Nucleic acid amplification method based on monodisperse framework material and application of nucleic acid amplification method

Abstract

The invention relates to a nucleic acid amplification method based on a monodisperse framework material and application thereof, and the nucleic acid amplification method comprises the following steps: mixing the monodisperse framework material with a PCR reaction system, and carrying out amplification according to a PCR program; wherein the monodisperse framework material comprises any one or a combination of at least two of a metal organic framework material, a mesoporous silicon material or a mesoporous carbon material, and the aperture of the monodisperse framework material is 3-7nm. The method provided by the invention can be suitable for various nucleic acid amplification technologies, especially for accurate quantification of low-abundance nucleic acid samples, and effectively prevents amplification products from diffusing outwards and the environment from being polluted.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid amplification, and in particular to a nucleic acid amplification method based on a monodisperse framework material and application thereof. Background Art

[0002] Nucleic acid amplification technology (such as PCR, LAMP, etc.) is a core method in molecular biology, genetic testing, and disease diagnosis. However, a prominent problem in traditional nucleic acid amplification technology is nucleic acid contamination. During the nucleic acid amplification process, the amplified product may diffuse outward and contaminate the laboratory environment, resulting in false positive results in subsequent experiments, seriously affecting the accuracy and reliability of the experiment. In the prior art, the main methods to prevent nucleic acid contamination include physical isolation, chemical inhibitors, etc., but these methods cannot completely eliminate the contamination risk caused by the overflow of amplified products.

[0003] CN114164064A discloses a nucleic acid pollution remover, which comprises the following raw materials in parts by weight: 0.1-3% of an acid-containing reagent, 0.1-3% of a chlorine-containing reagent, 5-10% of an oxidant, 0.1-2% of a stabilizer, 0.1-1% of nano titanium dioxide and deionized water.

[0004] In addition, with the development of genomic research and precision medicine, the demand for accurate quantification of low-copy number nucleic acids is increasing. Digital droplet PCR (ddPCR), as a highly sensitive and high-precision nucleic acid quantification technology, achieves quantification at the single-molecule level by dispersing nucleic acid molecules into droplets, with each droplet acting as an independent reaction chamber for amplification. However, ddPCR technology relies on complex droplet generation equipment, which is costly and cumbersome to operate.

[0005] CN110819523A discloses a digital PCR system and a digital PCR droplet formation method, the digital PCR system includes a droplet formation component and a droplet nozzle component, the droplet nozzle component is connected below the droplet formation component, the droplet formation component includes a cover plate and at least one annular step connected to the lower surface of the cover plate, the droplet nozzle component includes a plurality of droplet nozzles, wherein the upper surface of the droplet nozzle component, the lower surface of the cover plate and the annular step together form a droplet formation chamber, and a vaporization component is provided in the droplet nozzle for vaporizing the liquid layer of the digital PCR solution in the droplet nozzle and quickly pushing it into the droplet formation oil in the droplet formation chamber to form digital PCR droplets.

[0006] In summary, how to provide a nucleic acid amplification method that prevents PCR products from spreading and polluting the environment has become one of the problems that need to be solved urgently in this field. Summary of the invention

[0007] To solve the above technical problems, the present invention provides a nucleic acid amplification method based on a monodisperse framework material and its application. The multi-porous structure of the monodisperse material effectively restricts the movement of large molecules of the amplification product, preventing the escape of the amplification product. At the same time, the multi-porous structure is utilized as a micro-reaction chamber. By detecting whether there is an amplification product in each micro-reaction chamber, accurate nucleic acid digital quantitative analysis can be achieved, which is particularly suitable for the detection of low-abundance nucleic acid samples.

[0008] To achieve this purpose, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides a nucleic acid amplification method based on a monodisperse framework material, and the nucleic acid amplification method includes: mixing the monodisperse framework material with a PCR reaction system and performing amplification according to the PCR procedure;

[0010] Wherein, the monodisperse framework material includes any one or a combination of at least two of metal-organic framework materials, mesoporous silicon materials or mesoporous carbon materials, and the pore size of the monodisperse framework material is 3 to 7 nm (for example, it can be 3 nm, 3.5 nm, 4 nm, 4.5 nm, 5 nm, 5.5 nm, 6 nm, 6.5 nm or 7 nm, etc.).

[0011] The monodisperse framework material provided by the present invention has a unique multi-porous structure, and its pore size allows the smooth diffusion of amplification reaction components (such as enzymes, primers and small molecule reaction substrates, etc.), but restricts the free movement of the amplification product. Nucleic acid molecules bind to the interior of the pores through adsorption. These pores form a tiny physical barrier, thereby effectively preventing the outward diffusion of the amplification product and environmental pollution, and having potential advantages in preventing nucleic acid contamination. The present invention can also improve the adsorption capacity for nucleic acids by methods such as amino modification.

[0012] In addition, the monodisperse framework material can also provide a natural micro-reaction chamber for the nucleic acid amplification system, disperse the nucleic acid amplification into independent micro-reaction spaces, and achieve nucleic acid quantification similar to digital PCR. Finally, by detecting the fluorescence signal to determine whether there is an amplification product in each micro-reaction chamber, counting the number of amplification-positive reaction chambers, and combining with the Poisson distribution model, the initial copy number of nucleic acids in the sample can be accurately deduced to achieve accurate digital quantification of nucleic acids. This method avoids the complex droplet generation and processing process, and at the same time retains the advantages of high sensitivity and high precision of digital PCR.

[0013] Therefore, the method provided by the present invention can be applicable to various nucleic acid amplification technologies such as conventional PCR, qPCR, LAMP, RCA, etc. After the amplification is completed, the amplification products can be analyzed by gel electrophoresis, fluorescence detection or other detection means. Especially for the accurate quantification of low-abundance nucleic acid samples, the application of monodisperse materials in the nucleic acid amplification system can effectively solve the contamination problem in the prior art and simplify the operation process of digital quantification.

[0014] In addition to the monodisperse framework materials listed in the present invention, other porous materials with a highly uniform pore size distribution can also be applicable to the present invention. Furthermore, the monodisperse framework materials provided by the present invention can be obtained by appropriate synthesis methods such as sol-gel method, template method or self-assembly method to prepare materials with uniform particle size and adjustable pore structure. The specific preparation method has no limiting effect on the present invention.

[0015] Preferably, the porosity of the monodisperse framework material is 50% - 80% (for example, it can be 50%, 55%, 60%, 65%, 70%, 75% or 80%, etc.).

[0016] By adjusting the porosity of the monodisperse framework material and combining the ratio of the monodisperse framework material to the template DNA, the present invention greatly reduces the probability of DNA molecules binding to the surface of the monodisperse framework material and controls the DNA molecules inside the pores of the monodisperse framework material, so as to achieve the purpose of preventing the diffusion of nucleic acid molecules.

[0017] Preferably, the metal-organic framework material includes any one or a combination of at least two of ZIF-8, UiO-66, MIL-101, HKUST-1 or MOF-5.

[0018] Preferably, the particle size of the metal-organic framework material is 150 - 250 nm (for example, it can be 150 nm, 170 nm, 190 nm, 200 nm, 210 nm, 230 nm or 250 nm, etc.).

[0019] Preferably, the mesoporous silica material includes any one or a combination of at least two of MCM-41, SBA-15, MSN, KIT-6, FDU-12 or TUD-1.

[0020] Preferably, the particle size of the mesoporous silica material is 50 - 600 nm (for example, it can be 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm or 600 nm, etc.).

[0021] Preferably, the mesoporous carbon material includes any one or a combination of at least two of CMK-3, CMK-8, FDU-15, MCF, OMC or nitrogen-doped mesoporous carbon.

[0022] Preferably, the particle size of the mesoporous carbon material is 100 - 300 nm (for example, it can be 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, etc.).

[0023] Preferably, the PCR reaction system contains template DNA, primers, dNTPs, buffer, and DNA polymerase.

[0024] The template DNA referred to in the present invention refers to all DNA molecules added to the PCR reaction system before the PCR reaction, which contains the DNA sequence to be amplified by the PCR reaction. For example, the template DNA is derived from DNA molecules extracted from cells. There are multiple DNA molecules in the PCR reaction system, and the PCR reaction only occurs on some of the DNA molecules.

[0025] Preferably, the molar ratio of the monodisperse framework material to the template DNA is (10 - 50):1.

[0026] The specific point values of the above (10 - 50) can be selected as 10, 15, 20, 25, 30, 35, 40, 45, 50, etc.

[0027] Preferably, the PCR reaction system further contains a fluorescent probe.

[0028] Preferably, the PCR reaction system includes any one of the reaction systems of the conventional PCR method, the qPCR method, the LAMP method, the RCA method, and the SDA method.

[0029] Preferably, the reaction system of the conventional PCR method contains template DNA, primers, dNTPs, buffer, and Taq DNA polymerase.

[0030] Preferably, the reaction system of the qPCR method contains template DNA, primers, dNTPs, fluorescent probe, buffer, and Taq DNA polymerase.

[0031] Preferably, the reaction system of the LAMP method contains template DNA, primers, dNTPs, LAMP fluorescent probe, buffer, and Bst DNA polymerase.

[0032] Preferably, the reaction system of the RCA method contains template DNA, primers, dNTPs, RCA fluorescent probe, buffer, and Φ29 DNA polymerase.

[0033] Preferably, the reaction system of the SDA method contains template DNA, primers, dNTPs, SDA fluorescent probe, buffer, nicking enzyme, and Bst DNA polymerase.

[0034] Preferably, the PCR program includes any one of the reaction programs of the conventional PCR method, the qPCR method, the LAMP method, the RCA method, and the SDA method.

[0035] Preferably, the reaction program of the conventional PCR method includes 93 - 97 °C for 25 - 35 s, 53 - 57 °C for 25 - 35 s, 70 - 74 °C for 55 - 65 s, with 28 - 32 cycles.

[0036] The specific point values within the range of 93 - 97 °C can be selected as 93 °C, 94 °C, 95 °C, 96 °C, or 97 °C, etc.

[0037] The specific point values within the range of 25 - 35 s can be selected as 25 s, 27 s, 29 s, 30 s, 31 s, 33 s, or 35 s, etc.

[0038] The specific point values within the range of 53 - 57 °C can be selected as 53 °C, 54 °C, 55 °C, 56 °C, or 57 °C, etc.

[0039] The specific point values within the range of 70 - 74 °C can be selected as 70 °C, 71 °C, 72 °C, 73 °C, or 74 °C, etc.

[0040] The specific point values within the range of 55 - 65 s can be selected as 55 s, 57 s, 59 s, 60 s, 61 s, 63 s, or 65 s, etc.

[0041] The specific point values within the range of 28 - 32 can be selected as 28, 29, 30, 31, or 32, etc.

[0042] Preferably, the reaction program of the qPCR method includes 93 - 97 °C for 100 - 140 s; 93 - 97 °C for 10 - 20 s, 58 - 62 °C for 25 - 35 s, with 38 - 42 cycles.

[0043] The specific point values within the range of 93 - 97 °C can be selected as 93 °C, 94 °C, 95 °C, 96 °C, or 97 °C, etc.

[0044] The specific point values within the range of 100 - 140 s can be selected as 100 s, 105 s, 110 s, 115 s, 120 s, 125 s, 130 s, 135 s, or 140 s, etc.

[0045] The specific point values within the range of 10 - 20 s can be selected as 10 s, 12 s, 14 s, 15 s, 16 s, 18 s, or 20 s, etc.

[0046] The specific point values within the range of 58 - 62 °C can be selected as 58 °C, 59 °C, 60 °C, 61 °C, or 62 °C, etc.

[0047] The specific point values within the range of 25 to 35 s described above can be selected as 25 s, 27 s, 29 s, 30 s, 31 s, 33 s, 35 s, etc.

[0048] The specific point values within the range of 38 to 42 described above can be selected as 38, 39, 40, 41, 42, etc.

[0049] Preferably, the reaction program of the LAMP method includes a constant temperature reaction at 60 to 70 °C for 25 to 35 min.

[0050] The specific point values within the range of 60 to 70 °C described above can be selected as 60 °C, 62 °C, 64 °C, 65 °C, 66 °C, 68 °C, 70 °C, etc.

[0051] The specific point values within the range of 25 to 35 min described above can be selected as 25 min, 27 min, 29 min, 30 min, 31 min, 33 min, 35 min, etc.

[0052] Preferably, the reaction program of the RCA method includes a constant temperature reaction at 30 to 37 °C for 1 to 2 h.

[0053] The specific point values within the range of 30 to 37 °C described above can be selected as 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, etc.

[0054] The specific point values within the range of 1 to 2 h described above can be selected as 1 h, 1.2 h, 1.4 h, 1.5 h, 1.6 h, 1.8 h, 2 h, etc.

[0055] Preferably, the reaction program of the SDA method includes a constant temperature reaction at 37 to 42 °C for 1 to 2 h.

[0056] The specific point values within the range of 37 to 42 °C described above can be selected as 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, etc.

[0057] The specific point values within the range of 1 to 2 h described above can be selected as 1 h, 1.2 h, 1.4 h, 1.5 h, 1.6 h, 1.8 h, 2 h, etc.

[0058] In a second aspect, the present invention provides an application of the nucleic acid amplification method as described in the first aspect in preventing nucleic acid contamination.

[0059] In a third aspect, the present invention provides an application of the nucleic acid amplification method as described in the first aspect in nucleic acid quantitative detection.

[0060] The nucleic acid amplification method provided by the present invention has broad application prospects in the fields of genomic research, clinical diagnosis, detection of trace pathogens, detection of cancer gene mutations, etc. In addition, the nucleic acid amplification method provided by the present invention can also be applied to non-disease diagnosis fields, such as detecting environmental samples, preventing and controlling viruses, and related basic research such as cancer gene mutation analysis.

[0061] Preferably, the method for nucleic acid quantitative detection includes real-time fluorescence quantitative PCR or digital PCR.

[0062] In a fourth aspect, the present invention provides a method for nucleic acid quantitative detection based on the principle of digital PCR. The nucleic acid quantitative detection method includes: obtaining a PCR amplification product using the nucleic acid amplification method described in the first aspect, counting the number of positive fluorescence signals, and analyzing to obtain the initial copy number of the nucleic acid.

[0063] Preferably, the PCR reaction system contains template DNA, primers, dNTPs, fluorescent probes, buffer, and Taq DNA polymerase.

[0064] Preferably, the template DNA contains the target nucleic acid sequence of the primers.

[0065] Preferably, the molar ratio of the monodisperse framework material to the target nucleic acid sequence of the primers in the template DNA is (20-40):1.

[0066] In order to achieve absolute quantification of the target nucleic acid, each monodisperse framework material particle needs to bind at most one target nucleic acid. Too little monodisperse framework material will result in a smaller quantitative result.

[0067] Specific point values of the above (20-40) can be selected as 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, etc.

[0068] Preferably, the PCR program includes 93-97°C, 100-140 s; 93-97°C, 10-20 s, 58-62°C, 25-35 s, for 38-42 cycles.

[0069] Specific point values of the above 93-97°C can be selected as 93°C, 94°C, 95°C, 96°C, or 97°C, etc.

[0070] Specific point values of the above 100-140 s can be selected as 100 s, 105 s, 110 s, 115 s, 120 s, 125 s, 130 s, 135 s, or 140 s, etc.

[0071] Specific point values of the above 10-20 s can be selected as 10 s, 12 s, 14 s, 15 s, 16 s, 18 s, or 20 s, etc.

[0072] The specific point value of 58-62°C can be selected from 58°C, 59°C, 60°C, 61°C or 62°C, etc.

[0073] The specific point values of 25 to 35 seconds may be selected as 25s, 27s, 29s, 30s, 31s, 33s or 35s etc.

[0074] The specific point values of 38 to 42 mentioned above can be selected from 38, 39, 40, 41 or 42, etc.

[0075] Preferably, the analysis to obtain the initial copy number of the nucleic acid includes: combining a Poisson distribution model to infer the initial copy number of the nucleic acid in the sample.

[0076] After obtaining the number of positive fluorescent signals, the present invention can calculate the probability Pr(n) that each monodisperse framework material particle contains n target copies through Poisson distribution, that is, if the average number of target copies of each monodisperse framework material particle is C, then:

[0077]

[0078] Enter n = 0 to obtain the probability that the monodisperse framework material particle is empty for a given C value:

[0079] Pr(0)=e -C

[0080] For a population of monodisperse framework material particles, the observed fraction of empty monodisperse framework material particles (E) is an unbiased estimate of Pr(0), so:

[0081] E=e -C

[0082] According to the definition of the proportion E of empty monodisperse framework material particles, we have:

[0083]

[0084] Where N is the total number of particles of the monodisperse framework material, N neg is the number of negative monodisperse framework material particles, which is equal to the total number of particles minus the number of positive particles. Combining the above two equations, we can get:

[0085]

[0086] And C is the number of copies per monodisperse framework material particle, which needs to be converted into the number of copies per microliter:

[0087]

[0088] Wherein, Concentration is the concentration of the target nucleic acid in the original sample, with the unit of copies / μL, and V is the sample volume, with the unit of μL.

[0089] Other specific point values within the above numerical ranges can be selected, and will not be elaborated one by one here.

[0090] Compared with the prior art, the present invention has the following beneficial effects:

[0091] (1) The present invention realizes the effective prevention and control of nucleic acid contamination. The multi-porous structure of the monodisperse material effectively restricts the movement of macromolecular amplification products, prevents the escape of amplification products, and thus avoids the problem of nucleic acid contamination in the laboratory environment. Compared with the existing pollution prevention and control measures (such as physical isolation or chemical inhibitors), this method is more simple and effective.

[0092] (2) The present invention realizes the digital quantification of nucleic acids. By using the multi-porous structure as a micro reaction chamber, precise digital quantification analysis of nucleic acids can be achieved through the detection of whether there are amplification products in each micro reaction chamber, which is particularly suitable for the detection of low-abundance nucleic acid samples.

[0093] (3) The nucleic acid amplification method provided by the present invention has the characteristics of high sensitivity, high safety, simple operation, low cost, etc. Compared with ddPCR, it does not require complex droplet generation equipment, the experimental operation is more simple, and it retains the advantages of high sensitivity and accuracy of ddPCR, and is suitable for wide application in conventional laboratories. Description of the Drawings

[0094] Figure 1 It is a comparison diagram of the quantitative results of Example 4 and the quantitative results of the ddPCR method. Detailed Embodiments

[0095] To further illustrate the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with examples and drawings. It can be understood that the specific embodiments described herein are only used to explain the present invention, rather than limiting the present invention.

[0096] For those not specifying specific techniques or conditions in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product specifications. For the reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0097] Example 1

[0098] This example provides a nucleic acid amplification method based on monodisperse metal-organic framework material (MOF), including the following steps:

[0099] (1) A self-assembly reaction was carried out on a mixed solution of 2-methylimidazole and zinc chloride to synthesize ZIF-8 particles with a particle size of 200 nm, a pore size of 5 nm, a porosity of 50%-60%. Ultrasonic dispersion was used to ensure uniform distribution of the particles, and unreacted impurities were removed by centrifugal washing.

[0100] (2) A qPCR reaction system was prepared, including template DNA, primers, dNTPs, buffer, fluorescent probe, and Taq DNA polymerase. The template DNA was derived from the nucleic acid of tumor cells. The primer sequences were SEQ ID NO.1: 5’-AAGGCCTGCTGAAAATGACT-3’ and SEQ ID NO.2: 5’-GTCC TGCACCAGTAATATGC-3’, and the fluorescent probe was SEQ ID NO.3: 5’-FAM-TGGAGCTGG TGGCGT-MGB-3’, which was used to amplify the KRAS gene.

[0101] (3) The ZIF-8 particles were mixed with the qPCR reaction system, where the molar ratio of ZIF-8 particles to template DNA was 30:1. The positive reaction system with the target and the negative control system were placed in an open-top fluorescence quantitative PCR instrument for amplification reaction. The qPCR program included: pre-denaturation at 95°C for 2 min, 40 cycles of denaturation at 95°C for 15 s, annealing and extension at 60°C for 30 s, and the fluorescence signal was analyzed using qPCR software.

[0102] The detection results showed that the positive group produced a fluorescence signal, while no fluorescence signal was found in the negative control group, indicating that the ZIF-8 particles successfully prevented the external diffusion of the amplification products.

[0103] Example 2

[0104] This example provides a nucleic acid amplification method based on monodisperse mesoporous silica (MSN), which includes the following steps:

[0105] (1) Tetraethyl orthosilicate (TEOS) was used as the silicon source, and MSN particles with a particle size of 500 nm were synthesized by the sol-gel method under the catalysis of ammonia water. The template was removed by ablation to form a porous material with a pore size of 3 nm and a porosity of 40%-70%, and amino modification was carried out to improve its nucleic acid adsorption performance.

[0106] (2) Prepare a conventional PCR reaction system, including template DNA, primers, dNTPs, buffer, and Taq DNA polymerase. The template DNA is derived from HPV16, and the primer sequences are SEQ ID NO.4: 5’-CTCTTTGGCTGCCTAGTGAG-3’ and SEQ ID NO.5: 5’-GCGTGCAACATATTCA TCCG-3’, which are used to amplify the HPV16 L1 gene.

[0107] (3) Mix the MSN particles with the PCR reaction system, where the molar ratio of MSN particles to template DNA is 10:1. Open the lids of the positive reaction system with the target and the negative control system and place them in a thermal cycler for amplification reaction according to the conventional PCR program. The PCR program includes: denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 60 s, for a total of 30 cycles. Detect the amplification products by agarose gel electrophoresis.

[0108] The electrophoresis results show that no external contamination was detected in the negative control. Due to the porous structure of MSN restricting the movement of large molecules of the amplification products, the PCR products were confined in the positive reaction system.

[0109] Example 3

[0110] This example provides a nucleic acid amplification method based on monodisperse mesoporous carbon material (CMK-3), including the following steps:

[0111] (1) Prepare CMK-3 material by the hard template method. Use SBA-15 (mesoporous silica) as the template, introduce the carbon source, and remove the template after heat treatment to obtain CMK-3 particles with a porous structure, with a particle size of 200 nm, a pore size of 7 nm, and a porosity of 60%-80%.

[0112] (2) Prepare the LAMP reaction system, including template DNA, primers, dNTPs, LAMP fluorescent probe, Bst DNA polymerase, and buffer. The template DNA is derived from Escherichia coli. The primer sequences are SEQ ID NO.6: 5’-GGCGTTAAGTTGCAGGGTAT-3’, SEQ ID NO.7: 5’-TCAC GAGGCGCTACCTAA-3’, SEQ ID NO.8: 5’-CGGTTCGGTCCTCCAGTTAGTG TTTTCCCGAAACCCGGTGATCT-3’, SEQ ID NO.9: 5’-TAGCGGATGACTTGT GGCTGGTTTTTCGGGGAGAACCAGCTATC-3’, SEQ ID NO.10: 5’-ACCTTCAACCTGCCCATG-3’, SEQ ID NO.11: 5’-GTGAAAGGCCAATCAAACC-3’. The fluorescent probe is SEQ IDNO.12: 5’FAM-CGGTTCGGTCCTCCAGTTAGTGTTTTCCC GAAACCCGGTGATCT-3’, SEQ ID NO.13: 5’-GACCGAACCG-3’BHQ, which is used to amplify the 23S gene.

[0113] (3) Mix CMK-3 with the LAMP reaction system. The molar ratio of CMK-3 particles to template DNA is 50:1. Under the constant temperature condition of 65 °C, the positive reaction system with the target and the negative control system are subjected to the LAMP reaction with the lids open. The reaction time is 30 min, and the fluorescence signals collected in real-time by the fluorescence quantitative PCR instrument are analyzed.

[0114] The test results show that the positive group produces high-intensity fluorescence signals, while the negative group does not produce fluorescence signals. The porous structure of CMK-3 successfully inhibits the diffusion of the amplification products and avoids the external contamination of the LAMP amplification products.

[0115] Example 4

[0116] This embodiment provides a method for nucleic acid quantitative detection. ZIF-8 particles were prepared according to the method provided in Example 1, and a qPCR reaction system was formulated, including a DNA sample, primers, dNTPs, buffer, fluorescent probe, and Taq DNA polymerase. The nucleic acid sequences of the primers and fluorescent probe were the same as those in Example 1. The preparation method of the DNA sample included: the PCR product of the KRAS gene was obtained by agarose gel electrophoresis and then recovered and purified by gel extraction. The target sequences of primers SEQ ID NO.1, SEQ ID NO.2, and fluorescent probe SEQ ID NO.3 were contained on this DNA sample. The ZIF-8 particles were mixed with the qPCR reaction system, where the molar ratio of ZIF-8 particles to the DNA sample was 30:1. The qPCR program was the same as that in Example 1. After placing it in a fluorescence quantitative PCR instrument for amplification reaction, the number of positive fluorescence signals was counted, and combined with the Poisson distribution model, the initial copy number of nucleic acid in the sample was deduced.

[0117] Example 5

[0118] This embodiment provides a method for nucleic acid quantitative detection, which is only different from Example 4 in that the molar ratio of the monodisperse framework material to the DNA sample is 20:1.

[0119] Example 6

[0120] This embodiment provides a method for nucleic acid quantitative detection, which is only different from Example 4 in that the molar ratio of the monodisperse framework material to the DNA sample is 40:1.

[0121] Example 7

[0122] This embodiment provides a method for nucleic acid quantitative detection, which is only different from Example 4 in that the molar ratio of the monodisperse framework material to the DNA sample is 10:1.

[0123] Example 8

[0124] This embodiment provides a method for nucleic acid quantitative detection, which is only different from Example 4 in that the molar ratio of the monodisperse framework material to the DNA sample is 50:1.

[0125] Comparative Example 1

[0126] This comparative example provides a method for nucleic acid quantitative detection, which is only different from Example 4 in that the pore size of the ZIF-8 particles is 8 nm.

[0127] Test Example 1

[0128] In this embodiment, the DNA sample concentration was gradient-diluted from 5000 copies / μL to 10 copies / μL, and qPCR reaction was carried out with reference to Example 4 to analyze and obtain the initial copy number of nucleic acid, and the analysis results were compared with those of the ddPCR system. The results are asFigure 1 As shown, the method provided by the present invention has excellent consistency with the ddPCR method, and the intraclass correlation coefficient is 0.994.

[0129] Test Example 2

[0130] In this test example, the nucleic acid amplification methods provided in Examples 4-8 and Comparative Example 1 were used to detect the same sample to be tested, and at the same time, the ddPCR method was used to detect the sample to be tested. The obtained copy numbers of template DNA are shown in Table 1.

[0131] Table 1

[0132] Group Template DNA Concentration (copies / μL) Example 4 683 Example 5 675 Example 6 680 Example 7 389 Example 8 677 Comparative Example 1 462 ddPCR Group 689

[0133] It can be concluded from Table 1 that:

[0134] (1) By comparing Example 4 with the ddPCR group, it can be seen that the nucleic acid amplification method provided by the present invention can achieve absolute quantification of the target nucleic acid in the sample to be tested with high accuracy.

[0135] (2) By comparing Example 4 with Example 7, it can be seen that when the molar ratio of the monodisperse framework material to the DNA sample is small, that is, when the proportion of the monodisperse framework material is small, the probability of multiple DNAs binding to the same monodisperse framework material particle increases, and the effect of performing at most one amplification reaction on each monodisperse framework material particle cannot be achieved. Therefore, the obtained quantitative result is small.

[0136] (3) By comparing Example 4 with Example 8, it can be seen that when the molar ratio of the monodisperse framework material to the DNA sample is large, that is, when the proportion of the monodisperse framework material is large, the obtained quantitative results have no obvious difference, but it will cause a certain degree of waste of materials and increase the cost.

[0137] (4) By comparing Example 4 with Comparative Example 1, it can be seen that when the pore size of the particles is large, the probability of multiple DNAs binding to the same monodisperse framework material particle increases. Therefore, the obtained quantitative result is small.

[0138] In summary, the present invention provides a nucleic acid amplification method based on a monodisperse framework material. The multi-porous structure of the monodisperse material effectively restricts the macromolecular movement of the amplification product, thereby avoiding the problem of nucleic acid contamination. It can be applied to a variety of PCR reactions, especially suitable for the detection of low-abundance nucleic acid samples. Compared with ddPCR, it does not require complex droplet generation equipment and retains the advantages of high sensitivity and accuracy of ddPCR.

[0139] The applicant declares that the above description is only a specific embodiment 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 disclosure scope of the present invention.

Claims

1. A nucleic acid amplification method based on a monodisperse framework material, characterized in that, The nucleic acid amplification method includes: mixing a monodisperse framework material with a PCR reaction system and performing amplification according to a PCR program; Among them, the monodisperse framework material includes any one or a combination of at least two of a metal-organic framework material, a mesoporous silica material, or a mesoporous carbon material, and the pore diameter of the monodisperse framework material is 3 to 7 nm.

2. The nucleic acid amplification method according to claim 1, wherein The porosity of the monodisperse framework material is 50% to 80%; Preferably, the metal-organic framework material includes any one or a combination of at least two of ZIF-8, UiO-66, MIL-101, HKUST-1, or MOF-5; Preferably, the particle size of the metal-organic framework material is 150 to 250 nm; Preferably, the mesoporous silica material includes any one or a combination of at least two of MCM-41, SBA-15, MSN, KIT-6, FDU-12, or TUD-1; Preferably, the particle size of the mesoporous silica material is 50 to 600 nm; Preferably, the mesoporous carbon material includes any one or a combination of at least two of CMK-3, CMK-8, FDU-15, MCF, OMC, or nitrogen-doped mesoporous carbon; Preferably, the particle size of the mesoporous carbon material is 100 to 300 nm.

3. The nucleic acid amplification method according to claim 1 or 2, wherein The PCR reaction system contains template DNA, primers, dNTPs, buffer, and DNA polymerase; Preferably, the molar ratio of the monodisperse framework material to the template DNA is (10 to 50):1; Preferably, the PCR reaction system further contains a fluorescent probe; Preferably, the PCR reaction system includes any one of the reaction systems of the conventional PCR method, the qPCR method, the LAMP method, the RCA method, or the SDA method; Preferably, the reaction system of the conventional PCR method contains template DNA, primers, dNTPs, buffer, and Taq DNA polymerase; Preferably, the reaction system of the qPCR method contains template DNA, primers, dNTPs, fluorescent probe, buffer, and Taq DNA polymerase; Preferably, the reaction system of the LAMP method contains template DNA, primers, dNTPs, LAMP fluorescent probe, buffer, and Bst DNA polymerase; Preferably, the reaction system of the RCA method contains template DNA, primers, dNTPs, RCA fluorescent probe, buffer, and Φ29 DNA polymerase; Preferably, the reaction system of the SDA method contains template DNA, primers, dNTPs, SDA fluorescent probe, buffer, nicking enzyme, and Bst DNA polymerase.

4. The nucleic acid amplification method according to any one of claims 1 to 3, characterized in that, The PCR program includes any one of the reaction programs of the conventional PCR method, the qPCR method, the LAMP method, the RCA method, or the SDA method; Preferably, the reaction program of the conventional PCR method includes 93 to 97 °C, 25 to 35 s, 53 to 57 °C, 25 to 35 s, 70 to 74 °C, 55 to 65 s, for 28 to 32 cycles; Preferably, the reaction program of the qPCR method includes 93 - 97°C for 100 - 140 s; 93 - 97°C for 10 - 20 s, 58 - 62°C for 25 - 35 s, with 38 - 42 cycles; Preferably, the reaction program of the LAMP method includes an isothermal reaction at 60 - 70°C for 25 - 35 min; Preferably, the reaction program of the RCA method includes an isothermal reaction at 30 - 37°C for 1 - 2 h; Preferably, the reaction program of the SDA method includes an isothermal reaction at 37 - 42°C for 1 - 2 h.

5. Use of the nucleic acid amplification method according to any one of claims 1 - 4 in preventing nucleic acid contamination.

6. Use of the nucleic acid amplification method according to any one of claims 1 - 4 in nucleic acid quantitative detection.

7. The application according to claim 6, wherein The method for nucleic acid quantitative detection includes real-time fluorescence quantitative PCR or digital PCR.

8. A nucleic acid quantitative detection method based on the principle of digital PCR, characterized in that, The method for nucleic acid quantitative detection includes: obtaining a PCR amplification product using the nucleic acid amplification method according to any one of claims 1 - 4, counting the number of positive fluorescence signals, and analyzing to obtain the initial copy number of the nucleic acid.

9. The nucleic acid quantification detection method according to claim 8, wherein The PCR reaction system contains template DNA, primers, dNTPs, fluorescent probes, buffer, and Taq DNA polymerase; Preferably, the template DNA contains the target nucleic acid sequence of the primers; Preferably, the molar ratio of the monodisperse framework material to the target nucleic acid sequence of the primers in the template DNA is (20 - 40):1; Preferably, the PCR program includes 93 - 97°C for 100 - 140 s; 93 - 97°C for 10 - 20 s, 58 - 62°C for 25 - 35 s, with 38 - 42 cycles.

10. The nucleic acid quantitative detection method according to claim 8 or 9, characterized in that, The analysis to obtain the initial copy number of the nucleic acid includes: combining with the Poisson distribution model to deduce the initial copy number of the nucleic acid in the sample.

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