CN115521979A - Single-molecule nucleic acid detection method, product and related application - Google Patents
Single-molecule nucleic acid detection method, product and related application Download PDFInfo
- Publication number
- CN115521979A CN115521979A CN202211176430.XA CN202211176430A CN115521979A CN 115521979 A CN115521979 A CN 115521979A CN 202211176430 A CN202211176430 A CN 202211176430A CN 115521979 A CN115521979 A CN 115521979A
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- detection
- product
- reaction
- capture probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a detection method of single molecule nucleic acid, a product and related application, and relates to the technical field of single molecule detection. According to the invention, biotin modified dNTP is inserted into the extension of a target nucleic acid chain, binding sites of SA-fluorescent microspheres and the surfaces of magnetic beads are increased, and the detection sensitivity is improved to a single-molecule level.
Description
Technical Field
The invention relates to the technical field of single molecule detection, in particular to a detection method of single molecule nucleic acid, a product and related application.
Background
The ability to quantify nucleic acids in complex matrices with high sensitivity and accuracy is central to a wide range of applications from biomedical research to clinical therapy. Conventional nucleic acid detection technologies mainly include Polymerase Chain Reaction (PCR), real-time fluorescent quantitative PCR (qPCR), digital PCR (dPCR), fluorescence In Situ Hybridization (FISH), next generation sequencing technology (NGS), and the like.
The PCR technology is a technology for amplifying a target nucleic acid in vitro, and detects a target DNA qualitatively or quantitatively by designing a specific primer, and the schematic diagram of the principle is shown in FIG. 1 below.
The PCR technology is the most commonly used DNA amplification means at present, and the target fragment is amplified at an exponential speed through continuous cycles of three basic reaction steps of denaturation-annealing-extension. Denaturation is to heat the double-stranded DNA by heating and then dissociate the double-stranded DNA into single-stranded DNA which is used as a copied template; annealing is to reduce the temperature to about 55 ℃ so that the complementary sequences between the primer and the template DNA single strand can be paired and combined; the extension is to synthesize a new DNA single strand complementary to the template DNA strand according to the base complementary pairing and half-retention replication principles at the optimal temperature of DNA polymerase. The target sequence in the sample is amplified by one time for each cycle. Thus, after a few cycles, the number of target segments will exhibit an exponential growth. PCR techniques provide higher sensitivity for current nucleic acid detection, however, they have limited specificity in mutation identification, suffer from precision, are highly sensitive to polymerase inhibitors, and do not allow absolute quantification of target nucleic acids.
The real-time fluorescent quantitative PCR (qPCR) technology is characterized in that an intercalating dye or a fluorescent probe is added in a conventional PCR reaction, the fluorescence intensity in the PCR process is monitored in real time, and copy number levels of different samples are compared according to the difference of the number of cycles of reaching specific fluorescence intensity of a reaction system, so that the quantitative purpose is achieved. And comparing with a standard curve made by using a standard product, and accurately quantifying the sample. The qPCR technology has high sensitivity and specificity, and is the most common PCR technology in clinic at present.
Digital PCR (dPCR) mainly refers to two forms of digital PCR, one is droplet digital PCR technology (ddPCR), in which a single independent and closed reaction system is formed by generating monodisperse droplets, and the other is by dividing the reaction system by a high-precision microwell array. In digital PCR, a sample is divided into a plurality of extremely small droplets to perform PCR reactions, each reaction initially contains only 1 template according to the poisson distribution principle, and then the number of template molecules in the initial sample is determined by counting luminescent droplets, so that dPCR itself can complete absolute quantification, which is commonly used in detection work requiring precise quantification, and the schematic diagram of the principle is shown in fig. 2 below.
The FISH technology designs a fluorescence labeling specific nucleic acid probe through a nonradioactive in situ hybridization technology, performs in situ hybridization with a sample, quantifies target DNA according to a fluorescence signal, and has higher sensitivity and specificity, but the technology has high operation difficulty and higher cost.
The NGS technology, also known as next generation sequencing technology, is the core of an epoch-making revolution on the first generation sequencing technology, and the schematic diagram of the principle is shown in fig. 3, the NGS technology solves the limitation that one generation of sequencing can only determine one sequence at a time, and can simultaneously obtain dozens to millions of nucleic acid molecule sequences by one operation, but the obtained single sequence has a short length. The NGS technology is mainly accomplished by 4 steps of constructing a sequencing library, through a sequencing flow cell, bridge PCR amplification and denaturation, and sequencing.
The current nucleic acid detection technologies, including PCR, qPCR, dPCR, FISH, NGS technologies, etc., still require associated instrumentation, complex operational procedures and professional analysis processing; and two forms of digital PCR: the use of high precision microwell arrays as reaction carriers and by generating single and uniform water-in-oil droplets also has corresponding disadvantages: 1. the former has high requirements on manufacturing process and precision: the high-precision micropore array is complex to process, high in cost and not easy to popularize; 2. the latter requires specially matched reagents: surfactants are relied on to reduce the surface tension between droplets to prevent the droplets from fusing, and the current commercial surfactants generally cannot stabilize the droplets for a long time and prevent cross-contamination caused by the diffusion of hydrophobic small molecules between the droplets; 3. the detection time is longer: both rely on long thermal cycling amplification reactions.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a detection method of single-molecule nucleic acid, a product and related application.
The invention is realized by the following steps:
in a first aspect, embodiments of the present invention provide the use of a reagent combination for the manufacture of a product for single molecule nucleic acid detection, the reagent combination comprising: the kit comprises a solid phase carrier, a capture probe, dNTP modified with biotin, streptavidin coupled with a fluorescent marker and a PCR detection reagent; wherein the dNTP is selected from at least one of dATP, dTTP, dCTP, dGTP and dUTP.
In a second aspect, the present invention provides a method for detecting a single-molecule nucleic acid, which includes detecting a sample to be detected by using the reagent combination described in the previous embodiments.
In a third aspect, the present invention provides a kit for single molecule nucleic acid detection, the kit comprising the reagent combination described in the previous embodiments.
The invention has the following beneficial effects:
1. the detection time is short: the signal amplification depends on the DNA polymerase to amplify the target nucleic acid for multiple times, so that the overall fluorescence signal of the solution is gradually intensified and converted into the fixation of the fluorescent particles, and the overall optical signal of the solution is converted into the digital signal of a single high-fluorescence particle, so that the reaction time is greatly shortened.
2. The detection sensitivity is high: by inserting Biotin modified dNTP into the extension of a target nucleic acid chain, binding sites of the SA-fluorescent microspheres and the surface of a solid phase carrier are increased, and the detection sensitivity is improved to a single molecular level.
3. The requirement on detection equipment is low: the dependence of the current nucleic acid detection technology on matched equipment is eliminated; the fluorescent microspheres are used for converting single-molecule signals into single-fluorescent-microsphere signals to realize signal amplification, so that the requirements of detection equipment on an optical system are reduced.
4. The cost is low: does not need to use precision machining equipment, and has the advantages of easy integration, less reagent consumables, low cost and the like.
5. The application range is wide: the method is suitable for most nucleic acid detection systems, and has wide application potential in the fields of cell biology, molecular biology, clinical medicine and the like.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic diagram of the basic principle of PCR;
FIG. 2 is a schematic diagram of digital PCR (microdroplet/high precision microarray; nucleic acid molecule signal amplification; digital signal collection, statistics and analysis);
FIG. 3 is a schematic diagram of the NGS technology;
FIG. 4 is a flow chart of single molecule nucleic acid detection;
FIG. 5 is a graph showing the effect of reaction time on assay performance;
FIG. 6 is a biotin-dUTP concentration optimization;
FIG. 7 is a sensitivity validation of the detection method provided in example 1;
FIG. 8 is a linear range of the detection method provided in example 1;
FIG. 9 is a selective validation of the detection method provided in example 1;
FIG. 10 is a comparison of the detection method provided in example 1 with the detection time of qPCR.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
According to the invention, biotin (Biotin) modified dNTP is added, and a captured target nucleic acid chain is inserted into a sequence under the extension of polymerase; the Streptavidin (SA) -modified high-fluorescence microspheres are added, the PCR reaction without depending on long-time thermal cycling is realized through the specific combination of SA and Biotin, the limitation of a matched nucleic acid amplification instrument is eliminated, the signals of nucleic acid molecules are converted into the signals of the fluorescence microspheres, and the streptavidin-modified high-fluorescence microspheres have the advantages of high sensitivity, high accuracy, low cost, easy integration and the like.
In particular, embodiments of the present invention provide the use of a combination of reagents in the preparation of a product for single molecule nucleic acid detection, the combination of reagents comprising: the kit comprises a solid phase carrier, a capture probe, dNTP modified with biotin, streptavidin coupled with a fluorescent marker and a PCR detection reagent; wherein the dNTP is selected from at least one of dATP, dTTP, dCTP, dGTP and dUTP.
In some embodiments, the dNTP (biotin-modified dNTP) is selected from at least one of dATP, dTTP, dCTP, dGTP, and dUTP. Alternatively, the dNTP is dUTP, exemplified by dUTP, by adding Biotin (Biotin) modified dUTP, replacing a portion of the dTTP bases, and inserting the captured target nucleic acid strand into the sequence under extension by a polymerase; in other embodiments, the dNTP may be dATP, dTTP, dCTP and/or dGTP.
In some embodiments, the capture probe is immobilized to a surface of the solid support. The method of immobilizing the capture probe on the solid support can be performed based on the conventional technical knowledge in the art, for example, the 5-terminal of the capture probe sequence can be modified with (-NH) 2 ) And modifying NHS (N-hydroxysuccinimide) on the surface of the solid phase carrier, and mixing the NHS and the N-hydroxysuccinimide for reaction to obtain the solid phase carrier with the capture probe fixed on the surface.
In some embodiments, the solid support comprises any one of magnetic beads, polystyrene, polyvinyl chloride, PVDF membrane, cellulose, and gel.
In some embodiments, the solid support comprises a magnetic bead. The magnetic beads are used as solid phase carriers, and the concentration of the modified capture probe is improved by utilizing the characteristic that the magnetic beads have higher specific surface area; meanwhile, the magnetic beads are uniformly dispersed in the solution phase, so that the collision between the target nucleic acid in the sample solution and the capture probe on the surface of the magnetic beads is promoted, and the detection sensitivity of the specific recognition reaction is further improved. Other solid supports may also achieve similar effects.
The fluorescent label may be selected from existing fluorescent labels used for single molecule detection, in some embodiments, the fluorescent label comprises a nano-fluorescent particle; in other embodiments, micron-sized fluorescent particles may be included. Optionally, the nano-fluorescent particles comprise at least one of organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
In some embodiments, the fluorescent label comprises a colloid. Optionally, the colloid is selected from at least one of a colloidal metal, a disperse dye, a dye-labeled microsphere, and a dye-labeled latex. The colloidal metal is at least one selected from colloidal gold and colloidal selenium.
In some embodiments, the capture probe is capable of specifically binding to a target nucleic acid comprising any one of any single-stranded DNA, single-stranded RNA, double-stranded DNA, and double-stranded RNA.
In some embodiments, the number of base pairs for hybridization of the capture probe to the target nucleic acid is ≧ 10 pairs. The base pair number may be specifically any one of or a range between any two of 10 pairs, 12 pairs, 14 pairs, 16 pairs, 18 pairs, 20 pairs, 22 pairs, 24 pairs, 26 pairs, 28 pairs, 30 pairs, 32 pairs, 34 pairs, 36 pairs, 38 pairs, 40 pairs, 42 pairs, 44 pairs, 46 pairs, 48 pairs, 50 pairs, 52 pairs, 54 pairs, 56 pairs, 58 pairs, 60 pairs, 62 pairs, 64 pairs, 66 pairs, 68 pairs, 70 pairs, 72 pairs, 74 pairs, 76 pairs, 78 pairs and 80 pairs.
In some embodiments, the length of the capture probe is 10-80 bp, and within this length range, the effect described herein can be achieved, and the longer the length of the probe, the higher the specificity, and specifically, the longer the length of the probe, the more the capture probe can be 10bp, 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, or any two ranges therebetween.
In some embodiments, the product comprises a reagent or kit.
In some embodiments, the PCR detection reagents comprise: 4 kinds of dNTP and Mg 2+ DNA polymerase and PCR reaction buffer.
In some embodiments, the method of use of the product for single molecule nucleic acid detection comprises:
mixing the solid phase carrier fixed with the capture probe, the biotin-labeled dNTP, the PCR detection reagent and a sample to be detected at an annealing temperature for reaction;
after the reaction is finished, removing free dNTP modified with biotin in the reaction product, adding streptavidin coupled with a fluorescent marker in the reaction product, and incubating;
after removing the streptavidin coupled with the fluorescent marker free in the incubation product, the detection and/or analysis of the fluorescent signal is performed.
In some embodiments, the method for single molecule nucleic acid detection may be defined in the application instructions and included in the kit as one of the components in the combination of reagents.
In some embodiments, the annealing temperature is 4 to 50 ℃, and specifically may be any one or a range between any two of 4 ℃,5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃ and 50 ℃.
In some embodiments, the mixing reaction time is 2min to 2h, and specifically may be any one or a range between any two of 2min, 5min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min, 65min, 70min, 75min, 80min, 85min, 90min, 95min, 100min, 105min, 110min, 115min, and 120 min.
In some embodiments, the biotin-modified dNTP is used at a concentration of 0.02. Mu.M-2 mM, and specifically, may be used at any one or two ranges of 0.02. Mu.M, 0.05. Mu.M, 0.1. Mu.M, 0.2. Mu.M, 0.3. Mu.M, 0.4. Mu.M, 0.5. Mu.M, 0.6. Mu.M, 0.7. Mu.M, 0.8. Mu.M, 0.9. Mu.M, 1. Mu.M, 10. Mu.M, 50. Mu.M, 100. Mu.M, 150. Mu.M, 200. Mu.M, 250. Mu.M, 300. Mu.M, 350. Mu.M, 400. Mu.M, 450. Mu.M, 500. Mu.M, 550. Mu.M, 600. Mu.M, 650. Mu.M, 700. Mu.M, 750. M, 800. Mu.M, 850. M, 900. M, 950. M, 1mM, 1.1.1.1.2 mM, 1.3mM, 1.4mM, 1.5mM, 1.6mM, 1.7, 1.8mM, and 9.9 mM.
In some embodiments, the capture probe is at a working concentration of 0.02. Mu.M to 2mM.
In some embodiments, the amount of streptavidin conjugated to a fluorescent label is 1nM to 100. Mu.M.
The embodiment of the invention also provides a method for detecting the single-molecule nucleic acid, which comprises the step of detecting a sample to be detected by adopting the reagent combination in any embodiment.
In some embodiments, the step of detecting comprises a method for single molecule nucleic acid detection as described in any of the preceding embodiments.
In addition, the embodiment of the invention also provides a kit for detecting single-molecule nucleic acid, and the product comprises the reagent combination described in any embodiment of the invention.
The invention has the characteristics that:
1. breakthrough of detection principle level: compared with the traditional nucleic acid molecule detection method for realizing quantitative detection by measuring the integral optical characteristics of the solution, such as fluorescence quantitative PCR reaction, the single-molecule nucleic acid detection system constructed by the embodiment of the invention does not need to rely on multiple amplifications of DNA polymerase on the target nucleic acid in the detection principle level, thereby realizing the breakthrough in principle, so that the sensitivity and the accuracy of the single-molecule nucleic acid detection system are far beyond the traditional technology, and the single-molecule nucleic acid detection system has bright prospect in the fields of clinical application and scientific research frontier.
2. Breakthrough of application level: the novel monomolecular nucleic acid detection system constructed by the invention overcomes the sensitivity limitation of the traditional detection method, and realizes the high-sensitivity detection of low-abundance nucleic acid in clinical samples. The Lambda DNA is taken as a target nucleic acid, and a necessary novel research method is provided for the research and development of novel biomarkers and the research of a deeper single cell level and a single molecule level in the future.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
A method for detecting a single-molecule nucleic acid, which is represented by the following reaction scheme in FIG. 4, includes the following steps.
(1) Designing specific target nucleic acid probe aiming at target nucleic acid to be detected, extending target nucleic acid chain, and modifying (-NH) at 5-end of probe 2 ) A group; the sequence information of the capture probes and target nucleic acids used is shown in table 1.
TABLE 1 sequence information of Capture probes and target nucleic acids
(2) Diluting the probe obtained in the step (1) to 50 mu M with PBS (phosphate buffer solution) (pH7.4) to obtain 500 microliter of solution I;
(3) Adding the solution I into 5mgNHS (N-hydroxysuccinimide) modified magnetic beads to obtain Mb @ capture probe (magnetic beads modified with a capture probe);
(4) Using the PCR reagent, 2. Mu.l of the target nucleic acid, mb @ Capture probe in step (3), and 10. Mu.l of 10mM Biotin-dUTP were added to 1 XPCR mix and reacted at 60 ℃ for 5 minutes.
(5) After the reaction is finished, removing redundant Biotin-dUTP (Bio-engineering (Shanghai) Co., ltd.; product number: R0081)) by using a magnetic separation technique to obtain a solution II;
(6) Adding 20 microliter of SA-nano fluorescent microspheres (FG 0400SA, suzhou Dendu Biotechnology Co., ltd.) to the solution II, and incubating at 37 ℃ for 15-20 minutes;
(7) After the reaction is finished, washing for at least 3 times, separating the magnetic particles by a magnetic separation technology, and removing redundant SA-nano fluorescent microspheres to obtain a solution III;
(8) And after washing, carrying out technical, statistical and analysis on the fluorescent signal of the magnetic beads by the aid of a fluorescent microscope on the solution III.
(9) And (4) processing the fluorescence signal data obtained in the step (8) to accurately analyze the target nucleic acid to be detected on a single-molecule level.
Example 2
Based on the method provided in example 1, the reaction (step (4)) time was used as a single factor variable, multiple experimental groups were set, and the effect of different reaction times on the detection effect was verified. The results are shown in Table 2 and FIG. 5.
TABLE 2 test results
Remarking: copy/mL is the concentration of the target nucleic acid; parthenomber is the digital signal of the highly fluorescent particle.
Example 3
Based on the method provided by the embodiment 1, the concentration of biotin-dUTP is taken as a single-factor variable, a plurality of groups of experiment groups are set, and the influence of the concentration of biotin-dUTP on the detection effect is verified. The results are shown in Table 3 and FIG. 6.
TABLE 3 test results
| dUTP/μM | copy/mL | particle number |
| 0.5 | 5000 | 5014±360 |
| 1 | 5000 | 7512±480 |
| 2 | 5000 | 8671±560 |
| 5 | 5000 | 9157±740 |
| 10 | 5000 | 9216±850 |
| 20 | 5000 | 8512±741 |
| 40 | 5000 | 6521±458 |
Example 4
The sensitivity of the detection method provided in example 1 was verified, and the detection results are shown in table 4 and fig. 7.
TABLE 4 test results
| copy/ | particle number | |
| 1 | 2±0.25 | |
| 4 | 6±1.2 | |
| 20 | 38±2.6 | |
| 80 | 150±6.7 | |
| 200 | 390±10.2 | |
| 1000 | 1900±55.2 |
The linear range of the detection method provided in example 1 was verified and the results are shown in table 5 and fig. 8.
TABLE 5 detection results of the linear range
| copy/ | particle number | |
| 2 | 4±0.25 | |
| 20 | 38±2.6 | |
| 200 | 390±10.2 | |
| 1000 | 1900±55.2 | |
| 5000 | 4735±268 | |
| 20000 | 28975±1597 | |
| 100000 | 191027±12456 |
Example 5
The high selectivity of example 1 was verified.
The sequence information of the capture probes, the target nucleic acids and the mutant nucleic acids added to the test sample used in this example is shown in Table 6.
TABLE 6 sequence information
| Name(s) | Sequence (5 '-3') |
| Probe | NH2-GCT ACA GCA ACC TCA |
| WT | GGA TTC CGAT GGT GTC TGT TTG AGG TTG CTG TAGC |
| A→C | GGA TTC CGAT GGT GTC TGT TTGCGG TTG CTG TAGC |
| A→G | GGA TTC CGAT GGT GTC TGT TTG GGG TTG CTG TAGC |
| A→T | GGA TTC CGAT GGT GTC TGT TTG TGG TTG CTG TAGC |
Remarking: probe is a capture Probe; WT is the target nucleic acid and bases bold/underlined are the mutation sites in the mutant nucleic acid.
The results are shown in Table 7 and FIG. 9.
TABLE 7 test results
| copy/mL | particle number | |
| A→C | 5000 | 608±21 |
| A→G | 5000 | 512±15 |
| A→T | 5000 | 485±25 |
| WT | 5000 | 9157±349 |
Example 6
The detection time of the method of the invention is verified.
The detection time was determined based on the method provided in example 1, using probe 2 to detect target 2. Meanwhile, a control group was set based on qPCR, and the time required for completion of the detection was as shown in table 10 and fig. 10.
The embodiment of the control group is as follows: the final concentrations of target 2 in the PCR reaction solutions of each group, blank and Sample, were 0nM and 10pM, respectively, prepared according to the recipe shown in Table 8. Amplifying on a thermal cycling amplifier according to the following steps: (1) 94 ℃ for 3min; (2) circulating for 40 times at 94-56-72 ℃ for 30sec respectively; (3) 72 ℃,5min; (4) 4 ℃ for 3min.
TABLE 8 PCR reaction solution formulation (microliter)
| | Sample | |
| Target | ||
| 2 | 0 | 10pM |
| Forward primer | 0.2μM | 0.2μM |
| Reverse primer | 0.2μM | 0.2μM |
| TransStart Green qPCR Supermix (gold full type) | 1× | 1× |
TABLE 9 DNA sequences used in example 6
TABLE 10 detection time
| Time of | min |
| qPCR | |
| 90 | |
| Method for producing a |
5 |
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. Use of a combination of reagents for the preparation of a product for single molecule nucleic acid detection, wherein the combination of reagents comprises: the kit comprises a solid phase carrier, a capture probe, dNTP modified with biotin, streptavidin coupled with a fluorescent marker and a PCR detection reagent; wherein the dNTP is selected from at least one of dATP, dTTP, dCTP, dGTP and dUTP.
2. The use of claim 1, wherein the capture probe is immobilized on the surface of the solid support;
preferably, the solid phase carrier comprises any one of magnetic beads, polystyrene, polyvinyl chloride, a PVDF membrane, cellulose and gel;
preferably, the solid support comprises magnetic beads.
3. The use according to claim 1, wherein the dntps are selected from any one of dATP, dTTP, dCTP, dGTP and dUTP;
preferably, the fluorescent marker comprises a nano-fluorescent particle;
preferably, the nano fluorescent particles include at least one of organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles;
preferably, the fluorescent marker comprises a colloid;
preferably, the colloid is selected from at least one of a colloidal metal, a disperse dye, a dye-labeled microsphere, and a dye-labeled latex;
preferably, the colloidal metal is selected from at least one of colloidal gold and colloidal selenium.
4. The use of any one of claims 1 to 3, wherein the capture probe is capable of specifically binding to a target nucleic acid comprising any one of single-stranded DNA, single-stranded RNA, double-stranded DNA, and double-stranded RNA;
preferably, the base number of the hybridization pairing of the capture probe and the target nucleic acid is more than or equal to 10 pairs;
preferably, the length of the capture probe is 10-80 bp;
preferably, the product comprises a reagent or kit;
preferably, the PCR detection reagent comprises: 4 dNTPs and Mg 2+ DNA polymerase and PCR reaction buffer.
5. The use according to claim 4, wherein the method of the product for single molecule nucleic acid detection comprises:
mixing the solid phase carrier fixed with the capture probe, the dNTP modified with the biotin, the PCR detection reagent and a sample to be detected at an annealing temperature for reaction;
after the reaction is finished, removing free dNTP modified with biotin in the reaction product, adding streptavidin coupled with a fluorescent marker in the reaction product, and incubating;
after removing the streptavidin coupled with the fluorescent marker free in the incubation product, the detection and/or analysis of the fluorescent signal is performed.
6. Use according to claim 5, wherein the annealing temperature is between 4 and 50 ℃;
preferably, the time of the mixing reaction is 2 min-2 h.
7. The use according to claim 5, wherein the biotin-modified dNTP is used at a concentration of 0.02. Mu.M-2 mM.
8. A method for detecting a single-molecule nucleic acid, which comprises detecting a sample to be tested with the combination of reagents according to any one of claims 1 to 7.
9. The method according to claim 8, wherein the step of detecting comprises the method for detecting a single-molecule nucleic acid according to any one of claims 5 to 7;
preferably, the detection method is not directed towards the diagnosis or treatment of a disease.
10. A kit for single molecule nucleic acid detection, comprising the combination of reagents of any one of claims 1 to 7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211176430.XA CN115521979A (en) | 2022-09-26 | 2022-09-26 | Single-molecule nucleic acid detection method, product and related application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211176430.XA CN115521979A (en) | 2022-09-26 | 2022-09-26 | Single-molecule nucleic acid detection method, product and related application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115521979A true CN115521979A (en) | 2022-12-27 |
Family
ID=84700100
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211176430.XA Pending CN115521979A (en) | 2022-09-26 | 2022-09-26 | Single-molecule nucleic acid detection method, product and related application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115521979A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116926168A (en) * | 2023-09-18 | 2023-10-24 | 南方科技大学 | microRNA detection method for secondary signal amplification and application |
-
2022
- 2022-09-26 CN CN202211176430.XA patent/CN115521979A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116926168A (en) * | 2023-09-18 | 2023-10-24 | 南方科技大学 | microRNA detection method for secondary signal amplification and application |
| CN116926168B (en) * | 2023-09-18 | 2023-12-22 | 南方科技大学 | microRNA detection method for secondary signal amplification and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2017200433B2 (en) | Multivariate diagnostic assays and methods for using same | |
| JP4343682B2 (en) | Methods for the detection and quantification of analytes in complex mixtures | |
| EP1040202B9 (en) | Dna polymorphism identity determination using flow cytometry | |
| Gilboa et al. | Single-molecule analysis of nucleic acid biomarkers–A review | |
| JPH06505394A (en) | Nucleic acid classification by polymerase extension of oligonucleotides using terminator complexes | |
| AU2002327202A1 (en) | Methods for detection and quantification of analytes in complex mixtures | |
| JP2004524823A (en) | An improved method for monitoring gene expression on electrical microarrays | |
| CN106536735A (en) | Probe set for analyzing a dna sample and method for using the same | |
| CN113755558A (en) | Nucleic acid detection method based on liquid chip technology | |
| CN107523624B (en) | Multi-cross isothermal amplification method combining AUDG and self-avoiding molecule recognition system | |
| CN115521979A (en) | Single-molecule nucleic acid detection method, product and related application | |
| KR101820440B1 (en) | Method for detection of target nucleic acids | |
| KR20220130592A (en) | Highly sensitive methods for accurate parallel quantification of nucleic acids | |
| CN107250381A (en) | The normalized iterative bar code of DNA set and sequencing | |
| US11970736B2 (en) | Methods for accurate parallel detection and quantification of nucleic acids | |
| KR20180136078A (en) | Method of preparing a surface enhanced raman scattering nanocomplex for detecting pathogens | |
| CN110878342A (en) | Digital absolute quantitative analysis method for nucleic acid marker based on single fluorescent particle counting of flow cytometer | |
| CN117625763A (en) | High sensitivity method for accurately parallel quantification of variant nucleic acid | |
| KR20160097881A (en) | Non-Amplification Method for DNA Detection Using Gold Nanoparticle and Magnetic Bead Particle | |
| CN114350771A (en) | Construction method of SNaPshot multiple analysis system | |
| TW202411430A (en) | Methods for accurate parallel detection and quantification of nucleic acids | |
| CN114540548A (en) | Gold nano biosensor based on multi-cross constant temperature amplification | |
| Song et al. | Direct Detection of Bacterial DNA and viral RNA at Subfemtomolar Concentrations Using Single Molecule Arrays (Simoa) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |