WO2021232186A1 - 基于数字微流控平台富集核酸及构建测序文库的方法 - Google Patents

基于数字微流控平台富集核酸及构建测序文库的方法 Download PDF

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WO2021232186A1
WO2021232186A1 PCT/CN2020/090796 CN2020090796W WO2021232186A1 WO 2021232186 A1 WO2021232186 A1 WO 2021232186A1 CN 2020090796 W CN2020090796 W CN 2020090796W WO 2021232186 A1 WO2021232186 A1 WO 2021232186A1
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microfluidic
nucleic acid
amplification
product
library
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PCT/CN2020/090796
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English (en)
French (fr)
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任悍
崔淼
陈杨帆
汪元涛
江媛
陈奥
章文蔚
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深圳华大生命科学研究院
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Priority to PCT/CN2020/090796 priority Critical patent/WO2021232186A1/zh
Priority to CN202080088509.XA priority patent/CN114829627B/zh
Publication of WO2021232186A1 publication Critical patent/WO2021232186A1/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
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C12M1/38—Temperature-responsive control
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • the invention relates to the field of gene detection, in particular to a method for enriching nucleic acids based on a digital microfluidic platform and constructing a sequencing library.
  • NGS second-generation high-throughput sequencing technology
  • SNVs single nucleotide variants
  • CNVs copy number variants
  • gene fusion etc.
  • Multiplex PCR and hybrid capture technology are two types of widely used target area enrichment technologies.
  • the enrichment and amplification of the multiplex PCR technology is divided into two rounds of PCR.
  • the first round of PCR is mainly designed to design hundreds of pairs of PCR primers for the target region to simultaneously amplify different target regions.
  • the primers of the first round of PCR are At the same time, an identical sequence was designed for the second round of amplification.
  • the second round of PCR uses the same primer to amplify the enriched products in the first round, and the amplified library can be used for subsequent sequencing.
  • the primers used in the second round of PCR have partial sequences that are complementary to the primers used in the first round of PCR, as well as additional sequences for sequencing.
  • Hybrid capture technology uses a biotin-containing probe to hybridize the fragment containing the target region, and then uses a modified magnetic bead that specifically binds to biotin to adsorb and recover the captured probe, and finally uses the magnetic bead to purify the captured probe. Ways to wash away excess non-target regions fragments to obtain the desired target fragments.
  • the required starting DNA and reagents are determined based on the whole genome sequencing data, which causes a large amount of data waste and reduces the cost of the library construction process.
  • the present invention aims to solve one of the technical problems in the related technology at least to a certain extent, and provides a method for enriching nucleic acids based on a digital microfluidic platform and a method for constructing a sequencing library.
  • the provided methods for enriching nucleic acids rely on It achieves the purpose of enriching nucleic acids on the digital microfluidic platform, and is used in the construction of sequencing libraries and sequencing.
  • the application of this method only requires a small amount of nucleic acid samples to achieve the enrichment of nucleic acid samples, obtain a sequencing library, and apply it to the sequencing field; and with the help of a digital microfluidic platform, manual operations such as lengthy washing steps and manual operation control can be eliminated. To a specific temperature, etc., to achieve automatic operation.
  • Multiplex PCR and hybrid capture technology are two commonly used techniques for the enrichment of the target region of nucleic acid, which can be used for the enrichment of the target region of nucleic acid.
  • These technologies also have some shortcomings, such as the cumbersome steps of the enrichment process, involving multiple complicated manual operations, manual operations bring many disadvantages, and it is not easy to control the temperature. For example, the washing is unstable, which may easily lead to poor final library effects. .
  • hybrid capture technology Take hybrid capture technology as an example. In addition to its high cost, the most important thing is that most hybrid capture steps cannot avoid complicated operating procedures, including operation under a certain temperature control and multiple rounds of washing steps that are time-consuming and laborious.
  • the present invention provides a method for enriching nucleic acid based on a digital microfluidic platform and a method for constructing a sequencing library.
  • the method also uses a digital microfluidic platform to achieve nucleic acid enrichment, which can complete the enrichment process with a small amount of nucleic acid samples , And can not rely on manual operation, convenient for temperature control, and because the reaction system is reduced, the reaction time is shortened, so that rapid and low-cost automatic enrichment of nucleic acid molecules can be realized.
  • the stability of each batch operation can be guaranteed.
  • the present invention provides the following technical solutions:
  • the present invention provides a method for enriching nucleic acids based on a digital microfluidic platform.
  • the digital microfluidic platform includes a microfluidic chip and a microfluidic device.
  • the microfluidic chip Removably connected to the microfluidic device, the method includes: preparing microfluidic droplets on a microfluidic chip, the microfluidic droplets containing surfactants, silicone oils and nucleic acids; based on specific probes or specificity
  • the primer specifically captures or amplifies the target region of the nucleic acid in the microfluidic droplet to obtain an enriched nucleic acid product.
  • the nucleic acid enrichment method based on the digital microfluidic platform provided by the present invention is used to enrich the target area of nucleic acid, which solves the problem of complicated operation during the enrichment experiment of the target area, and does not require manual manual operations, including lengthy Washing steps, and manual operation at a specific temperature. This can reduce the time and errors caused by the experimental operation part. Moreover, the amount of DNA input required is less than other technologies, and the cost of reagents required for biochemical reactions is reduced.
  • the above-mentioned method for enriching nucleic acid based on a digital microfluidic platform may further include the following technical features:
  • the silicone oil is a low-viscosity silicone oil.
  • Low-viscosity silicone oil is easier when the electric field controls the movement of the droplets.
  • silicone oils below 10 cps are low-viscosity silicone oils.
  • it further includes the step of purifying the enriched nucleic acid product by using the microfluidic device.
  • the volume of the microfluidic droplet does not exceed 16 microliters.
  • the mass concentration of the surfactant in the microfluidic droplets does not exceed 0.5%, preferably 0.01% to 0.1%, more preferably 0.075%.
  • the content of surfactant is too high, it will seriously affect the progress of biochemical reactions.
  • Tween 20 with a mass concentration of 1% or more will seriously affect the progress of biochemical reactions, and the impact of 0.01% -0.1% of Tween 20 on biochemical reactions
  • the surfactant is Tween 20.
  • the microfluidic chip further includes:
  • a substrate on which an electrode matrix is arranged A substrate on which an electrode matrix is arranged
  • the operation area is located on the substrate, the operation area includes a sample application area, a storage area, a reaction area and a collection area, the sample application area, the storage area, the reaction area and the collection area
  • the districts are connected in turn.
  • the flow of droplets can be controlled by the electrode matrix in the substrate.
  • the movement of different microdroplets or microdroplets of different sizes can be controlled by the switch of the electrodes, so that different operations and processing of microdroplets can be realized without manual labor. operate.
  • the microfluidic device further includes: a temperature control module, the temperature control module is arranged opposite to the reaction zone, and the temperature control module is used to control the temperature of the reaction zone .
  • the microfluidic device further includes: a magnetron module, the magnetron module and the collection area are arranged directly opposite, the magnetron module is provided with a magnet, the magnetron The module controls the lifting of the magnet through a stepping motor.
  • a magnet is arranged in the magnetron module, and a stepping motor can be installed under the magnet, and then the up and down movement of the magnet can be controlled by the stepping motor. For example, when the magnet is above, that is, when it is close to the substrate, it can be used to attract magnetic beads; when the magnet is below, that is, when it is far away from the substrate, it can be used to release the magnetic beads, so as to achieve the purpose of purifying the reagent.
  • the microfluidic device further includes a water-cooling module, the water-cooling module is arranged directly opposite to the storage area through a metal plate, and the water-cooling module is used to control the temperature of the storage area.
  • the water-cooling module and the storage area are set directly opposite, and the water-cooling module can be always on to protect the reagents in the storage area from deactivation.
  • the metal plate is a Peltier plate.
  • the viscosity of the low-viscosity silicone oil may be 5 or less, for example, it may be 4.6
  • the microfluidic device further includes: a fluorescence detection module, the fluorescence detection module is connected to the reaction zone, and the fluorescence detection module is used for fluorescence detection of products in the reaction zone . Since the electrode matrix is provided on the substrate, the large droplets after the reaction can be divided into two or three by controlling the switch of the electrode matrix. The separated droplets can be used for fluorescence detection without Fluorescence detection can be realized by manual operation.
  • the digital microfluidic platform further includes: a data processing device configured to convert the reaction result of the microfluidic chip into electrical signal output.
  • a robotic arm is further provided on the data processing device, and the robotic arm is used to realize the automated operation of the microfluidic chip.
  • the nucleic acid is genomic DNA, and the genomic DNA is 5 ng or more.
  • the present invention provides a method for constructing a sequencing library, including: obtaining a target region enriched product of nucleic acid based on the method described in any one of the embodiments of the first aspect of the present invention; The target area is enriched with products and a library is constructed to obtain a sequencing library.
  • the above method for constructing a sequencing library may further include the following technical features:
  • it further includes: after preparing the microfluidic droplets on the microfluidic chip, performing end repair and adaptor ligation, and performing the first PCR amplification on the obtained ligation product, so as to construct a pre-capture DNA library ; Use specific probes to hybridize capture and purify the pre-capture DNA library, so as to obtain capture hybridization products;
  • a second PCR amplification is performed based on the captured hybrid product, so as to construct a captured DNA library.
  • the specific probe is a biotin-labeled specific probe, and streptavidin-labeled magnetic beads are used for the purification.
  • it further includes: after preparing the microfluidic droplets on the microfluidic chip, performing amplification using the first amplification primer to obtain the first amplification product, and the first amplification primer is suitable To amplify different regions of the nucleic acid; amplify the first amplification product with a universal primer to obtain a second amplification product, and the universal primer is partially complementary to the first amplification primer.
  • it further includes: thermally denaturing the second amplification product to obtain single-stranded circular DNA; and performing rolling circle amplification based on the single-stranded circular DNA to obtain DNA nano ball.
  • magnetic beads are used for purification.
  • Fig. 1 is a schematic cross-sectional view of a microfluidic chip according to an embodiment of the present invention.
  • Fig. 2 is a partial structural diagram of a digital microfluidic platform provided according to an embodiment of the present invention.
  • Fig. 3 is a schematic structural diagram of a digital microfluidic platform provided according to an embodiment of the present invention.
  • Fig. 4 is a schematic diagram of a method for exploring through off-chip conditions and adjusting to make it suitable for a microfluidic chip according to an embodiment of the present invention.
  • Fig. 5 is a flow chart of the biochemical reaction of hybridization and capture on a digital microfluidic platform according to an embodiment of the present invention.
  • Fig. 6 is a flow chart of the biochemical reaction of multiplex PCR on a digital microfluidic platform according to an embodiment of the present invention.
  • Fig. 7 is a detection result of real-time fluorescent quantitative PCR according to Example 1 of the present invention.
  • Fig. 8 is a graph showing the results of agarose gel electrophoresis detection of reaction products at different surfactant concentrations according to an embodiment of the present invention.
  • the “digital microfluidic platform” mentioned in this article is commonly understood in the art, and is a platform that relies on liquid droplets formed by the surface tension of the liquid and controls the movement of the droplets with the help of electric and/or magnetic fields.
  • a digital microfluidic platform may include a microfluidic chip.
  • it may also contain other operating instruments matched with the microfluidic chip, such as a microfluidic device, as long as it can control the movement of the droplets through an electric field and/or a magnetic field, and realize digital operation.
  • the microfluidic chip and the microfluidic device can be detachably connected, and the microfluidic chip can be connected to the microfluidic device as needed, and then the flow of the microfluidic droplets on the chip can be controlled by the microfluidic device.
  • the digital microfluidic platform can also contain other devices as needed, such as a data processing device, for directly converting the reaction results of the microfluidic chip into electrical signal output, so as to facilitate the control of the reaction process on the microfluidic chip.
  • the provided method for enriching a target region of nucleic acid based on a digital microfluidic platform is based on hybridization capture technology to complete the enrichment of the target region, so as to achieve a low starting amount and a small volume of nucleic acid. Automated enrichment. In at least some embodiments of the present invention, the provided method for enriching a target region of nucleic acid based on a digital microfluidic platform is based on multiplex PCR technology to complete the enrichment of the target region, so as to achieve a low starting amount and a small volume of nucleic acid. Automated enrichment. Prepare small-volume droplets (also called microfluidic droplets) on the microfluidic chip.
  • the microfluidic droplets contain surfactant, low-viscosity silicone oil and nucleic acid, and then use electrowetting to realize the movement and mixing of the microfluidic droplets.
  • the provided microfluidic chip includes a substrate, and an electrode matrix is provided on the substrate.
  • the substrate used can be a PCB substrate.
  • an insulating layer can be provided on the upper surface of the electrode matrix to prevent the droplets from conducting electricity so as to prevent the nucleic acids and proteins in the droplets from being affected by the current.
  • a hydrophobic coating can be provided on the surface of the microfluidic chip to help the formation of the micro-flow droplet.
  • the upper part of the microfluidic chip can be covered with a top layer.
  • hybrid capture technology based on the digital microfluidic platform to enrich nucleic acid target regions or construct sequencing libraries, the principle is to use fragmented nucleic acids to achieve hybridization capture of micro-volume nucleic acid target regions on a microfluidic chip. Including library construction and pre-amplification, denaturation and probe capture, washing and purification after hybridization, post-amplification, concentration detection, single-strand circularization and DNA nanosphere preparation.
  • the principle is to use nucleic acid to achieve the amplification and enrichment of a micro-volume nucleic acid target area on the microfluidic chip, including two Rounds of multiplex PCR enrichment and amplification, magnetic bead purification, concentration detection, single-stranded circularization and DNA nanosphere preparation and other steps.
  • the mentioned nucleic acid may be genomic DNA.
  • the provided method is suitable for multiple sequencing platforms, such as BGI platform, Illumina platform and so on.
  • the provided digital microfluidic platform includes: a microfluidic chip and a microfluidic device.
  • the microfluidic chip and the microfluidic device can be detachably connected.
  • the microfluidic chip can be fitted and connected with the microfluidic device .
  • the microfluidic chip contains an operation area, the operation area is located on the substrate, the operation area includes a sample application area, a storage area, a reaction area and a collection area, the sample application area, the storage area, the reaction area and the The collection areas are connected in sequence.
  • the microfluidic device can be provided with different modules, such as a magnetron module, a temperature control module, a water cooling module, etc., to assist the reactions in various stages completed on the microfluidic chip.
  • the temperature control module is arranged opposite to the reaction zone, and the temperature control module is used to control the temperature of the reaction zone.
  • the magnetron module is arranged directly opposite to the collection area, the magnetron module is provided with a magnet, and the magnetron module controls the lifting of the magnet through a stepping motor.
  • the water-cooling module is arranged directly opposite to the storage area through a metal plate, and the water-cooling module is used to control the temperature of the storage area.
  • the automatic enrichment of nucleic acid target regions or the automatic construction of sequencing libraries can be realized.
  • FIG. 2 shows a partial structure of the digital microfluidic platform
  • FIG. 3 also shows a schematic diagram of the structure of the digital microfluidic platform.
  • the microfluidic device may further contain a fluorescence detection module as required, the fluorescence detection module is connected to the reaction zone, and the fluorescence detection module is used for fluorescence detection of products in the reaction zone.
  • the hybrid capture technology is taken as an example to describe the method of enriching the nucleic acid target region based on the digital microfluidic platform or the method of constructing a sequencing library. This method can realize the automatic enrichment of the target area with low starting amount and small volume of nucleic acid.
  • the digital microfluidic platform is used to complete the automatic hybridization, capture, enrichment and library construction of nucleic acid target regions, which can be used for clinical diagnosis of target genes or nucleic acid target regions, as well as products such as all-exon capture sequencing.
  • the provided enrichment and purification process and library building process can be achieved under a system of less than 16 ⁇ L, and the system contains a certain concentration of surfactant.
  • the whole process can be automated without manual work.
  • the small-volume manual hybridization capture technology outside the chip was first developed. It performed better than the commonly used hybrid capture kits on the market in terms of the initial amount of DNA input, the amount of reagents used, and the time data, and the capture efficiency was equivalent (e.g. Shown in Figure 4).
  • the capture efficiency of this automated target area hybridization capture technology is equivalent to that of the commonly used kits on the market, and the capture uniformity performance is better, and
  • the initial amount of DNA required by this technology is 0.1-0.2 times that of conventional kits, and the amount of reagents required is also about 0.1 times that of conventional kits.
  • the automated library construction process saves labor costs and can reduce the contamination of the sample library by exogenous nucleic acids during the library construction process.
  • the provided capture library process is as follows:
  • the microfluidic chip loaded on the microfluidic device With low-viscosity silicone oil, and then mix the prepared small volume of surfactant-containing reaction reagents and the interrupted low-initiated DNA droplets Add the reagent storage area on the chip (the temperature of the storage area can be controlled by the water cooling module, for example, can be controlled at 4 degrees Celsius), and then click the run button in the program, the reaction reagents can be moved and mixed under the control of the electrode, using the micro The temperature control module and the magnetron module on the bottom of the flow control chip, namely the microfluidic device, realize the reaction temperature control and the magnetic bead purification of the sample.
  • the reagent storage area on the chip the temperature of the storage area can be controlled by the water cooling module, for example, can be controlled at 4 degrees Celsius
  • the reaction reagents can be moved and mixed under the control of the electrode, using the micro The temperature control module and the magnetron module on the bottom of the flow control chip, namely the microfluidic device,
  • the DNA in the droplet on the microfluidic chip first undergoes end repair, adapter connection, magnetic bead purification and pre-PCR amplification steps.
  • the fluorescence in the digital microfluidic device can be used.
  • the quantitative module extracts an appropriate amount of amplified product for the subsequent probe hybridization capture step. After hybridization and capture, magnetic beads with streptavidin are used to specifically bind to the biotin group on the probe, and after multiple rounds of automated washing processes, secondary amplification is performed, and the resulting product is the library after hybridization and capture. After continuing the single-strand circularization of the library and the preparation of DNA nanospheres, it can be taken out or directly connected to a sequencer for sequencing.
  • the above processes are all automated on the chip, and all reaction systems are less than 16 microliters and contain a certain amount of surfactant (as shown in Figure 5).
  • hybrid capture technology to enrich nucleic acid target regions based on digital microfluidic platforms or construct sequencing libraries has many beneficial effects, for example, it can be expressed as follows:
  • the small-volume and low-initiated hybrid capture technology of this example invention can reduce the cost of reagents by about 10 times, and can save precious starting DNA samples.
  • the provided method is a fully automated hybridization capture process. In addition to manual operations for loading reagent samples and removing products, no manual operations are required during the reaction.
  • the instruments and chips used are all portable devices, which can be widely used in clinical genetic diagnosis and rapid detection in hospitals, as well as whole exome sequencing, etc.
  • the initial amount of genomic DNA after the interruption of the commonly used hybrid capture library construction method is generally greater than 50ng.
  • the method provided by the present invention optimizes the biochemical system, and the required starting amount is only 5 ng.
  • the maximum reaction volume of each step in the conventional hybridization capture process is 200uL (washing step) and the minimum is 20uL (hybridization step).
  • the method provided by the present invention optimizes the biochemical system, and the reaction volume of the washing step can be less than 16uL.
  • the volume of the hybridization step is less than 10uL, and the capture efficiency will not be affected or even better.
  • the temperature control on the microfluidic chip is less accurate than the traditional PCR instrument. Therefore, we tested and debugged the temperature tolerance of each step (including hybridization and library building), and finally succeeded with the microfluidic chip. Combine.
  • the following uses multiplex PCR technology as an example to describe the method of enriching nucleic acid target regions or constructing a sequencing library based on a digital microfluidic platform. This method can realize the automatic enrichment of the target area with low starting amount and small volume of nucleic acid.
  • the digital microfluidic platform is used to complete automatic multiple PCR target area enrichment, and the enrichment library construction method can be applied to products such as clinical diagnosis of nucleic acid target areas.
  • Both the enrichment and purification process and the library building process can be realized in a system lower than 16uL, and the system contains a certain concentration of surfactant.
  • the whole process can be automated without manual work.
  • the off-chip small-volume multiplex PCR technology was first developed, which performed better than the commonly used hybrid capture kits on the market in terms of the initial amount of DNA input, the amount of reagents used, and the time, and the enrichment efficiency was comparable.
  • the amplification and library construction process of the method is as follows:
  • the reaction reagents can be moved and mixed under the control of the electrode, and at the same time, the temperature control module and the magnetron module at the bottom of the microfluidic chip can be used to control the reaction temperature and sample Purification of magnetic beads.
  • the DNA in the droplets on the microfluidic chip has passed through the first round of multiplex PCR (containing hundreds of pairs of primers) steps, magnetic bead purification, and the second round of amplification PCR (one pair of universal Primer) and magnetic beads purification steps, the product obtained is the library enriched in the target region after multiplex PCR.
  • the above processes are all automated on the chip, and all reaction systems are less than 16 microliters and contain a certain amount of surfactant (as shown in Figure 6).
  • the small-volume, low-initiating multiplex PCR technology of the present invention can reduce reagent costs by about 10 times, and can save precious starting DNA samples.
  • reaction processes provided by the present invention are all carried out on the chip, which can reduce the contamination of foreign nucleic acid to the library construction process.
  • the instruments and chips used in the present invention are all portable devices, which can be widely used in clinical genetic diagnosis and rapid detection in hospitals.
  • the provided method also solves some technical difficulties that are difficult to solve, which is manifested as:
  • the initial amount of genomic DNA required for conventional multiplex PCR enrichment methods is generally 10-20ng.
  • the method provided above optimizes the biochemical system, and the required starting amount is only 3ng.
  • the PCR amplification of the conventional multiplex PCR enrichment method is generally 50uL.
  • the method provided above can reduce the PCR volume to 6uL through the optimization of the biochemical system. Due to the excessive limitation of the number of primer pairs in multiplex PCR, small size may result in reduced specificity of PCR amplification. However, according to the PCR system design of this example, the specificity of primer amplification is not changed without changing the design of the number of primer pairs for multiplex PCR. There is no significant change and 7 mutation sites of positive BRCA1/2 can be effectively detected.
  • silicone oil generally does not affect biochemical reagents, but the ethanol used for magnetic bead purification will dissolve in silicone oil, so we use 20% polyethylene glycol 8000 and 2.5M sodium chloride solution as its replacement.
  • the PCR additives used are also changed to reagents that are insoluble in silicone oil.
  • the non-ionic surfactant Tween 20 is used as an additive. In the experiment, it was found that more than 1% of Tween 20 would seriously affect the progress of biochemical reactions. After testing, it was found that 0.01% -0.1% of Tween 20 had a low impact on biochemical reactions. To achieve the surface tension of digital microfluidics, we finally selected 0.075% Tween 20 as the surfactant to increase the surface tension of the droplets.
  • the digital microfluidic control has no current in the droplet due to the similar electrostatic shielding effect.
  • the fragment size of the sample DNA and the size of the primer fragment can be increased, and 1% polyethylene glycol is added when preparing the DNA nanospheres. 8000 protects it from directional movement causing droplets to adhere to the surface of silicone oil.
  • Example 1 Low-initiated and small-volume automated hybridization capture target area enrichment technology
  • Example 1 provides a method for hybridizing and capturing two target regions and a non-target region using a microfluidic platform.
  • the two target regions are the exon regions of the RUNX1 gene and the PRKG1 gene, and the non-target region is GAPDH The non-exon region of a gene.
  • the genome was interrupted using an ultrasonic interrupter, and the fragments after the interruption were selected using magnetic beads to obtain a DNA fragment with a main band of 280 bp.
  • step 2 After quantifying the DNA selected in step 1 or the extracted cell-free DNA (cfDNA), take 5 ⁇ L (5ng) droplets into the microfluidic chip, and load all the reaction reagents into the storage area, which is in the entire building. Maintain 4°C during storage.
  • reagents used below were purchased from the commercial kit MGIEAsy exome capture V5 probe reagent kit, manufactured by MGI, and the product number is 1000007746. Including end repair buffer, end repair enzyme mixture, ligase buffer, ligase, polymerase chain reaction mixture, RNA probe, RNase inhibitor and washing buffer, etc. Streptavidin magnetic beads M280 were purchased from Invitrogen, the product number is 00456577. Tween 20 at a final concentration of 0.075% was added to all reagents.
  • the magnetic beads AMPure XP used for magnetic bead purification were purchased from BECKMAN COULTER, the article number is 17775900. Tween 20 with a final concentration of 0.075% was added to the magnetic beads.
  • the washing reagent components used for magnetic bead purification are: final concentration 20% polyethylene glycol 8000, 2.5M sodium chloride solution and 0.075% Tween 20.
  • the amplified product is purified again with the separated magnetic bead reagent, and 3 ⁇ L TE buffer is separated for elution.
  • the eluted product is the pre-capture DNA library.
  • step 3.1 The eluted product obtained in step 3.1 is reacted at 95°C for 5 minutes in the central reaction zone, and then incubated at 65°C. Divide 3 ⁇ L of the probe mixture containing biotin-modified RNA probe and RNase inhibitor into the droplet incubated at 65°C and mix well.
  • step 3.3 Incubate the mixed droplets obtained in step 3.2 at 65°C for 16-24 hours.
  • step 3.3 Divide the product obtained in step 3.3 into 8 ⁇ L binding buffer resuspended M280 magnetic beads modified with streptavidin and mix thoroughly. Incubate for 45 minutes at room temperature in the central reaction zone.
  • the automated program controls the stepping motor to lift the magnet, and the product after the incubation in step 3.4 is used to recover the magnetic beads with a magnet, and the remaining liquid is removed. Separate 10 ⁇ L of washing buffer from the reagent storage to resuspend the magnetic beads (while the stepper motor lowers the magnet), move to the central reaction zone and incubate at room temperature for 30 minutes.
  • the automated program controls the stepper motor, and uses the magnet to recover the magnetic beads after the incubation product in step 3.5, and removes the remaining liquid. Separate 10 ⁇ L of strong washing buffer from the reagent storage to resuspend the magnetic beads, and incubate at 65°C for 10 minutes.
  • step 3.8 The automated program controls the stepper motor, the product after the incubation in step 3.7 is used to recover the magnetic beads with a magnet, the remaining liquid is removed, and 4uL of double distilled water is separated from the reagent storage to resuspend the magnetic beads. Finally, the capture hybrid product is obtained.
  • step 3 Divide the product obtained in step 3 into 6 ⁇ L of polymerase chain reaction mixture, mix thoroughly in the central reaction zone, and perform a PCR amplification reaction with a total volume of 10uL.
  • the amplification program is: 95°C pre-denaturation for 45 seconds; 12 cycles: 98°C denaturation for 15 seconds, 60°C annealing for 30 seconds, 72°C extension for 30 seconds; 72°C final extension for 1 minute.
  • step 4.1 The product obtained in step 4.1 is purified by magnetic beads, and finally eluted with TE buffer to obtain the captured and hybridized DNA library, which can be used for sequencing on the BGI sequencing platform.
  • the library can also be applied to other sequencing platforms such as Illumina for sequencing.
  • the BGI platform is taken as an example
  • the influence of the amount of surfactant Tween 20 on the results was studied, and the amount of surfactant Tween 20 used was adjusted to set the mass concentration of Tween 20 in the microfluidic droplets to 0.01% and 0.75, respectively. %, 0.1%, 0.5% and 1%, the experimental results show that 0.01%-0.1% basically has no effect on the biochemical reaction and can meet the surface tension of the digital microfluidic droplet. When the concentration reaches 1%, the biochemical reaction yield Severe decline or even basically impossible.
  • Tween 20 with a mass concentration of 1% or more will seriously affect the progress of biochemical reactions, while Tween 20 with a mass concentration of 0.01%-0.1% has a low impact on biochemistry and can achieve digital microfluidic control.
  • the surface tension, especially Tween 20 with a mass concentration of 0.075%, can be used as a surfactant to increase the surface tension of the droplets.
  • Embodiment 2 provides a method for multiplex PCR enrichment of a target area using a microfluidic platform, which includes the following steps:
  • microfluidic chip Insert the microfluidic chip into the microfluidic device, and fill the microfluidic chip with silicone oil. Using 1 ⁇ L (3ng) of human genomic DNA as raw material, it was injected into the microfluidic chip, and all reaction reagents were loaded into the storage area, which was maintained at 4°C during the entire library building process.
  • reagents used below are purchased from the commercial kit MGICare BRCA1/2 sequencing library preparation kit, the manufacturer is MGI. Including polymerase chain reaction mixture, multiple PCR primer mixture, universal primer mixture, PCR promoter, single-stranded loop preparation buffer, ligase, digestion mixture, stop buffer, DNB preparation primer, DNB preparation buffer , DNB prepares buffer enzymes and so on. Tween 20 at a final concentration of 0.075% was added to all reagents. The DNB preparation buffer was additionally added with a final concentration of 1% polyethylene glycol 8000.
  • the magnetic beads AMPure XP used for magnetic bead purification were purchased from BECKMAN COULTER, the article number is 17775900. Tween 20 with a final concentration of 0.075% was added to the magnetic beads.
  • the washing reagent components used for magnetic bead purification are: final concentration 20% polyethylene glycol 8000, 2.5M sodium chloride solution and 0.075% Tween 20.
  • the amplification program is: 95°C pre-denaturation for 10 minutes; 5 cycles: 95°C denaturation for 1 minute, 60°C annealing for 15 minutes, 72°C extension for 1 minute; 68°C final extension for 10 minutes.
  • the obtained product is purified using magnetic bead reagents separated by reagent storage, and a stepping motor is used to control the up and down of the magnet to control the adsorption of the magnetic beads.
  • the first round of amplification products are obtained after elution with 1 ⁇ L TE buffer.
  • the amplification procedure is: 95°C pre-denaturation for 10 minutes; 20 cycles: 95°C denaturation for 1 minute, 60°C annealing for 1 minute, 72°C extension for 1 minute; 68°C final extension for 10 minutes.
  • the obtained product is purified using magnetic bead reagents separated from the reagent storage, and a stepping motor is used to control the up and down of the magnet to control the adsorption of the magnetic beads.
  • the second round of amplification products are obtained after elution with 1 ⁇ L TE buffer.
  • step 3.5 Separate the magnetic beads from the storage reagent to purify the product obtained in step 3.4, use a stepper motor to control the up and down of the magnet to control the adsorption of the magnetic beads, and separate 3 ⁇ L TE buffer from the reagent storage to elute the magnetic beads.
  • step 3.6 After separating the 3 ⁇ LDNB preparation primer from the storage, add it to the product obtained in step 3.5 and mix thoroughly, 95°C for 1 min, 65°C for 1 min, and 40°C for 1 min.
  • the obtained product is a DNA nanosphere that can be used for sequencing on the BGI platform, that is, a sequencing library is obtained.
  • a small-volume manual multiple PCR method is used as a control, that is, all the above reactions are completed in a simulation system (centrifuge tube), and the temperature is controlled by a PCR instrument. All reagents are the same, 30-75uL silicone oil is added to the centrifuge tube to seal the droplets (see Figure 4 for the simulation system).
  • the normal volume manual multiple PCR method was used as a control.
  • the normal reaction process was completed in accordance with the instructions of the commercial kit MGICare BRCA1/2 sequencing library preparation kit. All reagents were not treated with Tween 20 and no silicone oil was added (see Figure 4). standard system).
  • the enriched library was sequenced using the BGISeq500 sequencer, and the experimental results are shown in Table 1 below:
  • the genome matching ratio in Table 1 refers to the ratio of the sequenced base sequence in the human reference genome; the capture ratio refers to the ratio of the sequenced base sequence in the two genes of BRCA1/2; average;
  • the relative proportion of sequencing depth is based on the depth measured by manual multiple PCR of normal volume as 1, and the proportion of the other two methods relative to 1. The higher the value, the more data volume. In this case, because it is an enriched library, the required data volume is not high, so the higher the data volume will cause waste); coverage means that the base sequence obtained by sequencing can cover BRCA1/ 2The degree of two gene regions; homogeneity refers to the uniformity of the base sequence obtained by sequencing covering different regions.

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Abstract

一种基于数字微流控平台富集核酸的方法及构建测序文库的方法。数字微流控平台包括微流控芯片和微流控装置，二者可拆卸地连接，基于该平台富集核酸的方法包括：在微流控芯片上制备微流滴，微流滴中含有表面活性剂、硅油和核酸；基于特异性探针对微流滴中所述核酸的目标区域进行特异性扩增，以便扩增产物，所述目标产物构成富集核酸。可以利用该富集产物进行文库的构建和测序。

Description

基于数字微流控平台富集核酸及构建测序文库的方法 技术领域
本发明涉及基因检测领域，具体涉及一种基于数字微流控平台富集核酸及构建测序文库的方法。
背景技术
近年来，二代高通量测序技术(NGS)广泛应用于许多研究及临床领域，NGS能够帮助鉴定遗传改变，包括单核苷酸变异(SNVs)，拷贝数变异(CNVs)以及基因融合等等。相比于常规NGS测序，目标区域富集后测序因其特异性、经济性及扩展性得到更为普遍的应用。为了获得覆盖区域中更高的测序深度并降低测序成本，我们需要设计高效率以及测序高均一度的捕获技术，这也使目标区域富集技术变得更加具有挑战性。
多重PCR和杂交捕获技术是两类广泛应用的目标区域富集技术。多重PCR技术的富集扩增分为两轮PCR进行，第一轮PCR主要是针对目标区域设计上百对PCR引物对所需不同的目标区域进行同时扩增，其第一轮PCR的引物上同时设计有一段相同的序列用于第二轮扩增。第二轮PCR使用同一种引物对第一轮富集到的产物进行扩增，扩增后得到的文库可用于后续测序。第二轮PCR使用的引物有部分序列与第一轮PCR使用的引物互补，同时带有额外的用于测序的序列。杂交捕获技术通过使用带有生物素的探针对含有目标区域的片段进行杂交，再利用修饰有能与生物素特异性结合的磁珠对捕获的探针进行吸附回收，最后利用磁珠纯化的方式洗涤掉多余的非目标区域的片段从而得到所需的目的片段。
然而在利用上述方法对目标区域富集建库时，所需要的起始DNA以及试剂等均是基于全基因组测序的数据而确定的，从而造成了大量数据的浪费，降低了建库过程中起始DNA的使用效率以及试剂的使用效率。因此，针对目标区域的富集技术仍需要进一步改进。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一，提供了一种基于数字微流控平台富集核酸的方法以及构建测序文库的方法，所提供的富集核酸的方法借助于数字微流控平台达到富集核酸的目的，并应用于构建测序文库和进行测序。应用该方法仅需要少量的核酸样本即可以实现核酸样本的富集，获得测序文库，应用于测序领域；而且借助于数字微流控平台可以摆脱手工操作，例如冗长的洗涤步骤，以及手工操作控制到特定的温度等，实现自动化操作。
多重PCR以及杂交捕获技术是两类常用的用于核酸的目标区域富集的技术，其可以用于 核酸的目标区域的富集。这些技术也存在一些缺点，例如其富集过程步骤繁琐，涉及多步复杂的人工操作，人工操作带来很多弊端，不容易控制温度，再例如，洗涤时不稳定，容易导致最终的文库效果差。以杂交捕获技术为例，其除了成本高以外，最关键在于绝大多数杂交捕获步骤无法避免复杂的操作流程，包括在某一温度控制下操作以及耗时费力的多轮洗涤步骤。
本发明提供了一种基于数字微流控平台富集核酸的方法以及构建测序文库的方法，还方法借助于数字微流控平台实现核酸的富集，其利用少量核酸样本就可以完成富集过程，而且可以不依托于人工操作，方便对于温度的控制，而且由于反应体系缩小，使得反应时间缩短，从而可以实现快速低成本自动化核酸分子目标区域的富集。同时，由于借助于数字微流控平台进行操作，能够保证各批次操作的稳定性。
具体而言，本发明提供了如下技术方案：
在本发明的第一方面，本发明提供了一种基于数字微流控平台富集核酸的方法，所述数字微流控平台包括微流控芯片和微流控装置，所述微流控芯片和所述微流控装置可拆卸地连接，所述方法包括：在微流控芯片上制备微流滴，所述微流滴含有表面活性剂、硅油和核酸；基于特异性探针或特异性引物对所述微流滴中所述核酸的目标区域进行特异性捕获或扩增，获得富集核酸产物。
利用本发明所提供的基于数字微流控平台富集核酸的方法对核酸的目标区域进行富集，解决了在目标区域富集实验过程中操作复杂的问题，不需要人工的手工操作，包括冗长的洗涤步骤，以及在特定温度下的手工操作。由此可以减少实验操作部分的时间以及引起的失误。而且所需DNA的投入量相比其他技术更少，并且减少了生化反应所需试剂的成本。
根据本发明的实施例，以上所述基于数字微流控平台富集核酸的方法可以进一步包括如下技术特征：
在本发明的一些实施例中，所述硅油为低粘度硅油。低粘度硅油的在电场控制液滴移动的时候更加容易。一般来说，10cps以下的硅油为低粘度硅油。
在本发明的一些实施例中，进一步包括利用所述微流控装置对所述富集核酸产物进行纯化的步骤。
在本发明的一些实施例中，所述微流滴的体积不超过16微升。
在本发明的一些实施例中，所述表面活性剂在所述微流滴中的质量浓度不超过0.5％，优选为0.01％～0.1％，更优先为0.075％。表面活性剂的含量过高时，会严重影响生化反应的进行，例如质量浓度为1％以上的吐温20会严重影响生化反应的进行，0.01％-0.1％的吐温20对生化造成的影响较低且能够达到数字微流控的表面张力，而质量浓度为0.075％吐温20可以显著增加液滴表面张力的表面活性剂。
在本发明的一些实施例中，所述表面活性剂为吐温20。
在本发明的一些实施例中，所述微流控芯片进一步包括：
基板，所述基板上设置有电极矩阵；
操作区，所述操作区位于所述基板上，所述操作区包括加样区、存储区、反应区和收集区，所述加样区、所述存储区、所述反应区和所述收集区依次相连。
通过基板中的电极矩阵可以控制液滴的流动，例如可以通过电极的开关控制不同微流滴或者不同大小的微流滴的移动，从而可以实现对微流滴的不同操作和处理，且无须人工操作。
在本发明的一些实施例中，所述微流控装置进一步包括：温度控制模块，所述温度控制模块与所述反应区正对设置，所述温度控制模块用于控制所述反应区的温度。
在本发明的一些实施例中，所述微流控装置进一步包括：磁控模块，所述磁控模块与所述收集区正对设置，所述磁控模块中设置有磁铁，所述磁控模块通过步进电机控制所述磁铁的升降。磁控模块中设置有磁铁，可以在磁铁的下部装有步进电机，然后通过步进电机控制磁铁的上下移动。例如，当磁铁处于上方时，即靠近基板时，可以用来吸取磁珠；当磁铁处于下方时，即远离基板时，可以用来释放磁珠，从而达到对试剂进行纯化的目的。
在本发明的一些实施例中，所述微流控装置进一步包括水冷模块，所述水冷模块通过金属板与所述存储区正对设置，所述水冷模块用于控制所述存储区温度。水冷模块与存储区正对设置，水冷模块可以一直处于开启的状态，用来保护存储区的试剂不会失活。根据本发明的实施例，所述金属板为帕尔贴板。
在本发明的一些实施例中，所述低粘度硅油的粘度可以为5以下，例如可以为4.6
在本发明的一些实施例中，所述微流控装置进一步包括：荧光检测模块，所述荧光检测模块与所述反应区相连，所述荧光检测模块用于对所述反应区产物进行荧光检测。由于基板上设置有电极矩阵，可以通过控制电极矩阵的开关，将反应后的大液滴一分为二，或者一分为三等等，分出的液滴可以用来进行荧光检测，而且不需要手动操作即可以实现荧光检测。
在本发明的一些实施例中，所述数字微流控平台进一步包括：数据处理装置，所述数据处理装置用于将所述微流控芯片的反应结果转换为电信号输出。根据本发明的实施例，所述数据处理装置上还设置有机械手臂，所述机械手臂用于实现所述微流控芯片的自动化操作。
在本发明的一些实施例中，所述核酸为基因组DNA，所述基因组DNA为5纳克以上。
在本发明的第二方面，本发明提供了一种构建测序文库的方法，包括：基于本发明第一方面任一实施例所述的方法获得核酸的目标区域富集产物；基于所述核酸的目标区域富 集产物，建库，以便获得测序文库。
根据本发明的实施例，以上构建测序文库的方法可以进一步包括如下技术特征：
在本发明的一些实施例中，进一步包括：在微流控芯片上制备微流滴后，进行末端修复、接头连接，所获得的连接产物进行第一PCR扩增，以便构建得到捕获前DNA文库；利用特异性探针对所述捕获前DNA文库进行杂交捕获和纯化，以便获得捕获杂交产物；
基于所述捕获杂交产物进行第二PCR扩增，以便构建得到捕获后DNA文库。
在本发明的一些实施例中，所述特异性探针为生物素标记的特异性探针，利用链霉亲和素标记的磁珠进行所述纯化。
在本发明的一些实施例中，进一步包括：在微流控芯片上制备微流滴后，利用第一扩增引物进行扩增，以便获得第一扩增产物，所述第一扩增引物适于对所述核酸的不同区域进行扩增；利用通用引物对所述第一扩增产物进行扩增，以便获得第二扩增产物，所述通用引物与所述第一扩增引物部分互补。
在本发明的一些实施例中，进一步包括：将所述第二扩增产物进行热变性，以便获得单链环状DNA；基于所述单链环状DNA进行滚环扩增，以便获得DNA纳米球。
在本发明的一些实施例中，在利用第一扩增引物扩增之后或者在进行利用通用引物对所述第一扩增产物进行扩增之后，分别利用磁珠进行纯化。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1是根据本发明的实施例提供的微流控芯片的横截面示意图。
图2是根据本发明的实施例提供的数字微流控平台的部分结构示意图。
图3是根据本发明的实施例提供的数字微流控平台的结构示意图。
图4是根据本发明的实施例提供的通过芯片外条件摸索，并调整使得适合于微流控芯片的方法示意图。
图5是根据本发明的实施例提供的杂交捕获在数字微流控平台上的生化反应流程图。
图6是根据本发明的实施例提供的多重PCR在数字微流控平台上的生化反应流程图。
图7是根据本发明的实施例1提供的实时荧光定量PCR的检测结果。
图8是根据本发明的实施例提供的不同表面活性剂浓度下反应产物的琼脂糖凝胶电泳检测结果图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
本文中所提到的“数字微流控平台”作本领域通常理解，是依靠于液体表面张力形成的液滴，借助于电场和/或磁场，控制液滴的移动的平台。这种数字微流控平台通过可以包括微流控芯片。另外还可以含有与微流控芯片搭配的其他操作仪器，例如微流控装置，只要能够通过电场和/或磁场，控制液滴移动，实现数字化操作即可。微流控芯片和微流控装置可以可拆卸地连接，可以根据需要将微流控芯片搭接到微流控装置中，然后通过微流控装置控制芯片上微流滴的流动。数字微流控平台还可以根据需要含有其他的装置，例如数据处理装置，用于微流控芯片的反应结果直接转换为电信号输出，从而便于控制微流控芯片上反应的进程。
在本发明的至少一些实施方式中，所提供的基于数字微流控平台富集核酸目标区域的方法，基于杂交捕获技术，来完成目标区域的富集，从而实现低起始量小体积核酸的自动化富集。在本发明的至少一些实施方式中，所提供的基于数字微流控平台富集核酸目标区域的方法，基于多重PCR技术，来完成目标区域的富集，从而实现低起始量小体积核酸的自动化富集。在微流控芯片上制备小体积液滴(也称为微流滴)，微流滴中含有表面活性剂、低粘度硅油和核酸，然后利用电浸润的方法实现微流滴的移动和混合(如图1所示)。如图1所示，所提供的微流控芯片包括基板，基板上设置有电极矩阵。所用到的基板可以为PCB基板。同时在电极矩阵的上表面还可以设置一层绝缘层，防止液滴导电以免液滴中的核酸和蛋白等受到电流影响。另外，为了方便形成微流滴，增大微流滴表面的表面张力，可以在微流控芯片的表面设置疏水涂层，从而帮助微流滴成形。微流控芯片上部可以加盖顶层。
以杂交捕获技术为例，基于数字微流控平台富集核酸目标区域或者构建测序文库，其原理是利用片段化的核酸在微流控芯片上实现微量体积的核酸的目标区域的杂交捕获，其中包括文库构建及前期扩增，变性及探针捕获，杂交后洗涤纯化，后期扩增，浓度检测，单链环化及DNA纳米球制备等步骤。
以多重PCR技术为例，基于数字微流控平台富集核酸目标区域或者构建测序文库，其原理是利用核酸在微流控芯片上实现微量体积的核酸目标区域的扩增富集，其中包括两轮多重PCR富集扩增，磁珠纯化，浓度检测，单链环化及DNA纳米球制备等步骤。
所提到的核酸可以为基因组DNA。所提供的方法适合于多个测序平台，例如BGI平台，illumina平台等。
所提供的数字微流控平台包括：微流控芯片和微流控装置，微流控芯片和微流控装置可以可拆卸地进行连接，例如微流控芯片可以和微流控装置嵌合连接，微流控芯片上含有操 作区，操作区位于基板上，操作区包括加样区、存储区、反应区和收集区，所述加样区、所述存储区、所述反应区和所述收集区依次相连。相应地，微流控装置中可以设置有不同的模块，例如磁控模块，温度控制模块，水冷模块等，用于辅助微流控芯片上所完成的各个不同阶段的反应。根据本发明的实施例，所述温度控制模块与所述反应区正对设置，所述温度控制模块用于控制所述反应区的温度。根据本发明的实施例，所述磁控模块与所述收集区正对设置，所述磁控模块中设置有磁铁，所述磁控模块通过步进电机控制所述磁铁的升降。根据本发明的实施例，所述水冷模块通过金属板与所述存储区正对设置，所述水冷模块用于控制存储区温度。由此可以实现核酸目标区域的自动化富集或者测序文库的自动化构建。为了方便理解，在图2中示出了数字微流控平台的部分结构，图3中也示出了数字微流控平台的结构示意图。另外，微流控装置还可以根据需要进一步含有荧光检测模块，所述荧光检测模块与所述反应区相连，所述荧光检测模块用于对所述反应区产物进行荧光检测。
下面以杂交捕获技术为例，对基于数字微流控平台的核酸目标区域富集的方法或者构建测序文库的方法进行说明。通过该方法可以实现低起始量小体积核酸的目标区域的自动化富集。
利用数字微流控平台完成核酸目标区域的自动化杂交捕获富集以及建库，可以用于目标基因或者核酸目标区域的临床诊断以及全外显子捕获测序等产品。所提供的富集纯化过程及建库过程均可在低于16μL的体系下实现，并且该体系中含有一定浓度的表面活性剂。整个过程可以在没有手工的情况下自动化实现。研究过程中，首先开发了芯片外小体积手工杂交捕获技术，其在DNA投入起始量、试剂使用量以及时间上数据表现均优于市面上常用的杂交捕获试剂盒，且捕获效率相当(如图4所示)。之后，我们将该技术应用于数字微流控平台实现自动化后，经试验证明，该自动化目标区域杂交捕获技术的捕获效率与现市面常用试剂盒的捕获效率相当且捕获均一度表现较好，并且该技术所需DNA的起始量是常规试剂盒的0.1-0.2倍，所需试剂用量也均为常规试剂盒的0.1倍左右。除此之外，该自动化建库流程节省了人力成本，并能够减少建库过程中外源核酸对样本文库的污染。在本发明的至少一些实施方式中，所提供的捕获建库过程如下：
首先，向装载在微流控装置上的微流控芯片中注满低粘度硅油，之后将配置好的小体积含表面活性剂的反应试剂及打断后的低起始量的DNA小液滴加入芯片上的试剂储存区(存储区的温度可以通过水冷模块进行控制，例如可以控制在4摄氏度)，之后点击程序中的运行按钮，反应试剂便能够在电极的控制下移动和混合，利用微流控芯片底部即微流控装置上的温度控制模块和磁控模块实现反应控温和样品的磁珠纯化。微流控芯片上的液滴 中的DNA在反应试剂和温度的帮助下首先经过了末端修复，接头连接，磁珠纯化及前期PCR扩增步骤，另外还可以利用数字微流控装置中的荧光定量模块抽取适量扩增产物进行后续的探针杂交捕获步骤。杂交捕获后使用带链霉亲和素磁珠与探针上的生物素基团特异性结合，经过多轮自动化洗涤过程后进行二次扩增，所得的产物即为杂交捕获后的文库。继续将该文库进行单链环化及DNA纳米球制备后即可取出或直接连接测序仪进行测序。上述过程均在芯片上自动化进行，所有反应体系均小于16微升并含有一定量的表面活性剂(如图5所示)。
应用杂交捕获技术，对基于数字微流控平台的核酸目标区域进行富集或者构建测序文库的，其具有诸多有益效果，例如可以表现为如下：
1、相比于市面上常用的杂交捕获产品，此示例发明的小体积低起始量杂交捕获技术可实现降低10倍左右的试剂成本，并能够节约珍贵的起始DNA样本。
2、所提供的方法是全自动化杂交捕获流程，除了加载试剂样本及取出产物需要手工操作外，反应过程中不需要任何手工操作。
3、所有的反应过程均在微流控芯片上进行，可降低外源核酸对建库过程中的污染。
4、所用到的仪器及芯片皆为便携式设备，可广泛应用于医院的临床基因诊断及快速检测，以及全外显子组测序等。
该方法同时解决了一些比较难解决的技术难点，例如：
1、DNA样本的不完整和低起始往往会导致杂交捕获文库的风险建库或建库失败，现阶段的杂交捕获常用建库方法的打断后基因组DNA起始量一般大于50ng。本发明所提供的方法通过对生化体系进行优化，所需起始量仅需5ng。
2、常规的杂交捕获过程中的各步骤的反应体积最大有200uL(洗涤步骤)，最小为20uL(杂交步骤)，本发明所提供的方法通过对生化体系的优化，洗涤步骤反应体积可小于16uL,杂交步骤体积小于10uL，并且捕获效率不会发生影响甚至更优。
3、微流控芯片上的温度控制相较于传统PCR仪精度会有所不够，因此我们对各步骤(包括杂交与建库)的温度耐性进行了测试与调试，最终与微流控芯片成功结合。
下面以多重PCR技术为例，对基于数字微流控平台进行核酸目标区域的富集或者构建测序文库的方法进行说明。通过该方法可以实现低起始量小体积核酸的目标区域的自动化富集。
利用数字微流控平台完成自动化多重PCR目标区域富集，该富集建库方法可以应用于核酸目标区域的临床诊断等产品。该富集纯化过程及建库过程均可在低于16uL的体系下实现，并且该体系中含有一定浓度的表面活性剂。整个过程可以在没有手工的情况下自动化 实现。研究过程中，首先开发了芯片外小体积多重PCR技术，其在DNA投入起始量、试剂使用量以及时间上数据表现均优于市面上常用的杂交捕获试剂盒，且富集效率相当。之后，将该技术应用于数字化液滴微流控平台实现自动化后，经试验证明，该多重PCR富集效率与现市面常用试剂盒的富集效率相当，且能够成功检测出阳性样品(BRCA1/2突变)中待检测基因位点，并且该技术所需DNA的起始量是常规试剂盒的0.1-0.2倍，所需试剂用量也均为常规试剂盒的0.1倍左右。除此之外，该自动化建库流程节省了人力成本，并能够减少富集及建库过程中外源核酸对样本文库的污染。在本发明的至少一些实施方式中，该方法的扩增建库过程如下：
首先，向装载在微流控装置上的微流控芯片中注满低粘度硅油，之后将配置好的小体积含表面活性剂的反应试剂及低起始量的基因组DNA小液滴加入微流控芯片上的存储区，之后点击程序中的运行按钮，反应试剂便能够在电极的控制下移动和混合，同时可以利用微流控芯片底部的温度控制模块和磁控模块实现反应控温和样品的磁珠纯化。微流控芯片上的液滴中的DNA在反应试剂和温度的帮助下先后经过了第一轮多重PCR(含有上百对引物)步骤，磁珠纯化，第二轮扩增PCR(一对通用引物)和磁珠纯化步骤，所得的产物即为多重PCR后目标区域富集的文库。另外，还可以利用数字微流控平台上的荧光定量的模块抽取适量文库可进行后续的单链环化及DNA纳米球制备后，即可取出或直接连接测序仪进行测序。上述过程均在芯片上自动化进行，所有反应体系均小于16微升并含有一定量的表面活性剂(如图6所示)。
该方法具有诸多优势，具体可以表现为：
1、相比于市面上常用的多重PCR目标区域富集产品，本发明的小体积低起始量多重PCR技术可实现降低10倍左右的试剂成本，并能够节约珍贵的起始DNA样本。
2、本发明所提供的的全自动化多重PCR流程，除了加载试剂样本及取出产物需要手工操作外，反应过程中不需要任何手工操作。
3、本发明所提供的反应过程均在芯片上进行，可降低外源核酸对建库过程中的污染。
4、本发明所用仪器及芯片皆为便携式设备，可广泛应用于医院的临床基因诊断及快速检测等。
所提供的方法同时解决了一些难以解决的技术难点，表现为：
1、常规多重PCR富集方法所需的基因组DNA起始量一般为10-20ng。上述所提供的方法通过对生化体系进行优化，所需起始量仅需3ng。
2、常规的多重PCR富集方法的PCR扩增一般为50uL，上述所提供的方法通过对生化体系的优化，PCR体积可降至6uL。由于多重PCR的引物对数过多限制，小体积可能会导致PCR扩增特异性降低，但根据此示例PCR体系设计，在不改变多重PCR引物对数设计的前提下， 引物扩增的特异性没有显著性改变并且能够有效检测出阳性BRCA1/2的7个突变位点。
另外，无论是基于数字微流控平台实现自动化多重PCR过程，还是实现杂交捕获的过程，其均需要在硅油中进行生化反应，并且要通过表面活性剂增加液滴的表面张力，并且不能忽略微弱电流的存在。因此通过生化调试及芯片测试解决了硅油、表面活性剂及电流对多重PCR步骤或者杂交捕获过程的影响。
测试发现硅油一般不会对生化试剂发生影响，但是磁珠纯化使用的乙醇会溶于硅油，所以我们使用20％聚乙二醇8000和2.5M氯化钠溶液作为其代替。此外，在多重PCR实验中，使用的PCR添加剂也改为不溶于硅油的试剂。
选用非离子表面活性剂吐温20作为添加剂，实验中发现1％以上的吐温20会严重影响生化反应的进行，经过测试发现0.01％-0.1％的吐温20对生化造成的影响较低且能够达到数字微流控的表面张力，最终我们选取0.075％吐温20作为增加液滴表面张力的表面活性剂。
另外，一般认为数字微流控由于类似静电屏蔽效应，液滴中不存在电流。但为了确保液滴移动时的微弱电流不会影响带电荷的DNA或蛋白质向表面移动，可以增加样品DNA的片段大小和引物片段大小，并在制备DNA纳米球的时候添加1％聚乙二醇8000保护其不会定向移动造成液滴附着在硅油表面。
下面将结合实施例对本发明的示例进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1 低起始量小体积自动化杂交捕获目标区域富集技术
实施例1提供了一种利用微流控平台对两个目标区域以及一个非目标区域进行杂交捕获的方法，两个目标区域分别为RUNX1基因以及PRKG1基因的外显子区域，非目标区域为GAPDH基因的非外显子区域。
包括如下步骤：
1、基因组DNA片段的制备方法：
以50ng人源基因组DNA为原料，使用超声打断仪对基因组进行打断，使用磁珠对打断后的片段进行片段选择，得到主带在280bp的DNA片段。
2、捕获前DNA文库构建方法：
2.1将微流控芯片插入微流控装置中，用硅油将芯片灌满。
2.2将步骤1片段选择后的DNA或提取的细胞游离DNA(cfDNA)定量后取5μL(5ng)液滴加入微流控芯片中，并将所有反应试剂加载到存储区，该存储区在整个建库过程中维 持4℃。
以下所用到的所有试剂均购自于商用试剂盒MGIEasy外显子组捕获V5探针试剂套装，厂家为华大智造，货号为1000007746。包括末端修复缓冲液，末端修复酶混合液，连接酶缓冲液，连接酶，聚合酶链式反应混合液，RNA探针，RNA酶抑制剂和洗涤缓冲液等。链霉亲和素磁珠M280购于Invitrogen，货号为00456577。所有试剂中加入了终浓度为0.075％的吐温20。
用于磁珠纯化的磁珠AMPure XP购于BECKMAN COULTER,货号为17775900。磁珠中加入了终浓度为0.075％的吐温20。
用于磁珠纯化的洗涤试剂成分为：终浓度20％聚乙二醇8000，2.5M氯化钠溶液以及0.075％吐温20。
2.3以下步骤均为程序控制电极开关从而控制液滴移动：
从存储区分出2μL末端修复缓冲液和1μL末端修复酶混合液，与DNA(cfDNA)液滴充分混匀后37℃反应30分钟，之后65℃反应15分钟。
2.4从存储区分出3μL含有接头的连接缓冲液和1μL连接酶加入步骤2.3得到的产物中，充分混匀后25℃孵育30分钟。
2.5从存储区分出磁珠试剂对步骤2.4得到的产物进行纯化，通过利用步进电机控制磁铁升降，完成磁珠的吸附、洗涤、释放和洗脱。从试剂储存区分出4uL的聚合酶链式反应混合液加入洗脱后得到的产物，对其进行总体积为6μL的聚合酶链式反应(PCR)扩增：98℃预变性3分钟；12个循环：98℃变性20秒，60℃退火15秒，72℃延伸30秒；72℃终延伸10分钟。该扩增反应同样在芯片的中央反应区进行。
扩增后的产物再次利用分出的磁珠试剂对其进行纯化，分出3μL TE缓冲液洗脱，洗脱后得到的产物即为捕获前DNA文库。
3、捕获杂交方法：
3.1从试剂储存区分出3μL COT-1DNA和3μL鲑鱼精DNA加入步骤2得到的产物充分混合后，分出磁珠试剂对其进行浓缩，之后分出含有DNA阻断核苷酸链的杂交缓冲液6uL对磁珠进行洗脱。
3.2将步骤3.1得到的洗脱产物于中央反应区95℃反应5分钟后65℃持续孵育。向65℃孵育的液滴中分入3μL含有生物素修饰的RNA探针和RNA酶抑制剂的探针混合液并充分混匀。
3.3将步骤3.2得到的混合液滴在65℃孵育16-24小时。
3.4将步骤3.3得到的产物分入8μL结合缓冲液重悬的修饰有链霉亲和素的M280磁珠并充分混匀。在中央反应区室温孵育45分钟。
3.5自动化程序控制步进电机抬升磁铁，将步骤3.4孵育后的产物使用磁铁对磁珠进行回收，去除其余液体。从试剂储存区分出10μL洗涤缓冲液重悬磁珠(同时步进电机降下磁铁)，移动至中央反应区室温孵育30分钟。
3.6自动化程序控制步进电机，将步骤3.5孵育后的产物使用磁铁对磁珠进行回收，去除其余液体。从试剂储存区分出10μL强力洗涤缓冲液重悬磁珠，65℃孵育10分钟。
3.7重复步骤3.6两次。
3.8自动化程序控制步进电机，将步骤3.7孵育后的产物使用磁铁对磁珠进行回收，去除其余液体，从试剂储存区分出4uL双蒸水重悬磁珠。最终得到捕获杂交产物。
4、捕获后文库构建方法：
4.1将步骤3得到的产物中分入6μL聚合酶链式反应混合液，在中央反应区充分混匀后进行总体积为10uL的PCR扩增反应。扩增程序为：95℃预变性45秒；12个循环：98℃变性15秒，60℃退火30秒，72℃延伸30秒；72℃终延伸1分钟。
4.2将步骤4.1得到的产物进行磁珠纯化，最终使用TE缓冲液洗脱后得到捕获杂交后的DNA文库，可用于BGI测序平台进行测序。(根据试剂盒和引物使用不同，该文库也可适用于其他测序平台如illumina进行测序，此处以BGI平台为例)
同时以不借助于微流控芯片进行富集作为对照实验(小体积手工操作富集)，即以上所有反应均在模拟体系(离心管)中完成，由PCR仪控制温度。所有试剂均保持一致，离心管中加入了30-75uL的硅油封住液滴(见图4模拟体系)。
实验结果如下：
使用实时荧光PCR对富集结果进行定量测定(包含两个目标区域和一个非目标区域)，所得结果如图7所示。图7中，未富集代表未作捕获杂交的文库，投入定量代表将三种类型的文库按照同一起始量进行qPCR鉴定。
从图7可以看出，与未富集文库相比，小体积手工操作富集文库和芯片自动化文库在两个目标区域均得到了很好的富集(qPCR曲线起峰较早，提前约3-4个循环)，在非目标区域没有得到富集甚至丢失(qPCR曲线起峰较晚)。这说明该生化方法能够成功通过杂交捕获的方法富集目标区域，而芯片自动化文库又因其自动化无需人工操作所以优势更加明显。
此外，正常反应流程按照商用试剂盒MGIEasy外显子组捕获V5探针试剂套装的说明书完成，所有试剂未作添加吐温20处理且不加硅油(见图4标准体系)。其结果与小体积手工操作一致(曲线重合)，因此未在实验结果中展示。
同时，研究了表面活性剂吐温20添加量对于结果的影响，调整所用到的表面活性剂吐 温20的添加量，分别设置为吐温20在微流滴中的质量浓度为0.01％、0.75％、0.1％、0.5％和1％，实验结果表明0.01％-0.1％基本对生化反应不会造成影响且能够满足数字微流控液滴的表面张力，当浓度到达1％时，生化反应产量严重下降甚至基本无法进行。
实验结果请见图7所示。
由此说明，发现质量浓度在1％以上的吐温20会严重影响生化反应的进行，而质量浓度为0.01％-0.1％的吐温20对生化造成的影响较低且能够达到数字微流控的表面张力，尤其是质量浓度为0.075％的吐温20可以作为增加液滴表面张力的表面活性剂。
实施例2 低起始量小体积自动化多重PCR目标区域富集技术
实施例2提供了一种采用微流控平台对目标区域进行多重PCR富集的方法，包括如下步骤：
将微流控芯片插入微流控装置中，用硅油将微流控芯片灌满。以1μL(3ng)人源基因组DNA为原料，将其注入微流控芯片中，并将所有反应试剂加载到存储区，该存储区域在整个建库过程中维持4℃。
以下所用到的所有试剂均购自于商用试剂盒MGICare BRCA1/2测序文库制备试剂盒，厂家为华大智造。包括聚合酶链式反应混合液，多重PCR引物混合液，通用引物混合液，PCR促进剂，单链环制备缓冲液，连接酶，消化混合液，终止缓冲液，DNB制备引物，DNB制备缓冲液，DNB制备缓冲酶等。所有试剂中加入了终浓度为0.075％的吐温20。其中DNB制备缓冲液中额外添加了终浓度1％聚乙二醇8000。
用于磁珠纯化的磁珠AMPure XP购于BECKMAN COULTER,货号为17775900。磁珠中加入了终浓度为0.075％的吐温20。
用于磁珠纯化的洗涤试剂成分为：终浓度20％聚乙二醇8000，2.5M氯化钠溶液以及0.075％吐温20。
以下步骤均为程序控制电极开关从而控制液滴移动：
1、第一轮PCR扩增及扩增后纯化方法
从试剂储存区分出1μL(3ng)人源基因组DNA溶液，3μL聚合酶链式反应混合液，1μL多重PCR引物混合液和1μLPCR促进剂移动至中央反应区并充分混匀，然后进行扩增。
扩增程序为：95℃预变性10分钟；5个循环：95℃变性1分钟，60℃退火15分钟，72℃延伸1分钟；68℃终延伸10分钟。所得产物使用试剂储存区分出的磁珠试剂进行纯化，利用步进电机控制磁铁升降从而控制磁珠的吸附，最终利用1μL TE缓冲液洗脱后得到第一轮扩增产物。
2、第二轮PCR扩增及扩增后纯化方法
从试剂储存区分出3μL聚合酶链式反应混合液，1μL通用引物混合液和1μLPCR促进剂加入步骤1所得产物中并充分混匀，然后进行扩增。
扩增程序为：95℃预变性10分钟；20个循环：95℃变性1分钟，60℃退火1分钟，72℃延伸1分钟；68℃终延伸10分钟。所得产物使用试剂储存区分出的磁珠试剂进行纯化，利用步进电机控制磁铁升降从而控制磁珠的吸附，最终利用1μL TE缓冲液洗脱后得到第二轮扩增产物。
3、DNA纳米球的制备
3.1利用电极控制步骤2中得到的产物移动到中央反应区进行95℃变性后冷却至室温。
3.2从存储区分出3μL单链环制备缓冲液和1μL连接酶缓冲液加入步骤3.1中得到的产物中并充分混匀后，37℃孵育30分钟。
3.3从存储区分出3μL消化混合液加入步骤3.2中得到的产物中并充分混匀，37℃孵育30分钟。
3.4从存储区分出1μL终止缓冲液加入步骤3.3中得到的产物中并充分混匀。
3.5从存储区分出磁珠试剂对步骤3.4中得到的产物进行纯化，利用步进电机控制磁铁升降从而控制磁珠的吸附，从试剂储存区分出3μL TE缓冲液洗脱磁珠。
3.6从存储区分出3μLDNB制备引物加入步骤3.5中得到的产物中并充分混合后，95℃1min，65℃ 1min，40℃ 1min。
3.7从试剂储存区分出6μL DNB制备混合液和1μL DNB制备聚合酶加入步骤3.6中得到的产物中后充分混匀。30℃孵育20分钟。
3.8从试剂储存区分出3μL终止缓冲液加入步骤3.7中得到的产物中并充分混匀。所得产物即为可用于BGI平台测序的DNA纳米球，即获得测序文库。
同时，以小体积手工操作多重PCR方法作为对照，即以上所有反应均在模拟体系(离心管)中完成，由PCR仪控制温度。所有试剂均保持一致，离心管中加入了30-75uL的硅油封住液滴(见图4模拟体系)。
以正常体积手工操作多重PCR的方法作为对照，正常反应流程按照商用试剂盒MGICare BRCA1/2测序文库制备试剂盒的说明书完成，所有试剂未作添加吐温20处理，且不加硅油(见图4标准体系)。
利用BGISeq500测序仪对富集后的文库进行测序，实验结果如下表1所示：
表1 测序数据分析结果
Figure PCTCN2020090796-appb-000001
其中表1中基因组匹配比例指的是测序所得的碱基序列在人类参考基因组上所占的比例；捕获比例是指测序所得的碱基序列在BRCA1/2两个基因上的所占比例；平均测序深度相对占比是以正常体积手工操作多重PCR所测得的深度为1，其他两种方法相对1的占比(平均测序深度指的是碱基序列在基因组各区域的平均覆盖次数，深度越高说明数据量越多。本案例中由于是富集文库，所需数据量要求不高，所以数据量越高将会造成浪费)；覆盖度是指测序所得的碱基序列能够覆盖BRCA1/2两个基因区域的程度；均一度是指测序所得的碱基序列覆盖不同区域的均一情况。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (20)

  1. 一种基于数字微流控平台富集核酸的方法，其特征在于，所述数字微流控平台包括微流控芯片和微流控装置，所述微流控芯片和所述微流控装置可拆卸地连接，
    所述方法包括：
    在微流控芯片上制备微流滴，所述微流滴含有表面活性剂、硅油和核酸；
    基于特异性探针或特异性引物对所述微流滴中所述核酸的目标区域进行特异性捕获或扩增，获得富集核酸产物。
  2. 根据权利要求1所述的方法，其特征在于，所述硅油为低粘度硅油。
  3. 根据权利要求1所述的方法，其特征在于，进一步包括利用所述微流控装置对所述富集核酸产物进行纯化的步骤。
  4. 根据权利要求1所述的方法，其特征在于，所述微流滴的体积不超过16微升。
  5. 根据权利要求1所述的方法，其特征在于，所述表面活性剂在所述微流滴中的质量浓度不超过0.5％，优选为0.01％～0.1％，更优选为0.075％。
  6. 根据权利要求1所述的方法，其特征在于，所述表面活性剂为吐温20。
  7. 根据权利要求1所述的方法，其特征在于，所述微流控芯片进一步包括：
    基板，所述基板上设置有电极矩阵；
    操作区，所述操作区位于所述基板上，所述操作区包括加样区、存储区、反应区和收集区，所述加样区、所述存储区、所述反应区和所述收集区依次相连。
  8. 根据权利要求1所述的方法，其特征在于，所述微流控装置进一步包括：
    温度控制模块，所述温度控制模块与所述反应区正对设置，所述温度控制模块用于控制所述反应区的温度。
  9. 根据权利要求1所述的方法，其特征在于，所述微流控装置进一步包括：
    磁控模块，所述磁控模块与所述收集区正对设置，所述磁控模块中设置有磁铁，所述磁控模块通过步进电机控制所述磁铁的升降。
  10. 根据权利要求1所述的方法，其特征在于，所述微流控装置进一步包括：
    水冷模块，所述水冷模块通过金属板与所述存储区正对设置，所述水冷模块用于控制所述存储区温度。
  11. 根据权利要求1所述的方法，其特征在于，所述金属板为帕尔贴板。
  12. 根据权利要求1所述的方法，其特征在于，所述微流控装置进一步包括：
    荧光检测模块，所述荧光检测模块与所述反应区相连，所述荧光检测模块用于对所述反应区产物进行荧光检测。
  13. 根据权利要求1所述的方法，其特征在于，所述数字微流控平台进一步包括：
    数据处理装置，所述数据处理装置用于将所述微流控芯片的反应结果转换为电信号输出；
    任选地，所述数据处理装置上还设置有机械手臂，所述机械手臂用于实现所述微流控芯片的自动化操作。
  14. 根据权利要求1所述的方法，其特征在于，所述核酸为基因组DNA，所述基因组DNA为5纳克以上。
  15. 一种构建测序文库的方法，其特征在于，包括：
    基于权利要求1～14中任一项所述的方法获得核酸的目标区域富集产物；
    基于所述核酸的目标区域富集产物，建库，以便获得测序文库。
  16. 根据权利要求15所述的方法，其特征在于，进一步包括：
    在微流控芯片上制备微流滴后，进行末端修复、接头连接，所获得的连接产物进行第一PCR扩增，以便构建得到捕获前DNA文库；
    利用特异性探针对所述捕获前DNA文库进行杂交捕获和纯化，以便获得捕获杂交产物；
    基于所述捕获杂交产物进行第二PCR扩增，以便构建得到捕获后DNA文库。
  17. 根据权利要求16所述的方法，其特征在于，所述特异性探针为生物素标记的特异性探针，利用链霉亲和素标记的磁珠进行所述纯化。
  18. 根据权利要求15所述的方法，其特征在于，进一步包括：
    在微流控芯片上制备微流滴后，利用第一扩增引物进行扩增，以便获得第一扩增产物，所述第一扩增引物适于对所述核酸的不同区域进行扩增；
    利用通用引物对所述第一扩增产物进行扩增，以便获得第二扩增产物，所述通用引物与所述第一扩增引物部分互补。
  19. 根据权利要求18所述的方法，其特征在于，进一步包括：
    将所述第二扩增产物进行变性，环化，以便获得单链环状DNA；
    基于所述单链环状DNA进行滚环扩增，以便获得DNA纳米球。
  20. 根据权利要求18所述的方法，其特征在于，在利用第一扩增引物扩增之后或者在进行利用通用引物对所述第一扩增产物进行扩增之后，分别利用磁珠进行纯化。
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