Application of lanthanide MOF in preparation of fluorescent pyrosequencing or nucleic acid detection kit

Abstract

The invention provides an application of lanthanide MOF in preparation of a fluorescent pyrosequencing or nucleic acid detection kit. The fluorescent lanthanide MOF adopted by the invention can be used for carrying out ratio self-calibration fluorescent pyrosequencing on DNA / RNA in biological samples (cells, tissues, blood, plasma, serum, urine and the like), so that the sequencing accuracy is improved; and the kit can also realize unmarked qPCR quantification and fluorescence self-correction isothermal amplification detection of DNA / RNA, has the advantages of high detection speed, high sensitivity, low cost and wide application range, can promote the innovation and progress of next-generation sequencing methods and instruments, and can also be used for detecting the DNA / RNA in the next-generation sequencing process and the next-generation sequencing instrument in the next-generation sequencing process. And the application of gene detection (including nucleic acid sequence detection, nucleic acid epigenetic modification detection and the like) in disease diagnosis can be promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid sequencing, nucleic acid sequence and epigenetic modification detection, and in particular to a pyrophosphate sequencing, qPCR and isothermal amplification detection method and kit based on self-calibration fluorescence. Background Art

[0002] At present, liquid biopsy based on biomarkers has become a research hotspot and future development trend in tumor diagnosis due to its advantages such as non-invasiveness, convenient specimen collection and powerful monitoring capabilities. Among them, the most important biomarkers are circulating total free DNA and RNA (cfDNA and cfRNA), which have extremely high clinical diagnostic value. The current mainstream diagnostic methods can be divided into two categories: one is DNA / RNA content analysis, which refers to the content monitoring of a certain section of a specific nucleic acid mutation sequence or a certain epigenetic modification (5fC, etc.) to reflect the progression of the disease. The representative gold standard method for this type of analysis is qPCR; the second is sequencing analysis of nucleic acid sequences, accurately analyzing the changes in sequences or epigenetic modifications in the whole genome, so as to have a deeper understanding of the occurrence and development of the disease and provide guidance for treatment decisions. Representative methods of this type of analysis include pyrophosphate sequencing, nanopore sequencing, etc.

[0003] However, the latest studies have shown that both qPCR content analysis and nucleic acid sequence analysis based on various sequencing methods have a common problem, that is, the low analysis accuracy caused by the lack of self-correction, which seriously limits the application value of these methods in clinical diagnosis. Therefore, it has become imperative to pursue innovative nucleic acid detection methods to improve the precision and accuracy of nucleic acid analysis in biomarker assessment and molecular diagnosis. We have noticed that pyrophosphate (PPi), as a key product in the nucleic acid amplification process, its content analysis is expected to be extended to accurately quantify the content of nucleic acid targets, including DNA / RNA sequence mutations and epigenetic modifications. The key difficulty lies in how to develop a new method that can accurately analyze the PPi content and use it in the content and sequence analysis of each nucleic acid.

[0004] Therefore, in order to achieve accurate, sensitive and highly selective PPi analysis and nucleic acid detection, it is necessary to develop a kit and method for pyrophosphate sequencing, qPCR and isothermal amplification detection of nucleic acids. Summary of the invention

[0005] The purpose of the present invention is to provide an application of a lanthanide MOF material in the preparation of a fluorescent pyrophosphate sequencing or nucleic acid detection kit. The inventors of the present application have found that the lanthanide MOF has a long fluorescence lifetime, has multiple luminescent centers, and can achieve accurate nucleic acid detection based on PPi self-correction sensing. The lanthanide MOF can achieve sequence analysis and content detection of DNA / RNA in various biological samples (blood, plasma, serum, urine, etc.), with high accuracy, high sensitivity, wide application range and low cost.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a use of a lanthanide MOF material in preparing a fluorescent pyrophosphate sequencing or nucleic acid detection kit is provided.

[0008] Furthermore, the nucleic acid detection kit includes one of a qPCR detection kit and an isothermal amplification detection kit; the lanthanide MOF material includes at least one of La-MOF, Ce-MOF, Pr-MOF, Nd-MOF, Pm-MOF, Sm-MOF, Eu-MOF, Gd-MOF, Tb-MOF, Dy-MOF, Ho-MOF, Er-MOF, Tm-MOF, Yb-MOF, and Lu-MOF, each of which has the characteristic long-life luminescence of its own lanthanide ion, and two or more lanthanide ions may exist simultaneously in the same lanthanide MOF, such as CeEu-MOF, CeTb-MOF, EuTb-MOF, etc.

[0009] In a second aspect of the present invention, a kit for DNA / RNA fluorescence detection is provided, the kit comprising: a lanthanide MOF material and reagents required for fluorescence detection.

[0010] Furthermore, the fluorescence detection includes one of nucleic acid qPCR detection, nucleic acid isothermal amplification detection, and pyrophosphate sequencing detection. The reagents required for the nucleic acid qPCR detection include: DNA primers, DNA templates, DNA polymerase, and amplification substrate dNTPs; the reagents required for the nucleic acid isothermal amplification detection include DNA templates, terminal deoxynucleotidyl transferase TdT enzyme, and amplification substrate dNTPs; the reagents required for the pyrophosphate sequencing detection include DNA templates, sequencing primers, dNTPs required for sequencing, and dual-emission lanthanide MOF.

[0011] Furthermore, the final concentration of the lanthanide MOF material is 10-1000 μg / mL.

[0012] In a third aspect of the present invention, a method for DNA / RNA fluorescent pyrophosphate sequencing is provided, the method comprising:

[0013] Pre-treating cells, tissues or body fluid samples to restore DNA / RNA to a free state, and obtaining a treated sample solution;

[0014] Using any nucleic acid extraction reagent to extract and purify the DNA / RNA in the treated sample solution;

[0015] The purified solution is subjected to label-free amplification detection to obtain sequencing results.

[0016] Furthermore, the body fluid sample includes at least one of whole blood, plasma, serum, urine, saliva, pleural effusion, cerebrospinal fluid, bile, lymph and ascites.

[0017] The lanthanide MOF self-correction fluorescent pyrophosphate sequencing method avoids the use of ATP sulfurylase, luciferase, substrate adenosine-5'-phosphosulfate (APS) and luciferin in the classical bioluminescent pyrophosphate sequencing method.

[0018] Furthermore, the final concentration of the lanthanide MOF material added is 5 to 500 μg / mL.

[0019] In a fourth aspect of the present invention, a method for qPCR detection of DNA / RNA is provided, the method comprising:

[0020] A qPCR reaction system was prepared to perform a qPCR reaction; after the qPCR reaction was completed, a lanthanide MOF was added, a fluorescence spectrophotometer was used to analyze the series fluorescence of the lanthanide MOF, a fluorescence standard curve of the lanthanide MOF was constructed, and the relative gene expression amount was calculated with the help of the fluorescence standard curve.

[0021] In a fifth aspect of the present invention, a method for isothermal amplification detection of DNA / RNA is provided, the method comprising:

[0022] Terminal deoxynucleotidyl transferase is used to extend the 3' end of the nucleic acid template in a reaction system containing dNTP. After the reaction is completed, lanthanide MOF is added to terminate the reaction, and the amplification reaction system is analyzed by a fluorescence detection system to construct a fluorescence curve of the lanthanide MOF, and the relative gene expression amount is calculated with the help of the fluorescence curve.

[0023] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0024] The present invention provides an application of a lanthanide MOF material in the preparation of a fluorescent detection kit for nucleic acids. In the application, after DNA / RNA is extracted from various biological samples, multi-dimensional nucleic acid information analysis can be achieved based on the luminescent lanthanide MOF, including nucleic acid sequence analysis (using the developed lanthanide MOF pyrophosphate sequencing method), nucleic acid abundance analysis (using the developed lanthanide MOF-based qPCR method) and nucleic acid isothermal amplification analysis (using TdT enzyme-assisted isothermal amplification-lanthanide MOF fluorescence analysis method). The premise for the realization of the above multi-dimensional information method is that the lanthanide MOF has excellent self-corrected fluorescence sensing performance for PPi. The specific reasons are: first, the lanthanide ions have an extremely high affinity for PPi, which is conducive to high sensitivity of sensing; secondly, the lanthanide MOF has multiple luminescence centers, which can realize single-sample multi-channel fluorescence self-correction analysis; finally, the lanthanide MOF prepared by reasonable design of ligand engineering, its luminescence properties can be regulated by PPi for fluorescence on, thereby achieving accurate and sensitive quantification. The specific advantages are as follows:

[0025] (1) Important improvements to the pyrophosphate sequencing method: On the one hand, lanthanide MOF materials have long-life fluorescence, the fluorescence signal is not easily affected by background interference, and the fluorescence tolerates photobleaching, which is the premise for accurate fluorescence quantification; on the other hand, there are differences in the response of multi-emission lanthanide MOFs to pyrophosphate, which produces a fluorescence self-calibration function, ensuring the high precision, high accuracy and high sensitivity of the pyrophosphate sequencing signal; More importantly, the traditional pyrophosphate sequencing method requires the use of enzymes such as ATP sulfurylase and luciferase, and relies on the substrates adenosine-5'-phosphosulfate (APS) and fluorescein. The use of these components not only increases the experimental cost, but also may affect the accuracy and stability of sequencing. The present invention realizes self-calibration and long-life fluorescence detection by introducing lanthanide MOF, thereby improving the classic pyrophosphate sequencing method, improving the accuracy of sequencing data, and having scientific and technological advancement and economic benefits.

[0026] (2) For the application of DNA / RNA qPCR analysis, the important improvement of this method is that there is no need for commercial nucleic acid fluorescent dyes with complex synthesis and high purification costs. This method uses luminescent lanthanide MOF to replace the key fluorescent fuel in qPCR. The specific advantages include but are not limited to simple preparation and purification, low cost, fluorescence tolerance to photobleaching, and dual-emission fluorescence self-calibration function. During the nucleic acid amplification process, the reaction can be quickly terminated without high temperature to destroy the polymerase, and then in situ fluorescence quantitative analysis can be performed.

[0027] (3) For label-free amplification detection of nucleic acids, an important improvement lies in extending the isothermal amplification method to new application scenarios, namely, the simple, highly sensitive, and precise quantitative analysis of DNA epigenetic modifications: after biotinylation of specific DNA epigenetic modifications, streptavidin magnetic beads can be used for enrichment, and then terminal deoxynucleotidyl transferase (TdT enzyme) can be used for isothermal amplification, thereby accurately quantifying extremely low-abundance DNA epigenetic modifications (5hmC, 5fC, 5caC, etc.).

[0028] (4) The method of the present invention can not only sequence or amplify the circulating free nucleic acids in various types of blood (including whole blood, plasma and serum) for quantitative analysis, but can also be applied to the enrichment of circulating free nucleic acids in various other types of body fluids, including but not limited to urine, saliva, pleural effusion, cerebrospinal fluid, bile, lymph, ascites, etc. This is mainly due to the long-life fluorescence of the lanthanide MOF as mentioned above, which is not interfered by biological background fluorescence, and therefore has a higher tolerance for various samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0030] Figure 1 Schematic diagram of the self-calibrated fluorescence response of the dual-emission lanthanide MOF of the present invention to PPi;

[0031] Figure 2 The extraction efficiency of ssDNA by three lanthanide MOF methods and the classic non-lanthanide MOF (Co-IRMOF-74-IV) method was compared by gel electrophoresis.

[0032] Figure 3 The advantages of dual-emission lanthanide MOF self-calibrated fluorescence detection of pyrophosphate are outstanding in precision and accuracy compared with single emission detection;

[0033] Figure 4 Schematic diagram of the process of using lanthanide MOF for self-calibrated fluorescence qPCR and isothermal amplification of nucleic acid detection;

[0034] Figure 5 This is a comparison of the consistency between the lanthanide MOF self-calibration fluorescence qPCR detection method and the classical qPCR detection method; where a is the Intensity value curve, b is the Ct value curve, and c is the I 546 Curve, d is I 616 curve;

[0035] Figure 6 The results of gel electrophoresis analysis of the amount of amplified products of 5fC-DNA chains assisted by TdT enzyme at gradient concentrations; the concentration of the substrate DNA template in Lane 1-Lane 9 in the figure decreases in a gradient manner;

[0036] Figure 7 The results of using lanthanide MOF for self-calibrated fluorescence isothermal amplification nucleic acid detection for DNA epigenetic modification content analysis were compared with the results of commercial kit analysis. DETAILED DESCRIPTION

[0037] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0038] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0039] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0040] The technical solution of the embodiment of the present application is to solve the above technical problems, and the overall idea is as follows:

[0041] The present invention is mainly divided into three parts. The first part is the application of lanthanide MOF materials in self-calibrated fluorescence pyrophosphate sequencing; the second part is the application of lanthanide MOF materials in self-calibrated fluorescence qPCR nucleic acid quantitative analysis; and the third part is the application of lanthanide MOF in self-calibrated fluorescence isothermal amplification nucleic acid detection.

[0042] Regarding the first part:

[0043] An embodiment of the present invention provides a method for self-calibration fluorescent pyrophosphate sequencing, the method comprising:

[0044] Verify the self-calibrated fluorescence response of lanthanide MOF to pyrophosphate, such as Figure 1 shown.

[0045] like Figure 2As shown, first amplify the DNA fragment and biotinylate the pyrophosphate template strand. First, separate the biotinylated single-stranded PCR amplicon through denaturation and hybridize it with a sequencing primer. The hybrid primer and single-stranded template are co-incubated with DNA polymerase and apyrase. Each time a dNTP is added, if the dNTP can pair with the template, a molecule of pyrophosphate will be released, which can be directly detected by the fluorescent Ln-MOF for self-calibration. After being recognized by the fluorescence detector, the number of consecutive NTPs in the sequence to be sequenced can be interpreted through the ratio fluorescence signal. At the same time, in this process, apyrase is a nucleotide degrading enzyme that continuously decomposes unbound nucleotides and ATP. When the decomposition is completed, another nucleotide will be introduced, so that the process continues, thereby achieving the purpose of accurately sequencing the entire sequence. Highlights of this method: It improves the classic pyrophosphate sequencing method and avoids the use of ATP sulfurylase and luciferase; it also avoids the use of substrate adenosine-5'-phosphosulfate (APS) and fluorescein; this sequencing method is based on the self-correction and long-life fluorescence of lanthanide MOF, and the sequencing data is more accurate. For RNA templates, first extract total RNA from cells and other samples and reverse transcribe it into cDNA, and the subsequent sequencing analysis method is consistent. The specific steps are as follows:

[0046] Step S1, verifying the dual emission response of lanthanide MOF to pyrophosphate, such as Figure 1 As shown, this is the basic chemical principle of self-calibrated fluorescent pyrosequencing;

[0047] In the step S1,

[0048] The lanthanide MOF material includes: at least one of La-MOF, Ce-MOF, Pr-MOF, Nd-MOF, Pm-MOF, Sm-MOF, Eu-MOF, Gd-MOF, Tb-MOF, Dy-MOF, Ho-MOF, Er-MOF, Tm-MOF, Yb-MOF, and Lu-MOF, and two or more lanthanide ions may exist simultaneously in the same lanthanide MOF, such as CeEu-MOF, CeTb-MOF, EuTb-MOF, etc.

[0049] The lanthanide MOF material is composed of an organic ligand and a lanthanide metal ion, and the structure type of the lanthanide MOF material includes but is not limited to a triclinic system, a monoclinic system, and a cubic system.

[0050] The organic ligands include terephthalic acid, o-phthalic acid, isophthalic acid, 1,3,5-benzenetricarboxylicacid, pyromellitic acid, biphenyl-3,3',5,5'-tetracarboxylic acid, [1,1':4',1']triphenyl-3,3',5,5"tetracarboxylic acid, acid), 1,1':4,1':4",1"-tetraphenyl-3,3",5,5"-tetracarboxylic acid and other organic ligands; the limited scope of organic ligands is not limited to this, and other organic ligands that can form lanthanide MOFs are also included in this scope.

[0051] As an optional embodiment, the preparation method of the lanthanide MOF material includes but is not limited to: the organic ligand is first dissolved in DMF, ethanol, water or a combination solution of the foregoing three solvents, and a certain amount of triethylamine is added as a deprotonation regulator; the lanthanide metal ion salt is dissolved in primary water, and then 1% volume of the lanthanide ion solution is taken out and added to the organic ligand solution for ultrasonication for 5-10 minutes, and then the remaining metal ion solution is merged into the organic ligand solution, and then a solvent thermal reaction is carried out under closed conditions (reaction temperature 120-160°C, reaction time 1-24h), and after the reaction is completed, it is naturally cooled to room temperature, centrifuged at 10000rpm, and washed with ethanol 2-3 times, and then the final solid product is obtained by reduced pressure drying or freeze drying technology.

[0052] The final concentration of the lanthanide MOF material added is 5-500 μg / mL. This concentration range is conducive to the signal collection and analysis of self-calibration fluorescence pyrophosphate sequencing.

[0053] Step S2: adding the lanthanide MOF material to the pyrophosphate sequencing system for sequential sequencing.

[0054] In the step S2,

[0055] The pyrophosphate sequencing system includes a hybridization primer, a single-stranded template, a DNA polymerase, apyrase, and a dNTP added each time sequencing is performed.

[0056] Step S3: Collect the fluorescence signals of sequencing and summarize and analyze the sequencing results.

[0057] Regarding the second part:

[0058] The embodiment of the present invention provides a method for self-calibration fluorescence qPCR nucleic acid quantitative analysis, the method comprising:

[0059] Taking DNA template as an example, the qPCR reaction system is 20 μL, including 10 μL 2x SYBR Green Master Mix, 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM), 1 μL cDNA template and 8.2 μL nuclease-free water. The qPCR reaction program is set as: 95°C pre-denaturation for 2 minutes, followed by 40 cycles (95°C denaturation for 15 seconds, 60°C annealing for 30 seconds, and 72°C extension for 30 seconds). Fluorescence signals are collected during the extension phase of each cycle. The data are analyzed using Ct values, and the relative gene expression is calculated by the 2^(-ΔΔCt) method.

[0060] After the qPCR reaction is completed, the lanthanide MOF material is added to the qPCR reaction system. There is no need to heat to destroy the polymerase. The lanthanide MOF can quickly terminate the amplification reaction. Then, a fluorescence spectrophotometer is used to analyze the series of fluorescence of the lanthanide MOF, and a fluorescence standard curve of the lanthanide MOF is constructed. The relative gene expression is calculated with the help of the fluorescence standard curve. And a consistency comparison is made with the commercial qPCR kit method. For RNA templates, total RNA is first extracted from cells and other samples and reverse transcribed into cDNA, and the subsequent analysis method is consistent.

[0061] Preferably, the specific lanthanide MOF material type with the best response is screened out based on the fluorescence change intensity before and after the lanthanide MOF amplification.

[0062] Obtain different types of lanthanide MOF materials (they can be prepared by existing methods, and the specific preparation method may include but is not limited to: the organic ligand is first dissolved in DMF or ethanol, and a certain amount of triethylamine is added as a deprotonation regulator; the lanthanide metal ions are dissolved in primary water, and then 1% volume of the lanthanide ion solution is taken out and added to the organic ligand solution for ultrasonication for 5 minutes, and then the remaining metal ion solution is combined with the organic ligand solution, and then a solvent thermal reaction is carried out under closed conditions, and the reaction is terminated to obtain)

[0063] The lanthanide MOF material includes at least one of La-MOF, Ce-MOF, Pr-MOF, Nd-MOF, Pm-MOF, Sm-MOF, Eu-MOF, Gd-MOF, Tb-MOF, Dy-MOF, Ho-MOF, Er-MOF, Tm-MOF, Yb-MOF, and Lu-MOF, and two or more lanthanide ions may exist simultaneously in the same lanthanide MOF, such as CeEu-MOF, CeTb-MOF, EuTb-MOF, etc. The preparation method thereof is the same as that in the first part.

[0064] As a specific implementation, the self-calibration fluorescence qPCR method is applicable to any target sequence that can be amplified, and only the corresponding primers need to be designed.

[0065] Regarding the third part:

[0066] An embodiment of the present invention provides a self-calibrated fluorescence isothermal amplification nucleic acid detection method, the method comprising:

[0067] Using double-stranded DNA as a template, without specific primers, terminal deoxynucleotidyl transferase (TdT enzyme) was used to extend the 3' end of the DNA template in a reaction system containing dNTP. The reaction system includes 1μL DNA template, 2μL 10xTdT buffer, 0.2μL TdT enzyme, 0.4μL 2.5mM dNTP and 16.4μL nuclease-free water, with a total volume of 20μL. After reacting at 37°C for 2 hours, lanthanide MOF was added to terminate the reaction, and the amplification reaction system was analyzed by a fluorescence detection system, and a fluorescent standard curve of lanthanide MOF was constructed, and the relative gene expression was calculated with the help of the fluorescent standard curve. Finally, the results of the commercial kit analysis were compared for consistency. For RNA templates, total RNA was first extracted from cells and other samples and reverse transcribed into cDNA, and the subsequent analysis methods were consistent.

[0068] The double-stranded DNA templates include various biotinylated DNA epigenetic modifications, including 5mC, 5hmC, 5fC, 5caC, etc.

[0069] The biotinylated DNA epigenetic modifications can be respectively captured by streptavidin magnetic beads, and then the above-mentioned primer-free TdT enzyme-assisted isothermal amplification is performed, and finally the self-calibrated fluorescence of the lanthanide MOF is used for quantitative analysis.

[0070] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0071] The present application will be described in detail below with reference to embodiments and experimental data.

[0072] Example 1: Lanthanide MOF for self-calibrated fluorescence pyrophosphate sequencing

[0073] 1. DNA fragment amplification and biotinylation

[0074] The DNA fragment to be sequenced is amplified using PCR technology, and a primer with a biotin tag is added to the PCR reaction to achieve biotinylation of the end of the amplicon. Due to the current lack of instruments based on the new mechanism of lanthanide MOF sensing, we used a method to simulate the pyrophosphate rate measurement process to simulate pyrophosphate sequencing and evaluate its specific improvements to the pyrophosphate sequencing method.

[0075] 1. Denature the PCR reaction product and separate the biotinylated single-stranded DNA template.

[0076] 2. Hybridization and enzyme treatment

[0077] (1) Hybridize the biotinylated single-stranded DNA template with a specific sequencing primer.

[0078] (2) The hybridized primer-template complex is incubated with DNA polymerase and apyrase.

[0079] 3. Pyrosequencing reaction

[0080] (1) Each time a deoxyribonucleotide triphosphate (dNTP) is added, if the dNTP is able to pair with the template, a molecule of pyrophosphate will be released under the action of the polymerase.

[0081] (2) The released pyrophosphate can be directly detected by the Ln-MOF fluorescence detector, and the ratio fluorescence signal can be used to interpret the number of consecutive dNTPs in the sequence to be sequenced.

[0082] 4. Fluorescence detection and signal analysis

[0083] (1) The Ln-MOF fluorescence detector has a self-calibration function, which can avoid background fluorescence interference and improve the reliability and accuracy of the signal.

[0084] (2) The ratio fluorescence signal analysis system will interpret the pairing status of each dNTP, thereby gradually constructing the DNA sequence to be sequenced.

[0085] 5. Nucleotide degradation and cycle sequencing

[0086] (1) Apyrase acts as a nucleotide degrading enzyme, continuously breaking down unbound dNTPs and ATP.

[0087] (2) After degradation is complete, the next dNTP is added and the above process is repeated until the entire sequence is sequenced.

[0088] 6. Sequencing of RNA templates

[0089] For RNA templates, total RNA is first extracted from cells or other samples, and reverse transcribed into cDNA using reverse transcriptase. The subsequent sequencing analysis method is consistent with the above DNA sequencing steps.

[0090] Since there is no instrument that can use this new material to perform sequencing, we used the "fluorescence method" to simulate the "pyrophosphate sequencing process". We used the fluorescence instrument to focus on the core of pyrophosphate sequencing, "pyrophosphate quantitative detection", to demonstrate the advantages of lanthanide MOF. Specific experimental data are as follows: Figure 2 (Simplified schematic diagram of the PPi sensing mimicking pyrosequencing process) and Figure 3 (Comparison of the advantages of dual-emission lanthanide MOF for accurate PPi quantitative analysis) shows: The standard curve of dual-emission lanthanide MOF is significantly better than the standard curve of single emission signal, which solves the core difficulty of PPi quantification, that is, it solves the core difficulty of pyrophosphate sequencing for accurate PPi quantitative analysis.

[0091] In summary, this method avoids the use of ATP sulfurylase and luciferase, and simplifies the sequencing reaction system. It does not require the use of APS and luciferin, reducing experimental costs and complexity. The self-calibration and long-life fluorescence based on lanthanide MOF significantly improves the accuracy and stability of sequencing data ( Figure 3 ).

[0092] Example 2: A new method for self-calibrated fluorescence qPCR nucleic acid detection using lanthanide MOF

[0093] 1. qPCR reaction system configuration

[0094] Prepare a 20 μL qPCR reaction system including the following components:

[0095] ○10μL 2x SYBR Green Master Mix

[0096] ○0.4μL forward primer (10μM)

[0097] ○0.4μL reverse primer (10μM)

[0098] ○1μL cDNA template

[0099] ○8.2μL nuclease-free water

[0100] 2. qPCR Reaction Procedure

[0101] 1. Set up the following reaction program in the qPCR instrument:

[0102] ○Pre-denaturation at 95℃ for 2 minutes

[0103] ○Perform 40 cycles:

[0104] ■ Denaturation at 95℃ for 15 seconds

[0105] ■ Annealing at 60℃ for 30 seconds

[0106] ■72℃ elongation for 30 seconds

[0107] 2. Collect fluorescence signals during the extension phase of each cycle.

[0108] 3. Data Analysis

[0109] (1) Use Ct value for preliminary data analysis.

[0110] (2) The relative gene expression was calculated by the 2^(-ΔΔCt) method.

[0111] 4. Introduction of lanthanide MOF for termination and fluorescence detection

[0112] After the qPCR reaction is completed, an appropriate amount of Ln-MOF material is added to the qPCR reaction system. Ln-MOF can quickly terminate the amplification reaction without heating to destroy the polymerase.

[0113] 5. Fluorescence Spectrophotometer Analysis

[0114] (1) The fluorescence of Ln-MOF was analyzed by fluorescence spectrophotometer.

[0115] (2) Construct the fluorescence curve of Ln-MOF.

[0116] (3) Calculate relative gene expression using fluorescence markers.

[0117] 6. Comparative Analysis

[0118] The results obtained by this method were compared with those of commercial qPCR kit methods to evaluate its accuracy and stability.

[0119] 7. Detection of RNA Template

[0120] For RNA templates, first extract total RNA from cells and other samples, and reverse transcribe the RNA into cDNA using reverse transcriptase. The subsequent qPCR and analysis steps are the same as those for DNA templates.

[0121] The experimental results are as follows Figure 4 and Figure 5 As shown, Figure 4 The upper part shows a schematic diagram of the PCR process using lanthanide MOFs. Figure 5The comparison between the standard curve of classical qPCR and the fluorescence standard curve of dual-emission lanthanide MOF is shown. The results show that the dual-emission lanthanide MOF has a linear response relationship similar to that of classical qPCR.

[0122] In summary, this method avoids the step of thermally destroying the polymerase and simplifies the operation process. Ln-MOF has a self-calibration function, which significantly improves the reliability and accuracy of the fluorescence signal. Based on the long-life fluorescence of Ln-MOF, the accuracy and stability of nucleic acid detection are improved. Compared with traditional methods, the method of the present invention is easy to operate and the detection results are more accurate ( Figure 3 The data demonstrate the self-calibration capability of dual-emission lanthanide MOFs, with the linearity of the PPi response being significantly stronger than that of the single emission).

[0123] Example 3: A lanthanide MOF for self-calibration fluorescence isothermal amplification nucleic acid detection method for detecting various DNA epitope modification contents

[0124] 1. Reaction system configuration

[0125] Prepare a 20 μL isothermal amplification reaction with the following components:

[0126] ○1μL double-stranded DNA template

[0127] ○2 μL 10x TdT buffer

[0128] ○0.2μL TdT enzyme

[0129] ○0.4μL 2.5mM dNTP

[0130] ○16.4μL nuclease-free water

[0131] 2. Isothermal Amplification Reaction

[0132] The reaction was carried out at 37°C for 2 hours, allowing the TdT enzyme to extend the 3' end of the DNA template in the reaction system containing dNTP.

[0133] 3. Introduce lanthanide MOF to terminate the reaction

[0134] After the reaction is completed, an appropriate amount of Ln-MOF material is added to terminate the amplification reaction.

[0135] 4. Fluorescence Detection and Data Analysis

[0136] 4.1 Analyze the amplification reaction system through the fluorescence detection system.

[0137] 4.2 Construction of the fluorescence curve of Ln-MOF.

[0138] 4.3 Calculate relative gene expression using fluorescence markers.

[0139] 5. Comparative Analysis

[0140] The results obtained by this method were compared with those of commercial kit analysis to evaluate its accuracy and stability.

[0141] 6. Detection of DNA epigenetic modifications by biotinylation

[0142] The double-stranded DNA templates of 6.1 include various biotinylated DNA epigenetic modifications, such as 5mC, 5hmC, 5fC, 5caC, etc.

[0143] 6.2 The biotinylated DNA epitope modification is captured by streptavidin magnetic beads.

[0144] 6.3 The fished DNA was subjected to the above-mentioned TdT enzyme-assisted isothermal amplification without primers.

[0145] Quantitative analysis was performed by self-calibrated fluorescence of Ln-MOF. Figure 6 and Figure 7 shown. Figure 6 The nucleic acid gel electrophoresis showed that as the concentration of the substrate DNA template decreased from high to low, the amount of nucleic acid after TdT enzyme amplification also changed significantly, showing a regular change. Figure 7 The data compares the quantitative analysis of 5fC-DNA by this method with that of the classic ELISA kit, proving that the method of the present invention has good detection precision and accuracy.

[0146] In summary, this method does not require specific primers and thermal cycling steps, which simplifies the operation process. Ln-MOF has a self-calibration function, which significantly improves the reliability and accuracy of the fluorescence signal. Based on the long-life fluorescence of Ln-MOF, the accuracy and stability of nucleic acid detection are improved. It is suitable for the detection of various DNA epigenetic modifications and has broad application prospects.

[0147] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0148] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0149] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of lanthanide MOF in the preparation of fluorescent pyrophosphate sequencing or nucleic acid detection kits.

2. The use according to claim 1, characterized in that: The nucleic acid detection kit includes one of a qPCR detection kit and an isothermal amplification detection kit; the lanthanide MOF includes at least one of La-MOF, Ce-MOF, Pr-MOF, Nd-MOF, Pm-MOF, Sm-MOF, Eu-MOF, Gd-MOF, Tb-MOF, Dy-MOF, Ho-MOF, Er-MOF, Tm-MOF, Yb-MOF, and Lu-MOF.

3. A kit for DNA / RNA fluorescence detection, characterized in that: The kit comprises: Lanthanide MOF materials and reagents required for fluorescence detection.

4. A kit for DNA / RNA fluorescence detection according to claim 3, characterized in that: The fluorescence detection includes one of nucleic acid qPCR detection, nucleic acid isothermal amplification detection, and pyrophosphate sequencing detection. The reagents required for the nucleic acid qPCR detection include: DNA primers, DNA template, DNA polymerase and amplification substrate dNTPs; The reagents required for the nucleic acid isothermal amplification detection include a DNA template, a terminal deoxynucleotidyl transferase TdT enzyme, and an amplification substrate dNTPs; The reagents required for the pyrophosphate sequencing detection include a DNA template, a sequencing primer, dNTPs required for sequencing, and a dual-emission lanthanide MOF.

5. A kit for DNA / RNA fluorescence detection according to claim 3, characterized in that: The final concentration of the lanthanide MOF material is 10-1000 μg / mL.

6. A method for DNA / RNA fluorescence pyrophosphate sequencing, characterized in that: The method comprises: Pre-treating cells, tissues or body fluid samples to restore DNA / RNA to a free state, and obtaining a treated sample solution; Using any nucleic acid extraction reagent to extract and purify the DNA / RNA in the treated sample solution to obtain a purified solution; The purified solution is subjected to label-free amplification detection to obtain sequencing results.

7. A method for DNA / RNA fluorescent pyrophosphate sequencing according to claim 6, characterized in that: The body fluid sample includes at least one of cells, tissues, whole blood, plasma, serum, urine, saliva, pleural effusion, cerebrospinal fluid, bile, lymph fluid, and ascites.

8. A method for qPCR detection of DNA / RNA, characterized in that: The method comprises: A qPCR reaction system was prepared to perform a qPCR reaction; after the qPCR reaction was completed, a lanthanide MOF was added, a fluorescence spectrophotometer was used to analyze the series fluorescence of the lanthanide MOF, a fluorescence standard curve of the lanthanide MOF was constructed, and the relative gene expression amount was calculated with the help of the fluorescence standard curve.

9. A method for isothermal amplification detection of DNA / RNA, characterized in that: The method comprises: Use terminal deoxynucleotidyl transferase to extend the 3' end of the nucleic acid template in a reaction system containing dNTPs; After the reaction is completed, lanthanide MOF is added to terminate the reaction, and the amplification reaction system is analyzed by a fluorescence detection system to construct a fluorescence standard curve of lanthanide MOF, and the relative gene expression amount is calculated with the help of the fluorescence standard curve.

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