CN111218498A

CN111218498A - Nucleic acid molecule detection kit without amplification and use method thereof

Info

Publication number: CN111218498A
Application number: CN201911252049.5A
Authority: CN
Inventors: 陈忠垒; 程鹏; 张敏超
Original assignee: Caike Suzhou Biotechnology Co Ltd
Current assignee: Caike Suzhou Biotechnology Co Ltd
Priority date: 2019-12-09
Filing date: 2019-12-09
Publication date: 2020-06-02

Abstract

The invention provides a nucleic acid molecule detection kit without amplification, which comprises: the surface of the microsphere is marked with a capture molecule, a detection molecule and a hybridization buffer solution; the capture molecule is a nucleotide chain or peptide nucleic acid molecule which is complementarily paired with the target nucleic acid molecule; the detection molecule is a mononucleotide or nucleotide chain coupled with a catalyst and complementarily paired with a target nucleic acid molecule. The nucleic acid molecule detection kit is characterized in that a catalyst is connected to trace nucleic acid molecules which are not amplified, the concentration of the catalyst is amplified to the maximum extent by utilizing the principle of a microreactor, a large amount of reactants in a catalytic solution react to realize signal amplification, the ultrahigh sensitivity in detection by using an amplification method is achieved, meanwhile, the kit is less polluted by the environment in detection, the simultaneous detection of multiple targets can be realized, and the detection result is accurate and reliable.

Description

Nucleic acid molecule detection kit without amplification and use method thereof

Technical Field

The invention belongs to the technical field of biological detection. In particular to a nucleic acid molecule detection kit and a use method thereof, in particular to a nucleic acid molecule detection kit without amplification and a use method thereof.

Background

Molecular diagnosis is to utilize molecular biology technology and method to study the existence, structure or expression regulation and control change of human endogenous (i.e. organism gene) or exogenous (e.g. virus, bacteria, etc.) biological molecules and molecular systems, and provide information and decision basis for prevention, prediction, diagnosis, treatment and outcome of diseases. Nucleic acid amplification technology has the widest application range in the field of molecular diagnostics.

Compared with the traditional clinical diagnosis method, the molecular diagnosis can make up for certain defects of the traditional clinical diagnosis method, for example, the molecular diagnosis can directly reveal the existence of pathogens, can objectively reflect the infection and activity conditions of the pathogens in a human body, and can be used as an effective monitoring means in clinical treatment. In addition, the molecular diagnosis can be used for detecting pathogens which are difficult to detect by the conventional detection method, for example, the problem of the window period from infection to antibody generation in the enzyme immunoassay technology can be overcome.

Therefore, molecular diagnostic techniques typified by PCR techniques are increasingly being used. The current commercial molecular diagnostics are mainly based on PCR amplification techniques combined with different detection techniques, and in particular typically include: a small amount of bacterial or viral DNA molecules are selectively and massively copied or the DNA obtained by reverse transcription of viral RNA molecules is selectively and massively copied, and a specific nucleic acid sequence is copied and modified by a designed primer so as to carry out detection. Common detection methods include second-generation gene sequencing, real-time fluorescence detection during amplification, capillary electrophoresis (first-generation sequencing), flow-type fluorescence detection, gene hybridization chip and the like.

However, the method of using PCR amplification technology for molecular diagnosis has some problems, such as: RNA reverse transcription takes a long time and affects PCR amplification structure; the DNA amplification consumes long time, has high requirements on equipment and needs to accurately control the temperature; specific amplification has a bias, amplification is easy to fail, and the requirements of partial detection items on the stability of the reagent are high; cross contamination is easy to occur, the existing clinical molecular test also needs highly skilled operators and has high requirements on fields, wherein the PCR clinical diagnosis also needs to carry out sample treatment, amplification and detection in a separated room, and in order to avoid high investment in the early stage of contamination and difficult cleaning after contamination, the whole laboratory has the problem of long-time false positive; the multi-index detection is difficult to carry out, and the sample requirement is high.

In order to solve the pollution problem in PCR amplification, in the prior art, seemingly GeneXpert and filiarray of Merrianga are taken as representatives, and each sample is detected by using a disposable integrated closed kit. The kit integrates all the steps of nucleic acid extraction, reverse transcription, amplification, detection and the like through the design of a microfluidic flow path, and the steps are mutually isolated and can only flow in one direction. The integrated design improves the analysis speed, and the time from the sample to the result is shortened to be less than one hour. However, the matched equipment can only process 1-2 kits at a time, and the sample flux of one equipment per day is only about 10, so that the clinical requirement cannot be met. And the integrated kit has complex and precise design and high manufacturing cost. The integrated solution cannot completely solve the contamination problem of PCR amplification in clinical molecular diagnostics because of its low throughput and high cost.

Therefore, there is an urgent need to develop a method for detecting nucleic acid molecules with high throughput and low cost, which does not require amplification, and has high detection sensitivity and accurate and reliable detection results.

Disclosure of Invention

In view of the problems in the prior art, the invention provides a nucleic acid molecule detection kit without amplification and a use method thereof, when the kit is used for detecting target nucleic acid molecules, PCR amplification is not needed, the phenomena of time consumption, volatile failure and the like caused by a PCR technology are avoided, and simultaneously, signals of the target molecules to be detected can be amplified by the kit, so that the ultrahigh sensitivity of amplification detection is achieved. In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides an amplification-free nucleic acid molecule detection kit, comprising:

the surface of the microsphere is marked with a capture molecule, a detection molecule and a hybridization buffer solution; the capture molecule is a nucleotide chain or peptide nucleic acid molecule which is complementarily paired with the target nucleic acid molecule; the detection molecule is a mononucleotide or nucleotide chain coupled with a catalyst and complementarily paired with a target nucleic acid molecule.

The nucleic acid molecule detection kit provided by the invention utilizes the coding microspheres with capture molecules to specifically bind with target nucleic acid molecules, and simultaneously utilizes the binding specificity of detection molecules and target nucleic acid molecules to form a composite structure of microspheres-capture molecules-target molecules-detection molecules-catalyst.

As a preferred technical solution of the present invention, the coded microspheres are microspheres coded by at least two luminescent materials.

Preferably, the luminescent material is selected from any one or a combination of more than two of organic fluorescent molecules, inorganic fluorescent molecules or quantum dots.

Preferably, the microspheres comprise magnetic nanoparticles.

Preferably, the surface of the encoded microspheres is chemically modified.

Preferably, the preparation method of the coding microsphere comprises the following steps: mixing at least two luminescent materials and microspheres in a high polymer material, dispersing a high polymer solution in a water phase to form uniform liquid drops through a multiple-coupling physical field, and then wrapping functional materials (such as the luminescent materials, magnetic nanoparticles and the like) in the liquid drops through a cross-linking polymerization reaction to obtain the coding microspheres.

In the invention, the preparation method of the coding microsphere specifically comprises the following steps:

(1) and (3) encoding: at least two fluorescence intensity signals are used as codes to facilitate the identification of the instrument, and are matched with code identification numbers, so that all samples identified by the numbers can be analyzed according to the numbers after being processed in the solution processed by the same sample.

Taking two fluorescence codes as an example, the machine can identify 6 intensities of each of fluorescence a and fluorescence B, and if the intensities of fluorescence a and fluorescence B are both 1, the coded microsphere can be numbered as A1B1, and so on, A1B2, A1B 3. Through the setting of multiple fluorescence codes, multi-index detection and high-throughput sequencing can be realized.

(2) Synthesizing microspheres: at least two fluorescent materials (organic fluorescent molecules, inorganic fluorescent molecules or quantum dots) and magnetic nanoparticles are mixed in a plurality of high molecular materials (different molecular weights and different functional groups) to be synthesized, a high molecular solution is dispersed in a water phase through a multiple coupling physical field to form uniform liquid drops (or called as micro-reaction groups), and then the fluorescent materials, the magnetic nanoparticles and the like are wrapped and buried in the high molecular liquid drops through cross-linking and polymerization reactions.

(3) Surface chemical modification: when the microsphere is prepared, an oligomeric polymer with reactivity can be added into a reaction solution, the oligomeric polymer is exposed on the surface of the microsphere through micro-phase separation, and the exposed oligomeric polymer can be used for further surface chemical modification reaction on the microsphere, so that the surface of the microsphere can have weak nonspecific affinity for nucleic acid molecules, and meanwhile, the specific coupling reaction activity is strong and the density is high.

(4) After identifying the conserved regions of target nucleic acid molecules (DNA or RNA, etc.), designing capture molecules with proper length, aiming at different applications, the capture molecules can be designed into single-stranded DNA or peptide nucleic acid molecules, covalently connecting reactive chemical functional groups at the tail ends (3 'or 5' ends) of the capture molecules, then reacting to the surfaces of microspheres, and the microspheres with the same number only aim at one target nucleic acid molecule, but can be coupled with a plurality of complementary paired capture molecules of a plurality of conserved regions or different regions of the same target nucleic acid molecule, thereby realizing the specific capture of nucleic acid.

The coded microsphere prepared by the method can resist under different reaction conditions (such as organic phase and high temperature), and finally, the controllable specific nucleic acid molecular probe can be connected to the surface of the microsphere.

In the invention, for the detection of single-site mutation, designed peptide nucleic acid molecules can be used as capture molecules to capture target nucleic acid molecules, peptide nucleic acid PNA molecules at mutation sites are left empty, complementary bases of the mutation sites are used for pairing after capture, and a catalytic reactant is coupled to the tail end of the mononucleotide.

For fragment detection, single-stranded DNA is fixed on the microsphere as a capture molecule binding part of a target nucleic acid molecule, and free single-stranded DNA molecules are used as detection molecules and bound at other positions of the target molecule, wherein the tail end of the free detection molecules is coupled with a catalytic reactant.

Preferably, the catalyst is any one or combination of more than two of galactosidase, alkaline phosphatase or horseradish peroxidase.

The composite microspheres which successfully capture target nucleic acid molecules and form microsphere-capture molecule-target molecule-detection molecule-catalyst are introduced into the microfluidic chip, and one catalyst molecule can catalyze reaction substrate molecules with multiple orders of magnitude through the reaction of a catalyst molecule catalytic solution at the tail end in a reaction substrate, so that the effective amplification of signals is realized. Taking galactosidase as an example, resorufin beta-D-galactopyranoside and dihydrofluorescein-di-beta-D-galactopyranoside are used as reaction substrates, and catalytic reaction can be carried out under the catalysis of galactosidase so as to realize signal amplification.

Preferably, the hybridization buffer is a hybridization buffer comprising sodium citrate at a molarity of 20-80mM (e.g., can be 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, or 80mM, etc.) and sodium chloride at a molarity of 500-800mM (e.g., can be 500mM, 550mM, 600mM, 650mM, 700mM, 750mM, or 800mM, etc.).

Preferably, the pH of the hybridization buffer is 6.8-7.4, and may be, for example, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, or the like

As a preferred technical scheme, the nucleic acid molecule detection kit further comprises a microporous plate chip, and the microporous plate chip is processed by adopting an injection molding production method or an MEMS method. Wherein the MEMS fabrication process is a generic term for nano-to millimeter-scale micro-structure fabrication processes.

The composite microsphere structure is introduced into the microporous plate chip for catalytic reaction, the composite microspheres exist in separate micropores, and even if only one catalyst molecule exists on the composite structure, the concentration of the composite microsphere structure is improved by multiple orders of magnitude, so that the amplification of signals can be realized under the condition of not using a nucleic acid amplification technology, and the detection has ultrahigh sensitivity.

Preferably, the volume of a single microwell on the microplate chip is (20-100). times.10^-15L, for example, may be 20X 10^-15L、30×10^-15L、40×10^-15L、50×10^-15L、60×10^-15L、70×10^-15L、80×10^-15L、90×10^-15L or 100X 10^-15L, and the like.

In the present invention, the density of the microwells on the microplate chip determines the amount of data collected and the dynamic range of detection. The densely packed microwells need to be clearly resolved by optical detection and ensure that there is only one or no microsphere in each microwell. Meanwhile, the microplate chip can use a disposable microplate or a reusable microplate chip; disposable microplate chips are produced by injection molding, and non-disposable microplate chips can be processed by a chip MEMS (Micro-Electro-Mechanical systems) method.

In a second aspect, a method of using the nucleic acid molecule detection kit of the first aspect, comprises the steps of:

(1) mixing a sample containing target molecules, coding microspheres marked with capture molecules on the surface, detection molecules and hybridization buffer solution to obtain composite microspheres and/or microspheres which are not combined with the target molecules;

(2) and introducing the composite microspheres and/or microspheres which are not combined with the target molecules into a microporous plate chip for reaction, and then applying external stimulation to detect coding signals and analyzing to obtain a detection result of the target molecules.

As a preferred embodiment of the present invention, the mixing in step (1) is carried out in an apparatus for diagnosing a nucleic acid molecule without amplification.

Preferably, the encoded microspheres with capture molecules labeled on their surface bind to one or not to the target nucleic acid molecules.

If single molecule detection with extremely low concentration is required, the proportion of the microspheres to the target molecules needs to be adjusted, and optimization is carried out according to Poisson distribution, so that only one or no target molecule is captured on one microsphere.

Preferably, the composite microspheres are coded microspheres with capture molecules labeled on the surfaces, which are combined with target molecules and detection molecules. The composite microsphere is a composite structure of microsphere-capture molecule-target molecule-detection molecule-catalyst.

As a preferred embodiment of the present invention, the method for introducing the composite microspheres into the microporous plate chip in step (2) is any one or a combination of two or more selected from gravity sedimentation, magnetic sedimentation, and electric field-induced sedimentation, and preferably electric field-induced sedimentation.

In order to ensure efficient and rapid assembly of microspheres and micropores in a one-to-one pairing manner, the following methods can be adopted:

(1) gravity settling: by utilizing the characteristic that the density of the microspheres is greater than that of water, the microspheres can naturally sink into the micropores by gravity;

(2) magnetic settling: because the microspheres can wrap the magnetic material, the microspheres can be manipulated into the micropores by using magnetic force;

(3) electric field induced sedimentation: because the dielectric constant of the microsphere is greatly different from that of the solution, the microsphere can be pushed into the micropore by applying a non-uniform-strength alternating electric field to generate dielectrophoresis force, and after the reaction detection is finished, the direction of the force can be changed by adjusting the frequency of the electric field to push the microsphere out of the micropore, so that the repeated use of the micropore is realized.

Preferably, the individual microwells on the microplate chip contain one or none of the composite microspheres and/or microspheres not bound to target molecules.

Preferably, the microwells on the microplate chip are sealed with an oil phase and/or a polymer film at the time of detection.

Preferably, the method for applying the external stimulus in step (2) is near-field excitation using fluorescence of at least two wavelengths.

In the present invention, if the traditional chemiluminescence catalysis mode is adopted, the concentration of the catalyst needs to be amplified to the maximum extent in order to achieve the unimolecular sensitivity without amplification. Therefore, in the invention, each composite microsphere structure is placed in a micropore by adopting a microreactor mode, and the volume of the micropore is 50 multiplied by 10^-15L or so, then using oil phase or high molecular film to seal and lock every micropore, so that every microsphere composite structure is in a single small volume, and the concentration of catalyst molecule on the composite structure is increasedIs many orders of magnitude higher and is sufficient to catalyze the luminescence of a single micropore.

As a preferred embodiment of the present invention, the non-amplified nucleic acid molecule diagnostic apparatus comprises a microsphere-based sample processing section and a microplate-based detection section.

Preferably, the microsphere-based sample processing part comprises a sample extraction module, a liquid transfer module, a mixing module, a magnetic suction module, a liquid pre-storage module and a liquid path cleaning module.

In the invention, the sample extraction module of the non-amplification nucleic acid molecule diagnosis equipment is used for cracking a biological sample, extracting nucleic acid molecules (DNA or RNA) released into a solution by adopting an automatic magnetic particle method or a disposable silica gel column mode, capturing the nucleic acid molecules on magnetic microspheres in a non-specific way, carrying out magnetic separation on the microspheres, washing and eluting the nucleic acid molecules on the surfaces of the microspheres by using water to obtain a pure nucleic acid solution; the liquid transfer module transfers the microspheres connected with the probes, the sample solution to be detected, the detection probes, the catalyst base solution and other reagents to the mixing module; the microspheres connected with molecules to be detected in the mixing module are hybridized with detection molecules (sequences); the magnetic module can wash and capture the microspheres for target molecules to be detected and detection molecules through enrichment; the liquid prestoring module is used for storing reaction solutions such as buffer solution and the like; the liquid path cleaning module cleans the liquid path to prevent cross contamination.

Preferably, the mixing in step (1) is performed in a mixing module of the apparatus for diagnosing nucleic acid molecules without amplification.

Preferably, the detection part based on the micro-porous plate comprises a liquid path control module, a multicolor fluorescence excitation module, a front scattering imaging module, a fluorescence emission filtering module, a magnetic absorption module and an alternating electric field control module.

The liquid path control module is used for introducing a solution containing composite microspheres into a clamp provided with a microporous plate and simultaneously carrying out oil sealing on the surface of the microporous plate; the multicolor fluorescence excitation module is used for exciting the multiple fluorescence coding microspheres and fluorescent substances released by substrate molecules under the action of a catalyst; obtaining a low-background fluorescent photo of the micropore plate after the microspheres are introduced through a front scattering imaging module; the fluorescence emission filtering module is used for distinguishing the coding microspheres and micropore brightness change (concentration and change of fluorescent substances released by substrate molecules under the action of a catalyst); the magnetic absorption module and the alternating electric field module are used for helping the microspheres to be led into or led out of the micropores.

Preferably, the non-amplified nucleic acid molecule diagnosis apparatus further comprises an automatic control module and an automatic image recognition analysis module.

Preferably, the image automatic recognition analysis module recognizes brightness of wells in the microplate chip.

All modules of the non-amplification nucleic acid molecule diagnosis equipment are automatically controlled through a bottom layer program, the software is integrated with an image automatic identification analysis module, a system automatically identifies the brightness in each micropore and obtains the distribution of the micropore brightness, whether a reaction occurs in each micropore or not is automatically judged, and the number of the microsphere corresponding to each reaction is automatically judged. And obtaining the dynamic detection range of the analog signal through brightness analysis, and taking the number of the reaction occurrences of the microspheres with the same number as the dynamic detection range of the digital signal.

As a preferred technical scheme, the use method of the nucleic acid molecule detection kit comprises the following steps:

(1) mixing a sample containing target molecules, coding microspheres marked with capture molecules on the surface, detection molecules and hybridization buffer solution in a mixing module of the non-amplification nucleic acid molecule diagnosis equipment, wherein the coding microspheres marked with the capture molecules on the surface are combined with one or not combined with the target nucleic acid molecules to obtain composite microspheres and/or microspheres not combined with the target molecules;

(2) and introducing the composite microspheres and/or the microspheres which are not combined with the target molecules into the microporous plate chip by an electric field induced sedimentation method, wherein single micropores on the microporous plate chip contain one or no composite microspheres and/or coded microspheres which are not combined with the target molecules, after reaction, near-field excitation is carried out by using fluorescence with at least two wavelengths, coding signals of the composite microspheres and/or the coded microspheres which are not combined with the target molecules are detected, and analysis is carried out by using an analysis module of a nucleic acid molecule diagnosis device without amplification, so as to obtain the detection result of the target molecules.

The recitation of numerical ranges herein includes not only the above-recited numerical values, but also any numerical values between any of the above-recited numerical ranges not recited, and for the sake of brevity and clarity, the present invention is not intended to be exhaustive of the specific numerical values encompassed within the range.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) according to the kit for detecting the nucleic acid molecules without amplification, the catalyst is connected to the trace nucleic acid molecules which are not amplified, the concentration of the catalyst is amplified to the maximum extent by utilizing the principle of a microreactor, a large amount of reactants in a catalytic solution react to realize the amplification of signals, and the ultrahigh sensitivity is achieved when the amplification method is used for detection;

(2) in the invention, the coded microspheres marked by a plurality of luminescent materials can realize the purpose of simultaneously detecting a plurality of nucleic acid molecules, and have less requirements on the amount of a sample, so that the molecular diagnosis is more efficient and faster; the detection kit is matched with the non-amplification nucleic acid molecular diagnosis equipment for use, so that on one hand, the cross contamination of the external operating environment can be avoided, on the other hand, the whole process of sample pretreatment and analysis can be realized, the accuracy of the analysis result is obviously improved, and the detection result is more reliable.

Drawings

FIG. 1 is a photograph of a microplate chip containing fluorescent-encoded microspheres taken by a nucleic acid molecule diagnosis device without amplification.

Detailed Description

The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings. The following examples are merely illustrative of the present invention and do not represent or limit the scope of the claims, which are defined by the claims.

In the following examples, the synthesis of fluorescence-encoded microspheres can be synthesized using the following method:

methacrylic acid, a styrene monomer, polymethyl methacrylate and a crosslinking agent (divinylbenzene) are mixed in chloroform to be used as a polymer solution, 4.5mL of the polymer solution is added with 0.5mL of rhodamine, 0.5mL of fluorescein and 90mg of nano magnetic particles, and then the polymer solution is placed in two reactors with 300mL of deionized water and sodium dodecyl sulfate to obtain emulsion.

Ultrasonic generators (ultrasonic pulses 50uJ and 100uJ) and sensors (4MHz, 6dB bandwidth, 4.4MHz and focusing length 10.5cm) are respectively arranged on two sides of a container for bearing the emulsion, and the real-time feedback closed-loop control of the ultrasonic waves through the sensors controls Fe in the microsphere emulsion₃O₄The magnetic nano particles are modulated, the magnetic particles absorb ultrasonic wave bands and keep the polarity orientation to be induced by the field of the external ultrasonic wave according to the principle of a magnetic resonance imaging device, and in order to keep the modulation efficiency of the induction, the sensor passes through Fe₃O₄Specific absorption spectrum of magnetic nano-particles can timely modulate energy of ultrasonic pulse so as to controllably modulate Fe₃O₄The orientation of the magnetic nano particles is adopted to obtain liquid drops with uniformly distributed functional particles and consistent dipole directions;

uniformly dispersing the solution into droplets of 10 mu m, adding an initiator azobisisobutyronitrile into the solution, heating for polymerization and crosslinking reaction, slowly dissolving chloroform in each droplet in water after 24 hours, volatilizing, polymerizing and crosslinking monomers, and finally forming the fluorescent coding microspheres.

Example 1

This example provides a nucleic acid molecule detection kit without amplification, which can detect influenza A virus nucleic acid molecules and influenza B virus nucleic acid molecules in a sample at the same time. The preparation method and the using method comprise the following steps:

two parts of fluorescent coding microspheres are taken, the microsphere 1 is marked by a complementary single-stranded DNA molecule (capture molecule 1) of an influenza A virus nucleic acid molecule, and the microsphere 2 is marked by a complementary single-stranded DNA molecule (capture molecule 2) of an influenza B virus nucleic acid molecule. The method comprises the following specific steps:

taking microsphere 1 as an example, 1mg of microsphere 1 is dispersed in 1mL of PBS buffer, 5mg of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 5mg of Sulfo-NHS (N-hydroxythiosuccinimide) are added, mixed uniformly and kept stirring for 10 minutes, then influenza A virus nucleic acid complementary single-stranded DNA molecules with amino at the 3' end are added, 10% BSA is added as a blocking agent, stirring is carried out for 30 minutes, then microsphere 1 is washed by magnetic separation and finally dispersed in hybridization buffer (50mM sodium citrate, pH 7.2, 750mM NaCl) to obtain fluorescent microsphere 1 marked by capture molecule 1, and the preparation method of fluorescent microsphere 2 marked by capture molecule 2 is the same as above.

Will contain 1X 10⁹Copies/microliter of influenza A virus and 1X 10⁹All nucleic acids of the copied/microliter influenza B virus culture solution are extracted by a magnetic particle method and are finally eluted in water. The method comprises the following specific steps: mu.L of lysis buffer (50mM Tris, pH 8.0, 4M guanidine hydrochloride, 1mM EDTA, 1% Triton-X100), 10. mu.L proteinase K (20mg/mL), was added to 200. mu.L of virus culture medium in a water bath at 55 ℃ for 10 minutes; adding 150 mu L of isopropanol into each sample, mixing and uniformly blowing, and adding 500 mu g of silicon dioxide magnetic beads with hydroxyl on the surfaces; after standing for five minutes, the magnetic beads were adsorbed on a magnetic frame, the supernatant was removed with a pipette, the magnetic beads were washed with 70% ethanol, and eluted with 50. mu.L of water.

Then adding 1mg/mL fluorescent microsphere 1 coupled with oligonucleotide probe and fluorescent microsphere 2, mixing in equal volume to obtain 0.2mL solution, adding 0.2mL hybridization buffer (50mM sodium citrate, pH 7.2, 750mM NaCl) and 0.2mL nucleic acid probe coupled with biotin, wherein the biotin molecule is specifically identified by streptavidin, the nucleic acid probe can be specifically combined with influenza A virus nucleic acid molecule or influenza B virus nucleic acid molecule, uniformly mixing for 5 minutes, then carrying out magnetic attraction and washing, adding 0.2mL streptavidin-beta-galactosidase conjugate and connecting to the nucleic acid probe as detection molecule, uniformly mixing for 5 minutes, then carrying out magnetic attraction and washing, and then dispersing magnetic beads into 100 mu M dihydrofluorescein-di-beta-D-galactopyranoside solution.

And (3) detection: adding a microsphere composite structure into a reactor with a microporous plate chip through microfluid, adding 10MHz alternating current, pushing microspheres into micropores, sealing the surfaces of the micropores with silicone oil, reacting for 2 minutes, exciting with 488nm wavelength light, photographing with an optical filter 1(512nm transmission, 20nm bandwidth), photographing with an optical filter 2(570nm transmission, 30nm bandwidth), exciting with 532nm wavelength light, photographing with an optical filter 3(615nm transmission, 30nm bandwidth), flowing ethanol, flowing cleaning fluid, changing the alternating current frequency to 10kHz, flowing cleaning fluid, and cleaning the reactor.

FIG. 1 is a photograph of a microplate containing fluorescence-encoded microspheres 1 obtained by a fluorescence excitation filter 1 with 488nm wavelength light for a non-amplified nucleic acid molecule diagnosis device. As can be seen from FIG. 1, the fluorescent microspheres 1 capturing the nucleic acids of the influenza A virus are uniformly dropped into the microwells, each microwell has only one microsphere and is limited in the microwell, and the labeled enzyme on the microspheres amplifies the fluorescent signal and is detected by the front scattering imaging module.

Data processing and concentration determination: the image processing software firstly identifies all magnetic beads, and classifies the fluorescent microspheres (fluorescent microsphere 1 and fluorescent microsphere 2) according to the fluorescent intensity under the optical filter 1 and the optical filter 2, because the unamplified concentration of the viral RNA is very low, most of the magnetic beads can not capture corresponding RNA to form composite microspheres and are conjugated and labeled by streptavidin-beta-galactosidase, and no corresponding fluorescent signal exists. The remaining magnetic beads can capture only one or a few target RNAs to form a sandwich complex and are conjugated and labeled by streptavidin-beta-galactosidase, and corresponding fluorescent signals are obtained.

The ratio of the total number of magnetic beads to which chemical signals were detected (fon) to the total number of labeled Enzyme molecules and the total number of magnetic beads (AEB, Average Enzyme per Bead) followed a Poisson distribution (AEB ═ ln (1-fon)). After linear fitting is carried out on the AEB value and the concentration value of the object to be measured to draw a calibration curve, the unknown sample is tested by the same method, the obtained signal value is brought into a standard curve by an interpolation method to measure the concentration of the object to be measured in the unknown sample, and the obtained concentration is 10⁵Copies/microliter (typically 10)⁴-10⁷Copy/microliter).

Sensitivity analysis and specificity analysis: influenza a virus nucleic acid and influenza b virus nucleic acid of known concentrations (copy number) were separately step-diluted and subjected to the above test, and a standard curve was plotted. The concentration (copy number) of the standard deviation on the standard curve after the background value plus three times of the background value is repeatedly measured is the sensitivity (detection limit) of the method; the specific analysis shows that the magnetic beads 1 can not detect the nucleic acid of the influenza A virus because of being coupled with the specific capture probe of the influenza A virus nucleic acid, and the magnetic beads 2 can not detect the nucleic acid of the influenza A virus.

Example 2

The embodiment provides an amplification-free nucleic acid molecule detection kit capable of simultaneously detecting miRNA miR-122 and miR-129 in a serum sample in a sample. The preparation method and the using method comprise the following steps:

two parts of fluorescence encoding microspheres are taken, wherein the microsphere 1 is marked by a complementary peptide nucleic acid molecule (capture molecule 1) of miRNA miR-122, and the microsphere 2 is marked by a complementary peptide nucleic acid molecule (capture molecule 2) of miRNA miR-129. The method comprises the following specific steps:

taking microsphere 1 as an example, dispersing 1mg of microsphere 1 in 1mL of PBS buffer solution, adding 5mg of EDC and 5mg of Sulfo-NHS, uniformly mixing and maintaining stirring for 10 minutes, then adding miR-122 complementary single-stranded peptide nucleic acid molecules with amino at the 3' end, then adding 10% BSA as a blocking agent, stirring for 30 minutes, then washing microsphere 1 through magnetic separation, finally dispersing in hybridization buffer solution (50mM sodium citrate, pH 7.2, 750mM NaCl) to obtain fluorescent microsphere 1 marked by capture molecule 1, and preparing fluorescent microsphere 2 marked by capture molecule 2.

Adding a solution containing 4M urea into the serum doped with the miR-122 and miR-129 standards to disperse miRNA in the solution, then adding a 1:1 mixed solution of fluorescent microspheres 1 and 2, adding 0.2mL of hybridization buffer solution and 0.2mL of nucleic acid probe coupled with biotin, uniformly mixing for 5 minutes, and then magnetically attracting and washing. Then 0.2mL of streptavidin-beta-galactosidase was added for conjugation, mixed evenly for 5 minutes, followed by magnetic attraction and washing.

And (3) detection: adding the microsphere composite structure into a reactor with a microporous plate chip through microfluid, adding 10MHz alternating current, pushing microspheres into micropores, sealing the surfaces of the micropores with silicone oil, reacting for 2 minutes, exciting with 488nm wavelength light, photographing with an optical filter 1, photographing with an optical filter 2, exciting with 532nm wavelength light, photographing with an optical filter 3, flowing ethanol, then flowing cleaning liquid, changing the alternating current frequency to 10kHz, then flowing cleaning liquid, and cleaning the reactor.

Data processing and concentration determination: the image processing software first identifies all the beads and classifies them according to the intensity of the fluorescence under filter 1 and filter 2. Because the concentration of miRNA which is not amplified is very low, most of magnetic beads can not capture corresponding RNA to form a sandwich complex and are conjugated and labeled by streptavidin-beta-galactosidase, and corresponding fluorescent signals do not exist. The remaining microspheres can capture only one or a few target RNAs to form composite microspheres and are conjugated and labeled by streptavidin-beta-galactosidase, and corresponding fluorescent signals are obtained.

The ratio of the total number of magnetic beads to which chemical signals were detected (fon) to the total number of labeled Enzyme molecules and the total number of magnetic beads (AEB, Average Enzyme per Bead) followed a Poisson distribution (AEB ═ ln (1-fon)). After linear fitting is carried out on the AEB value and the concentration value of the object to be detected to draw a calibration curve, the unknown sample is tested by the same method, the obtained signal value is brought into a standard curve by an interpolation method to measure the concentration of the object to be detected in the unknown sample, and the concentration of miRNA is 1 multiplied by 10⁴Copy/microliter.

Sensitivity analysis and specificity analysis: known concentrations (copy number) of miR-122 and miR-129 were separately step-diluted and subjected to the above-described test, and a standard curve was drawn. The concentration (copy number) of the standard deviation on the standard curve after the background value plus three times of the background value is repeatedly measured is the sensitivity (detection limit) of the method; the specificity analysis shows that the magnetic bead 1 can not detect the miR-129 due to the coupling of the miR-122 specific capture probe, and the magnetic bead 2 can not detect the miR-122 similarly.

As described above, the kit for detecting a nucleic acid molecule without amplification according to the present invention uses 2X 10 for detecting a viral nucleic acid molecule⁶-1×10⁹After the copied/microliter virus culture solution is extracted, or miRNA which is not extracted is directly detected, the final detectable concentration is 1 multiplied by 10⁴-10⁷Copy/microliter, the sensitivity for detecting virus nucleic acid can reach 3 x10³Copy/microliter, the sensitivity for detecting miRNA can reach 1.2 multiplied by 10³Copy/microliter, illustrating the use of microreaction in the present inventionThe principle of the reactor realizes the amplification of target molecule signals to be detected, achieves ultrahigh sensitivity when an amplification method is used for detection, is matched with non-amplification nucleic acid molecule diagnosis equipment, and has simple detection steps and accurate and reliable detection results.

The applicant declares that the present invention illustrates the detailed structural features of the present invention through the above embodiments, but the present invention is not limited to the above detailed structural features, that is, it does not mean that the present invention must be implemented depending on the above detailed structural features. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of selected components of the present invention, additions of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims

1. An amplification-free nucleic acid molecule detection kit, comprising:

the surface of the microsphere is marked with a capture molecule, a detection molecule and a hybridization buffer solution;

the capture molecule is a nucleotide chain or peptide nucleic acid molecule which is complementarily paired with the target nucleic acid molecule;

the detection molecule is a mononucleotide or nucleotide chain coupled with a catalyst and complementarily paired with a target nucleic acid molecule.

2. The nucleic acid molecule detection kit according to claim 1, wherein the encoded microspheres are microspheres encoded by at least two luminescent materials;

preferably, the luminescent material is selected from any one or a combination of more than two of organic fluorescent molecules, inorganic fluorescent molecules or quantum dots;

preferably, the microspheres comprise magnetic nanoparticles;

preferably, the surface of the encoded microspheres is chemically modified.

3. The nucleic acid molecule detection kit according to claim 1 or 2, wherein the preparation method of the encoded microsphere comprises the following steps:

mixing at least two luminescent materials and microspheres in a high polymer material, dispersing a high polymer solution in a water phase to form uniform liquid drops through a multiple-coupling physical field, and then wrapping the luminescent materials and magnetic nanoparticles in the liquid drops through a cross-linking polymerization reaction to obtain the coding microspheres.

4. The nucleic acid molecule detection kit according to any one of claims 1 to 3, wherein the catalyst is selected from the group consisting of galactosidase, alkaline phosphatase, and horseradish peroxidase;

preferably, the hybridization buffer comprises sodium citrate with a molar concentration of 20-80mM and sodium chloride with a molar concentration of 500-800 mM;

preferably, the pH of the hybridization buffer is 6.8-7.4;

preferably, the nucleic acid molecule detection kit further comprises a microplate chip;

preferably, the volume of a single microwell on the microplate chip is (20-100). times.10^-15L。

5. A method of using the nucleic acid molecule detection kit according to any one of claims 1 to 4, comprising the steps of:

(1) mixing a sample containing target nucleic acid molecules, coding microspheres marked with capture molecules on the surface, detection molecules and hybridization buffer solution to obtain composite microspheres and/or microspheres which are not combined with the target molecules;

6. The use of claim 5, wherein the mixing in step (1) is performed in a nucleic acid molecule diagnosis device without amplification;

preferably, the encoded microspheres with capture molecules labeled on the surface bind to one or not to the target nucleic acid molecules, and the single microwells on the microplate chip contain one or not the composite microspheres and/or microspheres not bound to target molecules;

preferably, the composite microspheres are coded microspheres with capture molecules labeled on the surfaces, which are combined with target molecules and detection molecules.

7. The use method according to claim 5 or 6, wherein the method for introducing the composite microspheres into the microporous plate chip in step (2) is selected from any one or a combination of two or more of gravity sedimentation, magnetic sedimentation or electric field-induced sedimentation;

8. The use of any of claims 5-7, wherein the non-amplified nucleic acid molecule diagnostic device comprises a microsphere-based sample processing part and a microplate-based detection part;

preferably, the microsphere-based sample processing part comprises a sample extraction module, a liquid transfer module, a mixing module, a magnetic suction module, a liquid pre-storage module and a liquid path cleaning module;

9. The use method according to any one of claims 5 to 8, wherein the non-amplified nucleic acid molecule diagnosis apparatus further comprises an image automatic recognition analysis module which recognizes brightness of microwells in the microplate chip.

10. Use according to any of claims 5-9, characterized in that it comprises the following steps: