A method for detecting PCR products

Abstract

The present invention belongs to the technical field of nucleic acid detection, and discloses a method for detecting PCR products, which is detected by an unmodified glass tube nanopore detection platform. The present invention uses the DNA fragment of the PCR amplified product as a signal transduction carrier, and indirectly detects the conserved genomic sequence of the virus by detecting the PCR product through the nanopore, replaces the fluorescence reading of the PCR amplicon with an electronic reading, eliminates the probe design and fluorescence labels, and at the same time overcomes the defect that fluorescence can detect at most 4 to 6 PCR products simultaneously, achieving single-molecule detection that cannot be achieved by fluorescence detection. The present invention can accurately and real-time achieve the individual detection and multiplex detection of single-molecule targets of PCR products, not only has high sensitivity at the single-molecule level, but also is simple, rapid and efficient in operation, and is a multi-target and low-cost detection platform.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and particularly relates to a method for detecting PCR products. Background Art

[0002] Infectious bacteria and viruses such as Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) Coronavirus (MERS-CoV), Ebola virus, SARS-CoV-2, dengue virus, and tuberculosis have brought catastrophic consequences and threats to human health. So far, the methods for diagnosing these pathogens mainly rely on the fluorescence readout of PCR amplicons of each unique gene of these pathogens. The development of the polymerase chain reaction (PCR) analysis method has made the detection of nucleic acids more sensitive. It is the commonly used nucleic acid amplification method so far and is considered the gold standard for nucleic acid detection. However, most of the current nucleic acid detection methods based on PCR (or amplification) rely on monitoring the signal intensity and color of fluorescence to quantitatively and qualitatively analyze nucleic acids, and are powerless for simultaneously detecting multiple targets. However, simultaneous detection of multiple targets is usually required in virus diagnosis and nucleic acid detection. Although oligonucleotide probes with sequence-specific fluorescent labels are used, such as color-coded molecular beacons, TaqMan, etc. This detection scheme where one color represents one target fundamentally limits the multiplexing ability of detection, and at most 4 to 6 target sequences can be detected simultaneously, and it is also difficult to reach the level of single-molecule detection.

[0003] Nanopores have the advantages of simple operation, high single-molecule resolution, and label-free, and have become a promising biomolecular sensing platform. Different from the situation where the product length is uncontrolled and leads to a wide distribution of blocked current peak amplitudes, the migration of double-stranded DNA with a fixed length through a nanopore will produce a definite distribution of blocked current peak amplitudes. This principle can be used to amplify a series of double-stranded products with a certain length, detected by a nanopore, and distinguished according to specific blocked amplitudes. Summary of the Invention

[0004] In view of the above problems, the present invention provides a method for detecting PCR products; by using an electrolyte buffer with a fixed salt concentration and a glass tube nanopore, a single-molecule, highly sensitive, stable and reliable, multiplex nanopore sensor platform is developed to detect and distinguish target nucleic acids by detecting PCR products, such as target virus conserved nucleic acid sequences with clinical diagnostic significance.

[0005] The object of the present invention is achieved by the following technical solutions:

[0006] A method for detecting PCR products, specifically detected by an unmodified glass tube nanopore detection platform.

[0007] Preferably, the method for detecting PCR products includes the following steps:

[0008] (1) PCR amplify the target gene and purify it;

[0009] (2) Dilute the purified DNA sample with the test buffer and add it to the glass tube nanopore detection platform for detection.

[0010] Preferably, the test buffer contains 4M LiCl, 10mM Tris, and 1mM EDTA, and its pH value is adjusted to 8.0 with HCl.

[0011] Preferably, the detection method of the glass tube nanopore detection platform includes the following steps:

[0012] Immerse the Ag / AgCl electrodes into the detection cell and the glass tube of the glass tube nanopore detection platform respectively, connect the patch clamp detection system, add the purified DNA sample to the detection cell, apply a bias voltage to ensure that the target DNA fragment can pass through the nanopore channel under the action of electrostatic force, and detect the amplified product DNA by detecting the blocking signal.

[0013] Preferably, the construction method of the glass tube nanopore detection platform includes the following steps:

[0014] Take a quartz glass tube and soak it in freshly prepared piranha solution to remove organic impurities, then wash the quartz glass tube with deionized water and place it in a vacuum drying oven for drying, and then install the treated quartz glass tube in a CO 2 -laser-actuated pipette puller and set two programs: (1) Heat 860, Filament 5, Velocity 50, Delay 140, and Pull 50; (2) Heat 860, Filament 4, Velocity 30, Delay 155, and Pull 250 to obtain a glass conical nanopore, inject 4M LiCl, 10mM Tris, 1mM EDTA electrolyte buffer solution into the tube, add the electrolyte buffer solution composed of 4M LiCl, 10mM Tris, 1mM EDTA into the centrifuge tube of the detection cell, and ensure that the nanopore is the only electrolyte connection channel to form a glass tube nanopore detection platform;

[0015] Among them, the volume ratio of sulfuric acid to hydrogen peroxide in the piranha solution is 7:3.

[0016] Preferably, the inner diameter of the quartz glass tube is 0.5mm and the outer diameter is 1mm.

[0017] The beneficial effects of the present invention are:

[0018] (1) The present invention discloses a detection method of a glass tube nanopore sensor for PCR products. The glass nanopore undergoes no modification process, has good signal-to-noise ratio, repeatability, selectivity, strong anti-interference ability and low pore-blocking rate. Within a certain range, the nanopore sensor used has no dependence on the size of the detection target. The translocation blockage amplitude of DNA in the nanopore shows a good linear relationship with the DNA length, and has a wide linear range. DNA as short as 199 bp and as long as 2996 bp can be detected and two PCR products with a length difference of 212 bp can be clearly distinguished. For the ability to detect very short DNA fragments, it can greatly improve the amplification efficiency of the target gene. According to the dependence of the signal intensity generated by translocation on the length of the target DNA, primer pairs with different amplification lengths can be designed to amplify the target gene to achieve simultaneous detection of multiple targets. Therefore, the present invention not only has high sensitivity at the single-molecule level, but also is simple, fast and efficient in operation, and further provides a multi-target and low-cost detection platform.

[0019] (2) The present invention uses the DNA fragment of the PCR amplification product as a signal transduction carrier, and indirectly detects the conserved genomic sequence of the virus by detecting the PCR product through the nanopore; since the size of the PCR product can be flexibly designed, it can greatly adapt to various different pore sizes of the sensor, avoiding the cumbersome process of adjusting the appropriate pore size to match different target molecules, and at the same time overcoming the defect that fluorescence can detect at most 4 to 6 PCR products simultaneously. At the same time, single-molecule detection that fluorescence detection cannot achieve is achieved. The present invention can accurately and real-time achieve the individual detection and multiplex detection of single-molecule targets of PCR products, which has important guiding significance for nucleic acid detection and virus diagnosis in clinical medicine. Brief Description of the Drawings

[0020] The present invention is further described with reference to the accompanying drawings. However, the embodiments in the drawings do not constitute any limitation to the present invention. For those of ordinary skill in the art, other drawings can also be obtained according to the following drawings without creative efforts.

[0021] Figure 1 is the schematic diagram of detecting PCR products by the glass tube nanopore detection platform of the present invention;

[0022] Figure 2 is the 2% agarose gel electrophoresis characterization diagram of the amplified double-stranded DNA fragment of 791 bp;

[0023] Figure 3 is the 2% agarose gel electrophoresis characterization diagram (from left to right) of the separately amplified double-stranded DNA fragments of 199 bp, 411 bp, 720 bp, 1104 bp, 1601 bp, 2220 bp, 2996 bp;

[0024] Figure 4 It is a 2% agarose gel electrophoresis characterization diagram of the concentration gradient of the amplified 791bp double-stranded DNA fragment Lambda DNA template;

[0025] Figure 5 It is a 2% agarose gel electrophoresis characterization diagram (from top to bottom) of the simultaneously amplified 199bp, 411bp, 720bp, 1104bp, 1601bp double-stranded DNA fragments;

[0026] Figure 6 It is a current-time trajectory diagram generated during the detection of the 791bp double-stranded DNA sample solution with / without a nanopore sensor;

[0027] Figure 7 It is a relationship diagram between the nanopore event rate and the concentration of the target 791bp DNA in the solution;

[0028] Figure 8 It is a relationship diagram between the nanopore event rate and the concentration of the template Lambda DNA in the solution;

[0029] Figure 9 It is a bar chart of the blockage amplitude - number of events of the translocation signal when the double-stranded DNA products of 7 lengths are detected separately;

[0030] Figure 10 It is a bar chart of the blockage amplitude - number of events of the translocation signal when the double-stranded DNA products of 7 lengths are detected simultaneously;

[0031] Figure 11 It is a bar chart of the blockage amplitude - number of events of the translocation signal when the corresponding primers of the double-stranded DNA of 5 lengths are amplified and detected simultaneously at equal concentrations. Detailed implementation manners

[0032] The present invention will be further described in conjunction with the following embodiments.

[0033] The embodiments of the present invention relate to a method for detecting PCR products based on a glass tube nanopore, comprising the following steps:

[0034] S1. Amplify a series of lengths of double-stranded DNA

[0035] The double-stranded DNA described above was obtained by amplifying a model genomic Lambda DNA template at a certain concentration with specific primers. The lengths of these double-stranded DNA fragments are 199bp, 411bp, 720bp, 791bp, 1104bp, 1601bp, 2220bp, and 2996bp respectively, and their sequences correspond to the nt2947 - 3145, nt1414 - 1824, nt3366 - 4085, nt20284 - 21074, nt5081 - 6184, nt7960 - 9560, nt1964 - 4183, and nt2846 - 5841 regions of Lambda DNA;

[0036] The specific amplification method includes the following steps:

[0037] (1) Place all single-stranded primer ssDNA dry powder in a centrifuge at 4000rpm for 60s, then dissolve it in pure water, and add a certain amount of pure water according to the required volume to make the final concentration 5μM for all;

[0038] (2) Amplification of 791bp double-stranded DNA: Add 5μL of 5μM forward primer for amplifying 791bp DNA, 5μL of 5μM reverse primer for amplifying 791bp DNA, 25μL of polymerase mixture, 2.5μL of 150pM Lambda DNA template, and 12.5μL of nuclease-free water into a 0.2mL PCR centrifuge tube;

[0039] (3) Amplification of 791bp DNA with a series of concentration templates: Add 5μL of 5μM forward primer for amplifying 791bp DNA, 5μL of 5μM reverse primer for amplifying 791bp DNA, 25μL of polymerase mixture into 9 0.2mL PCR centrifuge tubes, and add 2.5μL of nuclease-free water, 2.5μL of 15aM, 2.5μL of 150aM, 2.5μL of 1.5fM, 2.5μL of 15fM, 2.5μL of 150fM, 2.5μL of 1.5pM, 2.5μL of 15pM, 2.5μL of 150pM Lambda DNA template, and 12.5μL of nuclease-free water respectively;

[0040] (4) Separate amplification of double-stranded DNA of 7 lengths of 199bp, 411bp, 720bp, 1104bp, 1601bp, 2220bp, and 2996bp: Add 5μL of 5μM forward primer for amplifying DNA of a specific length, 5μL of 5μM reverse primer for amplifying DNA of a specific length, 25μL of polymerase mixture, 2.5μL of 150pM Lambda DNA template, and 12.5μL of nuclease-free water into a 0.2mL PCR centrifuge tube;

[0041] (5) Simultaneous amplification of double-stranded DNA of 5 lengths of 199bp, 411bp, 720bp, 1104bp, and 1601bp: Add 2 μL of 5 μM forward primer for amplifying 199bp DNA, 2 μL of 5 μM reverse primer for amplifying 199bp DNA, 2 μL of 5 μM forward primer for amplifying 411bp DNA, 2 μL of 5 μM reverse primer for amplifying 411bp DNA, 2 μL of 5 μM forward primer for amplifying 720bp DNA, 2 μL of 5 μM reverse primer for amplifying 720bp DNA, 2 μL of 5 μM forward primer for amplifying 1104bp DNA, 2 μL of 5 μM reverse primer for amplifying 1104bp DNA, 2 μL of 5 μM forward primer for amplifying 1601bp DNA, 2 μL of 5 μM reverse primer for amplifying 1601bp DNA, 25 μL of polymerase mixture, 2.5 μL of 150 pM Lambda DNA template, and 2.5 μL of nuclease-free water into a 0.2 mL PCR centrifuge tube.

[0042] (6) After vortexing and centrifuging the PCR centrifuge tubes containing the sample mixture in the above steps (2), (3), (4), and (5), place them in a BioRad T100 thermal cycler. Experience a denaturation step at 98°C for 30 s, and at 98°C for 10 s, 60°C for 10 s, 72°C (the holding times at 72°C in steps (2), (3), (4), and (7) are 30 s, 30 s, 1.5 min, and 1 min respectively). Run 35 cycles, and finally cool down to 12°C for storage; the amplified products are verified by 2% agarose gel electrophoresis, and the verification results are respectively referred to in Appendix Figure 2 See Figure 3 See Figure 4 See Figure 5 ;

[0043] (8) Purify the products obtained after amplification using a purification kit (QIAquick PCR Purification Kit) according to the operating instructions provided by the manufacturer;

[0044] S2. Glass tube nanopore detection

[0045] Dilute the DNA purified in step S1 with the test buffer and add it to the glass tube nanopore detection platform for detection. The test buffer contains 4 M LiCl, 10 mM Tris, 1 mM EDTA, and the pH value is adjusted to 8.0 with HCl. The specific detection method includes the following steps:

[0046] (1) Set up the glass tube nanopore detection platform:

[0047] Take quartz glass tubes with an inner diameter of 0.5 mm and an outer diameter of 1 mm, and soak them in freshly prepared piranha solution (V H2SO4 :V H2O2 = 7:3) for 2 hours to remove organic impurities, then wash the quartz glass tubes with deionized water, and place them in a vacuum drying oven at 70 °C for 20 minutes. Then, install the treated quartz glass tubes in a CO 2 -laser-actuated pipette puller and set two programs: (1) Heat 860, Filament 5, Velocity 50, Delay 140, and Pull 50; (2) Heat 860, Filament 4, Velocity 30, Delay 155, and Pull 250 to obtain glass conical nanopores. Inject 4M LiCl, 10 mM Tris, 1 mM EDTA electrolyte buffer solution into the tube, and add 4M LiCl, 10 mM Tris, 1 mM EDTA electrolyte buffer solution into the centrifuge tube of the detection cell to ensure that the nanopore is the only electrolyte connection channel, thereby forming the glass tube nanopore detection platform;

[0048] (2) Detection using the glass tube nanopore detection platform

[0049] Immerse Ag / AgCl electrodes into the detection cell and the glass tube of the glass tube nanopore detection platform respectively, and connect the patch clamp detection system;

[0050] Add the purified DNA sample obtained in step S1 into the detection cell, and apply a bias voltage of -1000 mV to ensure that the target DNA fragment can pass through the nanopore channel under the action of electrostatic force. By detecting the blocking signal, the detection of the amplified product DNA can be achieved.

[0051] Attached Figure 6 is a specific current-time trace diagram generated during the detection of the 791 bp DNA fragment sample solution with / without the analyte by the nanopore sensor; if the target DNA exists in the detection cell solution, it will be transferred through the nanopore under the action of the electric field, causing current blockage and generating a current blockage signal. On the contrary, if the target DNA does not exist, no substance can be detected passing through the nanopore, so no current blockage signal is generated.

[0052] Attached Figure 7Solutions of 791bp DNA fragments with different concentrations were added to the detection cell, and the corresponding current-time trajectory diagrams, the relationship diagrams between the translocation event rate and the concentration of DNA fragments in the solution were detected by the nanopore sensor; it can be seen from the scatter diagram that as the DNA concentration increases, the event rate in the solution increases linearly. It can be seen that the translocation event rate and the concentration of DNA fragments are linearly related, and quantitative analysis can be carried out.

[0053] Appendix Figure 8 Solutions of 791bp DNA fragments amplified under templates (Lambda DNA) with different concentrations were added to the detection cell and diluted 105 times, and the corresponding current-time trajectory diagrams and the relationship diagrams between the translocation event rate and the template concentration in the amplification system were detected by the nanopore sensor; it can be seen from the figure that as the template concentration increases, the event rate in the solution increases. In the range of template concentration from 75aM to 750fM, the event rate in the solution increases linearly. That is, when the template concentration is 75aM - 750fM, the translocation event rate and the concentration of the template are linearly related, and quantitative analysis of the template genome can be carried out.

[0054] Appendix Figure 9 It is a histogram of the blockage amplitude distribution obtained by separately detecting DNA fragments with 7 lengths of 199bp, 411bp, 720bp, 1104bp, 1601bp, 2220bp, and 2996bp. It can be seen from the histogram that DNA fragments with different lengths correspond to different amplitude regions. Therefore, 7 populations can be clearly seen in the figure, indicating that the blockage amplitude has high specificity and correlation with the DNA length, and it also further shows that multi-target simultaneous detection and differentiation of DNA with different lengths can be carried out.

[0055] Appendix Figure 10 It is the current-time trajectory diagram and the histogram of the blockage amplitude distribution obtained by mixing DNA fragments with 7 lengths of 199bp, 411bp, 720bp, 1104bp, 1601bp, 2220bp, and 2996bp in equal proportion and detecting. It can be seen from the histogram that the results are Figure 9 consistent with those in Appendix Figure 9 The conclusion obtained is that multi-target simultaneous detection can be carried out.

[0056] Appendix Figure 11Using the same template and equal primer concentration (200 nM), DNA fragments of 5 lengths, namely 199 bp, 411 bp, 720 bp, 1104 bp, and 1601 bp, were amplified simultaneously in a centrifuge tube, and the corresponding current-time trace diagrams and histograms of blockage amplitude distributions were obtained through nanopore detection. It can be seen from the histogram that the histogram contains 5 populations, which is completely consistent with the agarose gel electrophoresis verification results (5 bands) obtained by amplification. The position of each population is completely consistent with the population position when the 5 DNA fragments are detected individually, indicating that it is possible to simultaneously detect and distinguish different positions of the same gene or different gene fragments of a single virus, improving the accuracy of virus diagnosis. It is also possible to simultaneously detect the characteristic genes of different viruses, improving the detection efficiency and reducing the sampling volume.

[0057] In this example, a highly sensitive PCR amplification method was used. Using a model genomic Lambda DNA as a template, a series of DNA fragments of different lengths were obtained through PCR amplification, comprehensively verifying the length-dependent resolution of DNA double strands, ranging from 200 to 3000 base pairs. The study found that this glass tube nanopore has excellent ability to distinguish DNA double strands, with at least 200 base pairs. Since the peak distribution of the blocked current of DNA double strands with a length exceeding 2000 base pairs is wider, it is not suitable for multiplexing research. Therefore, the best choice for multiplexing research is a double-stranded length range of 200 - 2000 base pairs. The nanopore translocation of DNA double strands with a length between 250 - 2000 base pairs has the best current characteristics and significant performance in terms of quantitative and qualitative results. The results show that 5 PCR amplicons of different lengths are separated by more than 200 base pairs, corresponding to different fragments of the same Lambda genome and can be resolved simultaneously. The current signatures of DNA double strands of each designed length indicate that the longer the DNA double strand, the greater the median value of the blocked current peak amplitude. In addition, based on the length-resolution ability of the glass tube nanopore, various pathogenic bacteria and viruses in clinical samples, including SARS-CoV-2, can be identified at high resolution.

[0058] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention, rather than limiting the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for detecting nucleic acids based on a glass tube nanopore sensor, which is used for non-therapeutic and diagnostic purposes, characterized in that, it comprises the following steps: (1) Design primer pairs with different amplification lengths to perform PCR amplification on the target gene, and obtain a DNA sample after purification; (2) Dilute the purified DNA sample with a test buffer and add it to an unmodified glass tube nanopore detection platform for detection; The amplification length is 200 - 3000 base pairs; The test buffer contains 4M LiCl, 10mM Tris and 1mM EDTA, and its pH value is adjusted to 8.0 with HCl; The detection method of the glass tube nanopore detection platform comprises the following steps: Immerse the Ag / AgCl electrodes into the detection cell and the glass tube of the glass tube nanopore detection platform respectively, and connect the patch clamp detection system. Add the purified DNA sample to the detection cell, apply a bias voltage to ensure that the target DNA fragment can pass through the nanopore channel under the action of electrostatic force, and detect the amplified product DNA by detecting the blocking signal.

2. The method for detecting nucleic acids based on a glass tube nanopore sensor according to claim 1, characterized in that, the method for building the glass tube nanopore detection platform comprises the following steps: Take a quartz glass tube and soak it in freshly prepared piranha solution to remove organic impurities, then wash the quartz glass tube with deionized water and place it in a vacuum drying oven for drying. Then, install the treated quartz glass tube in a CO 2 -laser-actuated pipette puller and set two programs: (1) Heat 860, Filament 5, Velocity 50, Delay 140, and Pull 50; (2) Heat 860, Filament 4, Velocity 30, Delay 155, and Pull 250 to obtain a glass conical nanopore. Inject an electrolyte buffer solution of 4M LiCl, 10mM Tris, and 1mM EDTA into the tube. Add the electrolyte buffer solution composed of 4M LiCl, 10mM Tris, and 1mM EDTA into the centrifuge tube of the detection cell to ensure that the nanopore is the only electrolyte connection channel, forming a glass tube nanopore detection platform; wherein, the volume ratio of sulfuric acid to hydrogen peroxide in the piranha solution is 7:

3.

3. The method for detecting nucleic acids based on a glass tube nanopore sensor according to claim 2, characterized in that, the inner diameter of the quartz glass tube is 0.5mm and the outer diameter is 1mm.

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