Target nucleic acid rapid detection method based on solid nanopores

Abstract

The invention relates to a solid nanopore-based target nucleic acid rapid detection method, which comprises: (1) providing a specific probe matched with a target nucleic acid, the specific probe comprising a specific complementary sequence region capable of being complementarily paired with a target nucleic acid base and a non-specific complementary sequence region capable of forming a secondary structure; (2) mixing and incubating a sample to be tested and the specific probe; (3) detecting the current signal change of the incubation liquid in the step (2) by adopting a solid nanopore-based nucleic acid detection system, (4) enabling each continuous current drop to correspond to a via hole event, determining whether a via hole event which simultaneously contains a primary current drop and a secondary current drop and all or part of the secondary current drop is not at the two ends of the primary current drop exists or not, and if so, determining that the solid nanopore-based nucleic acid detection system does not detect the current signal change of the incubation liquid in the step (2). The target nucleic acid is contained. On the premise of being simple, rapid and low in cost, the nucleic acid detection specificity and sensitivity can be improved, then the accuracy is improved, and the method has wide potential market and application prospects.

Description

Technical Field

[0001] This invention relates to the field of target nucleic acid detection technology, and specifically to a rapid target nucleic acid detection method based on solid nanopores. Background Technology

[0002] The mainstream methodologies for detecting target nucleic acids include PCR-based and sequencing-based methods. Fluorescent PCR-based target nucleic acid detection is a quantitative nucleic acid detection method that combines traditional PCR with fluorescent labeling technology. Its core principle is to achieve precise quantitative analysis of target DNA or RNA by monitoring the accumulation of amplified products in real time through fluorescence signals, offering advantages such as high sensitivity and high specificity. However, its detection cycle is relatively long, generally requiring 3-4 hours from sample to result, and the detection equipment is relatively expensive, resulting in high costs. Sequencing-based nucleic acid detection can perform large-scale, high-precision sequence analysis of DNA or RNA, obtaining complete nucleic acid sequence information, thereby comprehensively analyzing gene mutations, pathogen typing, drug resistance genes, and microbial community composition. However, the detection process is complex and cumbersome, requiring complex library construction from sample to result, which is time-consuming and labor-intensive; it also relies on expensive sequencers, resulting in high costs.

[0003] Solid-state nanopores were initially used for protein detection, monitoring protein activity by detecting changes in current as proteins pass through the nanopore. Later, their application shifted to nucleic acid detection. Compared to PCR and sequencing, solid-state nanopores eliminate the need for amplification and fluorescence, reducing equipment costs. However, due to the rapid passage of individual target nucleic acids through the pore and the fact that any charged molecule can generate a signal, an excess of background nucleic acids can lead to a decreased signal-to-noise ratio. This results in poor specificity and low sensitivity for nucleic acid detection based on solid-state nanopores, limiting the large-scale clinical application of solid-state nanopore nucleic acid detection methods. Summary of the Invention

[0004] The purpose of this invention is to provide a rapid detection method for target nucleic acids based on solid nanopores that can improve the specificity and sensitivity of nucleic acid detection, thereby further improving the accuracy of detection results, while being simple, fast, and low-cost.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a rapid detection method for target nucleic acids based on solid-state nanopores, comprising the following steps:

[0007] (1) Provide a specific probe that matches the target nucleic acid, wherein the specific probe includes, from the 5' end to the 3' end, a first specific complementary sequence region that can pair with the bases of the target nucleic acid, a non-specific complementary sequence region that can form a secondary structure, and a second specific complementary sequence region that can pair with the bases of the target nucleic acid.

[0008] (2) Mix the sample to be tested with the specific probe and incubate;

[0009] (3) The current signal change of the incubation solution in step (2) is detected using a nucleic acid detection system based on solid nanopores. Each biomolecule in the incubation solution generates a continuous current drop when passing through the solid nanopore. When a nucleic acid molecule formed by the specific probe and the target nucleic acid or one of its sequences through base complementary pairing passes through the solid nanopore, the part of the nucleic acid molecule other than its secondary structure generates a first-order current drop when passing through the solid nanopore, and the secondary structure part generates a second-order current drop when passing through the solid nanopore. The minimum current value of the first-order current drop is greater than the minimum current value of the second-order current drop.

[0010] (4) Each consecutive current drop corresponds to a via event. It is determined whether there is a via event in the current signal change of the incubation solution within the preset detection time that simultaneously contains a first-level current drop and a second-level current drop, and all or part of the second-level current drop is not at both ends of the first-level current drop. If so, it is confirmed that the sample to be tested contains the target nucleic acid.

[0011] In practice, because nucleic acid molecules require a relatively long time to pass through solid-state nanopores, the resulting primary current drop generally appears as a continuous plateau. However, secondary structures pass through solid-state nanopores in a shorter time, resulting in a secondary current drop that generally appears as a peak downwards from the primary current drop or a plateau significantly shorter than the primary current drop, thus identifying it as a characteristic perforation event. However, in practice, some nucleic acid ends may curl to form their own secondary structures. When these secondary structures pass through solid-state nanopores, they also generate a secondary current drop, but this secondary current drop is located at the end of the primary current drop (including the front and / or back ends). Therefore, when determining specific perforation events, perforation events where the secondary current drop is entirely located at both ends of the primary current drop must be excluded.

[0012] Furthermore, the nonspecific complementary sequence region can form one or more of the secondary structures.

[0013] In some embodiments, the secondary structure is one or more of a ring structure, a T-shaped structure, a Y-shaped structure, and a cloverleaf structure. A ring structure (similar to an Ω structure) is preferred, as it offers improved specificity and sensitivity while having the lowest synthesis cost and being least likely to cause pore blockage compared to other secondary structures.

[0014] Furthermore, the secondary structure is a ring structure with a sequence length of 20 to 200 nt, such as 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt, 80 nt, 85 nt, 90 nt, 95 nt, 100 nt, 105 nt, 110 nt, 115 nt, 120 nt, 125 nt, 130 nt, 135 nt, 140 nt, 145 nt, 150 nt, 155 nt, 160 nt, 165 nt, 170 nt, 175 nt, 180 nt, 185 nt, 190 nt, 195 nt, or 200 nt.

[0015] In some specific embodiments, the non-specific complementary sequence region can only form a ring structure with a sequence length of 20 to 200 nt. When it passes through a solid nanopore, it can form a secondary current drop that is distinct from the primary current drop. When there is only one secondary structure, the longer the sequence length of the secondary structure, the more obvious the secondary current drop signal and the higher the detection sensitivity. However, the probe cost also increases accordingly. Preferably, the sequence length of the secondary structure does not exceed 150 nt, more preferably not more than 120 nt, and the most preferred length is 40 to 100 nt.

[0016] In some specific embodiments, the nonspecific complementary sequence region can form at least two ring structures with independent sequence lengths of 20-40 nt each, and a first linker sequence region is provided between adjacent ring structures. The first linker sequence region can be complementary to the target nucleic acid bases, and the sequence length of the first linker sequence region is 1-10 nt. According to existing experiments, in this manner, when passing through a solid nanopore, only one secondary current drop, distinct from the primary current drop, can be formed. That is, when the interval between two ring structures is less than 10 nt, it will not be distinguished as two secondary current drops in the electrical signal change. The secondary current drop formed by two or more ring structures is more easily distinguished from the primary current drop and can be identified compared to the secondary current drop formed by a single ring structure, resulting in higher detection sensitivity. Considering probe cost, the number of secondary structures is preferably no more than 10, more preferably no more than 5, and most preferably 2-4.

[0017] In some other specific embodiments, the nonspecific complementary sequence region includes a plurality of sequentially connected units capable of forming secondary structures and a second linker sequence region located between two adjacent units. The secondary structures that any two units can form are the same or different. The second linker sequence region can be complementary to the target nucleic acid bases and the sequence length of the second linker sequence region is greater than 10 nt. Each unit corresponds to a secondary current drop.

[0018] The sequence length of the second connection sequence region is preferably 40–120 nt, which facilitates the differentiation and statistical analysis of the number of secondary current drops in the same via event, avoids errors, and improves the accuracy of the detection results. Considering probe cost, the maximum interval between two adjacent units is preferably no more than 100 nt, more preferably no less than 50 nt, more preferably 50–100 nt, and more preferably 80–100 nt.

[0019] When multiple target nucleic acids need to be detected simultaneously, the non-specific complementary sequence regions of the specific probes corresponding to different target nucleic acids contain different secondary structures and / or different numbers of the units, so that different target nucleic acids generate different morphologies of continuous current drops, thereby enabling accurate differentiation of the specific vias corresponding to different target nucleic acids. Figure 1 This demonstrates the detection principle of one of the methods that can simultaneously detect three different types of nucleic acids.

[0020] Furthermore, the lengths of the first specific complementary sequence region and the second specific complementary sequence region are independently 20 to 80 nt, for example, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt or 80 nt.

[0021] Furthermore, the length of the nonspecific complementary sequence region is 20 to 200 nt, for example, 15 nt, 20 nt, 30 nt, 40 nt, 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, 100 nt, 110 nt, 120 nt, 130 nt, 140 nt, 150 nt, 160 nt, 170 nt, 180 nt, 190 nt or 200 nt.

[0022] In some embodiments, the incubation system includes 5× annealing buffer, the test sample, the specific probe, and nuclease-free water. The 5× annealing buffer is formulated as follows: 300–400 mM KCl, 25–75 mM Tris, 4–6 mM EDTA, with water as the solvent.

[0023] In some embodiments, the incubation procedure is as follows: maintain at 94-96°C for 3-5 minutes, and then cool down to 25°C at a cooling rate of 0.05-0.15°C / s.

[0024] In some implementations, the preset detection time in step (4) is 5 to 10 minutes.

[0025] In some implementations, a nucleic acid detection system based on solid-state nanopores includes a nanopore chip, electrodes, and an electrical signal data acquisition system.

[0026] Furthermore, the nanopore chip of this application can be an existing silicon nitride chip, preferably with a thickness of 10-50 nm, such as silicon nitride chips with thicknesses of 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 30 nm, 41 nm, 42 nm, 43 nm, 44 nm, 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, and 50 nm.

[0027] Furthermore, the nanoporous chip is a chip that has been cleaned by gaseous plasma.

[0028] Furthermore, the nanopore chip has through-holes with a pore size of 5–50 nm, such as 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, or 50 nm. In practical detection, nanopore chips with appropriate pore sizes can be selected based on nucleic acid length, probe length, and structure to obtain a higher proportion of characteristic through-hole events. According to previous experiments in this application, a pore size of 7–20 nm provides strong adaptability.

[0029] Specifically, the nanopore chip has a trans chamber on one side and a cis chamber on the other side. The trans chamber and the cis chamber are connected through the through-hole. The trans chamber and the cis chamber each contain a detection buffer and are respectively equipped with electrodes. The electrode of the trans chamber is connected to the positive terminal of an external power supply, and the electrode of the cis chamber is connected to the negative terminal of an external power supply, thereby forming a closed loop.

[0030] Furthermore, the detection buffer consists of 0.1–1 M KCl, 5–15 mM Tris, and 0.8–1.2 mM EDTA, with water as the solvent and a pH of 7.8–8.2.

[0031] Furthermore, the detection buffer solutions in the trans chamber and the cis chamber may be the same or different.

[0032] Using a detection buffer with a fold difference can increase the total number of via events and the number of characteristic via events during the detection time, making it particularly suitable for detecting low-concentration samples.

[0033] According to some specific embodiments, the KCl concentration of the detection buffer in the trans chamber is 3 to 40 times the KCl concentration of the detection buffer in the cis chamber, and the detection concentration of the sample can be reduced to 0.1 fM. The larger the difference, the lower the detectable concentration.

[0034] In some specific embodiments, the data acquisition system is a patch clamp amplifier.

[0035] Preferably, in step (3), the detection voltage is 50–800 mV, the sampling frequency is 20–500 kHz, and the filtering frequency is 2–10 kHz. In actual detection, the detection voltage, sampling frequency, and filtering frequency can be selected or adjusted within the above range to obtain the ideal current signal change pattern, which facilitates faster identification and statistical analysis of target feature via events.

[0036] The total number of via events increases with increasing detection voltage, but the higher the detection voltage, the faster the molecular via rate, which makes it easier to cause via blockage. In some specific embodiments, the detection voltage in step (3) is 100-300mV, the sampling frequency is 400-600kHz, and the filtering frequency is 2-10kHz.

[0037] According to some specific embodiments, the target nucleic acid is influenza B virus nucleic acid, and the nucleotide sequence of the corresponding specific probe is the nucleotide sequence of any one of SEQ ID NO: 2 to 4;

[0038] And / or, the target nucleic acid is influenza A virus nucleic acid, and the nucleotide sequence of the corresponding specific probe is the nucleotide sequence described in SEQ ID NO: 6.

[0039] This invention is based on solid-state nanopore technology. When the target nucleic acid in the sample to be tested binds to a specific probe, it generates a through-pore current signal that is different from that generated by other through-pore molecules. That is, the characteristic through-pore event is determined by the specific through-pore signal, thereby accurately identifying whether the sample to be tested contains the target nucleic acid.

[0040] The types of test samples in this invention include, but are not limited to, various biological samples that have been treated with cell lysis buffer, such as clinical samples treated with cell lysis buffer, such as swabs, blood, urine, secretions, pus, etc.

[0041] This invention can be used to quickly detect whether a test sample contains viruses and / or bacteria.

[0042] In the detection method of the present invention, single-stranded or double-stranded nucleic acids generate a first-order current drop, and the target nucleic acid that binds to the specific probe of the present invention will generate a second-order current drop. By statistically analyzing the through-hole events that generate the second-order current drop, the presence of the target nucleic acid and the relative content of the target nucleic acid in the nucleic acid of the sample to be tested can be quickly identified.

[0043] The detection method of the present invention is simple and fast to operate, requiring only three steps and taking less than one hour to complete the detection: incubation for about 15 to 25 minutes; sample detection through wells after incubation for about 5 to 10 minutes; and analysis of through-well data for about 5 to 10 minutes.

[0044] The detection method of the present invention provides more accurate detection results. It accurately identifies the target nucleic acid through a specific probe sequence, and at the same time, when the secondary structure formed passes through the nanopore, it generates a specific through-pore current signal, thereby accurately identifying the target nucleic acid.

[0045] The detection method of this invention is low-cost, uses inexpensive materials, and the probe molecules do not require expensive modification. The entire detection process does not involve enzymatic reactions, and the detection equipment is simple and cheaper than fluorescent PCR and sequencers.

[0046] A second aspect of the present invention provides a target nucleic acid detection kit based on solid nanopores, which includes the above-mentioned chip, the above-mentioned specific probe and detection reagent system, wherein the detection reagent system includes the above-mentioned 5× annealing buffer, the above-mentioned detection buffer and nuclease-free water.

[0047] A third aspect of the present invention provides a multi-detection system for influenza A / B viruses based on solid-state nanopores, comprising the aforementioned solid-state nanopore-based nucleic acid detection system, a specific probe corresponding to the influenza B virus nucleic acid (the nucleotide sequence of the specific probe for influenza B virus nucleic acid is any one of SEQ ID NO: 2-4) and the aforementioned specific probe corresponding to the influenza A virus nucleic acid (the nucleotide sequence of the specific probe for influenza A virus nucleic acid is the nucleotide sequence described in SEQ ID NO: 6), and a detection reagent system, wherein the detection reagent system comprises the aforementioned 5× annealing buffer, the aforementioned detection buffer, and nuclease-free water.

[0048] Due to the application of the above-mentioned technical solution, the present invention has the following advantages compared with the prior art:

[0049] The detection method of the present invention uses a specific probe that can specifically bind to the target nucleic acid and form a secondary structure, so that the target nucleic acid bound to it generates a two-stage current drop when passing through the pore, which is different from the single-stage current drop generated by single-stranded or double-stranded nucleic acids. By statistically analyzing the pore passing events that generate the two-stage current drop, the presence of the target nucleic acid and the relative content of the target nucleic acid in the nucleic acid of the sample to be tested can be quickly identified.

[0050] Furthermore, by combining secondary structure optimization, nanopore chip pretreatment, pore size selection, detection voltage selection, and detection buffer settings, the detection sensitivity and accuracy of detection results can be improved. Moreover, by using specific probes for different target nucleic acids in combination, the detection effect of simultaneously detecting two or more target nucleic acids can be achieved.

[0051] The detection method of the present invention is simple, fast, accurate and low cost, providing a better choice for applications with time-sensitive and price-sensitive needs in nucleic acid testing, and has broad potential market and application prospects. Attached Figure Description

[0052] Figure 1 A schematic diagram illustrating the principle of a method for the simultaneous and rapid detection of three target nucleic acids based on solid nanopores, provided by this invention;

[0053] Figure 2 This is a partial collection of data from the current signal change graph detected in Example 1;

[0054] Figure 3 This is a schematic diagram of the current signal changes of non-featured via events in the current signal change graph detected in Example 1.

[0055] Figure 4 This is a schematic diagram of the current signal changes of the characteristic via events of influenza B virus nucleic acid detected in Example 1.

[0056] Figure 5 This is a schematic diagram of the current signal changes of the interference feature via event in the current signal change graph detected in Example 1;

[0057] Figure 6 This is a partial collection of data from the current signal change graph detected in Example 2;

[0058] Figure 7 This is a schematic diagram of the current signal changes of non-featured via events in the current signal change graph detected in Example 2;

[0059] Figure 8 This is a schematic diagram of the current signal changes of the characteristic via events of influenza B virus nucleic acid in Example 2;

[0060] Figure 9 This is a partial collection of data from the current signal change graph detected in Example 3;

[0061] Figure 10 This is a schematic diagram of the current signal changes of non-featured via events in the current signal change graph detected in Example 3;

[0062] Figure 11This is a schematic diagram of the current signal changes of the characteristic via events of influenza B virus nucleic acid detected in Example 3.

[0063] Figure 12 This is a partial collection of data from the current signal change graph detected in Example 4;

[0064] Figure 13 This is a partial collection of data from the current signal change graph detected in Example 5;

[0065] Figure 14 This is a partial collection of data from the current signal change graph detected in Example 6;

[0066] Figure 15 This is a schematic diagram of the current signal changes of non-featured via events in the current signal change graph detected in Example 6.

[0067] Figure 16 This is a schematic diagram of the current signal changes of the characteristic via events of influenza B virus nucleic acid in Example 6;

[0068] Figure 17 This is a schematic diagram of the current signal changes of the characteristic via events of influenza A virus nucleic acid detected in Example 6. Detailed Implementation

[0069] The present invention will be further described below with reference to embodiments. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0070] Unless otherwise specified, all raw materials used in the following examples are commercially available.

[0071] Example 1: This example provides a method for rapid detection of influenza B virus nucleic acid using a nucleic acid detection system based on solid-state nanopores.

[0072] Samples to be tested: HeLa cell nucleic acid (negative control), artificially synthesized influenza B virus nucleic acid fragment (sequence corresponding to the influenza B virus-specific sequence screened from the NCBI nt database, numbered IVB_M), and influenza A / B virus multi-nucleic acid detection quality control (purchased from Ruifengkang Biomedical Technology Co., Ltd.).

[0073] A nucleic acid detection system based on solid nanopores includes detection equipment and detection reagent system.

[0074] Detection equipment: nanopore chip, electrodes, and electrical signal data acquisition system.

[0075] Nanopore Chip and Electrodes: A commercially available silicon nitride chip (12 μm thick) was used. The chip was cleaned with gaseous plasma using the Mobile Cubic Asher (MCA) developed by ibss Group Inc. The cleaning cycle was 30 W for 35 seconds. After plasma cleaning, nanopores with a diameter of 9 nm were fabricated via dielectric breakdown. One side of the chip is a trans chamber, and the other side is a cis chamber, connected by silicon nitride nanopores. Ag / AgCl electrodes are installed in both chambers. The Ag / AgCl electrode in the trans chamber is connected to the positive terminal of an external power supply, and the Ag / AgCl electrode in the cis chamber is connected to the negative terminal, thus forming a closed loop. When energized, the target nucleic acid of the sample travels from the cis chamber through the nanopores to the trans chamber, generating a captureable current signal change.

[0076] Electrical signal data acquisition system: The data acquisition system is a patch-clamp amplifier (Axopatch 200B, Molecular Devices, California, US), which acquires data at a sampling frequency of 500kHz and filters the signal with a 10kHz low-pass filter. The statistical software used is LabVIEW, or the analysis function of the software built into the data acquisition system (Clampex) can be used.

[0077] Detection reagent system: specific probe, detection buffer, 5× annealing buffer, nuclease-free water

[0078] Specific probe: Self-made. A specific complementary probe (IVB_P3) was designed and synthesized artificially on the influenza B virus specific sequence numbered IVB_M using Primer5. From 5' to 3', it consists of the first specific complementary sequence region (20nt in length), the non-specific complementary sequence region (secondary structure consists of two 20nt loops, with a 10nt first linker sequence region complementary to the target nucleic acid between the two loops), and the second specific complementary sequence region (36nt in length).

[0079] Detection buffer: 1M KCl, 10mM Tris and 1mM EDTA, solvent: water, pH=8.

[0080] 5× Annealing buffer: 70mM KCl, 10mM Tris, 1mM EDTA, solvent is water.

[0081] Nuclease-Free Water: Commercially available ultrapure water that does not contain DNase, RNase, protease, or endotoxin.

[0082] The detection method is as follows:

[0083] Pretreatment: Inject 80 μL of nuclease-free water into the trans and cis chambers of the chip, repeatedly pipetting and aspirating three times to discard the waste liquid, repeating this process three times. Then, rinse each chamber with 80 μL of detection buffer. Finally, add 50 μL of detection buffer to each chamber, adjust the power supply to apply a 200 mV voltage, and measure the blank signal using the data acquisition system.

[0084] Incubation: Add the test sample, specific probe, and 5× annealing buffer to the PCR tube according to the system shown in Table 1. Place the PCR tube in the PCR instrument and incubate according to the procedure shown in Table 2.

[0085] Table 1

[0086] Material Name Final concentration Volume μL 5× Annealing Buffer 5× 10 Nucleic acid of the sample to be tested 10^6copies / mL / 10Μm / 2000copies / mL / 500copies / mL X probe 100nM 5 Nuclease-free water \ 35-X total \ 50

[0087] Use nuclease-free water to adjust the nucleic acid concentration of the sample to the required final concentration.

[0088] Table 2

[0089]

[0090] First, the DNA is denatured and destranded at 95℃ for 3 minutes. Then, the temperature is lowered from the previous temperature to the next temperature at a rate of 0.1℃ / s. After reaching the next temperature, the temperature is held for 1 minute before the temperature is lowered again, until the temperature drops to 25℃.

[0091] Detection: Selectively add detection buffer to the incubation system for dilution. Take 50 μL of the diluted incubation system and add it to the CIS chamber. Perform through-hole detection according to the procedure shown in Table 3. Collect data through the data acquisition system, analyze through-hole events, and count through-hole events with significant current changes.

[0092] Table 3

[0093] parameter Parameter value Detection voltage 200mV Sampling frequency 500kHz Filtering 10kHz Sampling time 10min

[0094] Taking the IVB_M detection results as an example, some of the collected data in this embodiment can be found in [link to example]. Figure 2 Level 1 current drop via event, such as Figure 3 As shown, this is a non-characteristic via event; a characteristic via event containing both first-order current drop and second-order current drop is as follows: Figure 4 As shown, this is a characteristic perforation event of influenza B virus nucleic acid. An interfering characteristic event was detected in the HeLa cell nucleic acid sample, such as... Figure 5 As shown.

[0095] The statistical results of via events for different test samples in this embodiment are shown in Table 4.

[0096] Table 4

[0097]

[0098] Example 2: This example provides another method for rapid detection of influenza B virus nucleic acid using a solid-state nanopore-based nucleic acid detection system.

[0099] Test samples: HeLa cell nucleic acid (negative control), artificially synthesized influenza B virus nucleic acid fragment (sequence corresponding to the influenza B virus-specific sequence screened from the NCBI nt database, numbered IVB_M).

[0100] A nucleic acid detection system based on solid nanopores includes detection equipment and detection reagent system.

[0101] Testing equipment: chip, electrodes, and electrical signal data acquisition system.

[0102] Chip and Electrodes: A commercially available silicon nitride chip (12nm thick) was used to fabricate 10nm nanopores via dielectric breakdown. One side of the chip is a trans chamber, and the other side is a cis chamber, connected by silicon nitride nanopores. Ag / AgCl electrodes are placed in each chamber. The Ag / AgCl electrode in the trans chamber is connected to the positive terminal of an external power supply, and the Ag / AgCl electrode in the cis chamber is connected to the negative terminal, thus forming a closed loop. When energized, the target nucleic acid of the sample travels from the cis chamber through the nanopores to the trans chamber, generating a captureable change in current signal.

[0103] Electrical signal data acquisition system: Same as in Example 1.

[0104] Detection reagent system: specific probe, detection buffer, 5× annealing buffer, nuclease-free water

[0105] Specific probe: Self-made. A specific complementary probe (IVB_P1) was designed and synthesized on the influenza B virus specific sequence numbered IVB_M using Primer5. From 5' to 3', it consists of the first specific complementary sequence region (20nt in length), the non-specific complementary sequence region (secondary structure is a 20nt loop), and the second specific complementary sequence region (46nt in length).

[0106] Detection buffer: Same as in Example 1.

[0107] 5× Annealing buffer: Same as in Example 1.

[0108] Nuclease-Free Water: Same as in Example 1.

[0109] Detection method: Same as in Example 1.

[0110] Taking the IVB_M detection results as an example, some of the collected data in this embodiment can be found in [link to example]. Figure 6 Level 1 current drop via event, such as Figure 7 As shown, this is a non-characteristic via event; via events that simultaneously contain both first-order current drop and second-order current drop are as follows: Figure 8 As shown, this is a characteristic perforation event of influenza B virus nucleic acid.

[0111] The statistical results of via events for different test samples in this embodiment are shown in Table 5.

[0112] Table 5

[0113]

[0114] Example 3

[0115] This embodiment provides another method for rapid detection of influenza B virus nucleic acid using a solid-state nanopore-based nucleic acid detection system.

[0116] This embodiment is basically the same as Embodiment 1, except that the specific probe IVB_P3 is replaced with IVB_P2. All other operations and conditions are performed according to Embodiment 1. IVB_P2 is also self-made. A specific complementary probe (designated as IVB_P2) is designed and artificially synthesized on the influenza B virus specific sequence designated IVB_M using Primer5. From 5' to 3', it consists of the first specific complementary sequence region (20nt in length), the non-specific complementary sequence region (secondary structure is a 40nt loop), and the second specific complementary sequence region (46nt in length).

[0117] Taking the IVB_M detection results as an example, some of the collected data in this embodiment can be found in [link to example]. Figure 9 Level 1 current drop via event, such as Figure 10 As shown, this is a non-characteristic via event; via events that simultaneously contain both first-order current drop and second-order current drop are as follows: Figure 11 As shown, this is a characteristic perforation event of influenza B virus nucleic acid.

[0118] The statistical results of via events for different test samples in this embodiment are shown in Table 6.

[0119] Table 6

[0120]

[0121]

[0122] Example 4: This example provides a method for rapid detection of low-concentration influenza B virus nucleic acid using a nucleic acid detection system based on solid-state nanopores.

[0123] Test sample: Artificially synthesized influenza B virus nucleic acid fragment (sequence corresponding to the influenza B virus-specific sequence screened from the NCBI nt database, numbered IVB_M), at a concentration of 0.1 fM.

[0124] Specific probe: IVB_P1. The nucleic acid detection system based on solid-state nanopores includes the same detection equipment, detection reagent system, and testing methods as in Example 1.

[0125] Some of the collected data in this embodiment can be found in [link to example]. Figure 12 .

[0126] Example 5: This example provides a method for rapid detection of low-concentration influenza B virus nucleic acid using a solid-state nanopore-based nucleic acid detection system.

[0127] Test sample: Artificially synthesized influenza B virus nucleic acid fragment (sequence corresponding to the influenza B virus-specific sequence screened from the NCBI nt database, numbered IVB_M), at a concentration of 0.1 fM.

[0128] A nucleic acid detection system based on solid nanopores includes detection equipment and detection reagent system.

[0129] Testing equipment: Same as in Example 1.

[0130] Detection reagent system: specific probe, detection buffer, 5× annealing buffer, nuclease-free water

[0131] Specific probe: IVB_P1.

[0132] Detection buffer: 1M KCl, 10mM Tris and 1mM EDTA, in water, pH=8 (hereinafter referred to as 1M KCl solution); 0.2M KCl, 10mM Tris and 1mM EDTA, in water, pH=8 (hereinafter referred to as 0.2M KCl solution).

[0133] 5× Annealing buffer: Same as in Example 1.

[0134] Nuclease-Free Water: Same as in Example 1.

[0135] Detection method: Basically the same as in Example 1, except that the pretreatment step of "adding 50 μL of detection buffer to each of the two chambers" is changed to "adding 50 μL of 1M KCl solution to the trans chamber and 50 μL of 0.2M KCl solution to the cis chamber". All other operations and conditions are performed in accordance with Example 1.

[0136] Some of the collected data in this embodiment can be found in [link to example]. Figure 13 .

[0137] Table 6 shows a comparison of the statistical results of via events in Examples 4 and 5.

[0138] Table 6

[0139]

[0140] Example 6: This example provides a method for simultaneously and rapidly detecting influenza A virus nucleic acid and influenza B virus nucleic acid using a solid-state nanopore-based nucleic acid detection system.

[0141] Samples to be tested: Quality control materials for influenza A / B virus multi-nucleic acid detection (purchased from Ruifengkang Biomedical Technology Co., Ltd.), quality control materials for influenza A / B virus multi-nucleic acid detection.

[0142] The nucleic acid detection system based on solid nanopores is basically the same as in Example 1, except that the pore size of the nanopore is 7.5 nm.

[0143] The detection reagent system is basically the same as in Example 1, except that the specific probe is replaced with a mixture of specific probes (mixed probe P_Mix).

[0144] Specific probe mixture: In-house prepared. A specific influenza A virus sequence (IVA_M) was screened from the NCBI nt database. Using Primer 5, a specific complementary probe (IVA_P) was designed and synthesized based on this sequence. From 5' to 3', the sequence consisted of: a first specific complementary sequence region (22 nt in length), a non-specific complementary sequence region comprising two 28 nt loop secondary structural units, with a 50 nt second linker sequence complementary to the target nucleic acid between the two secondary structural units, and a second specific complementary sequence region (22 nt in length). IVA_P and IVB_P1 were mixed in equimolar amounts to prepare a probe mixture P_Mix.

[0145] The detection method is basically the same as in Example 1, except that the IVB_P3 probe in the incubation system is replaced with P_Mix. All other operations and condition parameters are the same as in Example 1.

[0146] Some of the collected data in this embodiment can be found in [link to example]. Figure 14Among them, non-characteristic via events such as Figure 15 As shown, the characteristic perforation events of influenza B virus nucleic acid are as follows: Figure 16 As shown, the characteristic perforation events of influenza A virus nucleic acid are as follows: Figure 17 As shown in Table 7, the statistical results of via events are presented.

[0147] Table 7

[0148]

[0149]

[0150] Example 7: Following the method in Example 6, the presence of influenza A and influenza B viruses in 10 clinical samples (throat swabs) was detected, and the results were compared with those from fluorescent PCR. The results are shown in Table 8. Fluorescent PCR detection was performed using a commercially available influenza A / B virus nucleic acid detection kit (fluorescent PCR method) according to its instructions.

[0151] Table 8

[0152]

[0153] Table 8 shows that the results of solid-state nanopore detection are consistent with those of fluorescent PCR detection. Compared with fluorescent PCR detection, solid-state nanopore detection is simpler, more efficient, and lower in cost.

[0154] The sequence information involved in the above embodiments is shown in Table 9.

[0155] Table 9

[0156]

[0157]

[0158] Note: The bolded part is the region where the loop is formed; the underlined part at the 5' end is the first specific complementary sequence region; the underlined part at the 3' end is the second specific complementary sequence region; the italicized part of IBV_P3 is the first connecting sequence region; and the italicized part of IAV_P is the second connecting sequence region.

[0159] The present invention has been described in detail above, with the aim of enabling those skilled in the art to understand and implement the invention. However, this description should not be construed as limiting the scope of protection of the invention. All equivalent changes or modifications made in accordance with the spirit and essence of the invention should be included within the scope of protection of the invention.

Claims

1. A rapid detection method for target nucleic acids based on solid-state nanopores, characterized in that, Includes the following steps: (1) Provide a specific probe that matches the target nucleic acid, wherein the specific probe includes, from the 5' end to the 3' end, a first specific complementary sequence region that can pair with the bases of the target nucleic acid, a non-specific complementary sequence region that can form a secondary structure, and a second specific complementary sequence region that can pair with the bases of the target nucleic acid. (2) Mix the sample to be tested with the specific probe and incubate; (3) The current signal change of the incubation solution in step (2) is detected using a nucleic acid detection system based on solid nanopores. Each biomolecule in the incubation solution generates a continuous current drop when passing through the solid nanopore. When a nucleic acid molecule formed by the specific probe and the target nucleic acid or one of its sequences through base complementary pairing passes through the solid nanopore, the part of the nucleic acid molecule other than its secondary structure generates a first-order current drop when passing through the solid nanopore, and the secondary structure part generates a second-order current drop when passing through the solid nanopore. The minimum current value of the first-order current drop is greater than the minimum current value of the second-order current drop. (4) Each consecutive current drop corresponds to a via event. It is determined whether there is a via event in the current signal change of the incubation solution within the preset detection time that simultaneously contains a first-level current drop and a second-level current drop, and all or part of the second-level current drop is not at both ends of the first-level current drop. If so, it is confirmed that the sample to be tested contains the target nucleic acid.

2. The rapid detection method for target nucleic acids based on solid-state nanopores according to claim 1, characterized in that, The non-specific complementary sequence region can form one or more of the secondary structures; And / or, the secondary structure is one or more of the following: ring structure, T-shaped structure, Y-shaped structure, and clover structure.

3. The rapid detection method for target nucleic acids based on solid-state nanopores according to claim 1, characterized in that, The secondary structure is a ring structure with a sequence length of 20 to 200 nt.

4. The rapid detection method for target nucleic acids based on solid-state nanopores according to claim 3, characterized in that, The non-specific complementary sequence region can only form a ring structure with a sequence length of 20nt to 200nt; Alternatively, the nonspecific complementary sequence region can form at least two circular structures with independent sequence lengths of 20 to 40 nt each, and a first linker sequence region is provided between two adjacent circular structures. The first linker sequence region can be complementary to the target nucleic acid bases and the sequence length of the first linker sequence region is 1 to 10 nt.

5. The rapid detection method for target nucleic acids based on solid-state nanopores according to claim 1, characterized in that, The nonspecific complementary sequence region includes multiple sequentially connected units capable of forming secondary structures and a second linker sequence region located between two adjacent units. Any two units can form the same or different secondary structures. The second linker sequence region is complementary to the target nucleic acid bases and has a sequence length greater than 10 nt. Each unit corresponds to a secondary current drop. When there are multiple target nucleic acids, the non-specific complementary sequence regions of the specific probes corresponding to different target nucleic acids contain different secondary structures and / or different numbers of the units.

6. The rapid detection method for target nucleic acids based on solid-state nanopores according to claim 1, characterized in that, The lengths of the first specific complementary sequence region and the second specific complementary sequence region are independently 20–80 nt, and / or, The length of the nonspecific complementary sequence region is 20–200 nt.

7. The rapid detection method for target nucleic acids based on solid-state nanopores according to claim 1, characterized in that, The incubation system includes 5× annealing buffer, the sample to be tested, the specific probe, and nuclease-free water. The 5× annealing buffer is formulated as follows: 300–400 mM KCl, 25–75 mM Tris, 4–6 mM EDTA, with water as the solvent. And / or, the incubation procedure is as follows: maintain at 94-96°C for 3-5 minutes, then cool down to 25°C at a cooling rate of 0.05-0.15°C / s; And / or, in step (4), the preset detection time is 5 to 10 minutes.

8. The rapid detection method for target nucleic acids based on solid-state nanopores according to claim 1, characterized in that, A nucleic acid detection system based on solid-state nanopores includes a nanopore chip, electrodes, and an electrical signal data acquisition system. The nanoporous chip is a silicon nitride chip with a thickness of 10–50 nm, and the nanoporous chip is a chip that has been cleaned by gaseous plasma. The nanoporous chip has through-holes with a pore size of 5–50 nm. One side of the nanoporous chip is a trans chamber, and the other side is a cis chamber. The trans chamber and the cis chamber are connected through the through-holes. The trans chamber and the cis chamber each contain a detection buffer solution and are respectively equipped with electrodes. The electrode of the trans chamber is connected to the positive terminal of an external power supply, and the electrode of the cis chamber is connected to the negative terminal of an external power supply, thereby forming a closed loop. The detection buffer solution consists of 0.1–1 M KCl, 5–15 mM Tris, and 0.8–1.2 mM EDTA, with water as the solvent and a pH of 7.8–8.

2. The detection buffer solutions in the trans chamber and the cis chamber may be the same or different.

9. The rapid detection method for target nucleic acids based on solid-state nanopores according to claim 8, characterized in that, The data acquisition system is a patch-clamp amplifier; And / or, in step (3), the detection voltage is 50 to 800 mV, the sampling frequency is 20 to 500 kHz, and the filtering frequency is 2 to 10 kHz.

10. The rapid detection method for target nucleic acids based on solid-state nanopores according to claim 8, characterized in that, The KCl concentration of the detection buffer in the trans chamber is 3 to 40 times that of the KCl concentration of the detection buffer in the cis chamber.

11. The rapid detection method for target nucleic acids based on solid-state nanopores according to claim 1, characterized in that, The target nucleic acid is influenza B virus nucleic acid, and the nucleotide sequence of the corresponding specific probe is the nucleotide sequence of any one of SEQ ID NO: 2 to 4; And / or, the target nucleic acid is influenza A virus nucleic acid, and the nucleotide sequence of the corresponding specific probe is the nucleotide sequence described in SEQ ID NO:

6.

12. A target nucleic acid detection kit based on solid-state nanopores, characterized in that, It includes the nanopore chip as described in claim 8, the specific probe and detection reagent system as described in any one of claims 1 to 5, wherein the detection reagent system includes the 5× annealing buffer as described in claim 7, the detection buffer as described in claim 8, and nuclease-free water.

13. A multi-detection system for influenza A / B viruses based on solid-state nanopores, characterized in that, It includes the solid-state nanopore-based nucleic acid detection system as described in any one of claims 8 to 10, the specific probe corresponding to influenza B virus nucleic acid as described in claim 11, the specific probe corresponding to influenza A virus nucleic acid as described in claim 11, and the detection reagent system, wherein the detection reagent system includes the 5× annealing buffer as described in claim 7, the detection buffer as described in claim 8, and nuclease-free water.

Images