WO2020103012A1 - 核酸分子检测方法、检测装置和检测系统 - Google Patents

核酸分子检测方法、检测装置和检测系统

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WO2020103012A1
WO2020103012A1 PCT/CN2018/116619 CN2018116619W WO2020103012A1 WO 2020103012 A1 WO2020103012 A1 WO 2020103012A1 CN 2018116619 W CN2018116619 W CN 2018116619W WO 2020103012 A1 WO2020103012 A1 WO 2020103012A1
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nucleic acid
acid molecule
voltage
detected
detection
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PCT/CN2018/116619
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English (en)
French (fr)
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赵静
赵霞
章文蔚
任悍
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深圳华大生命科学研究院
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Priority to PCT/CN2018/116619 priority Critical patent/WO2020103012A1/zh
Priority to CN201880099314.8A priority patent/CN113166703A/zh
Publication of WO2020103012A1 publication Critical patent/WO2020103012A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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

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  • the invention relates to the technical field of nucleic acid detection, in particular to a nucleic acid molecule detection method, detection device and detection system.
  • nucleic acid molecule length or concentration detection include the following methods: (1) Pulsed field gel electrophoresis detection, each detection takes about 16 hours, is time-consuming and expensive (about 30,000-90,000 yuan) ).
  • X-ray crystallography (X-ray crystallography), its main application is accurate structure detection, used to detect DNA length can be described as "big and small", the cost is too high, and it is not suitable for the simultaneous detection of a large number of nucleic acid fragments.
  • Agilent Genomics' Bioanalyser can accurately detect the integrity and concentration of nucleic acid molecules, but the analyzer itself requires about 38,000 US dollars, and the chip used for each test is about 50- 70 euros, expensive.
  • the prices of LabChip GX and GX II of Caliper Life Science are $ 3000 and $ 6900 respectively, and each test costs about $ 8.
  • Thermo Fisher Scientific's Qubit Fluorometric Quantitative Method and NanoDrop Spectrophotometer are two of the most widely used instruments for detecting nucleic acid concentration. However, according to the comparison experiment, it was found that the concentration of the same sample detected by nanodroplet and qubit fluorescence quantitative method differed by more than 2-3 times. The sensitivity and accuracy of nano-droplets for concentration detection are not ideal. Its biggest advantage is that it can detect the purity of DNA samples (OD260 / 280, OD260 / 230). In addition, these two kinds of detection equipment and the cost of each detection are not low. The price of a Qubit and NanoDrop instrument on the market is about 30,000 yuan and 150,000 yuan respectively. A test dose (dsDNA HS Assay kit, 500 assays) requires 4,210 yuan, and the average test cost for each DNA sample is 8.4 yuan. The cost of testing is relatively high.
  • the existing techniques for determining the length and concentration of nucleic acid molecules have the following four shortcomings: expensive, limited accuracy, too long test time, and test consumables can not be reused.
  • the invention utilizes the characteristics of the current change when the nucleic acid molecule passes through the nanopore to accurately quantify the length and / or concentration of the nucleic acid molecule fragment, thereby solving the problems of long detection time, high cost and limited accuracy.
  • the invention utilizes multiple detection cells on a detection device (such as a chip) to detect multiple samples at the same time, and the detection requires a small sample size, short time, low cost, and the detection device can be reused.
  • the present invention provides a nucleic acid molecule detection method, detection device and detection system.
  • an embodiment provides a nucleic acid molecule detection method, the method comprising: applying a voltage to a solution containing a nucleic acid molecule to be detected, so that the nucleic acid molecule to be detected is placed in the solution under the voltage The nanopore in the; detecting the current change when the nucleic acid molecule to be detected passes through the nanopore, and determining the length of the nucleic acid molecule to be detected and / or the concentration in the solution according to the current change.
  • the determining the length of the nucleic acid molecule to be detected according to the current change specifically includes: determining the speed at which the nucleic acid molecule to be detected passes through the nanopore; determining the duration of the current change; according to the speed and the duration Time determines the length of the nucleic acid molecule to be detected.
  • the determining the concentration of the nucleic acid molecule to be detected in the solution according to the current change specifically includes: determining a speed at which the nucleic acid molecule to be detected passes through the nanopore; determining that the nucleic acid molecule to be detected passes through the nanopore The total duration of time current change; determine the total number of bases of the nucleic acid molecule to be detected according to the speed and the total duration, and then determine the concentration of the nucleic acid molecule to be detected in the solution.
  • the above voltage is a millivolt level voltage.
  • the millivolt-level voltage is a voltage of 10 mV or more, preferably a voltage of 50 mV or more, more preferably a voltage of 100 mV or more, particularly preferably a voltage of 150 mV to 300 mV, and most preferably a voltage of 180 mV.
  • the above detection method includes: conducting the detected current change to the computer, and graphically presenting the above current change and the duration of the current change on the screen.
  • the amount of the nucleic acid molecule to be detected is in picogram (pg) grade.
  • the nucleic acid molecule to be detected is a single-stranded or double-stranded DNA or RNA molecule.
  • the nucleic acid molecule to be detected is a single-stranded DNA or RNA molecule.
  • an embodiment provides a nucleic acid molecule detection device used in the nucleic acid molecule detection method of the first aspect, the detection device comprising a detection cell for containing a solution containing the nucleic acid molecule to be detected, The detection cell is provided with a nanopore. When a voltage is applied to the solution, the nucleic acid molecule to be detected passes through the nanopore under the action of the voltage.
  • the detection device is a chip, and the detection cell is provided on the chip.
  • a plurality of detection cells are provided on the chip, and each of the detection cells is provided with the above-mentioned nanopores.
  • the aforementioned nanopore is a biological nanopore or a physical nanopore.
  • the above-mentioned biological nanopores are protein nanopores provided on the membrane constructed of biological materials; and the above-mentioned physical nanopores are nanopores provided on the membrane constructed of physical materials.
  • the membrane constructed by the above biological material is a phospholipid bilayer.
  • an embodiment provides a nucleic acid molecule detection system, the detection system including:
  • a voltage supply device for applying a voltage across the nanopores in the detection cell of the detection device, so that the nucleic acid molecules to be detected in the solution in the detection cell pass through the nanopore under the action of the voltage and cause a current change;
  • the current signal detection device is used to detect the current change when the nucleic acid molecule to be detected passes through the nanopore.
  • the voltage supply device described above provides millivolt-level voltage.
  • the millivolt-level voltage is a voltage of 10 mV or more, preferably a voltage of 50 mV or more, more preferably a voltage of 100 mV or more, particularly preferably a voltage of 150 mV to 300 mV, and most preferably a voltage of 180 mV.
  • the detection system further includes a computer, and the current signal detection device is a current sensor, and the current sensor transmits the detected current change to the computer.
  • the aforementioned computer graphically presents the received current changes and the duration of the current changes on the screen.
  • the invention utilizes the property that nucleic acid molecules cause current changes through nanopores to accurately quantify various nucleic acid molecules to achieve the advantages of low sample loading (pg nucleic acid), high accuracy, simultaneous operation of multiple samples, short test time and low cost. , It has a very wide range of applications in biological research and development.
  • FIG. 1 is a schematic diagram of the principle of a nucleic acid molecule detection device according to an embodiment of the present invention
  • FIG. 2 is a schematic diagram of the principle of a nucleic acid molecule detection system according to an embodiment of the present invention
  • FIG. 3 is a graph showing the length distribution of the ⁇ control DNA fragment in the pulse field detection in the embodiment of the present invention, where M1 and M2 represent DNA Marker; 1 represents the distribution of the ⁇ control DNA on the pulse field electrophoresis gel;
  • FIG. 4 is the length distribution and number of ⁇ control DNA fragments detected by the nanopore in the embodiment of the present invention, in which the longest detected DNA fragments are circled in an ellipse circle;
  • FIG. 5 is a partially enlarged view of FIG. 4 in which the longest DNA fragment detected is enclosed in an ellipse circle.
  • An embodiment of the present invention provides a nucleic acid molecule detection method.
  • the method includes: applying a voltage to a solution containing a nucleic acid molecule to be detected, so that the nucleic acid molecule to be detected passes through a nanopore placed in the solution under the voltage; The current change when the nucleic acid molecule to be detected passes through the nanopore is detected, and the length of the nucleic acid molecule to be detected and / or the concentration in the solution are determined according to the current change.
  • determining the length of the nucleic acid molecule to be detected according to the current change specifically includes: determining the speed at which the nucleic acid molecule to be detected passes through the nanopore; determining the duration of the current change; and determining the length of the nucleic acid molecule to be detected according to the speed and the upper duration length.
  • determining the concentration of the nucleic acid molecule to be detected in the solution according to the current change specifically includes: determining the speed at which the nucleic acid molecule to be detected passes through the nanopore; determining the total duration of the current change when the nucleic acid molecule to be detected passes through the nanopore; The total base number of the nucleic acid molecule to be detected is determined according to the speed and the total duration, and then the concentration of the nucleic acid molecule to be detected in the solution is determined.
  • the invention utilizes the property that nucleic acid molecules cause current changes through nanopores to accurately quantify various nucleic acid molecules, and the detection sample requires a low starting amount (generally only requires pg nucleic acid), and has high accuracy (errors are in a few or more than ten) Several bases), simultaneous operation of multiple samples, short test time and low cost, and the detection device (such as a detection chip) can be reused, which has a very wide range of applications in biological research and development.
  • an embodiment of the present invention provides a nucleic acid molecule detection device and system, which are used in the nucleic acid molecule detection method of the present invention.
  • the detection system includes a detection device, a voltage supply device and a current signal detection device.
  • the detection device includes a detection cell for containing a solution containing the nucleic acid molecule to be detected.
  • the detection cell is provided with a nanopore. When a voltage is applied to the solution, the nucleic acid molecule to be detected passes through the nanopore under the voltage. When the nucleic acid molecule to be detected passes through the nanopore, a current change is caused, and the length of the nucleic acid molecule to be detected and / or the concentration in the solution can be determined according to the current change.
  • each detection cell contains a nanopore
  • the nanopore can be a biological nanopore or a physical nanopore.
  • the biological nanopore may be a protein nanopore disposed on a membrane constructed by a biological material (for example, a phospholipid bilayer), and the physical nanopore may be a nanopore disposed on a membrane constructed of a physical material.
  • a voltage 180mV applied in Figure 1 is applied across the membrane (ie, at both ends of the nanopore), and the nucleic acid molecules will quickly move from the negative electrode through the nanopore to the positive electrode.
  • the speed at which acid molecules pass through the nanopore is related to the voltage applied across them. Generally, the higher the voltage, the faster the speed at which the acid molecules pass through the nanopore, but they are usually detected at the optimal voltage.
  • the voltage applied across the membrane can be adjusted through testing to find the optimal voltage and test the nucleic acid through-hole speed.
  • the voltage across the nanopore is provided by a voltage supply device, which is generally a millivolt (mV) voltage, such as a voltage above 10 mV, preferably a voltage above 50 mV, more preferably a voltage above 100 mV, and particularly preferably a voltage of 150 mV to 300 mV, most A voltage of 180mV is preferred.
  • mV millivolt
  • a protein nanopore is constructed on a bilayer of phospholipids, without any intervention, a voltage of 180 mV is applied, and the speed of nucleic acid molecules passing through the nanopore is about 10-12 bases / microsecond ( ⁇ s) .
  • the nucleic acid molecule detection device may be in various suitable forms.
  • a detection cell and a nanopore are provided on the chip. Multiple detection cells can be set on each chip, and the detection cells can be distributed in an array.
  • Such a nucleic acid molecule detection device is called a "nucleic acid molecule detection chip".
  • Other suitable formats for example, the detection cell and the nanowell are provided on a multi-well plate, such as a 96-well plate, a 384-well plate, etc.
  • the detection cell and the nanopore are provided on the chip to form the nucleic acid molecule detection chip of the present invention.
  • the nucleic acid molecule detection system of the embodiment of the present invention includes: the detection device of the present invention; and a voltage supply device for applying a voltage across the nanopores in the detection cell of the detection device to make the solution in the detection cell
  • the nucleic acid molecule to be detected passes through the nanopore under voltage and causes a current change; and a current signal detection device is used to detect the current change when the nucleic acid molecule to be detected passes through the nanopore.
  • the voltage supply device may be any power supply capable of supplying a suitable voltage level, for example, a voltage of millivolt level.
  • the current signal detection device may be any current sensor or the like capable of detecting a weak current such as picoampere (pA) current.
  • the nucleic acid molecule detection system further includes a computer connected to the detection device. The current sensor transmits the detected current change to the computer, and graphically receives the current change and the duration of the current change on the computer. Presented on the screen.
  • the current level changes with time on the computer screen, and the time for the nucleic acid molecule to pass through the nanopore is calculated according to the first and last nodes of the current level of each nucleic acid molecule.
  • Multiple nucleic acid molecules in the detection pool are sequentially Through the nanopore, a series of current changes are formed. According to this series of current changes, not only can the time for each nucleic acid molecule pass through the nanopore be identified, but also the total time for all nucleic acid molecules to pass through the nanopore can be identified. Given the speed of the nucleic acid molecule passing through the nanopore, the length of each nucleic acid molecule and the concentration of the nucleic acid molecule in the solution can be calculated separately. As shown in FIG.
  • a calculation program is set in the computer, and the calculation program can calculate the length of each nucleic acid molecule and the concentration of the nucleic acid molecule in the solution according to the acquired speed and passage time of the nucleic acid molecule through the nanopore, and the nucleic acid molecule The length and concentration are displayed graphically on the screen.
  • the nucleic acid molecule to be detected may be any single-stranded or double-stranded DNA or RNA molecule, such as DNA, mRNA, miRNA and other nucleic acid molecules, especially single-stranded DNA or RNA molecules.
  • the single-stranded DNA or RNA molecule has a simple structural form, and its moving speed in the electric field is not affected by the complex structure, so it is the most suitable nucleic acid molecule of the present invention.
  • double-stranded nucleic acid molecules can be converted into single-stranded nucleic acid molecules by suitable experimental methods, for example, double-stranded DNA (dsDNA) of different lengths is converted into single-stranded DNA (ssDNA), for example, heat denaturation or heat denaturation plus 5 % DMSO or heat denaturation plus 10% formamide or helicase for denaturation treatment.
  • dsDNA double-stranded DNA
  • ssDNA single-stranded DNA
  • the ends of the nucleic acid molecule fragments are modified so that the head and tail nodes of each nucleic acid molecule can be more accurately determined in terms of current changes.
  • the role of the end modification of nucleic acid molecule fragments is to introduce more obvious head and tail nodes in current changes.
  • the principle is that, for example, a linker (e.g., Poly (N)) or nano-scale protein modification can be added to the end (e.g., beginning and / or end) of the nucleic acid molecule so that the end (e.g., beginning and / Or the end), a series of phenomena with the same current level can be seen on the current (for example, when the connector Poly (N) is added), making the head and tail nodes more prominent.
  • a linker e.g., Poly (N)
  • nano-scale protein modification can be added to the end (e.g., beginning and / or end) of the nucleic acid molecule so that the end (e.g., beginning and / Or the
  • nucleic acid molecules such as DNA or methylated DNA with different degrees of damage
  • FFPE formalin-fixed and paraffin-embedded
  • A, C, T, and G can be added before detection Bases, fill and connect the damaged locations on the DNA, and then detect it, so that the current changes caused by the special labeled bases are different from ordinary A, C, T and G bases, and those nucleic acid molecules can be detected
  • the methylation site itself also generates a specific current signal, which can be amplified to modify the signal.
  • this modification is only a further improved embodiment, and it is not necessary to make such modification.
  • the invention is particularly suitable for the construction of sequencing libraries, such as the construction of libraries for second-generation sequencing or third-generation sequencing, for the precise determination of the integrity of nucleic acid molecule fragments and their concentrations.
  • the number of detected bases is shown in Table 1.
  • the total number of bases is 713014128.
  • the total number of moles calculated according to the formula is 0.24 * 10 -14 mol, the total mass is 1.52pg, and the concentration of the sample to be tested is 1.52pg / ⁇ L , Detection takes 1min.
  • This embodiment verifies that the detection method of the present invention has high sensitivity and short time.
  • the voltage can also use other set voltages, thereby further reducing the detection time.

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Abstract

一种核酸分子检测方法、检测装置和检测系统，该方法包括：向含有待检测核酸分子的溶液中施加电压，使待检测核酸分子在电压作用下通过置于溶液中的纳米孔；检测待检测核酸分子通过纳米孔时的电流变化，根据电流变化确定待检测核酸分子的长度和/或在溶液中的浓度。

Description

核酸分子检测方法、检测装置和检测系统 技术领域
本发明涉及核酸检测技术领域，具体涉及一种核酸分子检测方法、检测装置和检测系统。
背景技术
二代测序技术的发展促使测序文库构建技术也变得日新月异，文库构建前对DNA或mRNA的完整性及打断后核酸分子片段的大小及浓度都需要进行定量。而对于三代测序，核酸分子片段完整性及其浓度的精确测定更是不可或缺的检测指标。目前可用于核酸分子长度或浓度检测的技术有以下几种方法：(1)脉冲场凝胶电泳检测，每个检测所需时间为16小时左右，耗时长而且仪器昂贵(约3-9万元)。(2)X射线晶体学方法(X-ray crystallography)，其主要应用是精确的结构检测，用于检测DNA长度可谓“大材小用”，成本太高，而且也不适用于大量核酸片段同时检测。(3)安捷伦基因组学公司(Agilent Genomics)的生物分析仪(Bioanalyser)，可精确检测出核酸分子完整性及浓度，但仅分析仪本身就需要约3.8万美元，每次测试所用芯片约50-70欧元，价格昂贵。(4)圆规生命科学公司(Caliper Life Science)的LabChip GX和GX II，单纯分析仪的价格就分别为3000美元和6900美元，每次测试花费大约8美元。(5)赛默飞世尔公司(Thermo Fisher Scientific)的量子位荧光定量法(Qubit Fluorometric Quantitation)和纳米微滴(NanoDrop)分光光度计，是两款目前应用最为广泛的检测核酸浓度的仪器，但根据对比实验检测发现，同一样品采用纳米微滴和量子位荧光定量法检测的浓度相差2-3倍以上。纳米微滴对浓度检测的灵敏度及准确性并不理想，其最大优势是可以检测DNA样品的纯净度(OD260/280，OD260/230)。此外，这两种检测设备及每次检测费用都不低廉，目前市场上一 台Qubit和NanoDrop仪器的价格分别约3万元人民币和15万元人民币，Qubit检测试剂如双链DNA检测试剂盒500个检测剂量(dsDNA HS Assay kit，500assays)，需要4210元，平均每个DNA样品检测需8.4元，检测成本都较高。
综上所述，现有的核酸分子长度和浓度测定的技术分别存在以下四方面的不足：费用昂贵，精确度有限，测试时间太长以及测试耗材不可重复使用。
发明内容
本发明利用核酸分子通过纳米孔时引起电流变化的特征对核酸分子片段长度和/或浓度进行精准定量，从而解决检测时间长、成本高以及精确度有限等问题。本发明利用检测装置(如芯片)上的多个检测池可对多个样品同时检测，而且检测所需样本量少、时间短、成本低且检测装置可以重复使用。
基于此，本发明提供一种核酸分子检测方法、检测装置和检测系统。
根据第一方面，一种实施例中提供一种核酸分子检测方法，该方法包括：向含有待检测核酸分子的溶液中施加电压，使上述待检测核酸分子在上述电压作用下通过置于上述溶液中的纳米孔；检测上述待检测核酸分子通过上述纳米孔时的电流变化，根据上述电流变化确定上述待检测核酸分子的长度和/或在上述溶液中的浓度。
在优选实施例中，上述根据上述电流变化确定上述待检测核酸分子的长度，具体包括：确定上述待检测核酸分子通过上述纳米孔的速度；确定上述电流变化的持续时间；根据上述速度和上述持续时间确定上述待检测核酸分子的长度。
在优选实施例中，上述根据上述电流变化确定上述待检测核酸分子在上述溶液中的浓度，具体包括：确定上述待检测核酸分子通过上述纳米孔的速度；确定上述待检测核酸分子通过上述纳米孔时电流变化的总持续时间；根据上述速度和上述总持续时间确定上述待检测核酸分子的总碱基数，进而确定上述待检测核酸分子在上述溶液中的浓度。
在优选实施例中，上述电压是毫伏级电压。
在优选实施例中，上述毫伏级电压是10mV以上的电压，优选50mV以上的电压，更优选100mV以上的电压，特别优选150mV至300mV的电压，最优选180mV的电压。
在优选实施例中，上述检测方法包括：将检测到的电流变化传导到计算机上，并且将上述电流变化和电流变化的持续时间图形化地呈现在屏幕上。
在优选实施例中，上述待检测核酸分子的用量是皮克(pg)级别。
在优选实施例中，上述待检测核酸分子是单链或双链的DNA或RNA分子。
在优选实施例中，上述待检测核酸分子是单链的DNA或RNA分子。
根据第二方面，一种实施例中提供一种第一方面的核酸分子检测方法中使用的核酸分子检测装置，该检测装置包括检测池，该检测池用于容纳含有待检测核酸分子的溶液，上述检测池中设有纳米孔，当向上述溶液中施加电压时，上述待检测核酸分子在上述电压作用下通过上述纳米孔。
在优选实施例中，上述检测装置是芯片，上述检测池设置于上述芯片上。
在优选实施例中，上述芯片上设置有多个检测池，每个检测池中设有上述纳米孔。
在优选实施例中，上述纳米孔是生物纳米孔或物理纳米孔。
在优选实施例中，上述生物纳米孔是设置在生物材料构建的膜上的蛋白纳米孔；上述物理纳米孔是设置在物理材料构建的膜上的纳米孔。
在优选实施例中，上述生物材料构建的膜为磷脂双分子层。
根据第三方面，一种实施例中提供一种核酸分子检测系统，该检测系统包括：
第二方面的检测装置；
电压提供装置，用于在上述检测装置的检测池内的纳米孔两端施加电压，以使上述检测池内溶液中的待检测核酸分子在上述电压作用下通过上述纳米孔并引起电流变化；
电流信号检测装置，用于检测上述待检测核酸分子通过上述纳米孔时的电流变化。
在优选实施例中，上述电压提供装置提供毫伏级电压。
在优选实施例中，上述毫伏级电压是10mV以上的电压，优选50mV以上的电压，更优选100mV以上的电压，特别优选150mV至300mV的电压，最优选180mV的电压。
在优选实施例中，上述检测系统还包括计算机，上述电流信号检测装置是电流感应器，该电流感应器将检测到的电流变化传导到上述计算机。
在优选实施例中，上述计算机将接收到的电流变化和电流变化的持续时间图形化地呈现在屏幕上。
本发明利用核酸分子通过纳米孔引起电流变化的性质对各种核酸分子进行精确定量，实现测试上样量低(pg核酸)、精确度高、多样本同时操作、测试时间短及成本低的优势，在生物科研及研发等领域具有极为广泛的应用。
附图说明
图1为本发明实施例的核酸分子检测装置的原理示意图；
图2为本发明实施例的核酸分子检测系统的原理示意图；
图3为本发明实施例中脉冲场检测λ对照DNA片段长度分布结果图，其中M1和M2分别表示DNA Marker；1表示λ对照DNA在脉冲场电泳凝胶上的分布；
图4为本发明实施例中纳米孔检测λ对照DNA片段长度分布及数量，其中 椭圆圈内圈出了检测到的最长DNA片段；
图5为图4的部分放大图，其中椭圆圈内圈出了检测到的最长DNA片段。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。在以下的实施方式中，很多细节描述是为了使得本发明能被更好的理解。然而，本领域技术人员可以毫不费力的认识到，其中部分特征在不同情况下是可以省略的，或者可以由其他元件、材料、方法所替代。
另外，说明书中所描述的特点、操作或者特征可以以任意适当的方式结合形成各种实施方式。同时，方法描述中的各步骤或者动作也可以按照本领域技术人员所能显而易见的方式进行顺序调换或调整。因此，说明书和附图中的各种顺序只是为了清楚描述某一个实施例，并不意味着是必须的顺序，除非另有说明其中某个顺序是必须遵循的。
本发明的一种实施例中提供一种核酸分子检测方法，该方法包括：向含有待检测核酸分子的溶液中施加电压，使待检测核酸分子在电压作用下通过置于溶液中的纳米孔；检测待检测核酸分子通过纳米孔时的电流变化，根据电流变化确定待检测核酸分子的长度和/或在溶液中的浓度。
在优选实施例中，根据电流变化确定待检测核酸分子的长度，具体包括：确定待检测核酸分子通过纳米孔的速度；确定电流变化的持续时间；根据速度和上持续时间确定待检测核酸分子的长度。
在优选实施例中，根据电流变化确定待检测核酸分子在溶液中的浓度，具体包括：确定待检测核酸分子通过纳米孔的速度；确定待检测核酸分子通过纳米孔时电流变化的总持续时间；根据速度和总持续时间确定待检测核酸分子的总碱基数，进而确定待检测核酸分子在溶液中的浓度。
本发明利用核酸分子通过纳米孔引起电流变化的性质对各种核酸分子进行 精确定量，检测所需样本起始量低(一般仅需要pg核酸)，具有精确度高(误差在几个或十几个碱基左右)、多样本同时操作、测试时间短及成本低的优势，并且检测装置(例如检测芯片)可重复使用，在生物科研及研发等领域具有极为广泛的应用。
如图1和图2所示，本发明实施例提供一种核酸分子检测装置和系统，该检测装置和系统在本发明的核酸分子检测方法中使用。其中检测系统包括检测装置、电压提供装置和电流信号检测装置。检测装置包括检测池，该检测池用于容纳含有待检测核酸分子的溶液，检测池中设有纳米孔，当向溶液中施加电压时，待检测核酸分子在电压作用下通过纳米孔。待检测核酸分子通过纳米孔时，会引起电流变化，根据电流变化就可以确定待检测核酸分子的长度和/或在溶液中的浓度。
如图1所示，每个检测池中含有一个纳米孔，纳米孔可以是生物纳米孔或物理纳米孔。其中，生物纳米孔可以是设置在生物材料构建的膜(例如，磷脂双分子层)上的蛋白纳米孔，物理纳米孔可以是设置在物理材料构建的膜上的纳米孔。利用核酸分子带负电荷的性质，在膜两端(即纳米孔两端)施加电压(如图1中施加180mV电压)，核酸分子便会从负极迅速穿过纳米孔向正极移动。在没有向检测池中加入含有核酸分子的样品时，膜两端电流保持在初始电流水平；当加入检测样品时，核酸分子穿过纳米孔的瞬间，通过纳米孔的电流也会因该过程所引入的电阻而发生明显变化。由于每条核酸分子通过纳米孔都会有时间间歇，因此在电流变化水平线上每条核酸分子都会产生可辨识的首尾节点。辨识出每条核酸分子通过纳米孔的首尾节点，即得知每条核酸分子通过纳米孔的时间即通过时间，再知道核酸分子通过纳米孔速度，即可根据通过时间和速度计算出核酸分子每条核酸分子的长度。
酸分子通过纳米孔的速度与两端施加的电压有关，一般电压越高酸分子通过纳米孔的速度越快，但是通常在最佳电压下进行检测。可以通过测试对施加在膜两端的电压进行调整，找到最佳电压，测试出核酸通孔速度。纳米孔两端 的电压由电压提供装置来提供，一般是毫伏级(mV)电压，例如10mV以上的电压，优选50mV以上的电压，更优选100mV以上的电压，特别优选150mV至300mV的电压，最优选180mV的电压。发明人证实，180mV的电压是较理想的。在一个实施例中，在采用磷脂双分子层上构建蛋白纳米孔，无任何干预情况下，施加180mV的电压，核酸分子通过纳米孔的速度为大约10-12个碱基/微秒(μs)。
只需辨识出不同核酸分子的首尾节点(通过时间)，利用核酸分子的通过速度乘以通过时间即可得出核酸分子的准确长度，即核酸分子长度＝通过速度*通过时间(即首尾节点间时间)。
对于核酸分子的浓度，则可利用计算出的核酸分子的总碱基数除以6.02*10 23，再乘以650Da(g/mol)得到，即核酸分子的浓度＝核酸分子的总碱基数*650/(6.02*10 23)。
本发明实施例中，核酸分子检测装置可以是各种合适的形式，例如，如图2所示，将检测池和纳米孔设置在芯片上的形式。每个芯片上可以设置有多个检测池，检测池可以呈阵列分布。这样的核酸分子检测装置称为“核酸分子检测芯片”。其他合适的形式，例如，将检测池和纳米孔设置在多孔板上，例如96孔板、384孔板等。在优选实施例中，检测池和纳米孔设置在芯片上，形成本发明的核酸分子检测芯片。
如图2所示，本发明实施例的核酸分子检测系统，包括：本发明的检测装置；和电压提供装置，用于在检测装置的检测池内的纳米孔两端施加电压，以使检测池内溶液中的待检测核酸分子在电压作用下通过纳米孔并引起电流变化；以及电流信号检测装置，用于检测待检测核酸分子通过纳米孔时的电流变化。其中，电压提供装置可以是任何能够提供合适电压水平例如毫伏级电压的电源。电流信号检测装置可以是任何能够检测微弱电流例如皮安(pA)电流的电流感应器等。图2中，核酸分子检测系统还包括与检测装置相连的计算机，电流感应器将检测到的电流变化传导到计算机上，并在计算机上将接收到的电 流变化和电流变化的持续时间图形化地呈现在屏幕上。
如图2所示，计算机屏幕上可以呈现出随着时间变化的电流水平变化，根据每个核酸分子的电流水平首尾节点计算出该核酸分子通过纳米孔的时间，检测池中多个核酸分子依次通过纳米孔，形成一连串的电流变化，根据这一连串的电流变化不但可以辨识每个核酸分子通过纳米孔的时间，还可以辨识所有核酸分子全部通过纳米孔的总时间。在已知核酸分子通过纳米孔的速度的情况下，可分别计算出每个核酸分子的长度和核酸分子在溶液中的浓度。如图2所示，在计算机中设置计算程序，计算程序根据获取的核酸分子通过纳米孔的速度和通过时间，能够计算出每个核酸分子的长度以及溶液中核酸分子的浓度，并将核酸分子的长度和浓度图形化地呈现在屏幕上。
本发明实施例中，待检测核酸分子可以是任何单链或双链的DNA或RNA分子，如DNA、mRNA、miRNA等核酸分子，尤其是单链的DNA或RNA分子。单链的DNA或RNA分子的结构形式简单，其在电场中的移动速度不受复杂结构的影响，因此是最适合本发明的核酸分子。当然，通过合适的实验手段可以将双链的核酸分子转变成单链的核酸分子，例如将不同长度的双链DNA(dsDNA)转化为单链DNA(ssDNA)，例如热变性或热变性加5％DMSO或热变性加10％甲酰胺或利用解旋酶进行变性处理。
在一些实施例中，核酸分子片段末端进行修饰，从而在电流变化上更准确地判断出每条核酸分子的首尾节点。具体而言，核酸分子片段末端修饰的作用是，可以在电流变化上引入更明显的首尾节点。其原理是，例如，可以在核酸分子末端(例如，开头和/或结尾)加上接头(例如，Poly(N))或纳米级的蛋白修饰，这样核酸分子被检测的末端(例如，开头和/或结尾)，在电流上可以看到一连串电流水平一致的现象(例如，加有接头Poly(N)的情况下)，使得首尾节点更加突出。当然，这种修饰仅是一种进一步改进的实施方式，而不是必须进行这种修饰。每条核酸分子进入纳米孔中间都有一段间歇，可以在电流上识别出来，这种修饰是为了让首尾节点识别更确切。
此外，通过对不同程度损伤的DNA或甲基化DNA等核酸分子进行特异性标记，从而精确检测某种DNA损伤或修饰的DNA浓度或长度变化。例如，对于福尔马林固定和石蜡包埋(FFPE)样品，DNA上经常出现切口(nick)或缺口(gap)等损伤，可以在检测前加入带有化学修饰的A、C、T和G碱基，将DNA上的损伤位置进行填充连接，然后再进行检测，这样通过特殊标记的碱基引起的不同于普通A、C、T和G碱基的电流变化，即可检测出那些核酸分子存在缺失性损伤。对于甲基化DNA，甲基化位点本身也会产生特异性的电流信号，可以通过修饰将信号放大。然而，应当理解，这种修饰仅是一种进一步改进的实施方式，而不是必须进行这种修饰。
本发明尤其适用于测序文库构建，例如二代测序或三代测序的文库构建中，核酸分子片段完整性及其浓度的精确测定。
以下通过实施例对本发明进行详细描述，需要说明的是，该实施例仅是示例性的，不能理解为对本发明保护范围的限制。
实施例
(1)利用λ对照DNA进行初步验证试验：
取200ngλ对照DNA(全长为48502bp)利用脉冲场电泳检测其片段长度分布，如图3所示，DNA片段长度分布在20kbp以下。
(2)对同样的λ对照DNA样本，利用本发明的检测装置(采用生物纳米孔，即在磷脂双分子层上形成蛋白纳米孔)，对其DNA片段长度分布及样品浓度进行检测。
将λ对照DNA样品稀释至pg级别(Qubit检测其浓度“太低(too low)”，超出最低检测范围)，取1μL加入检测池中，检测池两端施加180mV电压，在此电压下，DNA通过纳米孔的速度为10个碱基/微秒(μs)，运行2min，检测运行一分钟后已无电流水平变化。与检测池连接的计算机屏幕显示如图4和图5所示，DNA片段长度分布在0-20kbp之间，平均长度为8045.2bp，检测到的最 长DNA片段为48288bp。
检测到的碱基数如表1所示，总碱基数为713014128个，根据公式计算其总摩尔数为0.24*10 -14mol，总质量为1.52pg，待检测样品浓度为1.52pg/μL，检测用时1min。
表1
Figure PCTCN2018116619-appb-000001
本实施例验证了本发明的检测方法灵敏度高，用时短。此外，电压还可使用其它设定电压，从而进一步缩短检测时间。
以上应用了具体个例对本发明进行阐述，只是用于帮助理解本发明，并不用以限制本发明。对于本发明所属技术领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。

Claims (17)

  1. 一种核酸分子检测方法，其特征在于，所述方法包括：向含有待检测核酸分子的溶液中施加电压，使所述待检测核酸分子在所述电压作用下通过置于所述溶液中的纳米孔；检测所述待检测核酸分子通过所述纳米孔时的电流变化，根据所述电流变化确定所述待检测核酸分子的长度和/或在所述溶液中的浓度。
  2. 根据权利要求1所述的检测方法，其特征在于，所述根据所述电流变化确定所述待检测核酸分子的长度，具体包括：确定所述待检测核酸分子通过所述纳米孔的速度；确定所述电流变化的持续时间；根据所述速度和所述持续时间确定所述待检测核酸分子的长度。
  3. 根据权利要求1所述的检测方法，其特征在于，所述根据所述电流变化确定所述待检测核酸分子在所述溶液中的浓度，具体包括：确定所述待检测核酸分子通过所述纳米孔的速度；确定所述待检测核酸分子通过所述纳米孔时电流变化的总持续时间；根据所述速度和所述总持续时间确定所述待检测核酸分子的总碱基数，进而确定所述待检测核酸分子在所述溶液中的浓度。
  4. 根据权利要求1至3任一项所述的检测方法，其特征在于，所述电压是毫伏级电压。
  5. 根据权利要求4所述的检测方法，其特征在于，所述毫伏级电压是10mV以上的电压，优选50mV以上的电压，更优选100mV以上的电压，特别优选150mV至300mV的电压，最优选180mV的电压。
  6. 根据权利要求1至3任一项所述的检测方法，其特征在于，所述检测方法包括：将检测到的电流变化传导到计算机上，并且将所述电流变化和电流变化的持续时间图形化地呈现在屏幕上。
  7. 根据权利要求1至3任一项所述的检测方法，其特征在于，所述待检测核酸分子的用量是皮克(pg)级别。
  8. 根据权利要求1至3任一项所述的检测方法，其特征在于，所述待检测核酸分子是单链或双链的DNA或RNA分子。
  9. 根据权利要求8所述的检测方法，其特征在于，所述待检测核酸分子是单链的DNA或RNA分子。
  10. 一种权利要求1至9任一项所述的核酸分子检测方法中使用的核酸分子检测装置，其特征在于，所述检测装置包括检测池，该检测池用于容纳含有待检测核酸分子的溶液，所述检测池中设有纳米孔，当向所述溶液中施加电压时，所述待检测核酸分子在所述电压作用下通过所述纳米孔。
  11. 根据权利要求10所述的检测装置，其特征在于，所述检测装置是芯片，所述检测池设置于所述芯片上。
  12. 根据权利要求10所述的检测装置，其特征在于，所述芯片上设置有多个检测池，每个检测池中设有所述纳米孔。
  13. 一种核酸分子检测系统，其特征在于，所述检测系统包括：
    权利要求10至12任一项所述的检测装置；
    电压提供装置，用于在所述检测装置的检测池内的纳米孔两端施加电压，以使所述检测池内溶液中的待检测核酸分子在所述电压作用下通过所述纳米孔并引起电流变化；
    电流信号检测装置，用于检测所述待检测核酸分子通过所述纳米孔时的电流变化。
  14. 根据权利要求13所述的检测系统，其特征在于，所述电压提供装置提供毫伏级电压。
  15. 根据权利要求14所述的检测系统，其特征在于，所述毫伏级电压是10mV以上的电压，优选50mV以上的电压，更优选100mV以上的电压，特别优选150mV至300mV的电压，最优选180mV的电压。
  16. 根据权利要求13所述的检测系统，其特征在于，所述检测系统还包括计算机，所述电流信号检测装置是电流感应器，该电流感应器将检测到的电流 变化传导到所述计算机。
  17. 根据权利要求16所述的检测系统，其特征在于，所述计算机将接收到的电流变化和电流变化的持续时间图形化地呈现在屏幕上。
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