WO2021114040A1 - 一种无扩增的核酸分子检测试剂盒及其使用方法 - Google Patents

一种无扩增的核酸分子检测试剂盒及其使用方法 Download PDF

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WO2021114040A1
WO2021114040A1 PCT/CN2019/124085 CN2019124085W WO2021114040A1 WO 2021114040 A1 WO2021114040 A1 WO 2021114040A1 CN 2019124085 W CN2019124085 W CN 2019124085W WO 2021114040 A1 WO2021114040 A1 WO 2021114040A1
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nucleic acid
microspheres
detection
molecule
molecules
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PCT/CN2019/124085
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French (fr)
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陈忠垒
程鹏
张敏超
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彩科(苏州)生物科技有限公司
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification

Definitions

  • the invention belongs to the technical field of biological detection. Specifically, it relates to a nucleic acid molecule detection kit and a method of use, and more particularly to a nucleic acid molecule detection kit without amplification and a method of use.
  • Molecular diagnosis is the use of molecular biology techniques and methods to study the existence, structure or expression regulation changes of human endogenous (ie, the body's own genes) or exogenous (such as viruses, bacteria, etc.) biomolecules and molecular systems, which are diseases Provide information and decision-making basis for prevention, prediction, diagnosis, treatment and outcome.
  • Nucleic acid amplification technology has the widest application range in the field of molecular diagnosis.
  • molecular diagnosis can make up for some of the shortcomings of traditional clinical diagnosis methods.
  • molecular diagnosis can directly reveal the existence of pathogens, can objectively reflect the infection and activity of pathogens in the human body, and can be used in clinical treatment. An effective means of monitoring.
  • molecular diagnosis can also detect pathogens that are difficult to detect by conventional detection methods. For example, it can overcome the problem of the window period from infection to antibody production in enzyme immunoassay technology.
  • PCR technology has been increasingly widely used.
  • the current commercial molecular diagnostics are mainly based on the combination of PCR amplification technology and different detection technologies. Specifically, they usually include: a small amount of bacterial or viral DNA molecules are selectively replicated in large quantities or viral RNA molecules are reverse transcribed. DNA is selectively replicated in large quantities, and specific nucleic acid sequences are replicated and modified by designed primers for detection.
  • Common detection methods include second-generation gene sequencing, real-time fluorescence detection during amplification, capillary electrophoresis (first-generation sequencing), flow fluorescence detection, and gene hybridization chips.
  • RNA reverse transcription takes a long time and affects the structure of PCR amplification
  • DNA amplification takes a long time, requires high equipment, and requires precise temperature control
  • specific amplification has Bias, amplification is easy to fail, and some test items have high requirements for reagent stability; cross-contamination is easy to occur, and the existing clinical molecular tests also require highly skilled operators and require high site requirements.
  • PCR clinical diagnosis is also Sample processing, amplification and testing must be carried out in separate rooms, and in order to avoid high investment in the early stage of contamination, and it is difficult to clean up after contamination, the entire laboratory will have a long-term false positive problem; it is difficult to perform multi-index testing, sample requirements high.
  • the kit integrates all the steps of nucleic acid extraction, reverse transcription, amplification, detection, etc. through the microfluidic flow path design. They are isolated from each other and can only circulate in one direction. This integrated design improves the analysis speed and shortens the time from sample to result to less than one hour.
  • the supporting equipment can only process 1-2 kits at a time, and the sample throughput of one equipment per day is only about 10, which is far from meeting clinical needs.
  • the design of the integrated kit is complicated and precise, and the manufacturing cost is high. The integrated solution cannot completely solve the pollution problem of PCR amplification in clinical molecular diagnosis because of its low throughput and high cost.
  • the present invention provides a nucleic acid molecule detection kit without amplification and a method of use thereof.
  • the kit does not need to undergo PCR amplification when detecting target nucleic acid molecules, thereby avoiding PCR technology. This results in phenomena such as time-consuming and prone to failure.
  • the kit can amplify the signal of the target molecule to be tested to achieve ultra-high sensitivity of amplification detection.
  • the present invention adopts the following technical solutions:
  • the present invention provides a nucleic acid molecule detection kit without amplification, the kit comprising:
  • the surface is marked with the coding microspheres of capture molecules, detection molecules and hybridization buffer;
  • the capture molecules are nucleotide chains or peptide nucleic acid molecules complementary to the target nucleic acid molecules;
  • the detection molecules are coupled with catalysts and A single nucleotide or chain of nucleotides that is a complementary pair of target nucleic acid molecules.
  • the nucleic acid molecule detection kit utilizes coded microspheres with capture molecules to specifically bind target nucleic acid molecules, and at the same time uses the binding specificity of detection molecules and target nucleic acid molecules to form a "microsphere-capture molecule-target molecule" -Detect the complex structure of molecule-catalyst.
  • the coded microspheres are microspheres coded by at least two luminescent materials.
  • the luminescent material is selected from any one or a combination of two or more of organic fluorescent molecules, inorganic fluorescent molecules or quantum dots.
  • the microspheres contain magnetic nanoparticles.
  • the surface of the coded microsphere is chemically modified.
  • the method for preparing the coded microspheres includes the following steps: mixing at least two luminescent materials and microspheres in a polymer material, and dispersing the polymer solution in the water phase through multiple coupling physical fields to form a uniform liquid Then, functional materials (such as luminescent materials, magnetic nanoparticles, etc.) are wrapped in the droplets through a cross-linking polymerization reaction to obtain the coded microspheres.
  • functional materials such as luminescent materials, magnetic nanoparticles, etc.
  • the preparation method of the coded microspheres specifically includes the following steps:
  • Code Use at least two fluorescence intensity signals as codes to facilitate the identification of the instrument, and are equipped with a coded identification number, so that all the samples identified by the number can be processed according to the number after processing in the same sample processing solution analysis.
  • A1B1B1B2 the intensity of fluorescence A and fluorescence B
  • A1B3 the coded microsphere
  • Microsphere synthesis Mix at least two fluorescent materials (organic fluorescent molecules, inorganic fluorescent molecules or quantum dots) with magnetic nanoparticles in a variety of (different molecular weights, different functional groups) polymer materials that need to be synthesized.
  • the coupled physical field disperses the polymer solution in the water phase to form uniform droplets (or “micro-reaction groups”), and then through cross-linking and polymerization reactions, the fluorescent materials and magnetic nanoparticles are wrapped and buried in the polymer In the droplets.
  • the capture molecule can be designed as a single-stranded DNA or peptide nucleic acid molecule and placed at the end of the capture molecule. (3' or 5'end) is covalently connected with reactive chemical functional groups, and then reacted to the surface of the microspheres.
  • the microspheres with the same number are only for one target nucleic acid molecule, but can be coupled with multiple conserved target nucleic acid molecules Nucleic acid-specific capture can be achieved through this step with a variety of capture molecules with complementary pairings in different regions or different regions.
  • the coded microspheres prepared by this method can be tolerated under different reaction conditions (such as organic phase, high temperature), and finally, controllable specific nucleic acid molecular probes can be connected to the surface of the microspheres.
  • the designed peptide nucleic acid molecule can be used as the capture molecule to capture the target nucleic acid molecule, the peptide nucleic acid PNA molecule at the mutation site is left blank, and the mutation site complementation is used after the capture
  • the bases are paired, and a catalytic reactant is coupled to the end of this single nucleotide.
  • single-stranded DNA can be immobilized on the microsphere as a capture molecule to bind part of the target nucleic acid molecule, and then free single-stranded DNA molecule can be used as a detection molecule to bind to other positions of the target molecule. The end of this free detection molecule is even Combined with catalytic reactants.
  • the catalyst is any one or a combination of two or more of galactosidase, alkaline phosphatase or horseradish peroxidase.
  • microspheres that successfully capture the target nucleic acid molecule and form "microsphere-capture molecule-target molecule-detection molecule-catalyst" are introduced into the microfluidic chip, and the reaction of the solution is catalyzed by the catalyst molecule on the end of the reaction substrate ,
  • the use of one catalyst molecule can catalyze multiple orders of magnitude of reaction substrate molecules, so as to achieve effective signal amplification.
  • galactosidase as the catalyst as an example, using resorufin beta-D-galactopyranoside, dihydrofluorescein-di-beta-D-galactopyranoside as the reaction substrate, galactosidase catalyzed It can catalyze the reaction and realize signal amplification.
  • the hybridization buffer includes sodium citrate at a molar concentration of 20-80 mM (for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM or 80 mM, etc.) and 500-800 mM (e.g., It can be 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM or 800 mM, etc.) sodium chloride.
  • 20-80 mM for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM or 80 mM, etc.
  • 500-800 mM e.g., It can be 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM or 800 mM, etc.
  • the pH of the hybridization buffer is 6.8-7.4, for example, it can be 6.8, 6.9, 7, 7.1, 7.2, 7.3 or 7.4, etc.
  • the nucleic acid molecule detection kit further includes a microplate chip, and the microplate chip is processed by an injection molding method or a MEMS method.
  • the MEMS manufacturing process is a general term for the processing technology of nanometer to millimeter scale microstructures.
  • the composite microspheres are introduced into the microplate chip for catalytic reaction.
  • the composite microspheres exist in individual micropores. Even if there is only one catalyst molecule on the composite structure, its concentration is increased by several orders of magnitude. Therefore, the nucleic acid is not used. In the case of amplification technology, the signal can be amplified and the detection has ultra-high sensitivity.
  • the volume of a single microwell on the microplate chip is (20-100) ⁇ 10 -15 L, for example, it can be 20 ⁇ 10 -15 L, 30 ⁇ 10 -15 L, 40 ⁇ 10 -15 L , 50 ⁇ 10 -15 L, 60 ⁇ 10 -15 L, 70 ⁇ 10 -15 L, 80 ⁇ 10 -15 L, 90 ⁇ 10 -15 L or 100 ⁇ 10 -15 L, etc.
  • the density of the micropores on the microplate chip determines the amount of collected data and the dynamic range of detection. Closely arranged micropores need to be clearly distinguished by optical detection, and to ensure that there is only one or no microsphere in each micropore.
  • microplate chips can use disposable microplates or reusable microplate chips; disposable microplate chips are produced by injection molding, and non-disposable microplate chips can be produced by MEMS (Micro -Electro-Mechanical System) method for processing.
  • MEMS Micro -Electro-Mechanical System
  • a method for using the nucleic acid molecule detection kit as described in the first aspect includes the following steps:
  • the composite microspheres and/or the microspheres that are not bound to the target molecule are introduced into the microplate chip for reaction, and then external stimuli are applied to detect and analyze the encoded signal to obtain the detection result of the target molecule.
  • the mixing in step (1) is carried out in a non-amplified nucleic acid molecule diagnostic equipment.
  • the coding microspheres labeled with capture molecules on the surface bind one or not to the target nucleic acid molecule.
  • the ratio of microspheres to target molecules needs to be adjusted, and optimized according to the Poisson distribution, so that only one or no target molecule can be captured on a microsphere.
  • the composite microspheres are coded microspheres labeled with capture molecules on the surface bound to the target molecule and the detection molecule.
  • the composite microsphere is a composite structure of "microsphere-capture molecule-target molecule-detection molecule-catalyst".
  • the method for introducing the composite microspheres into the microplate chip in step (2) is selected from any one or more of gravity sedimentation, magnetic sedimentation or electric field induced sedimentation The combination of electric field induced sedimentation is preferred.
  • a single microwell on the microplate chip contains one or no composite microspheres and/or microspheres that are not bound to target molecules.
  • the micropores on the microplate chip are sealed with oil phase and/or polymer film during detection.
  • the method for applying external stimuli in step (2) is to use fluorescence of at least two wavelengths for near-field excitation.
  • a microreactor is used to place each composite microsphere structure in a micropore, the volume of the micropore is about 50 ⁇ 10 -15 L, and then the oil phase or polymer film is used to seal each A micropore, so that each microsphere composite structure is in a single small volume, even if there is only one catalyst molecule on the composite structure, the concentration is increased by multiple orders of magnitude, which is enough to catalyze a single micropore to emit light.
  • the non-amplified nucleic acid molecule diagnostic device includes a sample processing part based on microspheres and a detection part based on microplates.
  • the microsphere-based sample processing part includes a sample extraction module, a liquid transfer module, a mixing module, a magnetic attraction module, a liquid pre-storage module, and a liquid path cleaning module.
  • the sample extraction module of the non-amplified nucleic acid molecule diagnostic equipment is used to lyse the biological sample, and the nucleic acid molecules (DNA or RNA) released into the solution are automatically magnetic particle method or disposable silica column method.
  • the nucleic acid molecules are captured non-specifically on the magnetic microspheres, the microspheres are magnetically separated and washed, and then the nucleic acid molecules on the surface of the microspheres are eluted with water to obtain a pure nucleic acid solution; the liquid transfer module is connected to the probe
  • the microspheres of the needle, the sample solution to be tested, the detection probe, the catalyst and the catalyst base liquid and other reagents are transferred to the mixing module;
  • the microspheres connected to the test molecule in the mixing module are hybridized with the detection molecule (sequence);
  • the magnetic attraction module The enrichment function can be used to wash the microspheres that capture the target molecule to be detected and the detection molecule;
  • the liquid pre-storage module is used to store the reaction solution such as buffer solution; the liquid path cleaning module cleans the liquid path to prevent cross-contamination.
  • the mixing in step (1) is carried out in the mixing module of the non-amplified nucleic acid molecule diagnostic equipment.
  • the microplate-based detection part includes a liquid path control module, a multicolor fluorescence excitation module, a forward scattering imaging module, a fluorescence emission filter module, a magnetic attraction module, and an alternating electric field control module.
  • the liquid path control module is used to introduce the solution containing the composite microspheres into the holder with the microwell plate, and at the same time oil seal the surface of the microplate;
  • the multi-color fluorescence excitation module is used to excite the multiple fluorescent coded microspheres and the substrate
  • the fluorescent substance released by the molecule through the action of the catalyst is obtained through the forward scattering imaging module;
  • the fluorescence emission filter module is used to distinguish the coded microspheres and the brightness changes of the micropores (substrate molecules).
  • the concentration of the fluorescent substance released by the catalyst and its change); the magnetic attraction module and the alternating electric field module are used to help the microspheres lead into or out of the micropores.
  • the non-amplified nucleic acid molecular diagnostic equipment further includes an automatic control module and an image automatic recognition and analysis module.
  • the image automatic recognition and analysis module recognizes the brightness of the micropores in the microplate chip.
  • All modules of the non-amplified nucleic acid molecular diagnostic equipment are automatically controlled by the underlying program, and the software integrates the image automatic recognition and analysis module.
  • the system automatically recognizes the brightness of each micropore and obtains the distribution of micropore brightness, and automatically judges each micropore. Whether there is a reaction in the well, the number of the microsphere corresponding to each reaction.
  • the dynamic detection range of the analog signal is obtained through the brightness analysis, and the number of microspheres with the same number is used as the dynamic detection range of the digital signal.
  • the method of using the nucleic acid molecule detection kit includes the following steps:
  • the sample containing the target molecule, the coded microspheres labeled with capture molecules on the surface, the detection molecule, and the hybridization buffer are mixed, and the surface of the coded microspheres labeled with capture molecules is mixed. Bind one or not to the target nucleic acid molecule to obtain composite microspheres and/or microspheres that are not bound to the target molecule;
  • the composite microspheres and/or the microspheres that are not bound to the target molecule are introduced into the microplate chip by an electric field induced sedimentation method, and a single microwell on the microplate chip contains one or not
  • the composite microspheres/or the coded microspheres not bound to the target molecule are used for near-field excitation after the reaction to detect the composite microspheres/or the coded microspheres not bound to the target molecule.
  • the encoded signal is analyzed by the analysis module of the non-amplified nucleic acid molecular diagnostic equipment to obtain the detection result of the target molecule.
  • the numerical range described in the present invention not only includes the above-exemplified point values, but also includes any point values between the above-mentioned numerical ranges that are not listed. Due to the limitation of space and for the sake of brevity, the present invention will not exhaustively list the stated values. The specific point value included in the range.
  • the present invention has at least the following beneficial effects:
  • the non-amplified nucleic acid molecule detection kit connects a catalyst to the unamplified trace nucleic acid molecule, and uses the principle of a microreactor to maximize the concentration of the catalyst to catalyze the A large number of reactants react to achieve signal amplification, achieving ultra-high sensitivity when using amplification methods;
  • the use of multiple luminescent materials for labeling coded microspheres can achieve the purpose of detecting multiple nucleic acid molecules at the same time, requiring less sample quantity, making molecular diagnosis more efficient and faster; the detection reagent
  • the cassette is used with non-amplified nucleic acid molecular diagnostic equipment. On the one hand, it can avoid cross-contamination of the external operating environment. On the other hand, it can realize the whole process of sample pretreatment and analysis. The accuracy of the analysis results is significantly improved, and the detection results More reliable.
  • Figure 1 is a photo of a microplate chip containing fluorescently coded microspheres taken by a non-amplified nucleic acid molecular diagnostic device.
  • the synthesis of fluorescent coded microspheres can be synthesized using the following methods:
  • An ultrasonic generator (ultrasonic pulse 50uJ, 100uJ) and a sensor (4MHz, 6dB bandwidth, 4.4MHz, focus length 10.5cm) are set on both sides of the container carrying the emulsion, and the real-time feedback closed loop of the sensor controls the ultrasonic wave in the microsphere emulsion.
  • This embodiment provides a non-amplified nucleic acid molecule detection kit that can simultaneously detect influenza A virus nucleic acid molecules and influenza B virus nucleic acid molecules in a sample.
  • the specific preparation method and use method include the following steps:
  • Microsphere 1 uses the complementary single-stranded DNA molecule of influenza A virus nucleic acid molecule (capture molecule 1) for labeling
  • microsphere 2 uses the complementary single-stranded DNA molecule of influenza B virus nucleic acid molecule (capture Molecule 2) is labeled.
  • the specific steps are:
  • Microsphere 1 Take Microsphere 1 as an example, disperse 1mg of Microsphere 1 in 1mL PBS buffer, add 5mg EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 5mg Sulfo-NHS (N-Hydroxythiosuccinimide), mix well and keep stirring for 10 minutes, then add the influenza A virus nucleic acid complementary single-stranded DNA molecule with an amino group at the 3'end, and then add 10% BSA as a blocking agent , Stir for 30 minutes, then magnetically separate and wash the microsphere 1, and finally disperse in hybridization buffer (50mM sodium citrate, pH 7.2, 750mM NaCl) to obtain the fluorescent microsphere 1 labeled with capture molecule 1, and the fluorescence labeled with capture molecule 2
  • hybridization buffer 50mM sodium citrate, pH 7.2, 750mM NaCl
  • nucleic acids containing 1 ⁇ 10 9 copies/ ⁇ l of influenza A virus and 1 ⁇ 10 9 copies/ ⁇ l of influenza B virus culture medium were extracted by magnetic particle method, and finally eluted in water.
  • the specific steps are as follows: Add 130 ⁇ L lysis buffer (50mM Tris, pH 8.0, 4M guanidine hydrochloride, 1mM EDTA, 1% Triton-X100), 10 ⁇ L proteinase K (20mg/mL), and water bath at 55°C for 10 minutes into 200 ⁇ L virus culture broth; After adding 150 ⁇ L of isopropanol to each sample, mix and pipette evenly and add 500 ⁇ g of silica magnetic beads with hydroxyl on the surface; after standing for five minutes, the magnetic beads are adsorbed on the magnetic stand, and the supernatant is removed with a pipette. Wash the magnetic beads with 70% ethanol and elute with 50 ⁇ L of water.
  • the microsphere composite structure is added to the reactor with the microplate chip through the microfluid, 10MHz AC is applied, the microspheres are pushed into the micropores, and then the surface of the micropores is sealed with silicone oil. After reaction for 2 minutes, use 488nm Wavelength light excitation, filter 1 (512nm transmission, 20nm bandwidth) to take photos, filter 2 (570nm transmission, 30nm bandwidth) to take photos, then use 532nm wavelength light to excite, filter 3 (615nm transmission, 30nm bandwidth) to take photos, After flowing into ethanol, flow into the cleaning solution, change the AC frequency to 10kHz, and then flow into the cleaning solution to clean the reactor.
  • Fig. 1 is a photo of a microwell plate containing fluorescently coded microspheres 1 obtained by a non-amplified nucleic acid molecule diagnostic device when taking a photo of a light excitation filter 1 with a wavelength of 488 nm. It can be seen from Figure 1 that the fluorescent microspheres 1 that captured the nucleic acid of influenza A virus fell uniformly into the micropores. There is only one microsphere in each micropore and it is confined in the micropores. The microspheres carry the labeling enzyme. The fluorescent signal is amplified and detected by the forward scatter imaging module.
  • the image processing software first identifies all magnetic beads, and classifies the fluorescent microspheres (fluorescent microsphere 1 and fluorescent microsphere 2) according to the intensity of fluorescence under filter 1 and filter 2, because of viral RNA
  • the unamplified concentration is very low, most of the magnetic beads cannot capture the corresponding RNA to form composite microspheres and are conjugated with streptavidin-beta-galactosidase, without corresponding fluorescent signal.
  • the remaining magnetic beads can only capture one or a few target RNAs to form a sandwich complex, which is conjugated with streptavidin-beta-galactosidase and has a corresponding fluorescent signal.
  • the ratio of the magnetic beads that produce chemical signals to the total magnetic beads (fon) and the ratio of the total number of labeled enzyme molecules to the total number of magnetic beads (AEB, Average Enzyme per Bead) follow the Poisson distribution (AEB -ln (1-fon)).
  • the resulting concentration of 105 copies / l typically 104-107 copies / l).
  • Sensitivity analysis and specificity analysis Dilute influenza A virus nucleic acid and influenza B virus nucleic acid of known concentration (copy number) separately and perform the above test, and draw a standard curve.
  • the background value plus the three times the background value after repeated measurement and the standard deviation corresponding to the concentration (copy number) on the standard curve is the sensitivity (detection limit) of this method; specific analysis shows that magnetic beads 1 is because of the coupling of influenza A virus
  • the nucleic acid-specific capture probe cannot detect the nucleic acid of the influenza B virus, and similarly, the magnetic bead 2 cannot detect the nucleic acid of the influenza A virus.
  • This embodiment provides a non-amplified nucleic acid molecule detection kit that can simultaneously detect miRNA miR-122 and miR-129 in a serum sample in a sample.
  • the specific preparation method and use method include the following steps:
  • Microsphere 1 is labeled with a complementary peptide nucleic acid molecule of miRNA miR-122 (capture molecule 1)
  • Microsphere 2 is labeled with a complementary peptide nucleic acid molecule of miRNA miR-129 (capture molecule 2). The specific steps are:
  • Microsphere 1 Take Microsphere 1 as an example. Disperse 1mg of Microsphere 1 in 1mL PBS buffer, add 5mg EDC and 5mg Sulfo-NHS, mix well and keep stirring for 10 minutes, then add miR-122 complementary monomer with amino group at the 3'end. Strand peptide nucleic acid molecules, then add 10% BSA as a blocking agent, stir for 30 minutes, then magnetically separate and wash the microspheres 1, and finally disperse in hybridization buffer (50 mM sodium citrate, pH 7.2, 750 mM NaCl) to obtain capture molecules 1 The preparation method of the labeled fluorescent microsphere 1 and the fluorescent microsphere 2 labeled with the capture molecule 2 is the same as the above.
  • microsphere composite structure is added to the reactor with microplate chip through microfluid, 10MHz alternating current is applied, the microspheres are pushed into the micropores, and then the surface of the micropores is sealed with silicone oil.
  • reaction for 2 minutes use 488nm Wavelength light excitation, filter 1 to take photos, filter 2 to take photos, then use 532nm wavelength light to excite, filter 3 to take photos, flow into ethanol and then flow into the cleaning solution, change the AC frequency to 10kHz, then flow into the cleaning solution, cleaning reaction Device.
  • the image processing software first identifies all magnetic beads, and classifies the magnetic beads according to the intensity of fluorescence under filter 1 and filter 2. Because the unamplified concentration of miRNA is very low, most of the magnetic beads cannot capture the corresponding RNA to form a sandwich complex and are conjugated with streptavidin-beta-galactosidase, and there is no corresponding fluorescent signal. The remaining microspheres can only capture one or a few target RNAs to form composite microspheres, which are conjugated with streptavidin-beta-galactosidase and have corresponding fluorescent signals.
  • the ratio of the magnetic beads that produce chemical signals to the total magnetic beads (fon) and the ratio of the total number of labeled enzyme molecules to the total number of magnetic beads (AEB, Average Enzyme per Bead) follow the Poisson distribution (AEB -ln (1-fon)).
  • the concentration of miRNA is 1 ⁇ 10 4 copies/ ⁇ l.
  • Sensitivity analysis and specificity analysis dilute miR-122 and miR-129 of known concentration (copy number) step by step, perform the above test, and draw a standard curve.
  • the background value plus the three times the background value after repeated measurement of the standard deviation corresponding to the concentration (copy number) on the standard curve is the sensitivity (detection limit) of this method; specific analysis shows that magnetic bead 1 is specific because of the coupling of miR-122 The capture probe could not detect miR-129, and similarly, Magnetic Bead 2 could not detect miR-122.
  • the non-amplified nucleic acid molecule detection kit uses 2 ⁇ 10 6 -1 ⁇ 10 9 copies/ ⁇ l of virus culture medium for extraction or direct detection when detecting viral nucleic acid molecules.
  • the final detectable concentration is 1 ⁇ 10 4 -10 7 copies/ ⁇ l
  • the sensitivity of detecting viral nucleic acid can reach 3 ⁇ 10 3 copies/ ⁇ l
  • the sensitivity of detecting miRNA can reach 1.2 ⁇ 10 3
  • the copy/microliter indicates that the principle of the microreactor is used in the present invention to realize the amplification of the target molecule signal to be tested, and achieve the ultra-high sensitivity when using the amplification method to detect.
  • the detection step is simple The test results are accurate and reliable.

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Abstract

一种无扩增的核酸分子检测试剂盒，试剂盒包括：表面标记有捕获分子的编码微球、检测分子和杂交缓冲液；捕获分子为与目标核酸分子互补配对的核苷酸链或肽核酸分子；检测分子为偶联有催化剂、并且与目标核酸分子互补配对的单核苷酸或核苷酸链。核酸分子检测试剂盒在未被扩增的痕量核酸分子上连接催化剂，利用微反应器的原理将催化剂的浓度最大程度地放大，以催化溶液中的大量反应物反应以实现信号的放大，达到使用扩增方法检测时的超高灵敏度，同时试剂盒检测时受环境污染较小，还可以实现多目标物的同时检测，检测结果准确可靠。

Description

一种无扩增的核酸分子检测试剂盒及其使用方法 技术领域
本发明属于生物检测技术领域。具体涉及一种核酸分子检测试剂盒及其使用方法，尤其涉及一种无扩增的核酸分子检测试剂盒及其使用方法。
背景技术
分子诊断是利用分子生物学的技术和方法研究人体内源性(即机体自身的基因)或外源性(如病毒、细菌等)生物分子和分子体系的存在、结构或表达调控变化，为疾病的预防、预测、诊断、治疗和转归提供信息和决策依据。核酸扩增技术在分子诊断领域中应用范围最广。
相比于传统的临床诊断方法，分子诊断可以弥补传统临床诊断方法的某些缺陷，比如，分子诊断能直接揭示病原体的存在，能客观反映病原体在人体内感染及活动情况，可以作为临床治疗中的一个有效监控手段。另外采用分子诊断还可以检测到常规检测方法难以检测到的病原体，例如可以克服酶免检测技术中从感染到抗体产生的窗口期问题。
因此，以PCR技术为代表的分子诊断技术得到日益广泛的应用。目前商业化的分子诊断主要基于PCR扩增技术及不同的检测技术结合，具体来说通常包括：将少量的细菌或病毒的DNA分子有选择性的大量复制或将病毒的RNA分子逆转录后的DNA有选择性的大量复制，通过设计好的引物将特定的核酸序列复制并修饰好以进行检测。常见的检测方式包括二代基因测序、扩增时的实时荧光检测、毛细管电泳(一代测序)、流式荧光检测和基因杂交芯片等方法。
然而PCR扩增技术实现分子诊断的方法存在一些问题，例如：RNA逆转录耗时长且影响PCR扩增结构；DNA扩增耗时长，对设备的要求高，需要精确控 制温度；特异性扩增有偏见，扩增易失败，且部分检测项目对试剂稳定性要求较高；极易出现交叉污染，现有的临床分子检验还需要高度熟练的操作人员，同时对场地要求高，其中PCR临床诊断还必须将样品处理、扩增和检测在分隔的房间内进行，而且为了避免污染前期投入较高，而且污染后难以清理，整个实验室会有长时间假阳性问题；难以做多指标检测，样品要求高。
为了解决PCR扩增中的污染问题，现有技术中以赛沛的GeneXpert和梅里埃的filmarray为代表，将每个样本都用一个一次性的一体化封闭试剂盒检测。试剂盒通过微流控流路设计集成了核酸核酸提取、逆转录、扩增、检测等所有的步骤，他们之间相互隔离且只能单向流通。这种一体化设计提高了分析速度，从样品到结果的时间缩短到一个小时以内。但配套的设备一次只能处理1-2个试剂盒，一台设备一天的样本通量只有10个左右，远不能满足临床需求。而且一体化试剂盒设计复杂精密，制造成本高。一体化解决方案因为其低通量和高成本无法完全解决临床分子诊断中PCR扩增的污染问题。
因此，迫切需要研发一种无需扩增、同时检测灵敏度较高、检测结果准确可靠的高通量、低成本的核酸分子检测方法。
发明内容
鉴于现有技术中存在的问题，本发明提供一种无扩增的核酸分子检测试剂盒及其使用方法，所述试剂盒在检测目标核酸分子时，不需要经过PCR扩增，避免了PCR技术导致的耗时且易失败等现象，同时所述试剂盒能够将待测目标分子的信号进行放大，达到扩增检测的超高灵敏度。为达此目的，本发明采用以下技术方案：
第一方面，本发明提供一种无扩增的核酸分子检测试剂盒，所述试剂盒包括：
表面标记有捕获分子的编码微球、检测分子和杂交缓冲液；所述捕获分子为与目标核酸分子互补配对的核苷酸链或肽核酸分子；所述检测分子为偶联有催化剂、并且与目标核酸分子互补配对的单核苷酸或核苷酸链。
本发明提供的核酸分子检测试剂盒，利用带有捕获分子的编码微球特异性地结合目标核酸分子，同时利用检测分子与目标核酸分子的结合特异性，形成“微球-捕获分子-目标分子-检测分子-催化剂”的复合结构。
作为本发明一种优选的技术方案，所述编码微球为至少两种发光材料编码的微球。
优选地，所述发光材料选自有机荧光分子、无机荧光分子或量子点中的任意一种或两种以上的组合。
优选地，所述微球中包含磁性纳米颗粒。
优选地，所述编码微球的表面经过化学修饰。
优选地，所述编码微球的制备方法包括如下步骤：将至少两种发光材料和微球混合在高分子材料中，通过多重耦合的物理场将高分子溶液分散在水相中形成均一的液滴，之后通过交联聚合反应将功能材料(例如发光材料、磁性纳米颗粒等)包裹在所述液滴中得到所述编码微球。
本发明中，所述编码微球的制备方法具体包括如下步骤：
(1)编码：以至少两种荧光强度信号作为编码以方便仪器进行识别，并配有编码识别号，从而在同一个样品处理的溶液中对所有编号识别的样品进行处理后还可依据编号进行分析。
以两种荧光编码为例，机器可以识别荧光A和荧光B各6个强度，若荧光A和荧光B强度均为1，可将此编码微球编号为A1B1，依此类推可编码A1B2，A1B3，...，A6B6，则编码一共可达6×6＝36种。通过多重荧光编码的设置，还 能够实现多指标检测和高通量测序。
(2)微球合成：将至少两种荧光材料(有机荧光分子、无机荧光分子或量子点)与磁性纳米颗粒混合在需要合成的多种(不同分子量、不同官能团)高分子材料中，通过多重耦合的物理场将高分子溶液分散在水相中形成均一的液滴(或称之为“微反应集团”)，之后通过交联及聚合反应将荧光材料和磁性纳米颗粒等包裹掩埋在高分子液滴中。
(3)表面化学修饰：在制备微球时，可以在反应溶液中添加有反应活性的寡聚高分子，所述寡聚高分子通过微小相分离暴露在微球表面，利用暴露出来的寡聚高分子，可以对所述微球进行进一步表面化学修饰反应，使微球表面能够对核酸分子非特异性亲和力弱，同时特异性偶联反应活性强且密度高。
(4)将目标核酸分子(DNA或RNA等)的保守区识别出后，设计长短适度的捕获分子，针对不同应用，捕获分子可设计为单链DNA或肽核酸分子，并在捕获分子的末端(3’或5’端)共价连接可反应的化学官能团，之后反应到微球表面，同一个编号的微球只针对一种目标核酸分子，但可偶联与同一目标核酸分子多个保守区或不同区域的互补配对的多种捕获分子，通过此步骤可实现核酸特异性抓取。
通过此方法制备得到的编码微球可在不同反应条件(如有机相、高温)下耐受，最终可实现可控的特异性核酸分子探针连接在微球表面。
本发明中，对于单位点突变的检测，可使用设计好的肽核酸分子作为捕获分子抓取目标核酸分子，在突变位点的肽核酸PNA分子留空，在抓取后使用突变位点的互补碱基进行配对，这一单核苷酸的末端偶联有催化反应物。
针对片段的检测，可使用单链DNA固定在微球上作为捕获分子结合部分目标核酸分子，再使用游离单链DNA分子作为检测分子结合在目标分子的其他位 置，这一游离的检测分子末端偶联有催化反应物。
优选地，所述催化剂为半乳糖苷酶、碱性磷酸酶或辣根过氧化物酶中的任意一种或两种以上的组合。
将成功捕获目标核酸分子并且形成“微球-捕获分子-目标分子-检测分子-催化剂”的复合微球导入微流控芯片中，在反应底物中通过末端带有的催化剂分子催化溶液的反应，利用一个催化剂分子能够催化多个数量级的反应底物分子，从而实现信号的有效放大。以半乳糖苷酶为催化剂为例，使用试卤灵beta-D-吡喃半乳糖苷，二氢荧光素-二-beta-D-吡喃半乳糖苷作为反应底物，半乳糖苷酶催化下能够催化反应发生并且实现信号放大。
优选地，所述杂交缓冲液为所述杂交缓冲液包括摩尔浓度为20-80mM(例如可以是20mM、30mM、40mM、50mM、60mM、70mM或80mM等)的柠檬酸钠和500-800mM(例如可以是500mM、550mM、600mM、650mM、700mM、750mM或800mM等)的氯化钠。
优选地，所述杂交缓冲液的pH为6.8-7.4，例如可以是6.8、6.9、7、7.1、7.2、7.3或7.4等
作为本发明一种优选的技术方案，所述核酸分子检测试剂盒还包括微孔板芯片，所述微孔板芯片采用注塑生产法或MEMS法进行加工。其中MEMS制造工艺是纳米至毫米尺度微结构加工工艺的通称。
将复合微球构导入微孔板芯片中进行催化反应，复合微球都存在于单独的微孔中，即使复合结构上只有一个催化剂分子，其浓度也提高了多个数量级，因此在不使用核酸扩增技术的情况下就能够实现信号的放大，使检测具有超高的灵敏度。
优选地，所述微孔板芯片上单个微孔的体积为(20-100)×10 -15L，例如可以是 20×10 -15L、30×10 -15L、40×10 -15L、50×10 -15L、60×10 -15L、70×10 -15L、80×10 -15L、90×10 -15L或100×10 -15L等。
本发明中，微孔板芯片上微孔的密度决定了所采集的数据量及检测动态范围。密排的微孔需要光学检测能清晰分辨，并保证每一个微孔中只有一个或没有微球。同时，微孔板芯片可使用一次性的微孔板或可重复使用的微孔板芯片；一次性使用的微孔板芯片采用注塑生产，非一次性的微孔板芯片可通过芯片MEMS(Micro-Electro-Mechanical System)方法进行加工。
第二方面，一种如第一方面所述的核酸分子检测试剂盒的使用方法，包括如下步骤：
(1)将含有目标分子的样品、表面标记有捕获分子的编码微球、检测分子和杂交缓冲液混合，得到复合微球和/或未与目标分子结合的微球；
(2)将所述复合微球和/或未与目标分子结合的微球导入微孔板芯片中反应，而后施加外界刺激检测编码信号并分析，得到目标分子的检测结果。
作为本发明一种优选的技术方案，步骤(1)中所述混合于无扩增核酸分子诊断设备中进行。
优选地，所述表面标记有捕获分子的编码微球结合一个或不结合所述目标核酸分子。
如需进行极低浓度的单分子检测，则需要调整微球与目标分子的比例，依据泊松分布进行优化，使一个微球上只抓取一个或不抓取目标分子。
优选地，所述复合微球为与目标分子和检测分子结合的表面标记有捕获分子的编码微球。所述复合微球为“微球-捕获分子-目标分子-检测分子-催化剂”的复合结构。
作为本发明一种优选的技术方案，步骤(2)中将所述复合微球导入所述微 孔板芯片的方法选自重力沉降、磁力沉降或电场诱导沉降中的任意一种或两种以上的组合，优选为电场诱导沉降。
为了保证高效快捷地将微球与微孔一一配对组装，可采用以下几种方法：
(1)重力沉降：利用微球密度大于水的特性，重力可使微球自然沉入微孔中；
(2)磁力沉降：由于微球可包裹磁性材料，可使用磁力将微球操纵入微孔中；
(3)电场诱导沉降：由于微球的介电常数与溶液相差极大，可通过施加非匀强交变电场产生介电泳力将微球推入微孔中，在反应检测结束后，可通过调整电场频率改变力的方向将微球推出微孔从而实现微孔重复使用。
优选地，所述微孔板芯片上单个微孔中含有一个或不含所述复合微球和/或未与目标分子结合的微球。
优选地，检测时所述微孔板芯片上的微孔使用油相和/或高分子薄膜进行封闭。
优选地，步骤(2)中所述施加外界刺激的方法为使用至少两种波长的荧光进行近场激发。
本发明中若采用传统的化学发光催化方式，为了达到不扩增的单分子灵敏度，需要将催化剂的浓度最大程度地放大。所以，本发明中采用微反应器的方式将每一个复合微球结构放置在一个微孔中，微孔的体积在50×10 -15L左右，之后使用油相或高分子薄膜等封锁住每一个微孔，这样每一个微球复合结构都在一个单独的小体积中，即使复合结构上只有一个催化剂分子浓度也提高了多个数量级，足够催化单个微孔发光。
作为本发明一种优选的技术方案，所述无扩增核酸分子诊断设备包括基于 微球的样品处理部分和基于微孔板的检测部分。
优选地，所述基于微球的样品处理部分包括样品提取模块、液体转移模块、混合模块、磁吸模块、液体预存模块和液路清洗模块。
本发明中，所述无扩增核酸分子诊断设备的样品提取模块是用来将生物样本裂解，将释放到溶液中的核酸分子(DNA或者RNA)采用自动磁颗粒法或一次性硅胶柱的方式进行核酸提取，将核酸分子非特异性地捕获到磁性微球上，将微球磁性分离并清洗后用水将微球表面的核酸分子洗脱，获得纯净的核酸溶液；液体转移模块是将连有探针的微球，待测的样品溶液，检测探针，催化剂和催化剂底液等试剂转移至混合模块；混合模块中连有待测分子的微球与检测分子(序列)进行杂交；磁吸模块可以通过富集作用来洗涤捕获有待测目标分子以及检测分子的微球；液体预存模块用于存放缓冲液等反应溶液；液路清洗模块清洗液路防止交叉污染。
优选地，步骤(1)中所述混合于无扩增核酸分子诊断设备的混合模块中进行。
优选地，所述基于微孔板的检测部分包括液路控制模块、多色荧光激发模块、前散射成像模块、荧光发射滤光模块、磁吸模块和交变电场控制模块。
其中，液路控制模块用以导入含有复合微球的溶液进入放有微孔板的夹具中，同时将微孔板表面进行油封；多色荧光激发模块用以激发多重荧光编码微球以及底物分子通过催化剂作用释放出来的荧光物质；通过前散射成像模块得到低背景的导入微球后的微孔板荧光照片；荧光发射滤光模块用以区分编码微球以及微孔亮度变化(底物分子通过催化剂作用释放出来的荧光物质的浓度及其变化)；磁吸模块和交变电场模块用以帮助微球导入或导出微孔。
优选地，所述无扩增核酸分子诊断设备还包括自动控制模块和图像自动识 别分析模块。
优选地，所述图像自动识别分析模块识别所述微孔板芯片中微孔的亮度。
所述无扩增核酸分子诊断设备的所有模块均通过底层程序自动控制，软件集成图像自动识别分析模块，系统自动识别每一个微孔中的亮度并得到微孔亮度的分布，自动判断每个微孔中是否有反应发生，每一个反应对应的微球编号。通过亮度分析得到模拟信号的动态检测范围，通过同一编号微球有反应发生的个数作为数字信号的动态检测范围。
作为本发明一种优选的技术方案，所述核酸分子检测试剂盒的使用方法包括如下步骤：
(1)在无扩增核酸分子诊断设备的混合模块将含有目标分子的样品、表面标记有捕获分子的编码微球、检测分子和杂交缓冲液混合，所述表面标记有捕获分子的编码微球结合一个或不结合所述目标核酸分子，得到复合微球和/或未与目标分子结合的微球；
(2)将所述复合微球和/或未与目标分子结合的微球通过电场诱导沉降的方法导入所述微孔板芯片中，所述微孔板芯片上单个微孔中含有一个或不含所述复合微球/或未与目标分子结合的编码微球，反应后使用至少两种波长的荧光进行近场激发，检测所述复合微球/或未与目标分子结合的编码微球的编码信号并利用无扩增核酸分子诊断设备的分析模块进行分析，得到目标分子的检测结果。
本发明所述的数值范围不仅包括上述例举的点值，还包括没有列举出的上述数值范围之间的任意的点值，限于篇幅及出于简明的考虑，本发明不再穷尽列举所述范围包括的具体点值。
与现有技术相比，本发明至少具有以下有益效果：
(1)本发明提供的无扩增核酸分子检测试剂盒，在未被扩增的痕量核酸分 子上连接催化剂，利用微反应器的原理将催化剂的浓度最大程度地放大，以催化溶液中的大量反应物反应以实现信号的放大，达到使用扩增方法检测时的超高灵敏度；
(2)本发明中，使用多种发光材料的进行标记的编码微球，可以实现同时检测多种核酸分子的目的，对样品的量要求较少，使分子诊断更加高效快捷；所述检测试剂盒配合无扩增核酸分子诊断设备使用，一方面可以避免外界操作环境的交叉污染，另一方面，可以实现从样品的预处理及分析的全过程，其分析结果的准确度明显提高，检测结果更加可靠。
附图说明
图1为无扩增核酸分子诊断设备拍摄的含有荧光编码微球的微孔板芯片照片。
具体实施方式
下面结合附图并通过具体实施方式来进一步说明本发明的技术方案。但下述的实例仅仅是本发明的简易例子，并不代表或限制本发明的权利保护范围，本发明的保护范围以权利要求书为准。
以下实施例中，荧光编码微球合成可以使用如下方法合成：
将甲基丙烯酸、苯乙烯单体、聚甲基丙烯酸甲酯和交联剂(二乙烯基苯)混合在氯仿中作为高分子溶液，取4.5mL高分子溶液加入0.5mL罗丹明和0.5mL荧光素和90mg纳米磁颗粒，之后将其置于两个有300mL去离子水和十二烷基硫酸钠的反应器中，得到乳液。
在承载乳液的容器的两侧分别设置超声波发生器(超声脉冲50uJ，100uJ)及传感器(4MHz，6dB带宽，4.4MHz，聚焦长度10.5cm)，通过传感器的实时反馈闭环控制超声波对微球乳液中的Fe 3O 4磁性纳米颗粒进行调制，以磁共 振成像装置原理，磁性颗粒对超声波段吸收并保持极性朝向被外加超声波的场诱导，为了保持诱导进行的调制效率，传感器通过Fe 3O 4磁性纳米颗粒特定的吸收波谱及时调解超声脉冲的能量从而可控调解Fe 3O 4磁性纳米颗粒的朝向，得到功能性颗粒均匀分布、偶极方向一致的液滴；
将溶液均匀分散为10μm的液滴，在溶液中加入引发剂偶氮二异丁腈并加热进行聚合交联反应，24小时之后，每个液滴中的氯仿缓慢溶于水中并挥发，单体聚合并交联，最终形成荧光编码微球。
实施例1
本实施例提供一种可以同时检测样本中的甲型流感病毒核酸分子和乙型流感病毒核酸分子的无扩增核酸分子检测试剂盒。具体制备方法及使用方法包括如下步骤：
取两份荧光编码微球，微球1使用甲型流感病毒核酸分子的互补单链DNA分子(捕获分子1)进行标记，微球2使用乙型流感病毒核酸分子的互补单链DNA分子(捕获分子2)进行标记。具体步骤为：
以微球1为例，将1mg微球1在1mL PBS缓冲液中分散，加入5mg EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)和5mg Sulfo-NHS(N-羟基硫代琥珀酰亚胺)，混匀并维持搅拌10分钟，之后加入3’端有氨基的甲型流感病毒核酸互补单链DNA分子，之后加入10％BSA作为封闭剂，搅拌30分钟，之后通过磁性分离洗涤微球1，最终分散在杂交缓冲液(50mM柠檬酸钠，pH 7.2，750mM NaCl)中得到捕获分子1标记的荧光微球1，捕获分子2标记的荧光微球2的制备方法同上。
将含有1×10 9拷贝/微升的甲型流感病毒和1×10 9拷贝/微升乙型流感病毒培养液的全部核酸利用磁颗粒方法进行提取，最终洗脱在水中。具体步骤为： 在200μL病毒培养液中加入130μL裂解液(50mM Tris，pH 8.0，4M盐酸胍，1mM EDTA，1％Triton-X100)，10μL蛋白酶K(20mg/mL)，55℃水浴10分钟；每个样品中加入150μL异丙醇后，混合吹打均匀后加入500μg表面带有羟基的二氧化硅磁珠；静置五分钟后将磁珠在磁力架上吸附，用移液枪移去上层清液，用70％乙醇洗涤磁珠，50μL水洗脱。
之后加入偶连了寡聚核苷酸探针的1mg/mL的荧光微球1和荧光微球2等体积混合，得到溶液0.2mL，加入0.2mL杂交缓冲液(50mM柠檬酸钠，pH 7.2，750mM NaCl)和0.2mL的偶联有生物素的核酸探针，所述生物素分子被链霉亲和素特异性识别，所述核酸探针可以特异性结合甲型流感病毒核酸分子或乙型流感病毒核酸分子，均匀混合5分钟，之后磁吸并洗涤，再加入0.2mL的链霉亲和素-beta-半乳糖苷酶共轭连接在核酸探针上作为检测分子，均匀混合5分钟，之后磁吸并洗涤，再将磁珠分散到100μM二氢荧光素-二-beta-D-吡喃半乳糖苷溶液中。
检测：将微球复合结构通过微流体加入有微孔板芯片的反应器中，加10MHz交流电，微球被推入微孔中，之后用硅油将微孔表面封闭，反应2分钟后，使用488nm波长光激发，滤光片1(512nm透射，20nm带宽)拍照，滤光片2(570nm透射，30nm带宽)拍照，再使用532nm波长光激发，滤光片3(615nm透射，30nm带宽)拍照，流入乙醇后再流入清洗液，改变交流电频率至10kHz，之后流入清洗液，清洗反应器。
图1为无扩增核酸分子诊断设备在488nm波长光激发滤光片1拍照时得到的含有荧光编码微球1的微孔板照片。由图1可知，捕获了甲流病毒核酸的荧光微球1均匀的落到了微孔里，每个微孔中只有一个微球而且被限制在了微孔中，微球上带有的标记酶放大了荧光信号并被前散射成像模块检测到。
数据处理和浓度测定：图像处理软件首先识别所有磁珠，根据滤光片1和滤光片2下荧光的强度将荧光微球进行分类(荧光微球1和荧光微球2)，因为病毒RNA未经扩增浓度很低，大部分磁珠不能捕获相应RNA形成复合微球并被链霉亲和素-beta-半乳糖苷酶共轭标记，无相应的荧光信号。剩余磁珠只能捕获一个或少数几个目标RNA形成夹心复合物并被链霉亲和素-beta-半乳糖苷酶共轭标记，有相应的荧光信号。
检测到产生化学信号的磁珠占总磁珠的比例值(fon)与标记酶分子的总数和磁珠总数的比例值(AEB，Average Enzyme per Bead)之间遵从泊松分布(AEB＝-ln(1-fon))。将AEB值和待测物浓度值进行线性拟合绘制校准曲线后，用相同的方法测试未知样品，得到的信号值用内插法带入标准曲线即测得未知样品中待测物的浓度，所得浓度为10 5拷贝/微升(一般为10 4-10 7拷贝/微升)。
灵敏度分析和特异性分析：将已知浓度(拷贝数)的甲型流感病毒核酸和乙型流感病毒核酸分别阶梯稀释并进行上述测试，并绘制标准曲线。背景值加上三倍背景值反复测量后标准偏差在标准曲线上对应的浓度(拷贝数)即为本方法的灵敏度(检出限)；特异性分析显示磁珠1因为偶连甲型流感病毒核酸特异性的捕获探针，检测不到乙型流感病毒的核酸，同理磁珠2也检测不到甲型流感病毒的核酸。
实施例2
本实施例提供一种可以同时检测样本中血清样本中的miRNA miR-122和miR-129的无扩增核酸分子检测试剂盒。具体制备方法及使用方法包括如下步骤：
取两份荧光编码微球，微球1使用miRNA miR-122的互补肽核酸分子(捕获分子1)进行标记，微球2使用miRNA miR-129的互补肽核酸分子(捕获分 子2)进行标记。具体步骤为：
以微球1为例，将1mg微球1在1mL PBS缓冲液中分散，加入5mg EDC和5mg Sulfo-NHS，混匀并维持搅拌10分钟，之后加入3’端有氨基的miR-122互补单链肽核酸分子，之后加入10％BSA作为封闭剂，搅拌30分钟，之后通过磁性分离洗涤微球1，最终分散在杂交缓冲液(50mM柠檬酸钠，pH 7.2，750mM NaCl)中得到捕获分子1标记的荧光微球1，捕获分子2标记的荧光微球2的制备方法同上。
在掺杂miR-122和miR-129标品的血清中加入含有4M尿素的溶液使miRNA分散在溶液中，之后加入荧光微球1和荧光微球2的1:1混合溶液，加入0.2mL杂交缓冲液和0.2mL的偶联有生物素的核酸探针，均匀混合5分钟，之后磁吸并洗涤。再加入0.2mL的链霉亲和素-beta-半乳糖苷酶共轭，均匀混合5分钟，之后磁吸并洗涤。
检测：将微球复合结构通过微流体加入有微孔板芯片的反应器中，加10MHz交流电，微球被推入微孔中，之后用硅油将微孔表面封闭，反应2分钟后，使用488nm波长光激发，滤光片1拍照，滤光片2拍照，再使用532nm波长光激发，滤光片3拍照，流入乙醇后再流入清洗液，改变交流电频率至10kHz，之后流入清洗液，清洗反应器。
数据处理和浓度测定：图像处理软件首先识别所有磁珠，根据滤光片1和滤光片2下荧光的强度将磁珠分类。因为miRNA未经扩增浓度很低，大部分磁珠不能捕获相应RNA形成夹心复合物并被链霉亲和素-beta-半乳糖苷酶共轭标记，无相应的荧光信号。剩余微球只能捕获一个或少数几个目标RNA形成复合微球并被链霉亲和素-beta-半乳糖苷酶共轭标记，有相应的荧光信号。
检测到产生化学信号的磁珠占总磁珠的比例值(fon)与标记酶分子的总数 和磁珠总数的比例值(AEB，Average Enzyme per Bead)之间遵从泊松分布(AEB＝-ln(1-fon))。将AEB值和待测物浓度值进行线性拟合绘制校准曲线后，用相同的方法测试未知样品，得到的信号值用内插法带入标准曲线即测得未知样品中待测物的浓度，miRNA的浓度在1×10 4拷贝/微升。
灵敏度分析和特异性分析：将已知浓度(拷贝数)的miR-122和miR-129分别阶梯稀释并进行上述测试，并绘制标准曲线。背景值加上三倍背景值反复测量后标准偏差在标准曲线上对应的浓度(拷贝数)即为本方法的灵敏度(检出限)；特异性分析显示磁珠1因为偶连miR-122特异性的捕获探针，检测不到miR-129，同理磁珠2也检测不到miR-122。
综上所述，本发明提供的无扩增核酸分子检测试剂盒，在检测病毒核酸分子时，利用2×10 6-1×10 9拷贝/微升的病毒培养液进行提取后，或直接检测不经过提取的miRNA，最终可检测出浓度为1×10 4-10 7拷贝/微升，检测病毒核酸的灵敏度可达3×10 3拷贝/微升，检测miRNA的灵敏度可达1.2×10 3拷贝/微升，说明本发明中利用微反应器的原理实现待测目标分子信号的放大，达到使用扩增方法检测时的超高灵敏度，配合无扩增核酸分子诊断设备，其检测步骤简单，检测结果准确可靠。
申请人声明，本发明通过上述实施例来说明本发明的详细结构特征，但本发明并不局限于上述详细结构特征，即不意味着本发明必须依赖上述详细结构特征才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明所选用部件的等效替换以及辅助部件的增加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (11)

  1. 一种无扩增的核酸分子检测试剂盒，其特征在于，所述试剂盒包括：
    表面标记有捕获分子的编码微球、检测分子和杂交缓冲液；
    所述捕获分子为与目标核酸分子互补配对的核苷酸链或肽核酸分子；
    所述检测分子为偶联有催化剂、并且与目标核酸分子互补配对的单核苷酸或核苷酸链。
  2. 根据权利要求1所述的核酸分子检测试剂盒，其特征在于，所述编码微球为至少两种发光材料编码的微球；
    优选地，所述发光材料选自有机荧光分子、无机荧光分子或量子点中的任意一种或两种以上的组合；
    优选地，所述微球中包含磁性纳米颗粒；
    优选地，所述编码微球的表面经过化学修饰。
  3. 根据权利要求1或2所述的核酸分子检测试剂盒，其特征在于，所述编码微球的制备方法包括如下步骤：
    将至少两种发光材料和微球混合在高分子材料中，通过多重耦合的物理场将高分子溶液分散在水相中形成均一的液滴，之后通过交联聚合反应将发光材料与磁性纳米颗粒包裹在所述液滴中得到所述编码微球。
  4. 根据权利要求1-3任一项所述的核酸分子检测试剂盒，其特征在于，所述催化剂选自半乳糖苷酶、碱性磷酸酶或辣根过氧化物酶中的任意一种或两种以上的组合；
    优选地，所述杂交缓冲液包括摩尔浓度为20-80mM的柠檬酸钠和500-800mM的氯化钠；
    优选地，所述杂交缓冲液的pH为6.8-7.4。
  5. 根据权利要求1-3任一项所述的核酸分子检测试剂盒，其特征在于，所 述核酸分子检测试剂盒还包括微孔板芯片；
    优选地，所述微孔板芯片上单个微孔的体积为(20-100)×10 -15L。
  6. 一种如权利要求1-4任一项所述的核酸分子检测试剂盒的使用方法，其特征在于，包括如下步骤：
    (1)将含有目标核酸分子的样品、表面标记有捕获分子的编码微球、检测分子和杂交缓冲液混合，得到复合微球和/或未与目标分子结合的微球；
    (2)将所述复合微球和/或未与目标分子结合的微球导入微孔板芯片中反应，而后施加外界刺激检测编码信号并分析，得到目标分子的检测结果。
  7. 根据权利要求5所述的使用方法，其特征在于，步骤(1)中所述混合于无扩增核酸分子诊断设备中进行；
    优选地，所述表面标记有捕获分子的编码微球结合一个或不结合所述目标核酸分子，所述微孔板芯片上单个微孔中含有一个或不含所述复合微球和/或未与目标分子结合的微球；
    优选地，所述复合微球为与目标分子和检测分子结合的表面标记有捕获分子的编码微球。
  8. 根据权利要求5或6所述的使用方法，其特征在于，步骤(2)中将所述复合微球导入所述微孔板芯片的方法选自重力沉降、磁力沉降或电场诱导沉降中的任意一种或两种以上的组合；
    优选地，步骤(2)中所述施加外界刺激的方法为使用至少两种波长的荧光进行近场激发。
  9. 根据权利要求5-7任一项所述的使用方法，其特征在于，所述无扩增核酸分子诊断设备包括基于微球的样品处理部分和基于微孔板的检测部分；
    优选地，所述基于微球的样品处理部分包括样品提取模块、液体转移模块、 混合模块、磁吸模块、液体预存模块和液路清洗模块；
    优选地，所述基于微孔板的检测部分包括液路控制模块、多色荧光激发模块、前散射成像模块、荧光发射滤光模块、磁吸模块和交变电场控制模块。
  10. 根据权利要求5-8任一项所述的使用方法，其特征在于，所述无扩增核酸分子诊断设备还包括图像自动识别分析模块，所述图像自动识别分析模块识别所述微孔板芯片中微孔的亮度。
  11. 根据权利要求5-9任一项所述的使用方法，其特征在于，所述使用方法包括如下步骤：
    (1)在无扩增核酸分子诊断设备的混合模块将含有目标分子的样品、表面标记有捕获分子的编码微球、检测分子和杂交缓冲液混合，所述表面标记有捕获分子的编码微球结合一个或不结合所述目标核酸分子，得到复合微球和/或未与目标分子结合的微球；
    (2)将所述复合微球和/或未与目标分子结合的微球通过电场诱导沉降的方法导入所述微孔板芯片中，所述微孔板芯片上单个微孔中含有一个或不含所述复合微球/或未与目标分子结合的编码微球，反应后使用至少两种波长的荧光进行近场激发，检测所述复合微球/或未与目标分子结合的编码微球的编码信号并利用无扩增核酸分子诊断设备的分析模块进行分析，得到目标分子的检测结果。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117388227A (zh) * 2023-10-18 2024-01-12 博瑞生物医药（苏州）股份有限公司 使用检测微球测定样品中目标分子浓度的方法、计算机可读介质和分析设备

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130244895A1 (en) * 2010-08-20 2013-09-19 Eth Zurich, Eth Transfer Microwell Arrays for Direct Quantification of Analytes on a Flat Sample
CN103834726A (zh) * 2014-01-24 2014-06-04 湖南工程学院 基于微流控微珠阵列芯片和dna聚合酶介导引物延伸技术的微小核糖核酸检测方法
JP2015073523A (ja) * 2013-10-11 2015-04-20 国立大学法人 東京大学 核酸の検出方法、検出プローブ、マイクロアレイ、核酸検出キット、核酸‐検出プローブ‐捕捉プローブ複合体、核酸固定化担体、及び流体デバイス
CN105807047A (zh) * 2016-04-15 2016-07-27 上海交通大学 基于核酸序列编码的elisa检测芯片及其制备和应用
CN106047677A (zh) * 2016-05-19 2016-10-26 沈阳今唐基因与医学技术研究院 检测单细胞中核酸的微流控芯片及检测单细胞中核酸的方法
CN106282323A (zh) * 2015-05-29 2017-01-04 南京理工大学 基于聚胸腺嘧啶为模板生成铜纳米粒子的高灵敏度dna荧光分析方法
CN107058584A (zh) * 2017-06-07 2017-08-18 浙江殷欣生物技术有限公司 一种核酸杂交化学发光检测方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130244895A1 (en) * 2010-08-20 2013-09-19 Eth Zurich, Eth Transfer Microwell Arrays for Direct Quantification of Analytes on a Flat Sample
JP2015073523A (ja) * 2013-10-11 2015-04-20 国立大学法人 東京大学 核酸の検出方法、検出プローブ、マイクロアレイ、核酸検出キット、核酸‐検出プローブ‐捕捉プローブ複合体、核酸固定化担体、及び流体デバイス
CN103834726A (zh) * 2014-01-24 2014-06-04 湖南工程学院 基于微流控微珠阵列芯片和dna聚合酶介导引物延伸技术的微小核糖核酸检测方法
CN106282323A (zh) * 2015-05-29 2017-01-04 南京理工大学 基于聚胸腺嘧啶为模板生成铜纳米粒子的高灵敏度dna荧光分析方法
CN105807047A (zh) * 2016-04-15 2016-07-27 上海交通大学 基于核酸序列编码的elisa检测芯片及其制备和应用
CN106047677A (zh) * 2016-05-19 2016-10-26 沈阳今唐基因与医学技术研究院 检测单细胞中核酸的微流控芯片及检测单细胞中核酸的方法
CN107058584A (zh) * 2017-06-07 2017-08-18 浙江殷欣生物技术有限公司 一种核酸杂交化学发光检测方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117388227A (zh) * 2023-10-18 2024-01-12 博瑞生物医药（苏州）股份有限公司 使用检测微球测定样品中目标分子浓度的方法、计算机可读介质和分析设备
CN117388227B (zh) * 2023-10-18 2024-05-31 博瑞生物医药（苏州）股份有限公司 使用检测微球测定样品中目标分子浓度的方法、计算机可读介质和分析设备

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