0100] After extracting whole blood RNA, a hydrogel LAMP reaction system was prepared. The 25 μL reaction system of hydrogel LAMP was as follows: 12.5 μL of WarmStart LAMP 2X Master Mix (NEB), 2.5 μL of 10× primer mixture, 0.5 μL of LAMP Fluorescent Dye (NEB), PEG hydrogel system (1.92 mg of 4Arm-PEG-AC (Mw: 10000), 1.32 mg of SH-PEG-SH (Mw: 3400)), 1 μL of M-MLV reverse transcriptase, 2.5 μL of the DNA of the sample to be tested, and finally made up to 25 μL with sterile water. After the prepared system was mixed evenly, it was sealed in a 200 μL centrifuge tube and allowed to gel at room temperature for 5 min. After gelling, the hydrogel was placed in a PCR amplifier for amplification (the LAMP reaction conditions were amplification at 65 °C for 25 min). After amplification was completed, the sample was installed on an electric translation stage through a tube clamp, and a light sheet fluorescence microscope was used for fluorescence signal detection. The software hAmplicon_count-master was used to count the fluorescence amplification points.

[0101] Comparing the experimental results with the clinical tuberculosis diagnosis results, a high degree of consistency was obtained, and the area under the ROC curve was 0.9655, as Figure 11 shown.

[0102] Example 11

[0103] This example uses different hydrogel LAMP systems for quantitative analysis of CXCL10 mRNA in clinical whole blood samples.

[0104] After extracting whole blood RNA, prepare the hydrogel LAMP reaction system. The 25 μL reaction system of hydrogel LAMP is as follows: 12.5 μL of WarmStart LAMP 2X Master Mix (NEB), 2.5 μL of 10× primer mixture, 0.5 μL of LAMP Fluorescent Dye (NEB), pHEMA hydrogel system (17.2 mmol HEMA, 0.5 mmol EGDMA, 0.105 mmol APS) or PNIPAM hydrogel system (1.04 mg NIPAM, 0.05 mg K2S2O8, 0.01 mg MBAA) or PAM hydrogel system (2.5 mg AM, 0.31 mg PEGDA, 0.03 mg HMPP) or P(AMPS-AM) hydrogel system (0.88 mg AMPS, 1.76 mg AM, 0.08 mg MBAA, 0.22 mg HMPP), 1 μL of M-MLV reverse transcriptase, 2.5 μL of the DNA of the sample to be tested, and finally make up to 25 μL with sterile water. After the prepared system is mixed evenly, seal it in a 200 μL centrifuge tube and let it gel at room temperature for 5 min. After gelling, place the hydrogel in a PCR amplifier for amplification (the LAMP reaction condition is amplification at 65 °C for 25 min). After amplification, install the sample on the electric translation stage through a tube clamp, use a light sheet fluorescence microscope to detect the fluorescence signal, and count the fluorescence amplification points using the software hAmplicon_count-master.

[0105] Compare the experimental results with the clinical tuberculosis diagnosis results. There is a high consistency, and the areas under the ROC curves are 0.9793, 0.9751, 0.9872, and 0.9699, as Figure 12 shown.

[0106] Comparative example

[0107] This comparative example provides a quantitative analysis of the 23S ribosomal gene of Escherichia coli by the hydrogel LAMP system. The difference from Example 9 is only that APS in the pHEMA hydrogel system is replaced with HMPP in equal amount, or the dosage of MBAA in the PNIPAM hydrogel system is reduced by half, or HMPP in the PAM hydrogel system is replaced with APS in equal amount, or HMPP in the P(AMPS-AM) hydrogel system is replaced with APS in equal amount. The experimental results are compared with the results of analyzing the same samples using the ddPCR system, and the intraclass correlation coefficient (ICC) results are shown in Table 2.

[0108] Table 2

[0109]

[0110] In summary, the reagent system designed for absolute quantification of nucleic acids in the present invention includes a designed specific solid-phase carrier reagent, which can form a specific hydrogel system. The specific porous structure can effectively support the nucleic acid amplification reaction, uniformly confine the sample to be tested and the amplification reaction system in the microholes for reaction, effectively achieve high-throughput rapid independent reactions, and facilitate fluorescence imaging. The specific initial copy number of target nucleic acid molecules in the sample can be calculated according to the number of fluorescent bright spots in the hydrogel, realizing absolute quantification without additional physical structures and making standard curves, significantly reducing costs and simplifying the process.

[0111] The applicant declares that the above description is only a specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by any person skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the public disclosure scope of the present invention.

Claims

1. A reagent system for absolute quantitative detection of nucleic acids, characterized in that, The reagent system includes a solid-phase carrier reagent, and the solid-phase carrier reagent includes at least one of the following reagent combinations: (1) including poly(ethylene glycol) acrylate compounds and poly(ethylene glycol) thiol compounds; (2) including poly(ethylene glycol) maleimide compounds and poly(ethylene glycol) thiol compounds; (3) including N-isopropylacrylamide, a first crosslinking agent, and a first initiator; (4) including 2-hydroxyethyl methacrylate, a second crosslinking agent, a second initiator, and a co-initiator; (5) including acrylamide, a third crosslinking agent, and a third initiator; (6) including acrylamide, N,N'-methylenebisacrylamide, a fourth crosslinking agent, and a fourth initiator; (7) including 2-acrylamido-2-methylpropanesulfonic acid and acrylamide.

2. The reagent system for absolute quantification detection of nucleic acid according to claim 1, wherein The reagent system further includes primers for the target nucleic acid molecule and reagents for nucleic acid amplification reaction.

3. The reagent system for absolute quantitative detection of nucleic acids according to claim 1 or 2, characterized in that, The poly(ethylene glycol) acrylate compounds include poly(ethylene glycol) acrylate compounds containing at least 2 acrylate groups; Preferably, the poly(ethylene glycol) acrylate compounds include at least one of poly(ethylene glycol) diacrylate, three-arm poly(ethylene glycol) acrylate, four-arm poly(ethylene glycol) acrylate, and eight-arm poly(ethylene glycol) acrylate; Preferably, the poly(ethylene glycol) maleimide compounds include poly(ethylene glycol) maleimide compounds containing at least 2 maleimide groups; Preferably, the poly(ethylene glycol) maleimide compounds include at least one of bis(maleimide) poly(ethylene glycol), three-arm poly(ethylene glycol) maleimide, four-arm poly(ethylene glycol) maleimide, six-arm poly(ethylene glycol) maleimide, and eight-arm poly(ethylene glycol) maleimide; Preferably, the poly(ethylene glycol) thiol compounds include poly(ethylene glycol) thiol compounds containing at least 2 thiol groups; Preferably, the poly(ethylene glycol) thiol compounds include bis(thiol) poly(ethylene glycol) and / or four-arm poly(ethylene glycol) thiol; Preferably, the mass ratio of the poly(ethylene glycol) acrylate compounds to bis(thiol) poly(ethylene glycol) is (1-30):(10-1).

4. The reagent system for absolute quantification detection of nucleic acid according to any one of claims 1-3, characterized in that, The first crosslinking agent includes N,N'-methylenebisacrylamide; Preferably, the first initiator includes potassium persulfate; Preferably, the mass ratio of the N-isopropylacrylamide, the first crosslinking agent, and the first initiator is (80-98):(0.5-5):(0.1-1).

5. The reagent system for absolute quantification detection of nucleic acid according to any one of claims 1-4, characterized in that, The second crosslinking agent includes ethylene glycol dimethacrylate; Preferably, the second initiator includes ammonium persulfate; Preferably, the co-initiator includes N,N,N',N'-tetramethylethylenediamine; Preferably, the mass ratio of the 2-hydroxyethyl methacrylate, the second crosslinking agent, the second initiator, and the co-initiator is (70-95):(0.5-5):(0.1-1):(0.1-1).

6. The reagent system for absolute quantification detection of nucleic acid according to any one of claims 1-5, characterized in that, The third crosslinking agent includes poly(ethylene glycol) diacrylate; Preferably, the third initiator includes 2-hydroxy-2-methylpropiophenone; Preferably, the mass ratio of the acrylamide, the third crosslinking agent, and the third initiator is (70-90):(5-20):(0.1-1); Preferably, the mass ratio of the 2-acrylamido-2-methylpropanesulfonic acid to acrylamide is 1:(2-10); Preferably, the fourth crosslinking agent includes tetramethylethylenediamine, and the fourth initiator includes ammonium persulfate; Preferably, the mass ratio of acrylamide, N,N-methylenebisacrylamide, the fourth crosslinking agent and the fourth initiator is (152-228):(8-12):(0.1-2):(0.1-2).

7. Use of the nucleic acid absolute quantification detection system according to any one of claims 1-6 in the preparation of a nucleic acid absolute quantification kit and / or nucleic acid absolute quantification detection.

8. A nucleic acid absolute quantification detection kit, characterized in that, The kit includes the nucleic acid absolute quantification detection system according to any one of claims 1-6.

9. A method for absolute quantification of nucleic acids, characterized in that, The nucleic acid absolute quantification method includes: Preparing the nucleic acid absolute quantification detection system according to any one of claims 1-7, performing a nucleic acid amplification reaction, performing fluorescence imaging on the reaction product, calculating the number of fluorescent bright spots, and calculating the copy number of the target nucleic acid molecule according to the number of fluorescent bright spots.

10. The absolute nucleic acid quantification method according to claim 9, wherein The nucleic acid amplification reaction includes any one of loop-mediated isothermal amplification reaction, recombinase polymerase amplification reaction, polymerase chain reaction, rolling circle amplification reaction or other common nucleic acid amplification reactions in the art.

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