WO2022246781A1 - 基于电浸润的crispr的核酸检测系统及其方法 - Google Patents

基于电浸润的crispr的核酸检测系统及其方法 Download PDF

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WO2022246781A1
WO2022246781A1 PCT/CN2021/096620 CN2021096620W WO2022246781A1 WO 2022246781 A1 WO2022246781 A1 WO 2022246781A1 CN 2021096620 W CN2021096620 W CN 2021096620W WO 2022246781 A1 WO2022246781 A1 WO 2022246781A1
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nucleic acid
reaction
module
crispr
reaction solution
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French (fr)
Inventor
章文蔚
江媛
王冀
席凤
刘传
汪为茂
崔路漫
纪泽阳
郭苗苗
章登位
任悍
兰茜
王欧
汪晓珏
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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    • C—CHEMISTRY; METALLURGY
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    • 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
    • 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 belongs to the field of biological analysis and detection, and in particular relates to a nucleic acid detection system and method based on electroinfiltration CRISPR.
  • DNA and RNA are the main genetic material of nucleic acid, and they are also the most direct detection objects for most pathogens, especially viruses, when they enter the human body.
  • Pathogens as antigens, stimulate the body to produce antibodies, which serve as the main body of protein detection.
  • This type of immunodiagnostic kit is also the mainstream of domestic diagnostic kits.
  • the metabolite detection method belongs to the biochemical detection method of the pathogen. This detection method is actually the detection of the metabolic enzyme of the pathogen.
  • Molecular diagnosis is only applicable when the pathogen has multiplied to a certain extent in the host and can be isolated and cultured in vitro. Molecular diagnosis has advantages that the other two diagnoses do not have: (1) High accuracy. Molecular diagnosis is the detection of pathogenic genetic material, which is highly specific and characteristic.
  • RT-PCR reverse transcription PCR
  • RT-qPCR real-time quantitative PCR
  • LAMP loop-mediated isothermal amplification assays
  • POCT point-of-care testing
  • RT-PCR and RT-qPCR design primers according to the virus-specific genome sequence for nucleic acid amplification. The detection is fast and sensitive, but the target sites generally do not exceed 5, and the requirements for primer design are relatively high. It is greatly affected by reaction conditions and systems, and has a high rate of false negatives and false positives.
  • kits based on viral nucleic acid detection methods do not include nucleic acid extraction, because nucleic acid extraction mostly requires manual operation, which is easy to introduce operational errors and pollution.
  • the automatic nucleic acid purification and fluorescent PCR analysis systems that appear on the market such as Anada9850 or NGS automatic preparation systems such as MGISP-100 adopt the form of large pipetting stations, which realize the full automation of sample sampling, nucleic acid extraction, gene amplification or NGS library construction , but the equipment is expensive.
  • the automated nucleic acid extraction instrument alone is 300,000-350,000 yuan
  • the fluorescent PCR instrument is 250,000-350,000 yuan. Since the whole system is an open system, there is still the problem of contamination between samples.
  • CRISPR-Cas is the acquired immune system of bacteria and archaea, which use RNA-guided nucleases to recognize and degrade foreign nucleic acids.
  • CRISPR-Cas9 family protein
  • Cas12a and Cas13 have gene editing properties that are very different from the well-known Cas9, they still have extremely high specificity for the target sequence.
  • Cas12a only needs one RucV to catalyze the enzymatic cleavage of two strands at the same time, and once the target sequence is combined, Cas12a has the activity of endonuclease that degrades single-stranded DNA of any sequence (including trans-single-stranded DNA).
  • DETECTR DNA Endonuclease Targeted CRISPR Trans-Reporter
  • SHERLOCK specific high-sensitivity enzyme reporter unlocking
  • the detection sensitivity is as high as attomole level (this level can already detect single nucleic acid strands in milliliters of blood), without the need for nucleic acid extraction, and the detection time can be carried out within 2 hours without professional operation.
  • This CRISPR The emergence of related effector proteins points out a new direction for novel rapid molecular diagnostics.
  • Electrowetting is an emerging cutting-edge technology for automated biological sample processing and detection. It creates, moves, mixes and separates one or more tiny droplets on a fully enclosed chip by fabricating and controlling a set of electrodes with hydrophobic surfaces. The whole process is automatically operated in a closed environment, completely avoiding environmental and operational pollution. At the same time, the small amount of reaction system (100nl-5 ⁇ L) not only reduces the amount of samples and reagents, but also speeds up the reaction speed. This technology does not require complicated and expensive mechanical pipetting equipment, and all control and feedback are realized through fully automatic electronic circuits, which allows scientists to operate droplets as if writing a program, and realize tens of hundreds of steps of complex fluid, incubation and optics. Detect operation.
  • the present invention provides a CRISPR-based nucleic acid detection system and method based on electroinfiltration, which uses a highly closed Microfluidic technology, combined with the double matching method of RT-PCR (reverse transcription PCR) and then CRISPR to reduce the false positive rate, using the method of droplet distribution after amplification in the same tube to reduce the false negative rate, improving the sensitivity and throughput quantity.
  • RT-PCR reverse transcription PCR
  • the first aspect of the present invention relates to a nucleic acid detection system based on electrowetting CRISPR, comprising: an electrowetting chip module, a CRISPR reaction module, a signal capture module, and a control and analysis module; wherein:
  • the electrowetting chip module includes: a reagent storage area, a biochemical reaction area, a fluorescent photographing area, a driving system, and a microchannel between each area;
  • the CRISPR reaction module includes: a CRISPR reaction solution containing a fluorescent probe, a protein reaction solution containing Cas12a, and a gRNA reaction solution containing gRNA; the protein reaction solution, gRNA reaction solution and CRISPR reaction solution all contain surfactants and enzymes buffer;
  • the control and analysis module drives the nucleic acid sample to be tested and the CRISPR reaction module to enter the biochemical reaction area through the microchannel through the drive system, and the nucleic acid sample to be tested and the CRISPR reaction module are mixed and generated in the biochemical reaction area.
  • a fluorescent signal is generated after the reaction; the fluorescent signal is captured and collected by the signal capture module in the fluorescence photographing area, and then input to the control and analysis module for analysis.
  • reaction conditions in which the CRISPR reaction module enters the biochemical reaction zone through a microchannel to mix and react are conventional in the art, for example, 48° C. for 20 minutes.
  • the CRISPR reaction module and the nucleic acid sample to be tested are stored in the reagent storage area before the reaction.
  • the protein reaction solution, the gRNA reaction solution and the CRISPR reaction solution all contain surfactants and enzyme buffers.
  • the surfactant is Tween-20, such as Tween-20 with a final concentration of 0.05-0.1%, and the enzyme buffer is 10 ⁇ NEB buffer 3.1.
  • the sequence of the gRNA includes sequences shown in one or more of SEQ ID NO: 6, 9, 10, 11 and 12; preferably the sequence of the gRNA also includes The sequences shown in SEQ ID NO: 13 and 14.
  • the sequence of the gRNA is as shown in SEQ ID NO: 6, 10, 11 or 12, or as shown in SEQ ID NO: 6, 10, 13 and 14, or as shown in SEQ ID NO : 9, 10, 13 and 14.
  • the nucleic acid sample to be tested is DNA or RNA; preferably, the DNA is cDNA obtained by reverse transcription PCR.
  • the signal capture module can be a conventional signal capture and collection device in the art, preferably including a fluorometer, a fluorescence photometer or a fluorescence spectrophotometer.
  • the control and analysis module can be a conventional analysis device in the field, preferably including a computer, a tablet or a numerical control device.
  • the protein reaction solution also includes Cas12a protein, RNase inhibitor, glycerol and nuclease-free water
  • the gRNA reaction solution also includes gRNA, RNase inhibitor and nuclease-free water
  • the CRISPR reaction solution also includes fluorescent probes.
  • the fluorescent probe can be a DNA probe with a fluorescent light emitting group FAM and a fluorescent quenching group BHQ1 at the 5' end and the 3' end respectively, such as Fam-TTATT-Q.
  • the protein reaction solution includes 0.25-1 ⁇ M, preferably 0.5 ⁇ M Cas12a protein, 4,000-10,000 unit/mL, preferably 5,340 unit/mL RNase inhibitor, 10-30%, preferably 15.69% glycerol, 0.05-0.5% (w/v) Preferably 0.1% (w/v) Tween-20 and 10 ⁇ NEB buffer 3.1.
  • the gRNA reaction solution includes: gRNA of 0.1-0.5 ⁇ M, preferably 0.3 ⁇ M, 10 ⁇ NEB buffer 3.1, RNase inhibitor of 2,000-10,000 unit/mL, preferably 4,000 unit/mL, 0.05-0.5% (w/v), preferably 0.1 % (w/v) of Tween-20.
  • the CRISPR reaction solution includes: 10-100 ⁇ M, preferably 25 ⁇ M fluorescent probe.
  • the protein reaction solution is prepared from a CRISPR protein stock solution and a CRISPR protein dilution solution at a ratio of 1:1, wherein each 0.1 mL of the CRISPR protein stock solution includes: 10 ⁇ L of 10 ⁇ M Cas12a protein, 26.7 ⁇ L 40,000unit/mL RNase inhibitor, 10 ⁇ L 10 ⁇ NEB buffer 3.1, 52.3 ⁇ L 60% (v/v) glycerol and 1 ⁇ L 10% (w/v) Tween-20;
  • Each 0.5 mL of the CRISPR protein dilution includes: 50 ⁇ L of 10 ⁇ NEB buffer 3.1, 5 ⁇ L of 10% (w/v) Tween-20 and 445 ⁇ L of nuclease-free water;
  • Each 0.5mL gRNA reaction solution includes: 75 ⁇ L 2 ⁇ M gRNA, 50 ⁇ L 10 ⁇ NEBbuffer3.1, 50 ⁇ L 40,000unit/mL RNase inhibitor, 5 ⁇ L 10% (w/v) Tween-20 and 320 ⁇ L nuclease-free water;
  • Each 1 mL of the CRISPR reaction solution includes: 250 ⁇ L of 100 ⁇ M fluorescent probe, 200 ⁇ L of 10 ⁇ NEB buffer 3.1 and 10 ⁇ L of 10% (w/v) Tween-20.
  • the nucleic acid detection system further includes GAPDH gRNA as shown in SEQ ID NO:5.
  • the nucleic acid detection system further includes a nucleic acid amplification reaction module; after the nucleic acid sample to be tested undergoes an amplification reaction in the nucleic acid amplification reaction module, a single droplet is uniformly dispersed into A plurality of droplets, for example, 3 droplets.
  • the nucleic acid amplification reaction module can perform nucleic acid amplification by RT-PCR, multiple RT-PCR or RT-qPCR.
  • the nucleic acid amplification reaction module for example, performs nucleic acid amplification by RT-PCR.
  • the nucleic acid amplification reaction module includes a temperature control sub-module and a nucleic acid amplification reaction system
  • the nucleic acid amplification reaction system includes an experimental reaction solution I, an experimental reaction solution II and a RT-PCR primer solution
  • the experimental reaction solution I includes Hot start enzyme, Alpha RTase, RNase inhibitor and 10 ⁇ Solution I buffer
  • the experimental reaction solution II includes hot start enzyme buffer, dNTP, Tween-20 and PF68.
  • the nucleic acid detection system further includes a positive template standard and/or a negative template standard.
  • the positive template standard includes artificially synthesized viral RNA and internal reference RNA.
  • the internal reference RNA is GAPDH, ⁇ -actin or 18sRNA.
  • every 100 ⁇ L of the experimental reaction solution I includes:
  • Each 150 ⁇ L of the experimental reaction solution II includes:
  • Each 100 ⁇ L of the positive template standard includes: 1 ⁇ L of 10ng/ ⁇ L artificially synthesized viral RNA or 10 ⁇ L of 10ng/ ⁇ L internal reference RNA, and the balance is water; each 100 ⁇ L of the negative template standard includes: 10 ⁇ L of 10ng/ ⁇ L internal reference RNA , the balance is water;
  • Each 225 ⁇ L of the RT-PCR primer solution includes:
  • RNA upstream primer 0.5 ⁇ L 100 ⁇ M internal reference RNA upstream primer, 0.5 ⁇ L 100 ⁇ M internal reference RNA downstream primer, 1.75 ⁇ L 100 ⁇ M RNA upstream primer of the nucleic acid sample to be tested and 1.75 ⁇ L 100 ⁇ M RNA downstream primer of the nucleic acid sample to be tested, the balance is water;
  • reaction conditions for RT-PCR are as follows: 10 ⁇ L reaction system includes 2 ⁇ L experimental reaction solution I and 3 ⁇ L experimental reaction solution II, 1 ⁇ L nucleic acid sample to be tested, 2.25 ⁇ L RT-PCR primers and 1.75 ⁇ L water.
  • the reaction program of RT-PCR is 50°C for 10 minutes; for example, the reaction program of PCR after RT-PCR is the first stage of 95°C for 5 minutes, the second stage of 94°C for 10 seconds, and 57°C for 20 seconds, a total of 30-40 minutes. For example, 38 cycles, the third stage is 68°C for 2 minutes.
  • the nucleic acid detection system also includes a nucleic acid extraction module, and the sample to be tested is extracted from the sample to be tested by the nucleic acid extraction module.
  • the nucleic acid extraction module includes a magnetic bead control area and a nucleic acid extraction system.
  • the magnetic bead control area includes a sample well for accommodating a sample, and electrodes connecting the sample well and the magnetic field area.
  • the sample is extracted by magnetic beads through a nucleic acid extraction system in the magnetic bead control area.
  • the magnetic bead extraction can be a routine operation in the art, and generally includes a complete process of grabbing and releasing the magnetic beads, washing the magnetic beads, and eluting nucleic acids from the magnetic beads.
  • the nucleic acid extraction system includes: nucleic acid extraction lysis solution, nucleic acid extraction binding solution, nucleic acid extraction washing solution 1, nucleic acid extraction washing solution 2, and nucleic acid extraction eluent;
  • the nucleic acid extraction lysate of every 1mL includes:
  • the nucleic acid extraction binding solution per 1 mL includes:
  • the nucleic acid extraction washing liquid 1 of every 5mL comprises:
  • the nucleic acid extraction washing liquid 2 of every 5mL comprises:
  • the nucleic acid extraction eluent of every 5mL comprises:
  • the magnetic bead control area is located on the electrowetting chip module, the nucleic acid extraction system is stored in the reagent storage area; the nucleic acid in the sample to be tested is extracted in the magnetic bead control area , to obtain the nucleic acid sample to be tested.
  • the sample to be tested is a blood sample or a body fluid sample such as a saliva sample.
  • the sample to be tested contains virus, and the virus is dsDNA virus, ssDNA virus, dsRNA virus, ssRNA virus, DNA retrovirus or RNA retrovirus; preferably dsRNA virus or ssRNA virus, such as COVID- 19 viruses.
  • the second aspect of the present invention provides a method for detecting nucleic acid for non-diagnostic purposes, which uses the nucleic acid detection system as described in the first aspect, and includes the following steps:
  • the control and analysis module drives the CRISPR reaction module and the nucleic acid sample to be tested into the electrowetting chip module through the drive system, and forms droplets respectively; for example, the CRISPR reaction solution, protein reaction solution and The gRNA reaction solution forms droplets respectively;
  • the fluorescent signal is captured and collected by the signal capture module in the fluorescent photographing area and then input to the control and analysis module for analysis; when the intensity of the fluorescent signal exceeds the negative control, it is judged that there is a target nucleic acid.
  • the negative control can be conventional in the art, such as an internal reference gene.
  • (1) also includes (02): amplifying the nucleic acid sample to be tested in the biochemical reaction zone through the nucleic acid amplification reaction module; thus (1) forming droplets on the amplified nucleic acid sample to be tested;
  • (01) is also included before (02): performing nucleic acid extraction on the sample to be tested in the magnetic bead control zone through the nucleic acid extraction reaction;
  • a nucleic acid amplification reaction module is added to the same droplet of the nucleic acid sample to be tested for nucleic acid amplification.
  • the droplets of the gRNA reaction solution are first mixed with the droplets of the CRISPR reaction solution, and then mixed with the droplets of the protein reaction solution.
  • Nucleic acid extraction is performed in the magnetic bead control area, and the driving system drives the nucleic acid sample to be tested and each module into the biochemical reaction area to form droplets and mix them; wherein, nucleic acid amplification is performed in the biochemical reaction area
  • the steps are all controlled by computer, tablet or numerical control equipment.
  • the method for detecting nucleic acid may be for non-diagnostic purposes, that is, it may be applied to laboratory scientific research or nucleic acid detection in environmental samples, for example:
  • the "detection method for non-diagnostic purposes" of the present invention can detect samples collected in the environment (including human secretions) in preventive medicine, and judge whether the environment is polluted, so as to decide whether to block the environment, suspend business, Disposal methods such as killing and disinfecting, and decide whether to upgrade infectious disease prevention and control measures for this region, city, or even a higher level. Prevent infectious diseases from the perspective of public health, and prevent the virus remaining in the environment from becoming a source of infection.
  • Those skilled in the art can, for example, test water samples to determine the transmission time and extent of the virus.
  • Brazil detected the new coronavirus in sewer water samples collected in 2019, earlier than the report of the world's first confirmed case, which can provide more reference for the traceability and transmission time of the virus.
  • an autopsy on a deceased person in California showed that he tested positive for the novel coronavirus, three weeks earlier than the first reported death from the novel coronavirus in the United States.
  • Detection by using the anti-new coronavirus antibody of the present invention can be used in virology research to detect whether the integration of viral nucleic acid has occurred, and can monitor and timely warn whether the integration of the virus occurs from the perspective of preventive medicine.
  • a third aspect of the present invention provides a computer-readable medium, the computer-readable storage medium stores a computer program, wherein, when the computer program is executed by a processor, the functions or functions of the nucleic acid detection system as described in the first aspect are realized.
  • the method for detecting nucleic acid for non-diagnostic purposes according to the second aspect of the present invention.
  • a fourth aspect of the present invention provides an electronic device, which includes a memory and a processor; the memory includes a computer program stored therein that can run on the processor; wherein, when the processor executes the computer program, the The steps of the method for detecting nucleic acid for non-diagnostic purposes according to the second aspect of the present invention.
  • the microfluidic system used in the present invention can quickly generate multiple micro-reaction droplets, which can greatly improve the pathogen detection throughput while performing efficient and rapid detection, and can simultaneously detect multiple pathogens with DNA or RNA as the main body, And the fully enclosed environment of microfluidics greatly reduces the contamination rate between the environment and samples.
  • Fig. 1 is a schematic diagram of the nucleic acid detection method of the present invention.
  • Figure 2 is a schematic diagram of the primer sites of the internal reference genes of the examples.
  • Figure 3 is a schematic diagram of the primer sites of the new coronavirus in Example 1.
  • FIG. 4 is a schematic diagram of PCR amplification results in Example 1.
  • Fig. 5 is a schematic diagram of the cleavage activity of PCR products amplified by GAPDH primers by gRNA-GAPDH in Example 1.
  • FIG. 6 is a schematic diagram of the cleavage activity of PCR products amplified by ORF1ab primers by gRNA-GAPDH of Example 1.
  • Fig. 7 is a schematic diagram of the cleavage activity of the PCR product amplified by the gRNA-ORF1ab of Example 1 to the ORF1ab primer.
  • Fig. 8 is a schematic diagram of the cleavage activity of PCR products amplified by GAPDH primers by gRNA-ORF1ab of Example 1.
  • FIG. 9 is a schematic diagram of the cleavage activity of the PCR product amplified by the negative control gRNA of Example 1 to GAPDH/ORF1ab primers.
  • Fig. 10 is a schematic diagram of the cleavage activity of the positive template standard in Example 1.
  • Fig. 11 is a schematic diagram of the cleavage activity of the negative template standard in Example 1.
  • Fig. 12 is a schematic diagram of ultrapure water cutting activity in Example 1.
  • Figure 13 is a schematic diagram of the detection results of different gRNAs and combinations.
  • Figure 14 is a schematic diagram of the test results of Example 1 and the commercial kit.
  • Fig. 15 is a schematic diagram of the chip microstructure of the present invention.
  • FIG. 16 is a schematic diagram of chip verification results in Embodiment 1.
  • Figure 17 is a schematic diagram of the detection effect of standard products.
  • FIG. 18 is a schematic structural diagram of the electronic device of Embodiment 2.
  • FIG. 1 The detection principle of the embodiment is shown in FIG. 1 .
  • the preservation solution is VTM (Viral Transport Medium) preservation solution, comprising: Hank's balanced salt solution (pH is 7.2 ⁇ 7.4), 1000IU/mL penicillin, 1000IU/mL streptomycin, 0.5% (w/v) bovine serum white Egg white, 0.016% (w/v) phenol red and 50% (v/v) glycerol.
  • VTM Virtual Transport Medium
  • the preservation solution can also be a phosphate buffer solution, including: 137mM sodium chloride, 2.7mM potassium chloride, 10mM sodium dihydrogen phosphate, 2mM potassium dihydrogen phosphate, with a pH of 7.4.
  • a phosphate buffer solution including: 137mM sodium chloride, 2.7mM potassium chloride, 10mM sodium dihydrogen phosphate, 2mM potassium dihydrogen phosphate, with a pH of 7.4.
  • RNA HS assay kit Thermo Fisher Scientific, Q32855.
  • 3.2 Human RNA is Universal Human Reference RNA (UHRR), purchased from Agilent, Germany.
  • UHRR the universal human reference RNA standard (Universal Human Reference RNA, Agilent Co.Ltd.), was selected as the negative control standard for in vitro testing. Based on the abundance of human gene expression, the RNA expressed by GAPDH gene, ⁇ -actin gene and 18S gene were selected as the experimental internal reference.
  • GAPDH (or G3PDH), namely glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase), is widely distributed in cells in various tissues.
  • ⁇ -actin is responsible for the expression of an important skeleton protein in cells, and has a high expression abundance in human cells.
  • 18S RNA is an important component of human ribosomal RNA, with the highest content in the total RNA of human cells. The main purpose of selecting these internal reference genes is to test the reliability of the experimental protocol and avoid false positives caused by experimental operations.
  • Prime Primer v5.0 software to calculate and optimize, different primer sites were designed for the three genes, as shown in Figure 2.
  • the sites in the figure can be used as internal reference genes in the examples, and the sites designed for internal reference primers include but are not limited to those sites shown in the above figure.
  • a pair of primers in GAPDH is taken as an example to carry out the experiment.
  • the primer sequence information is as follows:
  • GAPDH-F TTCATTGACCTCAACTACATGGTTTAC (SEQ ID NO: 1)
  • GAPDH-R GATTTTGGAGGGATCTCGCTCCTG (SEQ ID NO: 2)
  • the new coronavirus namely COVID-19 or 2019-nCoV, is a single-stranded RNA coronavirus. Based on the published gene sequence (GenBank: MN908947.3), multiple sets of detection sites for different genes of the new coronavirus were designed. The primer design As shown in Figure 3.
  • these sites can be used as detection genes in the examples, and the sites for primer design of detection genes include but are not limited to those sites shown in the figure above.
  • an experiment is carried out by taking a pair of primer sites on ORF1ab as an example.
  • the primer sequence information is as follows:
  • ORF1ab-F CCCTGTGGGTTTTACACTTAA (SEQ ID NO: 3)
  • ORF1ab-R ACGATTGTGCATCAGCTGA (SEQ ID NO: 4)
  • gRNA GuideRNA
  • GuideRNA that is, guide RNA
  • gRNA is one of the important components to realize CRISPR fluorescence detection.
  • gRNA activates Cas protein activity and realizes downstream fluorescence detection through complementary pairing with the DNA target fragment sequence.
  • the sequence specificity of gRNA determines the specificity of its detection results. It is easy to know that the sequence of the gRNA should be complementary to the sequence of the amplified product to be detected.
  • Figure 2 for the human internal reference, the corresponding gRNA sequences were designed based on the sites in Figure 2.
  • the corresponding gRNA sequences were designed respectively.
  • gRNA-GAPDH and gRNA-ORF1ab were used as detection targets in the examples, and the designed sites of gRNAs for detection targets include but are not limited to those sites shown in the figure.
  • the gRNA sequence information is as follows:
  • gRNA 203 N gene, target site position 20604.
  • gRNA 4.2 S gene, target site position 24831:
  • gRNA 004.1 ORF1ab gene, target site position 978:
  • gRNA 004.2 ORF1ab gene, target site position 1085):
  • gRNA 11.1 ORF1ab gene, target site position 29083:
  • gRNA 11.2 ORF1ab gene, target site position 2915:
  • the 5 ⁇ CRISPR reaction solution prepared here can be adjusted to 2 ⁇ or 10 ⁇ CRISPR reaction solution with any multiple concentration according to the formula, and be diluted proportionally when used as needed.
  • Amplification template 1Ultrapure water; 2Negative control; 3Positive control.
  • RT-PCR was performed on different concentrations of UHRR with GAPDH primers, and the PCR product was cut with gRNA-GAPDH, which confirmed that gRNA-GAPDH had cutting activity on the PCR product, and the signal value increased with the increase of UHRR input, as shown in Figure 5 .
  • gRNA-GAPDH has no cutting activity on RT-PCR products amplified by ORF1ab primers using viral cDNA as a template, as shown in FIG. 6 .
  • gRNA-ORF1ab has no cutting activity for RT-PCR products amplified with GAPDH primers using UHRR as a template, as shown in FIG. 8 .
  • the negative control gRNA-84 has no cleavage activity on the amplification products 1ng/ ⁇ L GAPDH and 1ng/ ⁇ L ORF1ab of other PCR primers in this example, which can be used to define the background reference fluorescence value.
  • the sequence of the gRNA-84 is shown in SEQ ID NO: 16.
  • the positive template standard 3 used in Section 7 was amplified by multiplex RT-PCR, and cut with gRNA-ORF1ab, gRNA-GAPDH and gRNA-84, respectively. The results are shown in Figure 10, where gRNA-84 is reported as a negative result, gRNA-GAPDH and gRNA-ORF1ab are reported as a positive result, and the signal is significantly higher than that of gRNA-84, as expected.
  • the RT-PCR negative template standard 2 used in Section 7 was amplified by multiple RT-PCR, and then cut with gRNA-ORF1ab, gRNA-GAPDH and gRNA-84 respectively. The results are shown in Figure 11. Among them, gRNA-84 and gRNA-ORF1ab were reported as negative results, and gRNA-GAPDH was reported as positive results, which were in line with expectations.
  • the aforementioned other gRNAs (SEQ ID NO: 7-15) were detected individually or in combination according to the above method, and the results showed that the sequence was as shown in SEQ ID NO: 6, 10, 11 or 12
  • the multiplex PCR and gRNA of the gRNAs shown, the gRNAs whose sequences are shown in SEQ ID NO: 6, 10, 14 and 15, and the gRNAs whose sequences are shown in SEQ ID NO: 9, 10, 14 and 15 can be used for the identification of samples , and has good detection ability.
  • the experiment was carried out using the CRISPR kit (Shanghai Tolo Harbor Biotechnology Co., Ltd., 32108-01), and the results are shown in FIG. 14 . It shows that this embodiment improves the detection ability compared with the existing kit.
  • the lower surface of the digital microfluidic chip is composed of several control units.
  • the voltage of the adjacent control unit terminal on the chip is controlled to make it the voltage of the ground terminal on the upper surface of the chip.
  • the same or form a pressure difference (commonly known as "on/off") to achieve the purpose of driving liquid deformation or droplet movement.
  • Each control unit can control 1 microliter droplets, and multiple control units can control larger volume droplets. Moving the droplets and cooperating with other elements, such as heating elements, water cooling devices, and magnets equipped with stepping motors, can ensure the smooth progress of reagent storage, reagent extraction, reagent movement, biochemical reactions, and magnetic bead purification.
  • the digital microfluidic chip is filled with silicone oil, and all the reagents required for this reaction are sequentially loaded into the reagent storage areas M1-M8 and L1-L8 according to FIG. 15 .
  • the throat swab sample of the person to be tested and add it to 1mL of lysate, soak for at least 5min. Properly mix the lysate containing the throat swab and resuspend the magnetic bead extract.
  • gRNA-84 negative control gRNA-84
  • GAPDH internal reference gRNA-GAPDH GAPDH internal reference
  • COVID-19 target gRNA-ORF1ab COVID-19 target gRNA-ORF1ab
  • the CRISPR protein can activate the fluorescent probe in the cutting reaction solution to produce fluorescence.
  • the droplet will move to the fluorescence photography area to take pictures, and judge whether the sample contains the new coronavirus nucleic acid according to the fluorescence intensity of the droplet.
  • Figure 16 shows the experimental results of using the simulated negative sample (containing only the universal human reference RNA) and the simulated positive sample (containing the universal human reference RNA and the RNA containing the partial region sequence of the new coronavirus) after the reaction on the chip and taking fluorescence photos.
  • the detection sensitivity of this example reaches 200 copies/mL, that is, the detection limit is 0.2 copies/ ⁇ L, and the expected positive agreement rate with qPCR detection is 91.67%, and the expected negative agreement rate is 100%, which proves that the experiment is successful.
  • This embodiment provides an electronic device, which can be expressed in the form of a computing device (for example, it can be a server device), including a memory, a processor, and a computer program stored on the memory and operable on the processor, wherein the processing
  • a computing device for example, it can be a server device
  • the non-diagnostic nucleic acid detection method in Embodiment 1 of the present invention can be realized when the computer program is executed by the computer.
  • FIG. 18 shows a schematic diagram of the hardware structure of this embodiment, and the electronic device 9 specifically includes:
  • At least one processor 91 at least one memory 92, and a bus 93 for connecting different system components, including the processor 91 and the memory 92, wherein:
  • the bus 93 includes a data bus, an address bus, and a control bus.
  • the memory 92 includes a volatile memory, such as a random access memory (RAM) 921 and/or a cache memory 922 , and may further include a read only memory (ROM) 923 .
  • RAM random access memory
  • ROM read only memory
  • Memory 92 also includes programs/utilities 925 having a set (at least one) of program modules 924 including, but not limited to, an operating system, one or more application programs, other program modules, and program data, examples of which are Each or some combination of these may include implementations of network environments.
  • the processor 91 executes various functional applications and data processing by running the computer program stored in the memory 92, such as the non-diagnostic nucleic acid detection method in Embodiment 1 of the present invention.
  • Electronic device 9 may further communicate with one or more external devices 94 (eg, keyboards, pointing devices, etc.). Such communication may occur through input/output (I/O) interface 95 .
  • the electronic device 9 can also communicate with one or more networks (such as a local area network (LAN), a wide area network (WAN) and/or a public network such as the Internet) through the network adapter 96 .
  • Network adapter 96 communicates with other modules of electronic device 9 via bus 93 .
  • other hardware and/or software modules may be used in conjunction with electronic device 9, including but not limited to: microcode, device drivers, redundant processors, external disk drive arrays, RAID (array of disks) systems, tape drives, and data backup storage systems.
  • An embodiment of the present invention provides a computer-readable storage medium on which a computer program is stored.
  • the program is executed by a processor, the steps of the method for detecting nucleic acid for non-diagnostic purposes in Embodiment 1 of the present invention are implemented.
  • the readable storage medium that can be used more specifically may include, but is not limited to: portable disk, hard disk, random access memory, read-only memory, erasable programmable read-only memory, optical storage device, magnetic storage device or any of the above-mentioned the right combination.
  • the present invention can also be implemented in the form of a program product, which includes program code, and when the program product runs on the terminal device, the program code is used to make the terminal device execute The steps of the method for detecting nucleic acid for non-diagnostic purposes in Example 1 of the present invention.
  • the program code for executing the present invention can be written in any combination of one or more programming languages, and the program code can be completely executed on the user equipment, partially executed on the user equipment, as an independent
  • the package executes, partly on the user device and partly on the remote device, or entirely on the remote device.

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Abstract

一种基于电浸润的CRISPR的核酸检测系统及其方法。所述核酸检测系统包括：电浸润芯片模块、CRISPR反应模块、信号捕获模块和控制与分析模块；其中：所述电浸润芯片模块包括：试剂储存区、生化反应区、荧光拍照区和驱动系统，以及各区之间的微通道；所述CRISPR反应模块包括：含有荧光探针的CRISPR反应液、含有Cas12a的蛋白反应液和含有gRNA的gRNA反应液；所述蛋白反应液、gRNA反应液和CRISPR反应液均包含表面活性剂和酶缓冲液；

Description

基于电浸润的CRISPR的核酸检测系统及其方法 技术领域
本发明属于生物分析检测领域，具体涉及一种基于电浸润的CRISPR的核酸检测系统及其方法。
背景技术
分子诊断、免疫诊断和生化诊断分别对应了DNA和RNA、蛋白质、代谢物三个层面，同时代表病原感染后初期、中期和末期的三个不同阶段。DNA和RNA为核酸主要的遗传物质，也是大多数病原尤其是病毒在进入人体初期最直接的检测物。而病原作为抗原刺激人体产生的抗体，则作为蛋白质检测的主体，此类免疫诊断试剂盒也是当今国内诊断试剂盒的主流。代谢物检测方法属于病原的生化检测方法，这种检测方法实际是检测病原的代谢酶，仅适用于当病原在宿主体内繁殖至一定程度且可以在体外分离培养的情况。分子诊断有着其他两种诊断没有的优点：(1)极高的准确性，分子诊断是对病原遗传物质的检测，具有极高的专一性和特征性，虽然说抗原抗体的也有较高的特异性，但也曾出现不同病原的激发相同免疫抗原的病例，而纵使是相同病原的不同基因型都可以通过分子诊断区分；(2)可早期诊断，正如前文所述，分子诊断所检测的DNA和RNA是病原进入宿主后最直接的产物，而免疫诊断则必须等到病原在宿主(人体)内增殖到一定程度，才会产生免疫反应，在体内出现抗体；以乙型肝炎与丙型肝炎的检测为例，酶联免疫检测的是病毒抗体，但当人体感染病毒初期，有一段时间是没有抗体的；(3)检测所需起始量低，尤其核酸本身具有复制放大能力，也使得分子诊断更加灵敏、特异、快捷，相较于免疫诊断，免疫诊断所需样本量大，信噪比低，于此分子诊断具有显著优势。分子诊断方法通过不同层次、不同维度的多个指标为医生的临床决策提供“精确”支持和依据。
现有分子诊断的常用技术包括灵敏度核酸扩增试验(NAAT)，例如反转录PCR(RT-PCR)、实时定量PCR(RT-qPCR)、基于环介导的等温扩增检测(LAMP)、基于DNA微阵列检测或基于测序检测，以及即时检测(POCT)、NGS全基因组测序。其中，RT-PCR和RT-qPCR根据病毒特异的基因组序列设计引物进行核酸扩增，检测快速且灵敏度高，但是靶向位点一般不超过5个，引物设计要求较高，实际扩增检测中受反应条件和体系影响较大，有较高的假阴性和假阳性率。NGS检测可以通过全基因组测序，精确且高通量地检测病原菌，且可监测病毒的变异，但是此方法耗时费力，成本较高，且需要特定的 高通量测序仪器，难以满足快检确诊的需求。目前基于病毒核酸检测方法的试剂盒都没有包括核酸提取，因核酸提取大多需要手工操作，容易引入操作误差和污染。市面上出现的自动核酸提纯及荧光PCR分析系统如Anada9850或者NGS自动化制备系统如MGISP-100采用的是大型移液站形式，其实现了样本取样、核酸提取、基因扩增或NGS建库全程自动化，但是仪器费用昂贵，仅自动化核酸提取仪就达30-35万人民币，荧光PCR仪器则在25-35万。由于整个系统属于开放式系统，仍存在样本间污染的问题。
因此，寻找新型的检测手段进行快速检测、降低分子诊断的门槛是未来分子诊断的发展方向。为了实现这一手段，美国华裔科学家张峰和美国科学家Jennifer A.Doudna先后发现了CRISPR(clustered regularly interspaced short palindromic repeats)相关家族蛋白，Type V的Cas12a及Type VI的Cas13a和Cas13b。CRISPR-Cas是细菌与古细菌的获得性免疫系统，它们利用RNA导向的核酸酶来识别和降解外源的核酸。为大家熟知的是CRISPR-Cas9家族的蛋白，因为它具有在guide-RNA(gRNA)的引导下由自身的两个的结构域RucV和HNH准确切割与guide-RNA互补部分的双链DNA的性质，已经被作为基因编辑的工具广泛应用。Cas12a和Cas13虽然有着和大众所熟知的与Cas9极为不同的基因编辑性质，但却依然具有对目标序列极高的专一性。Cas12a只需要一个RucV就可以同时催化两条链的酶切，并且一旦结合了目标序列，Cas12a就有了降解任意序列单链DNA(包括反式单链DNA)的内切酶的活性。美国科学家们分别根据Cas12a和Cas13略有不同的性质分别开发了针对双链DNA且具有普适性的分子诊断技术DETECTR(DNA Endonuclease Targeted CRISPR Trans-Reporter)和SHERLOCK(specific high-sensitivity enzymatic reporter unlocking)，检测灵敏度高达埃摩尔级别(此级别已经可以检测毫升血液中存在的单核酸链)，而不需要进行核酸提取，检测时间在2小时之内且不需要专业人员操作就可以进行，这种CRISPR相关效应蛋白的出现为新型快速分子诊断指明了新的方向。
电浸润微流体技术(Electrowetting)是新兴的自动化生物样品处理和检测前沿技术。它通过制造并控制一组表面疏水的电极，在全封闭的芯片上可以产生、移动、混合并且分离一个和多个微小的液滴。全流程在封闭环境自动操作，完全避免环境和操作污染，同时微量的反应体系(100nl-5μL)既减小了样品和试剂用量，又加快了反应速度。这项技术无需复杂昂贵的机械式移液设备，所有控制和反馈都通过全自动电子电路实现，这使得科学家可以像写程序一样操作液滴，实现几十上百步复杂的流体、孵育和光学检测操作。
目前，尚未有将电浸润与CRISPR技术结合以实现高灵敏度，并且高通量的简单、 快速有效的核酸检测的报道。
发明内容
针对现有技术中缺乏高灵敏度，并且高通量的简单、快速有效的核酸检测方法的不足，本发明提供一种基于基于电浸润的CRISPR的核酸检测系统及其方法，其采用高封闭性的微流控技术，结合先RT-PCR(反转录PCR)后CRISPR的两重匹配的方法减少假阳性率，使用同管扩增后液滴分配的方法减少假阴性率，提高了灵敏度与通量。
本发明通过以下技术方案解决上述技术问题：
本发明的第一方面涉及一种基于电浸润的CRISPR的核酸检测系统，包括：电浸润芯片模块、CRISPR反应模块、信号捕获模块和控制与分析模块；其中：
所述电浸润芯片模块包括：试剂储存区、生化反应区、荧光拍照区和驱动系统，以及各区之间的微通道；
所述CRISPR反应模块包括：含有荧光探针的CRISPR反应液、含有Cas12a的蛋白反应液和含有gRNA的gRNA反应液；所述蛋白反应液、gRNA反应液和CRISPR反应液均包含表面活性剂和酶缓冲液；
所述控制与分析模块通过驱动系统驱动待测核酸样品与所述CRISPR反应模块经过微通道进入所述生化反应区，所述待测核酸样品与CRISPR反应模块在所述生化反应区中混合并发生反应后产生荧光信号；所述荧光信号在荧光拍照区通过所述信号捕获模块捕获收集后输入所述控制与分析模块进行分析。
优选地，所述CRISPR反应模块通过微通道进入所述生化反应区中混合并发生反应的反应条件为本领域常规，例如为48℃、20min。
在本发明一较佳实施方案中，所述CRISPR反应模块和待测核酸样品在反应前贮存于所述试剂储存区。
所述蛋白反应液、gRNA反应液和CRISPR反应液均包含表面活性剂和酶缓冲液。
在本发明一较佳实施方案中，所述表面活性剂为Tween-20，例如终浓度为0.05～0.1％的Tween-20，所述酶缓冲液为10×NEB buffer 3.1。
在本发明一较佳实施方案中，所述gRNA的序列包括如SEQ ID NO：6、9、10、11和12的一种或多种所示的序列；优选所述gRNA的序列还包括如SEQ ID NO：13和14所示的序列。
在本发明的具体实施方案中，所述gRNA的序列如SEQ ID NO：6、10、11或12所示，或者如SEQ ID NO：6、10、13和14所示，或者如SEQ ID NO：9、10、13和14所 示。
所述待测核酸样品为DNA或RNA；优选所述DNA为经过反转录PCR得到的cDNA。
所述信号捕获模块可为本领域常规信号捕获收集装置，优选包括荧光仪、荧光光度计或荧光分光光度计。
所述控制与分析模块可为本领域常规分析装置，优选包括计算机、平板或数控设备。
在本发明一较佳实施方案中，所述蛋白反应液还包括Cas12a蛋白、RNase抑制剂、甘油以及无核酸酶水，所述gRNA反应液还包括gRNA、RNase抑制剂及无核酸酶水，所述CRISPR反应液还包括荧光探针。
所述荧光探针可为5’端和3’端分别具有荧光发光基团FAM和荧光淬灭基团BHQ1的DNA探针，例如为Fam-TTATT-Q。
所述蛋白反应液包括0.25～1μM优选0.5μM的Cas12a蛋白、4,000～10,000unit/mL优选5,340unit/mL的RNase抑制剂、10～30％优选15.69％甘油、0.05～0.5％(w/v)优选0.1％(w/v)的Tween-20和10×NEB buffer 3.1。
所述gRNA反应液包括：0.1～0.5μM优选0.3μM的gRNA、10×NEB buffer 3.1、2,000～10,000unit/mL优选4,000unit/mL的RNase抑制剂、0.05～0.5％(w/v)优选0.1％(w/v)的Tween-20。
所述CRISPR反应液包括：10～100μM优选25μM的荧光探针。
在本发明一更佳实施方案中，所述蛋白反应液由CRISPR蛋白贮存液和CRISPR蛋白稀释液按1∶1比例配制而成，其中，每0.1mL所述CRISPR蛋白贮存液包括：10μL 10μM的Cas12a蛋白、26.7μL 40,000unit/mL的RNase抑制剂、10μL 10×NEB buffer 3.1、52.3μL 60％(v/v)的甘油和1μL 10％(w/v)的Tween-20；
每0.5mL所述CRISPR蛋白稀释液包括：50μL的10×NEB buffer 3.1、5μL的10％(w/v)的Tween-20和445μL的无核酸酶水；
每0.5mL所述gRNA反应液包括：75μL 2μM的gRNA、50μL 10×NEBbuffer3.1、50μL 40,000unit/mL的RNase抑制剂、5μL 10％(w/v)的Tween-20和320μL的无核酸酶水；
每1mL所述CRISPR反应液包括：250μL 100μM的荧光探针、200μL 10×NEB buffer 3.1和10μL 10％(w/v)的Tween-20。
在本发明一较佳实施方案中，所述核酸检测系统还包括如SEQ ID NO：5所示的GAPDH gRNA。
在本发明一较佳实施方案中，所述核酸检测系统还包括核酸扩增反应模块；所述待 测核酸样品在所述核酸扩增反应模块发生扩增反应后，将单个液滴均匀分散为多个液滴，例如为3个液滴。
所述核酸扩增反应模块可通过RT-PCR、多重RT-PCR或RT-qPCR进行核酸扩增。
所述核酸扩增反应模块例如通过RT-PCR进行核酸扩增。
所述核酸扩增反应模块包括控温子模块和核酸扩增反应体系，所述核酸扩增反应体系包括实验反应液I、实验反应液II和RT-PCR引物溶液，所述实验反应液I包括热启动酶、Alpha RTase、RNase抑制剂和10×Solution I缓冲液，所述实验反应液II包括热启动酶缓冲液、dNTP、Tween-20和PF68。
在本发明一较佳实施方案中，所述核酸检测系统还包括阳性模板标准品和/或阴性模板标准品。
所述阳性模板标准品包括人工合成病毒RNA和内参RNA。
所述内参RNA为GAPDH、β-actin或18sRNA。
在本发明一实施方案中，每100μL所述实验反应液I包括：
7.5μL的HotStart HiTaq、4μL的Alpha RTase、5μL的RNase抑制剂和50μL的10×Solution I缓冲液；
每150μL所述实验反应液II包括：
100μL的5×HotStart HiTaq缓冲液、4μL 25mM的dNTP、2.5μL的10％(v/v)Tween-20和5μL的10％(w/v)PF68，余量为水；
每100μL所述阳性模板标准品包括：1μL 10ng/μL的人工合成病毒RNA或10μL 10ng/μL的内参RNA，余量为水；每100μL所述阴性模板标准品包括：10μL 10ng/μL的内参RNA，余量为水；
每225μL所述RT-PCR引物溶液包括：
0.5μL 100μM的内参RNA上游引物、0.5μL 100μM的内参RNA下游引物、1.75μL 100μM的待测核酸样品的RNA上游引物和1.75μL 100μM的待测核酸样品的RNA下游引物，余量为水；
例如，所述RT-PCR的反应条件为：10μL反应体系包括2μL实验反应液I和3μL实验反应液II、1μL待测核酸样品、2.25μL RT-PCR引物及1.75μL水。
所述RT-PCR的反应程序为50℃ 10分钟；例如RT-PCR后的PCR的反应程序为第一阶段95℃ 5分钟，第二阶段94℃ 10秒，57℃ 20秒，共30-40个循环例如38个循环，第三阶段68℃ 2分钟。
所述核酸检测系统还包括核酸提取模块，所述待测样品通过核酸提取模块提取由待 测样品获得。
所述核酸提取模块包括磁珠控制区与核酸提取体系。
所述磁珠控制区包括容纳样品的样本孔，以及连接所述样本孔和磁场区域的电极。所述样本在所述磁珠控制区内通过核酸提取体系进行磁珠提取。
所述磁珠提取可为本领域常规操作，通常包括抓取和释放磁珠、清洗磁珠、以及从磁珠上洗脱核酸的完整流程。
在本发明一较佳实施方案中，所述核酸提取体系包括：核酸提取裂解液、核酸提取结合液、核酸提取洗涤液1、核酸提取洗涤液2和核酸提取洗脱液；
每1mL所述核酸提取裂解液包括：
0.02mL 1M，pH为7.5的Tris-HCl、0.02mL0.5M，pH为8.0的EDTA、30％(w/v)的盐酸胍、0.02mL的10％(w/v)Triton X-100和0.01mL的10％(w/v)的Tween-20，余量为无核酸酶水；
每1mL所述核酸提取结合液包括：
0.4mL的50％(w/v)PEG6000、0.5mL 5M的NaCl、0.027mL的10％(w/v)Tween-20、0.002mL 0.5M的柠檬酸水溶液和0.07mL 10×稀释后的VDO磁珠，余量为无核酸酶水；
每5mL所述核酸提取洗涤液1包括：
0.1mL 1M，pH为7.5的Tris-HCl、0.5mL 5M的NaCl、0.05mL的10％(w/v)Tween-20和1.5mL的50％(w/v)PEG6000，余量为水；
每5mL所述核酸提取洗涤液2包括：
0.1mL 1M，pH为7.5的Tris-HCl、0.15mL 5M的NaCl、0.05mL的10％(w/v)Tween-20和2mL的50％(w/v)PEG6000，余量为水；
每5mL所述核酸提取洗脱液包括：
0.05mL 1M，pH为7.5的Tris-HCl和0.05mL的10％(w/v)Tween-20，余量为水；
在本发明一更佳实施方案中，所述磁珠控制区位于电浸润芯片模块上，所述核酸提取体系储存于所述试剂储存区；在所述磁珠控制区提取待测样品中的核酸，获得待测核酸样品。
所述待测样品为血液样本或者体液样本例如唾液样本。
所述待测样品中包含病毒，所述病毒为dsDNA病毒、ssDNA病毒、dsRNA病毒、ssRNA病毒、DNA反转录病毒或RNA反转录病毒；较佳地为dsRNA病毒或ssRNA病毒，例如COVID-19病毒。
本发明的第二方面提供一种非诊断目的的检测核酸的方法，其使用如第一方面所述的核酸检测系统，并包括以下步骤：
(1)控制与分析模块通过驱动系统驱动CRISPR反应模块和待测核酸样品进入所述电浸润芯片模块，并分别形成液滴；例如，所述CRISPR反应模块中的CRISPR反应液、蛋白反应液和gRNA反应液分别形成液滴；
(2)混合所述CRISPR反应模块形成的液滴和所述待测核酸样品形成的液滴，在生化反应区中发生反应后产生荧光信号；
(3)所述荧光信号在荧光拍照区通过所述信号捕获模块捕获收集后输入控制与分析模块进行分析；当所述荧光信号的强度超过阴性对照则判断存在目标核酸。
所述阴性对照可为本领域常规，例如内参基因。
在本发明一较佳实施方案中，所述(1)前还包括(02)：通过所述核酸扩增反应模块在所述生化反应区中对待测核酸样品进行扩增；由此(1)对扩增后的待测核酸样品形成液滴；
在本发明一更佳实施方案中，所述(02)前还包括(01)：通过所述核酸提取反应在所述磁珠控制区中对待测样品进行核酸提取；
例如，所述(02)中，在所述待测核酸样品的同一液滴内加入核酸扩增反应模块进行核酸扩增。
所述(1)的CRISPR反应模块中，先由gRNA反应液的液滴与CRISPR反应液的液滴混合，再与蛋白反应液的液滴混合。
在所述磁珠控制区中进行核酸提取，驱动系统驱动待测核酸样品及各模块进入所述生化反应区、形成液滴与液滴混合；其中，在所述生化反应区中进行核酸扩增的步骤均通过计算机、平板或数控设备控制。
所述检测核酸的方法可以是非诊断目的的，即可以应用于实验室科学研究或环境样本中的核酸检测，例如：
1.预防医学研究和公共卫生政策的制定
本领域技术人员知晓，现代医学分为两部分：预防医学和临床医学。本发明“非诊断目的的检测方法”，在预防医学中可以对环境中采集的样本(包括人体分泌物)进行检测，对环境是否被污染进行判断，从而决策是否对环境进行封锁、暂停营业、消杀等处置方式，并决策是否对这一地区、城市甚至更高层面升级传染病防控措施。从公共卫生角度出发对传染病进行预防，避免该环境残留的病毒成为传染源。
2.科学研究领域
(1)基础医学和生态学
本领域技术人员可以例如对水样进行检测，以判断病毒的传播时间和广泛度。再例如，巴西在2019年采集的下水道水样中检测出新冠病毒，早于世界第一例确诊病例的报道，可为病毒的溯源和传播时间提供更多的参考依据。又例如，美国加州在对死者进行尸检显示，其新冠病毒检测呈阳性，比美国报道的首例新冠肺炎死亡病例早三周。
(2)病毒学方面产品的研发
本领域技术人员知晓，当多种病毒在同一宿主体内同时感染，可能产生病毒突变，包括病毒自身核酸的重组，或来自不同病毒的核酸的重组。若出现病毒的突变，尤其当其发生在秋冬流感季，则情况更为严重。利用本发明的抗新冠病毒的抗体进行检测，可用于病毒学的研究，检测是否产生了病毒核酸的整合，可从预防医学的角度对病毒是否产生整合进行监测和及时预警。
本发明的第三方面提供一种计算机可读介质，所述计算机可读存储介质存储有计算机程序，其中，所述计算机程序被处理器执行时实现如第一方面所述核酸检测系统的功能或本发明的第二方面所述的非诊断目的的检测核酸的方法。
本发明的第四方面提供一种电子设备，其包括存储器和处理器；所述存储器包括存储在其中的可在处理器上运行的计算机程序；其中，所述处理器执行所述计算机程序时实现如本发明的第二方面所述的非诊断目的的检测核酸的方法的步骤。
本发明的积极有益效果：
(1)通过双重靶向区域筛选(针对病原核酸序列的扩增引物和CRISPRgRNA设计)降低假阳性率，通过设计针对病毒基因组的多个扩增片段以及多个CRISPR探针片段，在不增加时间成本和人力成本的条件下更有效果地减少假阴性率，避免当样品核酸浓度过低无法进行有效全长反转录获得实时荧光定量PCR目标序列，或者出现目标序列变异无法反转录出单个cDNA产物而导致实时荧光定量PCR产生假阴性的情况；
(2)将扩增过程和信号放大过程分离，使用PCR分别对目标片段进行扩增和快速的Cas效应蛋白的序列识别和反式单链DNA的切割达到特异的信号放大作用，相比传统的qPCR方法可提高检测的灵敏度；
(3)本发明使用的微流控系统能够快速产生多个微反应液滴，在高效快速进行检测的同时更大地提高了病原检测通量，能同时检测DNA或RNA为主体的多个病原，且微流控的全封闭环境大大降低了环境和样品之间的污染率。
附图说明
图1为本发明核酸检测方法示意图。
图2为实施例内参基因引物位点示意图。
图3为实施例1新冠病毒引物位点示意图。
图4为实施例1的PCR扩增结果示意图。
图5为实施例1的gRNA-GAPDH对GAPDH引物扩增的PCR产物切割活性示意图。
图6为实施例1的gRNA-GAPDH对ORF1ab引物扩增的PCR产物切割活性示意图。
图7为实施例1的gRNA-ORF1ab对ORF1ab引物扩增的PCR产物切割活性示意图。
图8为实施例1的gRNA-ORF1ab对GAPDH引物扩增的PCR产物切割活性示意图。
图9为实施例1阴性对照gRNA对GAPDH/ORF1ab引物扩增的PCR产物切割活性示意图。
图10为实施例1阳性模板标准品切割活性示意图。
图11为实施例1阴性模板标准品切割活性示意图。
图12为实施例1超纯水切割活性示意图。
图13为不同gRNA及组合的检测结果示意图。
图14为实施例1与商业试剂盒检测结果示意图。
图15为本发明芯片微结构示意图。
图16为实施例1芯片验证结果示意图。
图17为标准品检测效果示意图。
图18为实施例2的电子设备结构示意图。
具体实施方式
使用的试剂和材料见下表：
  厂家 货号
VDO磁珠 苏州为度生物技术有限公司 MS01H
Carrier RNA 杭州新景生物试剂开发有限公司 4003130
RT-PCR试剂盒 深圳华大智造生物科技股份有限公司 1000023286
CRISPR试剂盒 上海吐露港生物科技有限公司 32108-01
qPCR仪 赛默飞世尔科技 Stepone Plus
实施例的检测原理如图1所示。
实施例1
第一部分 试管实验调试
第一节 获取样本
1.获取带有保存液的咽拭子。
按照咽拭子采集标准方法进行采样。具体步骤如下：
1.1核对信息，将身份条形码黏贴于拭子采集管管壁上。
1.2执行手卫生，检查戴手套，准备压舌板和咽拭子。
1.3深吸气，(摘口罩)张嘴屏气，压舌板轻压舌面暴露咽部。
1.4在咽后壁和扁桃体轻轻刮取样本。
1.5嘱咐患者屏气戴上口罩后，再进行正常呼吸。
1.6将咽拭子放入有保存液的采集管中，扭断拭子，盖上管盖。
1.7将带有咽拭子头的采集管放置在56℃中灭活30分钟，至此获得符合下游提取要求的病毒保存液样本。
所述保存液为VTM(Viral Transport Medium)保存液，包括：Hank’s平衡盐溶液(pH为7.2～7.4)、1000IU/mL青霉素、1000IU/mL链霉素、0.5％(w/v)牛血清白蛋白、0.016％(w/v)苯酚红和50％(v/v)甘油。
所述保存液还可为磷酸盐缓冲液，包括：137mM氯化钠、2.7mM氯化钾、10mM磷酸二氢钠、2mM磷酸二氢钾，pH为7.4。
2.获取不带有保存液的干咽拭子
按照修改后的咽拭子采集方法进行采样。具体步骤如下：
2.1核对信息，将身份条形码黏贴于拭子采集管管壁上。
2.2执行手卫生，检查戴手套，准备压舌板和咽拭子。
2.3深吸气，(摘口罩)张嘴屏气，压舌板轻压舌面暴露咽部。
2.4在咽后壁和扁桃体轻轻刮取样本。
2.5嘱咐患者屏气戴上口罩后，再进行正常呼吸。
2.6将咽拭子放入空的采集管中，扭断拭子，盖上管盖。
2.7将带有咽拭子头的收集管放置在56℃中灭活30分钟，至此获得符合下游提取要求的干咽拭子。
3.以人源RNA与人工合成的病毒RNA的混合物为实验阳性标准品，以人源RNA为实验阴性标准品，步骤如下：
3.1人工合成病毒RNA
3.1.1将需要检测的病毒序列(GenomeNet登录号NC_045512.2区域13321-15459)送至外包核酸合成服务商(北京六合华大基因科技有限公司)进行合成。
3.1.2将合成的质粒和pET28a表达载体分别进行双酶切后纯化，获得目的片段和线性化带有粘性末端的表达载体。
3.1.3将目的片段连接至表达载体上。
3.1.4使用T7 RiboMAX TM快速大量RNA制备系统(Promega，货号P1320)进行体外转录。
3.1.5将转录后得到的RNA进行琼脂糖凝胶电泳检测，并使用Qubit RNA HS assay kit(赛默飞世尔科技，Q32855)进行定量。
3.1.6将定量和质控完成后的RNA保存至-80℃冰箱，至此完成病毒RNA样本制备。
3.2人源RNA为Universal Human Reference RNA(UHRR)，采购自德国Agilent公司。
第二节 引物设计
1.人源RNA内参引物设计：
选取UHRR，即通用人源参考RNA标准品(Universal Human Reference RNA，Agilent Co.Ltd.)，作为体外测试的阴性对照标准品。基于人类基因表达的丰度，分别选取GAPDH基因、β-actin基因和18S基因所表达的RNA作为实验内参。
GAPDH(或G3PDH)，即甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase)，广泛分布于各种组织中的细胞。β-actin负责表达细胞中一种重要的骨架蛋白，在人体细胞中有较高的表达丰度。18S RNA为人核糖体RNA的重要组成成分，在人体细胞的总RNA中含量最高。选取这些内参基因的主要目的是为了检测本实验方案的可靠性，避免因实验操作导致的假阳性出现。
通过使用Prime Primer v5.0软件计算与优化，针对三种基因分别设计了不同的引物位点，如图2所示。经测试，图中的位点均可作为内参基因应用到实施例中，且内参引物设计的位点包括但不限于上图中展示的这些位点。本实施例以GAPDH中的一对引物为例展开实验。
引物序列信息如下：
GAPDH-F：TTCATTGACCTCAACTACATGGTTTAC(SEQ ID NO：1)
GAPDH-R：GATTTTGGAGGGATCTCGCTCCTG(SEQ ID NO：2)
2.病毒RNA检测引物位点设计：
新型冠状病毒，即COVID-19或2019-nCoV，为单链RNA冠状病毒，基于已经发表的基因序列(GenBank：MN908947.3)设计了多组针对新冠病毒不同基因的检测位点，其引物设计如图3所示。
经测试，这些位点均可作为检测基因应用到实施例中，且检测基因的引物设计的位点包括但不限于上图中展示的这些位点。
本实施例以ORF1ab上的一对引物位点为例展开实验。
引物序列信息如下：
ORF1ab-F：CCCTGTGGGTTTTACACTTAA(SEQ ID NO：3)
ORF1ab-R：ACGATTGTGCATCAGCTGA(SEQ ID NO：4)
3.Guide RNA序列设计：
GuideRNA(gRNA)，即向导RNA，是实现CRISPR荧光检测的重要组分之一。gRNA通过与DNA目标片段序列的互补配对，激活Cas蛋白活性并实现下游的荧光检测。gRNA的序列特异性决定了其检测结果的特异性。容易得知，gRNA的序列应当与所要检测的扩增产物的序列互补配对。如图2所示，针对人源内参，基于图2中位点，分别设计了其对应gRNA序列。针对已发表的病毒序列，基于图3中位点，分别设计了其对应gRNA序列。
经测试，这gRNA均可作为检测靶点应用到实施例中，且检测靶点的gRNA设计的位点包括但不限于图中展示的这些位点。基于本实施例中使用的GAPDH和ORF1ab引物，我们以gRNA-GAPDH和gRNA-ORF1ab为例展开实验。
gRNA序列信息如下：
gRNA-GAPDH：
Figure PCTCN2021096620-appb-000001
gRNA-ORF1ab：
Figure PCTCN2021096620-appb-000002
gRNA E8.1：
Figure PCTCN2021096620-appb-000003
gRNA E8.2：
Figure PCTCN2021096620-appb-000004
gRNA 203(N基因，靶位点位置20604)：
Figure PCTCN2021096620-appb-000005
gRNA 4.2(S基因，靶位点位置24831)：
Figure PCTCN2021096620-appb-000006
gRNA 004.1(ORF1ab基因，靶位点位置978)：
Figure PCTCN2021096620-appb-000007
gRNA 004.2(ORF1ab基因，靶位点位置1085)：
Figure PCTCN2021096620-appb-000008
gRNA 11.1(ORF1ab基因，靶位点位置2903)：
Figure PCTCN2021096620-appb-000009
gRNA 11.2(ORF1ab基因，靶位点位置2915)：
Figure PCTCN2021096620-appb-000010
第三节 试剂配制准备
1.提取试剂配制。
按照表1配制核酸提取裂解液：
表1 核酸提取裂解液
Figure PCTCN2021096620-appb-000011
按照表2配制核酸提取结合液：
表2 核酸提取结合液(1mL)
Figure PCTCN2021096620-appb-000012
按照表3配制核酸提取洗涤液1：
表3 核酸提取洗涤液1(5mL)
Figure PCTCN2021096620-appb-000013
按照表4配制核酸提取洗涤液2：
表4 核酸提取洗涤液2(5mL)
Figure PCTCN2021096620-appb-000014
按照表5配制核酸提取洗脱液：
表5 核酸提取洗脱液(5mL)
Figure PCTCN2021096620-appb-000015
2.RT-PCR试剂配制。
按照表6进行实验反应液I的配制：
表6 实验反应液I
Figure PCTCN2021096620-appb-000016
Figure PCTCN2021096620-appb-000017
按照表7进行实验反应液II的配制：
表7 实验反应液II
Figure PCTCN2021096620-appb-000018
按照表8和表9进行RT-PCR阳性和阴性模板标准品的配制：
表8 RT-PCR阳性模板标准品
Figure PCTCN2021096620-appb-000019
表9 RT-PCR阴性模板标准品
Figure PCTCN2021096620-appb-000020
按照表10进行RT-PCR引物溶液的配制：
表10 RT-PCR引物溶液
Figure PCTCN2021096620-appb-000021
3.CRISPR试剂配制。
按照表11-14进行CRISPR试剂配制：
表11 5×CRISPR反应液(1mL)
Figure PCTCN2021096620-appb-000022
本领域技术人员皆知，此处配制的5×CRISPR反应液可依配方调整为2×或10×等任意倍数浓度的CRISPR反应液，并根据需要在使用的时候按比例稀释。
表12 CRISPR蛋白贮存液(0.1mL)
Figure PCTCN2021096620-appb-000023
表13 CRISPR蛋白稀释液(0.5mL)
Figure PCTCN2021096620-appb-000024
表14 gRNA反应液(0.5mL)
Figure PCTCN2021096620-appb-000025
第四节 提取实验操作步骤
1、咽拭子保存液提取流程：
1.1将Carrier RNA从冷冻包装盒中取出，解冻后置于冰上。
1.2向含有100μL核酸提取裂解液(含30mg盐酸胍)的离心管中加入灭活后的100μL病毒保存液，得到病毒液，颠倒混匀后室温孵育30分钟。
1.3孵育完成后，从上一步离心管中取121μL裂解后的病毒液，加入新的离心管中，再向新的离心管中加入77μL核酸提取结合液，以及2μL Carrier RNA。颠倒混匀，室温静置孵育10分钟。
1.4室温孵育结束后，将样品管置于磁力架上至澄清，弃掉上清。
1.5加入10μL的核酸提取洗涤液1，振荡混匀离心，置于磁力架至澄清，弃掉上清。
1.6加入10μL的核酸提取洗涤液2，振荡混匀离心，置于磁力架至澄清，弃掉上清。
1.7加入5μL核酸提取洗脱液(Nucleic Acid Extractor Eluent，NEE)，振荡混匀后，置于冰上备用。
2、干拭子提取流程：
2.1将Carrier RNA从冷冻保藏的试剂盒中取出，解冻后置于冰上。
2.2向含有咽拭子头的管中加入1mL核酸提取裂解液，颠倒混匀后室温孵育30分钟。
2.3孵育完成后，从上一步保存管中取120μl核酸提取裂解液，加入新的离心管中，再向新的离心管中加入77μl核酸提取结合液，以及1μl Carry RNA。颠倒混匀，室温静置孵育10分钟。
2.4室温孵育结束后，将样品管置于磁力架上至澄清，弃掉上清。
2.5加入10μL的核酸提取洗涤液1，振荡混匀离心，置于磁力架至澄清，弃掉上清。
2.6加入10μL的核酸提取洗涤液2，振荡混匀离心，置于磁力架至澄清，弃掉上清。
2.7加入5μL核酸提取洗脱液(NEE)，振荡混匀后，置于冰上备用。
3、实验阳性/阴性标准品提取流程：
3.1将Carrier RNA从冷冻保藏的试剂盒中取出，解冻后置于冰上。
3.2将阳性标准品/阴性标准品从-80℃冰箱中取出，置于冰上解冻。解冻后梯度稀释至需要的浓度，例如从10 10copies/mL稀释至10copies/mL。
3.3取一个新的离心管，加入上一步稀释好的标准品后，补核酸提取裂解液至120μl，再向其中加入77μl核酸提取结合液，以及1μl Carry RNA。颠倒混匀，室温静置孵育10 分钟。
3.4室温孵育结束后，将样品管置于磁力架上至澄清，弃掉上清。
3.5加入10μL的核酸提取洗涤液1，振荡混匀离心，置于磁力架至澄清，弃掉上清。
3.6加入10μL的核酸提取洗涤液2，振荡混匀离心，置于磁力架至澄清，弃掉上清。
3.7加入5μL核酸提取洗脱液(NEE)，振荡混匀后，置于冰上备用。
第五节 RT-PCR实验步骤
1、取3个250μl的PCR反应管，分别标记①、②和③。
2、分别准确吸取2μl实验反应液I和3μl实验反应液II，置于①、②和③PCR管中充分混匀。
3、准确吸取1μl RT-PCR阳性模板标准品，加入①管的反应混合液中；准确吸取1μl RT-PCR阴性模板标准品，加入②管的反应混合液中；准确吸取1μl超纯水，加入③管的反应混合液中。
4、向上述三种混合液中分别加入2.25μl RT-PCR引物及1.75μl超纯水，充分震荡混匀。
5、小心加入30μl硅油进行液封。
将PCR管置于PCR扩增仪上，设定如下反应程序
Figure PCTCN2021096620-appb-000026
6、反应结束后，小心弃去上层硅油，吸取5μl反应液进行琼脂糖电泳检测。
7、配置4％琼脂糖凝胶，加入步骤6中的反应产物及DNA染料，在160伏特电压下运行45分钟。拍照分析实验结果。
第六节 CRISPR实验步骤
1、Off Chip荧光检测
1.1取1μL如表12所示的CRISPR蛋白贮存液和1μL如表13所示CRISPR蛋白稀释液混合，配制蛋白反应液，冰上放置。
1.2取1μL所述蛋白反应液和如表14所示1μL gRNA反应液混匀，在室温下孵育10分钟。
1.3在1.2得到的混合溶液中加入1μL如表11所示的5×CRISPR反应液，与2μL阳性对照或阴性对照品的PCR产物混匀，在酶标仪或qPCR仪上48℃反应20min，每分钟采集FAM荧光信号值。
2、On Chip荧光检测
2.1每组样品使用一张独立包装芯片，加入3.5mL真空包装硅油后推入检测器。
2.2如图15所示，将3μL如第五节所述得到的RT-PCR的DNA产物和CRISPR buffer的混合液加入DF仪器chip的S1孔上，将2μL蛋白反应液和gRNA反应液加到S2孔上；
2.3按照设定程序进行样品混合，在生化反应区加热(反应温度48℃，20min)后样品依次移至荧光拍照区，设置相机曝光时间0.125秒，在荧光模式下进行拍照，记录结果。
第七节 实验结果
1、RNA提取
由于咽拭子本身刮取到的细胞和病毒数量较低，提取后无法进行质控，提取产物直接随磁珠进入到下一步多重PCR实验中。
2、多重PCR扩增
如图4所示，琼脂糖凝胶电泳结果显示，在323bp和119bp处有明显扩增，分别与GAPDH和ORF1ab的PCR产物对应。扩增用模板：①超纯水；②阴性对照；③阳性对照。
3、CRISPR检测
3.1内参gRNA-GAPDH
a)以GAPDH引物，对不同浓度UHRR进行RT-PCR，用gRNA-GAPDH切割PCR产物，证实gRNA-GAPDH对PCR产物具有切割活性，且信号值随UHRR投入量提高而增强，如图5所示。
b)gRNA-GAPDH对用病毒cDNA为模板，ORF1ab引物扩增的RT-PCR产物没有切割活性，如图6所示。
3.2病原核酸序列区域目的序列ORF1ab
a)以ORF1ab引物，对不同浓度病毒cDNA进行RT-PCR，用gRNA-ORF1ab切割PCR产物，验证该gRNA具有切割活性，且切割信号随cDNA投入量提高而增强，如图 7所示。
b)gRNA-ORF1ab对于以UHRR为模板用GAPDH引物扩增的RT-PCR产物没有切割活性，如图8所示。
3.3病原核酸序列阴性对照
如图9所示，阴性对照gRNA-84对本实施例中其它PCR primer的扩增产物1ng/μL GAPDH以及1ng/μL ORF1ab均未产生切割活性，可用于定义背景参比荧光值。
所述gRNA-84的序列如SEQ ID NO：16所示。
SEQ ID NO：16：
Figure PCTCN2021096620-appb-000027
3.4多重RT-PCR及CRISPR检测
将第七节中所用的阳性模板标准品③进行多重RT-PCR扩增，分别用gRNA-ORF1ab、gRNA-GAPDH和gRNA-84进行切割。结果如图10所示，其中gRNA-84报告为阴性结果，gRNA-GAPDH和gRNA-ORF1ab报告为阳性结果，信号显著高于gRNA-84，和预期一致。
将第七节中所用的RT-PCR阴性模板标准品②进行多重RT-PCR扩增，之后分别用gRNA-ORF1ab、gRNA-GAPDH和gRNA-84进行切割，结果如图11所示。其中gRNA-84和gRNA-ORF1ab报告为阴性结果，gRNA-GAPDH报告为阳性结果，和预期相符。
将第七节中所用的1μL超纯水①进行多重RT-PCR扩增，之后分别用gRNA-ORF1ab、gRNA-GAPDH和gRNA-84进行切割，结果如图12所示。gRNA-84、gRNA-ORF1ab和gRNA-GAPDH均报告为阴性结果，和预期一致。
如图13中a、b和c所示，将前述其他gRNA(SEQ ID NO：7～15)按照上述方法单独或者组合进行检测，结果表明序列如SEQ ID NO：6、10、11或12所示的gRNA、序列如SEQ ID NO：6、10、14和15所示的gRNA、以及序列如SEQ ID NO：9、10、14和15所示的gRNA的多重PCR及gRNA可用于样本的鉴定，且具有良好的检测能力。
按照说明书，使用CRISPR试剂盒(上海吐露港生物科技有限公司，32108-01)进行实验，结果如图14所示。表明，本实施例相比现有试剂盒，提升了检测能力。
第二部分 芯片实验验证部分
如图15所示，数字微流控芯片下表面由若干控制单元组成，芯片放入数字微流控机器后，通过控制芯片上相邻控制单元端的电压，使之与芯片上表面的接地端的电压相同或形成压差(俗称“通/断”)，来达到驱动液体变形或者液滴移动的目的。每个控制单元 可以控制1微升的液滴，多个控制单元可以控制更大体积的液滴。移动液滴并配合其他元件，例如加热元件、水冷装置和装有步进电机的磁铁，可以保证试剂保存、试剂取液、试剂移动、生化反应和磁珠纯化的顺利进行。
首先将数字微流控芯片用硅油灌满，根据图15将本反应所需所有试剂依次加载到试剂储存区M1-M8和L1-L8。取待检测人员的咽拭子样品加入1mL的裂解液中，浸泡至少5min。适当混匀含有咽拭子的裂解液并重悬混匀磁珠提取液，取120μL含有咽拭子的裂解液上清加入灭菌的1.5mL离心管中，依次加入77μL捕获磁珠提取液，使用100μL移液枪吹打10-15次充分混匀。将混匀后的液体分4次加载在数字微流控芯片上的4个样品加样孔S1-S4中(每次50μL，最后一次全部加入)。
启动数字微流控仪器，待系统自检后，将数字微流控芯片载入数字微流控仪器中并运行。在数字微流控仪器的控制下，将依次进行上述样本提取、RT-PCR和CRISPR反应。
在CRISPR切割反应中，使用了三种不同的gRNA，分别是阴性对照gRNA-84、GAPDH内参gRNA-GAPDH和COVID-19靶标gRNA-ORF1ab，分别对应不识别任何区域、识别GAPDH内参(该内参位于人类基因组上)以及识别新冠病毒的特异序列。一旦gRNA找到了能够识别的区域，CRISPR蛋白便能够激活切割反应液中的荧光探针从而产生荧光。CRISPR反应10min后，液滴将移动到荧光拍照区进行拍照，根据液滴的荧光强度判断样本是否含有新型冠状病毒核酸。
图16为使用模拟阴性样本(仅含有通用人类参照RNA)以及模拟阳性样本(含有通用人类参照RNA和含新冠病毒部分区域序列的RNA)在芯片上反应后荧光拍照的实验结果。
从结果上可看出，对于模拟阳性样本，含GAPDH内参gRNA-GAPDH液滴和含COVID-19靶标gRNA-ORF1ab液滴的荧光强度均大于阴性对照，结果呈报阳性；对于模拟阴性样本，仅含GAPDH内参gRNA液滴的荧光强度大于阴性对照，而含COVID-19靶标gRNA-ORF1ab液滴的荧光强度小于阴性对照，结果呈报阴性。如图17所示，本实施例的检测灵敏度达到200copies/mL，即检测限为0.2copies/μL，与qPCR检测的预期阳性一致率为91.67％，预期阴性一致率为100％，证明实验成功。
实施例2
1.电子设备
本实施例提供了一种电子设备，电子设备可以通过计算设备的形式表现(例如可以为服务器设备)，包括存储器、处理器及存储在存储器上并可在处理器上运行的计算机程序，其中处理器执行计算机程序时可以实现本发明实施例1中的非诊断目的的检测核酸 的方法。
图18示出了本实施例的硬件结构示意图，电子设备9具体包括：
至少一个处理器91、至少一个存储器92以及用于连接不同系统组件(包括处理器91和存储器92)的总线93，其中：
总线93包括数据总线、地址总线和控制总线。
存储器92包括易失性存储器，例如随机存取存储器(RAM)921和/或高速缓存存储器922，还可以进一步包括只读存储器(ROM)923。
存储器92还包括具有一组(至少一个)程序模块924的程序/实用工具925，这样的程序模块924包括但不限于：操作系统、一个或者多个应用程序、其它程序模块以及程序数据，这些示例中的每一个或某种组合中可能包括网络环境的实现。
处理器91通过运行存储在存储器92中的计算机程序，从而执行各种功能应用以及数据处理，例如本发明实施例1中非诊断目的的检测核酸的方法。
电子设备9进一步可以与一个或多个外部设备94(例如键盘、指向设备等)通信。这种通信可以通过输入/输出(I/O)接口95进行。并且，电子设备9还可以通过网络适配器96与一个或者多个网络(例如局域网(LAN)，广域网(WAN)和/或公共网络，例如因特网)通信。网络适配器96通过总线93与电子设备9的其它模块通信。应当明白，尽管图中未示出，可以结合电子设备9使用其它硬件和/或软件模块，包括但不限于：微代码、设备驱动器、冗余处理器、外部磁盘驱动阵列、RAID(磁盘阵列)系统、磁带驱动器以及数据备份存储系统等。
应当注意，尽管在上文详细描述中提及了电子设备的若干单元/模块或子单元/模块，但是这种划分仅仅是示例性的并非强制性的。实际上，根据本申请的实施方式，上文描述的两个或更多单元/模块的特征和功能可以在一个单元/模块中具体化。反之，上文描述的一个单元/模块的特征和功能可以进一步划分为由多个单元/模块来具体化。
2.计算机可读存储介质
本发明实施例提供了一种计算机可读存储介质，其上存储有计算机程序，程序被处理器执行时实现本发明实施例1中非诊断目的的检测核酸的方法的步骤。
其中，可读存储介质可以采用的更具体可以包括但不限于：便携式盘、硬盘、随机存取存储器、只读存储器、可擦拭可编程只读存储器、光存储器件、磁存储器件或上述的任意合适的组合。
在可能的实施方式中，本发明还可以实现为一种程序产品的形式，其包括程序代码，当所述程序产品在终端设备上运行时，所述程序代码用于使所述终端设备执行实现本发 明实施例1中非诊断目的的检测核酸的方法的步骤。
其中，可以以一种或多种程序设计语言的任意组合来编写用于执行本发明的程序代码，所述程序代码可以完全地在用户设备上执行、部分地在用户设备上执行、作为一个独立的软件包执行、部分在用户设备上部分在远程设备上执行或完全在远程设备上执行。虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (10)

  1. 一种基于电浸润的CRISPR的核酸检测系统，其特征在于，其包括：电浸润芯片模块、CRISPR反应模块、信号捕获模块和控制与分析模块；其中：
    所述电浸润芯片模块包括：试剂储存区、生化反应区、荧光拍照区和驱动系统，以及各区之间的微通道；
    所述CRISPR反应模块包括：含有荧光探针的CRISPR反应液、含有Cas12a的蛋白反应液和含有gRNA的gRNA反应液；所述蛋白反应液、gRNA反应液和CRISPR反应液均包含表面活性剂和酶缓冲液；
    所述控制与分析模块通过驱动系统驱动待测核酸样品与所述CRISPR反应模块经过微通道进入所述生化反应区，所述待测核酸样品与CRISPR反应模块在所述生化反应区中混合并发生反应后产生荧光信号；所述荧光信号在荧光拍照区通过所述信号捕获模块捕获收集后输入所述控制与分析模块进行分析。
  2. 如权利要求1所述的核酸检测系统，其特征在于，所述反应的条件为48℃、20min；
    和/或，所述CRISPR反应模块和待测核酸样品在反应前贮存于所述试剂储存区，
    和/或，所述表面活性剂为Tween-20，例如终浓度为0.05～0.1％的Tween-20，所述酶缓冲液为10×NEB buffer 3.1；
    和/或，所述gRNA的序列包括如SEQ ID NO：6、9、10、11和12的一种或多种所示的序列；优选所述gRNA的序列还包括如SEQ ID NO：13和14所示的序列；例如所述gRNA的序列如SEQ ID NO：6、10、11或12所示，或者如SEQ ID NO：6、10、13和14所示，或者如SEQ ID NO：9、10、13和14所示；
    和/或，所述待测核酸样品为DNA或RNA；优选所述DNA为经过反转录PCR得到的cDNA；
    和/或，所述信号捕获模块包括荧光仪、荧光光度计或荧光分光光度计；
    和/或，所述控制与分析模块包括计算机、平板或数控设备；
    较佳地，所述蛋白反应液还包括Cas12a蛋白、RNase抑制剂、甘油以及无核酸酶水，所述gRNA反应液还包括gRNA、RNase抑制剂及无核酸酶水，所述CRISPR反应液还包括荧光探针；例如：所述荧光探针为5’端和3’端分别具有荧光发光基团FAM和荧光淬灭基团BHQ1的DNA探针，例如为Fam-TTATT-Q；所述蛋白反应液包括0.25～1μM优选0.5μM的Cas12a蛋白、4,000～10,000unit/mL优选5,340unit/mL的RNase抑制剂、10～30％优选15.69％甘油、0.05～0.5％(w/v)优选0.1％(w/v)的Tween-20和10×NEB buffer 3.1；所述gRNA反应液包括：0.1～0.5μM优选0.3μM的gRNA、10×NEB buffer 3.1、 2,000～10,000unit/mL优选4,000unit/mL的RNase抑制剂、0.05～0.5％(w/v)优选0.1％(w/v)的Tween-20；所述CRISPR反应液包括：10～100μM优选25μM的荧光探针；
    更佳地，所述蛋白反应液由CRISPR蛋白贮存液和CRISPR蛋白稀释液按1∶1比例配制而成，其中，每0.1mL所述CRISPR蛋白贮存液包括：10μL 10μM的Cas12a蛋白、26.7μL 40,000unit/mL的RNase抑制剂、10μL 10×NEB buffer 3.1、52.3μL 60％(v/v)的甘油和1μL 10％(w/v)的Tween-20；每0.5mL所述CRISPR蛋白稀释液包括：50μL的10×NEB buffer 3.1、5μL的10％(w/v)的Tween-20和445μL的无核酸酶水；
    每0.5mL所述gRNA反应液包括：75μL 2μM的gRNA、50μL 10×NEB buffer 3.1、50μL 40,000unit/mL的RNase抑制剂、5μL 10％(w/v)的Tween-20和320μL的无核酸酶水；
    每1mL所述CRISPR反应液包括：250μL 100μM的荧光探针、200μL 10×NEB buffer 3.1和10μL 10％(w/v)的Tween-20。
  3. 如权利要求2所述的核酸检测系统，其特征在于，所述核酸检测系统还包括如SEQ ID NO：5所示的GAPDH gRNA；
    和/或，所述核酸检测系统还包括核酸扩增反应模块；所述待测核酸样品在所述核酸扩增反应模块发生扩增反应后，将单个液滴均匀分散为多个液滴，例如为3个液滴；
    较佳地，所述核酸扩增反应模块通过RT-PCR、多重RT-PCR或RT-qPCR进行核酸扩增；
    更佳地，所述核酸扩增反应模块通过RT-PCR进行核酸扩增；所述核酸扩增反应模块包括控温子模块和核酸扩增反应体系，所述核酸扩增反应体系包括实验反应液I、实验反应液II和RT-PCR引物溶液，所述实验反应液I包括热启动酶、Alpha RTase、RNase抑制剂和10×Solution I缓冲液，所述实验反应液II包括热启动酶缓冲液、dNTP、Tween-20和PF68。
  4. 如权利要求1～3任一项所述的核酸检测系统，其特征在于，所述核酸检测系统还包括阳性模板标准品和/或阴性模板标准品；
    较佳地，所述阳性模板标准品包括人工合成病毒RNA和内参RNA；
    所述内参RNA为GAPDH、β-actin或18sRNA；
    更佳地，每100μL所述实验反应液I包括：
    7.5μL的HotStart HiTaq、4μL的Alpha RTase、5μL的RNase抑制剂和50μL的10×Solution I缓冲液；
    每150μL所述实验反应液II包括：
    100μL的5×HotStart HiTaq缓冲液、4μL 25mM的dNTP、2.5μL的10％(v/v)Tween-20和5μL的10％(w/v)PF68，余量为水；
    每100μL所述阳性模板标准品包括：1μL 10ng/μL的人工合成病毒RNA或10μL 10ng/μL的内参RNA，余量为水；每100μL所述阴性模板标准品包括：10μL 10ng/μL的内参RNA，余量为水；
    每225μL所述RT-PCR引物溶液包括：
    0.5μL 100μM的内参RNA上游引物、0.5μL 100μM的内参RNA下游引物、1.75μL 100μM的待测核酸样品的RNA上游引物和1.75μL 100μM的待测核酸样品的RNA下游引物，余量为水；
    进一步更佳地，所述RT-PCR的反应条件为：10μL反应体系包括2μL实验反应液I和3μL实验反应液II、1μL待测核酸样品、2.25μL RT-PCR引物及1.75μL水；例如RT-PCR的反应程序为50℃ 10分钟；例如RT-PCR后的PCR的反应程序为第一阶段95℃ 5分钟，第二阶段94℃ 10秒，57℃ 20秒，共30-40个循环例如38个循环，第三阶段68℃ 2分钟。
  5. 如权利要求1～4任一项所述的核酸检测系统，其特征在于，所述核酸检测系统还包括核酸提取模块，所述待测样品通过核酸提取模块提取由待测样品获得；
    所述核酸提取模块包括磁珠控制区与核酸提取体系；
    较佳地，所述核酸提取体系包括：核酸提取裂解液、核酸提取结合液、核酸提取洗涤液1、核酸提取洗涤液2和核酸提取洗脱液；
    每1mL所述核酸提取裂解液包括：
    0.02mL 1M，pH为7.5的Tris-HCl、0.02mL 0.5M，pH为8.0的EDTA、30％(w/v)的盐酸胍、0.02mL的10％(w/v)Triton X-100和0.01mL的10％(w/v)的Tween-20，余量为无核酸酶水；
    每1mL所述核酸提取结合液包括：
    0.4mL的50％(w/v)PEG6000、0.5mL 5M的NaCl、0.027mL的10％(w/v)Tween-20、0.002mL 0.5M的柠檬酸水溶液和0.07mL 10×稀释后的VDO磁珠，余量为无核酸酶水；
    每5mL所述核酸提取洗涤液1包括：
    0.1mL 1M，pH为7.5的Tris-HCl、0.5mL 5M的NaCl、0.05mL的10％(w/v)Tween-20和1.5mL的50％(w/v)PEG6000，余量为水；
    每5mL所述核酸提取洗涤液2包括：
    0.1mL 1M，pH为7.5的Tris-HCl、0.15mL 5M的NaCl、0.05mL的10％(w/v)Tween-20和2mL的50％(w/v)PEG6000，余量为水；
    每5mL所述核酸提取洗脱液包括：
    0.05mL 1M，pH为7.5的Tris-HCl和0.05mL的10％(w/v)Tween-20，余量为水；
    更佳地，所述磁珠控制区位于电浸润芯片模块上，所述核酸提取体系储存于所述试剂储存区；在所述磁珠控制区提取待测样品中的核酸，获得待测核酸样品。
  6. 如权利要求1～5任一项所述的核酸检测系统，其特征在于，所述待测样品为血液样本或者体液样本例如唾液样本；
    和/或，所述待测样品中包含病毒，所述病毒为dsDNA病毒、ssDNA病毒、dsRNA病毒、ssRNA病毒、DNA反转录病毒或RNA反转录病毒；较佳地为dsRNA病毒或ssRNA病毒，例如COVID-19病毒。
  7. 一种非诊断目的的检测核酸的方法，其特征在于，其使用如权利要求1～6任一项所述的核酸检测系统，并包括以下步骤：
    (1)控制与分析模块通过驱动系统驱动CRISPR反应模块和待测核酸样品进入所述电浸润芯片模块，并分别形成液滴；例如，所述CRISPR反应模块中的CRISPR反应液、蛋白反应液和gRNA反应液分别形成液滴；
    (2)混合所述CRISPR反应模块形成的液滴和所述待测核酸样品形成的液滴，在生化反应区中发生反应后产生荧光信号；
    (3)所述荧光信号在荧光拍照区通过所述信号捕获模块捕获收集后输入控制与分析模块进行分析；当所述荧光信号的强度超过阴性对照则判断存在目标核酸；
    较佳地，所述(1)前还包括(02)：通过所述核酸扩增反应模块在所述生化反应区中对待测核酸样品进行扩增；由此(1)对扩增后的待测核酸样品形成液滴；
    更佳地，所述(02)前还包括(01)：通过所述核酸提取反应在所述磁珠控制区中对待测样品进行核酸提取；
    进一步更佳地，所述(02)中，在所述待测核酸样品的同一液滴内加入核酸扩增反应模块进行核酸扩增。
  8. 如权利要求7所述的方法，其特征在于，所述(1)的CRISPR反应模块中，先由gRNA反应液的液滴与CRISPR反应液的液滴混合，再与蛋白反应液的液滴混合；优选地，在所述磁珠控制区中进行核酸提取，驱动系统驱动待测核酸样品及各模块进入所述生化反应区、形成液滴与液滴混合；其中，在所述生化反应区中进行核酸扩增的步骤均通过计算机、平板或数控设备控制。
  9. 一种电子设备，其包括存储器和处理器；所述存储器包括存储在其中的可在处理器上运行的计算机程序；其特征在于，
    所述处理器执行所述计算机程序时实现如权利要求7或8所述的非诊断目的的检测核酸的方法。
  10. 一种计算机可读介质，所述计算机可读存储介质存储有计算机程序，其中，所述计算机程序被处理器执行时实现如权利要求7或8所述的非诊断目的的检测核酸的方法的步骤。
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