CN113684246A - High-sensitivity nucleic acid detection method without amplification and application thereof - Google Patents
High-sensitivity nucleic acid detection method without amplification and application thereof Download PDFInfo
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Abstract
The invention discloses a nucleic acid detection method, which comprises the following steps: providing a sample to be detected, magnetic beads, a primary antibody and a secondary antibody; mixing a sample to be detected, magnetic beads, primary antibodies and secondary antibodies for reaction to form a magnetic bead complex; and detecting according to the detection label of the magnetic bead complex to obtain a detection result. The nucleic acid detection method according to the embodiment of the application has at least the following beneficial effects: the magnetic beads are used as a reaction substrate and a minimum reaction unit of the whole reaction, and can play a role in concentration and amplification in subsequent immunoreaction. The magnetic bead labeled nucleotide chain and the nucleic acid segment to be detected in the sample to be detected are mutually matched to form a hybrid duplex, the hybrid duplex is captured through a primary antibody, and the hybrid duplex is detected through a secondary antibody, so that the specificity and the sensitivity of detection are increased, and the nucleic acid segment to be detected does not need to be amplified additionally.
Description
Technical Field
The application relates to the technical field of nucleic acid detection, in particular to a high-sensitivity nucleic acid detection method without amplification and application thereof.
Background
The hybridization technology based on the base complementary pairing principle is the earliest developed nucleic acid diagnosis technology, and the basic idea is that complementary pairing of oligonucleotide chains is utilized, and a probe and single-stranded nucleic acid to be detected in a sample are hybridized and complemented to generate a unique read signal, so that the detection of a known sequence is realized. The nucleic acid hybridization technology is mature, the cost is low, but the sensitivity is relatively low, and the high-sensitivity nucleic acid detection cannot be met. Therefore, the conventional method still requires a PCR amplification reaction for the target fragment. The complex temperature cycle required by the amplification process has the possibility of aerosol pollution, and is easy to cause false positive results. Therefore, it is necessary to provide a nucleic acid detection method without amplification with high sensitivity.
Disclosure of Invention
The present application is directed to solving at least one of the problems in the prior art. To this end, the present application proposes a highly sensitive, amplification-free nucleic acid detection method and its applications.
The present application also aims to provide a nucleic acid detection kit.
In a first aspect of the present application, there is provided a nucleic acid detection method comprising the steps of:
providing a sample to be detected, a magnetic bead, a primary antibody and a secondary antibody, wherein the magnetic bead is marked with a nucleotide chain, the nucleotide chain is used for complementary pairing with a nucleic acid fragment to be detected in the sample to be detected to form a hybrid duplex, the primary antibody is used for specific combination with the hybrid duplex, the secondary antibody is used for specific combination with the primary antibody, and the secondary antibody is provided with a detection mark;
mixing a sample to be detected, magnetic beads, primary antibodies and secondary antibodies for reaction to form a magnetic bead complex;
and detecting according to the detection label of the magnetic bead complex to obtain a detection result.
The nucleic acid detection method according to the embodiment of the application has at least the following beneficial effects:
(1) the magnetic beads are used as a reaction substrate and a minimum reaction unit of the whole reaction, and can play a role in concentration and amplification in subsequent immunoreaction; moreover, after various reaction reagents are mixed to participate in the reaction, the magnetic beads can also accelerate the immunoreaction and reduce the reaction time. In addition, magnetic beads can be further used for separation, washing and concentration.
(2) The magnetic bead labeled nucleotide chain and the nucleic acid segment to be detected in the sample to be detected are mutually matched to form a hybrid duplex, the hybrid duplex is captured through a primary antibody, and the hybrid duplex is detected through a secondary antibody, so that the specificity and the sensitivity of detection are increased, and the nucleic acid segment to be detected does not need to be amplified additionally.
The magnetic beads refer to magnetic particles which can exhibit strong magnetism under the action of an external magnetic field, and generally include internal magnetic particles and shells (non-limiting examples of which include polymer shells, silica shells and the like) wrapping the external sides of the magnetic particles, and also include "sandwich" magnetic beads formed by the internal and external polymer layers and the intermediate magnetic particles. The hybrid duplex is a double strand formed by combining a single strand of the nucleic acid fragment to be detected and a single strand of the nucleotide strand labeled on the magnetic bead through complementary bases on the two strands. The primary antibody is a capture antibody for specifically binding the hybridization duplex, and after the hybridization duplex is formed by the nucleic acid fragment to be detected and the nucleotide chain on the magnetic bead, the primary antibody is specifically bound with the antigen-antibody, so that a corresponding binding site is provided for the secondary antibody with the detection label. The secondary antibody is a detection antibody for specifically binding to the primary antibody, and the primary antibody has a part of other binding sites after binding to the hybridization duplex, and can be specifically bound to the secondary antibody, thereby forming a magnetic bead complex such as magnetic bead- (nucleotide chain-nucleic acid fragment to be detected) -primary anti-secondary antibody-detection label. The detection label refers to an optional label in immunoassay, and non-limiting examples thereof are radioisotopes, enzymes, fluorescent substances, chemiluminescent substances, bioluminescent substances, pigment molecules, and the like.
In some embodiments of the present application, the manner of detection is: and packaging the magnetic bead complex to form a liquid drop, and detecting according to the detection mark in the liquid drop to obtain a detection result. The magnetic bead complex in the liquid drop is used as the minimum reaction unit in the reaction process by the mode of packaging to form the liquid drop, so that the reaction speed is further accelerated, and the reaction time is shortened. The encapsulation may be performed by digital droplet microfluidics, using microfluidic chip processing, as is well known in the art.
In some embodiments of the present application, the volume of the droplet is (1-10). times.10-15And L. The signal of the detection label or the signal generated by the reaction of the detection label is further concentrated into a signal which can be directly detected by confining the magnetic bead complex in a droplet of about the size of a flight-rise, and furtherThe detection limit is reduced by one step.
In some embodiments of the present application, the method for encapsulating a complex of magnetic beads comprises the steps of: providing an aqueous solution of the oil phase and the magnetic bead complex; the oil phase is taken as a mobile phase, the aqueous solution of the magnetic bead complex is taken as a disperse phase, and the water-in-oil droplets are formed by encapsulation under the action of pressure and/or shearing force. The packaging method utilizes the capillary action of the oil phase to shear the aqueous solution of the magnetic bead complex, so that the dispersed phase is intercepted by the mobile phase to form a droplet for packaging the magnetic bead complex.
In some embodiments of the present application, the oil phase further comprises a surfactant. The droplets formed are made more stable by the addition of surfactants in the oil phase.
In some embodiments of the present application, S3 is: and analyzing to obtain the concentration of the nucleic acid fragment to be detected in the sample to be detected according to the quantity of the liquid drops encapsulated with the magnetic bead complex. Under the protection of the surfactant, the liquid drops can not be fused and exchanged with substances in the reaction process, and finally the complete and independent liquid drop form is kept, so that the liquid drops can be directly detected without designing a microporous structure with the size corresponding to the size of the liquid drops to sink into the microporous structure. Moreover, because the microporous structure is not required to be designed for partition and isolation, the size of the liquid drops in the detection area can be more uniform and has higher density, so that the detection is convenient. In addition, the droplets with the volume can ensure that only one magnetic bead is in one droplet, so the content or the concentration of the nucleic acid fragment to be detected in the sample to be detected can be judged by directly comparing the proportion of the droplet encapsulated with the magnetic bead complex to the total number of the droplets.
In some embodiments of the present application, the detectable label is an enzyme. Enzyme linked immunosorbent assay is used to make enzyme on the magnetic bead complex react with the provided substrate to develop color and generate light signal for detection. Compared with other immunodetection methods, the method has the advantages of good specificity, high sensitivity and lower environmental pollution.
In some embodiments of the present application, the enzyme is selected from at least one of β -galactosidase, horseradish peroxidase, alkaline phosphatase, urease, glucose oxidase.
In some embodiments of the present application, the secondary antibody is a porous nanomaterial immobilized with an antibody, the porous nanomaterial supporting a plurality of detection labels, the antibody being for specifically binding to the primary antibody. The porous nano material (such as a metal organic framework material, a covalent organic framework material and the like) has the characteristics of large specific surface area, high porosity and the like, and can further amplify detection signals by loading a plurality of detection marks on the porous nano material, so that the detection sensitivity is improved.
In some embodiments of the present application, the magnetic beads have a diameter of 0.5 to 2 μm.
In a second aspect of the present application, there is provided a nucleic acid detection kit, comprising:
the magnetic bead is marked with a nucleotide chain, and the nucleotide chain is used for complementary pairing with the nucleic acid segment to be detected to form a hybrid duplex;
a primary antibody for specific binding to the hybrid duplex;
the secondary antibody is used for being specifically combined with the primary antibody, and is also provided with a detection label;
the micro-fluidic chip is provided with a reaction chamber and a detection chamber which are communicated with each other, the reaction chamber is used for allowing magnetic beads, primary antibodies and secondary antibodies to react to form a magnetic bead complex, and the detection chamber is used for detecting signals of detection marks.
The nucleic acid detection kit according to the embodiment of the application has at least the following beneficial effects:
the traditional biochemical reaction has higher requirements on conditions such as time, temperature, mixing efficiency and the like, and a plurality of biochemical reaction functional areas can not be directly connected in sequence because the conditions required by different functional areas are different when the microfluidic chip is used for carrying out reaction. The reaction in the scheme does not need complex temperature circulation, and all parts can be integrated and connected in sequence, so that the manual operation is reduced, and the possibility of pollution can be further reduced. On the other hand, the magnetic beads are used as a reaction substrate and a minimum reaction unit of the whole reaction, so that the concentration and amplification effects can be achieved in the subsequent immunoreaction, the immunoreaction speed is accelerated, and the reaction time is shortened. The magnetic bead labeled nucleotide chain and the nucleic acid segment to be detected in the sample to be detected are mutually matched to form a hybrid duplex, the hybrid duplex is captured through a primary antibody, and the hybrid duplex is detected through a secondary antibody, so that the specificity and the sensitivity of detection are increased, and the nucleic acid segment to be detected does not need to be amplified additionally.
In some embodiments of the present application, the microfluidic chip further includes a droplet formation region, the droplet formation region is provided with an oil phase inlet for injecting the oil phase, a water phase inlet for injecting the aqueous solution of the magnetic beads, and an outlet for forming the droplets.
Additional aspects and advantages of the present application will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the present application.
Drawings
FIG. 1 is a schematic diagram of a complex of magnetic beads according to an embodiment of the present application.
Fig. 2 is a schematic diagram of a microfluidic chip according to an embodiment of the present application.
Fig. 3 is a partial enlarged view of the position a in fig. 2 of the present application.
FIG. 4 is a picture taken by a CCD camera of the droplet generation in the droplet generation region for different diameters of the outlet of FIG. 3 of the present application.
Fig. 5 is a schematic diagram of a microfluidic chip according to an embodiment of the present application, which is detected by an enzyme chain immunoassay method.
Fig. 6 is a fluorescence image of samples of different concentrations in example 1 of the present application.
FIG. 7 is a standard curve plotted for different concentrations of standard in example 1 of the present application.
Reference numerals: magnetic bead 100, streptavidin 110, biotin 120, nucleotide chain 130, nucleic acid segment to be detected 140, primary antibody 150, secondary antibody 160, detection mark 170, droplet generation region 210, first liquid inlet 211, second liquid inlet 212, third liquid inlet 213, mixed reaction region 220, reaction flow channel 221, detection region 230, detection chamber 231, waste liquid port 232, first oil phase inlet 310, second oil phase inlet 320, water phase inlet 330, outlet 340, and droplet 350.
Detailed Description
The conception and the resulting technical effects of the present application will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, and not all embodiments, and other embodiments obtained by those skilled in the art without inventive efforts based on the embodiments of the present application belong to the protection scope of the present application.
The following detailed description of embodiments of the present application is provided for the purpose of illustration only and is not intended to be construed as a limitation of the application.
In the description of the present application, the meaning of a plurality is one or more, the meaning of a plurality is two or more, and the above, below, exceeding, etc. are understood as excluding the present number, and the above, below, within, etc. are understood as including the present number. If the first and second are described for the purpose of distinguishing technical features, they are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.
In the description of the present application, unless otherwise expressly limited, terms such as set, mounted, connected and the like should be construed broadly, and those skilled in the art can reasonably determine the specific meaning of the terms in the present application by combining the detailed contents of the technical solutions.
In the description of the present application, reference to the description of the terms "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Referring to fig. 1, a structure of a magnetic bead complex generated in a nucleic acid detecting method according to an embodiment of the present disclosure is shown, the magnetic bead complex including a magnetic bead 100, a nucleotide chain 130, a nucleic acid fragment to be detected 140, a primary antibody 150, and a secondary antibody 160. The nucleotide chain 130 is connected with the magnetic bead 100 and connected with the nucleic acid fragment to be detected according to the base complementary pairing principle to form a hybrid duplex, while the primary antibody 150 can be specifically combined with the hybrid duplex through antigen-antibody interaction, and the secondary antibody 160 is specifically combined with the primary antibody 150 through antigen-antibody interaction.
In the above examples, the magnetic bead 100 is a magnetic particle, and in some embodiments, the magnetic bead 100 includes an inner magnetic particle and a polymer shell layer wrapping the outer magnetic particle.
In some embodiments, the nucleotide strand 130 is an RNA probe capable of base complementary pairing with the nucleic acid fragment 140 to be detected to form a hybrid duplex, and in some embodiments, the hybrid duplex is DNA: RNA hybridizes double-stranded.
In some embodiments, the nucleotide chain 130 is modified with a connecting element, which can be connected to the magnetic bead 100. For example, the connecting element may be biotin 120, and streptavidin 110 is modified on the magnetic bead 100, and the nucleotide chain 130 is fixed on the magnetic bead 110 through the connection between the streptavidin 110 and the biotin 120.
In the above embodiment, the secondary antibody 160 has a detection label 170 thereon, and the specific binding of the secondary antibody 160 to the primary antibody 150 allows the detection label 170 to be linked to the magnetic bead complex for detection. In some embodiments, the detection label 170 can be a radioisotope, an enzyme, a fluorescent substance, a chemiluminescent substance, a bioluminescent substance, a pigment molecule, or the like. In some preferred embodiments, the detection marker 170 is an enzyme. Compared with other immunoassay methods, the enzyme-linked immunoassay has good specificity, high sensitivity and lower environmental pollution, and the enzyme can be at least one of beta-galactosidase, horseradish peroxidase, alkaline phosphatase, urease, glucose oxidase and the like.
Referring to fig. 2, a schematic diagram of a microfluidic chip used in packaging and detection processes in one embodiment of the present application is shown. The microfluidic chip has a flow channel layer, which in some embodiments includes a droplet generation region 210, a mixing reaction region 220, and a detection region 230. The droplet generation region 210 is provided with a first liquid inlet 211 and a second liquid inlet 212, and the first liquid inlet 211 and the second liquid inlet 212 are respectively intersected at a position A through a connected flow channel and communicated with the mixed reaction region 220. The first inlet 211 is used for introducing an aqueous solution of the magnetic bead complex into the droplet generation region 210, and the second inlet 212 is used for introducing an oil phase into the droplet generation region 210. The mixing reaction region 220 is provided therein with a reaction chamber formed by the reaction flow channel 221, and the droplets generated in the droplet generation region 210 enter the reaction flow channel 221, in which reaction raw materials react with each other. After the reaction is finished, the droplets and the magnetic bead complex wrapped in the droplets are sent to the detection chamber 231 of the detection region 230 for dispersion, and the content of the nucleic acid fragments to be detected is determined by detecting whether the magnetic beads in the droplets have detection marks or not. In some specific embodiments, the apparatus further comprises a third liquid inlet 213, and the third liquid inlet 213 is close to the first liquid inlet 211 and is used for introducing other raw materials participating in the reaction into the droplet generation region 210. In some embodiments, the third inlet 213 and the first inlet 211 are merged before a, and the mixed liquid forming the water phase participates in the formation of the liquid droplets. For example, when the detection marker is an enzyme, a substrate for enzyme reaction may be introduced into the microfluidic chip through the third inlet 213, so that the substrate can be mixed with the enzyme for reaction. After the detection is finished, the waste is discharged from a waste liquid port 232 connected with the detection chamber 231, and the magnetic beads in the waste are separated and cleaned by an external magnetic field, so that the recovery is finished.
Referring to fig. 3, an enlarged view of a portion of the application at position a in fig. 2 is shown. Referring to fig. 2, the flow channel connected to the second liquid inlet 212 forms a first oil phase inlet 310 and a second oil phase inlet 320, the flow channel connected to the first liquid inlet 211 forms a water phase inlet 330, the first oil phase inlet 310 and the second oil phase inlet 320 are oppositely disposed and are connected to the water phase inlet 330 to form a junction region and an outlet 340, the oil phase fed from the second liquid inlet 212 is injected into the junction region through the first oil phase inlet 310 and the second oil phase inlet 320, the aqueous solution of the magnetic bead complex is injected through the water phase inlet 330, and in the junction region, the oil phase intercepts the flowing aqueous solution of the magnetic bead complex under the pressure and shear force and wraps the intercepted aqueous solution to form water-in-oil droplets 350, and then is fed into the reaction chamber 220 through the outlet 340 under the pressure.
In some embodiments, the volume of the droplets is controlled to be (1-10). times.10-15And L. After the magnetic bead complex is limited to the droplets with the size of about flying, the signal of the detection label or the signal generated by the reaction of the detection label is concentrated into a signal which can be directly detected, so that the detection limit can be further reduced, and the sensitivity and the accuracy of detection can be improved. In the microfluidic chip, the droplet size can be controlled by adjusting the flow rate of the liquid at each inlet and the difference between the components and the ratio of the components in the water phase and the oil phase, or by adjusting the size of the outlet to adjust the size of the generated droplet. Referring to FIG. 4, from a to c are photographs of the droplet sizes at 10 μm, 20 μm and 30 μm in diameter of the outlet in FIG. 3, respectively. It can be seen that as the size of the outlet 340 increases, the diameter of the resulting droplet 350 increases. Thus, the size of the droplets may be adjusted by at least any of the above or other means known in the art.
Referring to fig. 5, a schematic diagram of a microfluidic chip for detection by the enzyme chain immunoassay method in the embodiment of the present application is shown. Where a represents droplets that are transferred into the detection chamber and are uniformly arranged, and since the volume of the droplet is small, only one magnetic bead can be encapsulated in a single droplet, the droplets are divided into droplets in which a magnetic bead complex is encapsulated and droplets in which a magnetic bead complex is not contained. In the droplet containing the magnetic bead complex, the nucleotide strands on the magnetic bead complex form a hybridization duplex, allowing the primary and secondary antibodies to bind to the magnetic beads and carry the corresponding detection labels (olivary labels in the partial droplet in a). While other droplets do not have detectable detection marks therein. b is a color development result chart of the droplets in a. After the magnetic beads in the liquid drops wrapped with the magnetic bead complex are mixed with the substrate, a color reaction occurs, so that the liquid drops can detect corresponding optical signals; in contrast, the other droplets are not colored and remain as they are because they have no detectable detection marks. c represents the quantitative count of the corresponding result in b. Referring to fig. 5 c, because the volume of the droplet is smaller, only one magnetic bead is wrapped in one droplet, so that the number of the droplets emitting light in the detection chamber can be counted directly, or can be further compared with the total number of the droplets, so as to obtain the content of the nucleic acid fragment to be detected in the sample.
In some embodiments, because the detection method of the enzyme chain immunity is adopted, the requirements of each part of the microfluidic chip on the reaction temperature are not too strict, so that the generation, mixing, reaction and droplet imaging of the droplets can be connected by adopting a continuous flow design mode through a micro-channel, the advantages of small reagent consumption and high detection efficiency of the microfluidic chip are brought into play, and the analysis and detection cost and the artificial interference are greatly reduced. In the mixed reaction area of the liquid drops, the generated liquid drops are fully mixed by designing a plurality of mixing units in the flow channel and matching with the spiral flow channel, and the temperature of the area can be further adjusted by an instrument, so that the combination of a substrate and an enzyme is promoted, an enzymatic fluorescent substrate is generated, the whole liquid drops generate corresponding fluorescence, and finally the liquid drops are imaged in the detection area.
The present example provides a nucleic acid detection method, including the steps of: s1: providing a sample to be detected, magnetic beads, a primary antibody and a secondary antibody; s2: mixing a sample to be detected, magnetic beads, a primary antibody and a secondary antibody for reaction to obtain the magnetic bead complex; s3: and detecting according to the detection label of the magnetic bead complex to obtain a detection result. The nucleic acid detection method adopts magnetic beads as a reaction substrate and a minimum reaction unit of the whole reaction, and can play a role in concentration and amplification in the subsequent immunoreaction of a primary antibody and a secondary antibody; moreover, after various reaction reagents are mixed to participate in the reaction, the magnetic beads can also accelerate the immunoreaction and reduce the reaction time. The hybrid duplex is captured by the primary antibody, and is detected by the secondary antibody, so that the specificity and the sensitivity of detection are increased, and the nucleic acid fragment to be detected does not need to be amplified additionally.
In some embodiments, the magnetic beads participate in the reaction during the detection process in the form of encapsulation in a droplet. The magnetic beads in the liquid drops are used as the minimum reaction unit in the reaction process by a mode of packaging to form the liquid drops, so that the speed of immunoreaction is further accelerated, and the reaction time is reduced. In some preferred embodiments, the droplet formation is encapsulated in a microfluidic chip process.
In some embodiments, the droplet encapsulating the magnetic bead has a volume of (1-10) x 10-15And L. By generating and confining the magnetic bead complex in a droplet of about the size of a flight, the signal of the detection label or the signal generated by the reaction of the detection label can be further concentrated, so that a signal that is not easily detected can be more easily detected, thereby further lowering the detection limit.
In some embodiments, the magnetic beads are encapsulated by the microfluidic chip, an oil phase is used as a mobile phase, an aqueous solution containing the magnetic beads is used as a dispersed phase, and the magnetic beads are encapsulated by pressure and/or shear force to form a water-in-oil droplet. The oil phase may be liquid hydrocarbon, ester, etc., such as fluorine oil, silicone oil, mineral oil, vegetable oil, petroleum ether, etc. In some preferred embodiments, the oil phase further comprises a surfactant, non-limiting examples of which may be span, triton, EM 90, fluorocarbon surfactants, and the like. The formed liquid drops are more stable by adding the surfactant into the oil phase, so that the liquid drops can not be fused and subjected to substance exchange in the subsequent reaction process and can always keep a complete and independent liquid drop form. Therefore, when detecting, the liquid drop can be directly detected without designing a micropore structure corresponding to the size of the liquid drop and sinking the liquid drop into the micropore structure by means of magnetic force and the like. Meanwhile, due to the fact that a microporous structure is not needed to be divided and isolated, the size of liquid drops in the detection area can be more uniform and has higher density, detection can be facilitated, and higher flux can be provided.
In some embodiments, the detectable label on the secondary antibody is an enzyme. Enzyme linked immunosorbent assay is used to make enzyme on the magnetic bead complex react with the provided substrate to develop color and generate light signal for detection. Compared with other immunodetection methods, the method has the advantages of good specificity, high sensitivity and lower environmental pollution. Non-limiting examples of enzymes useful as detection markers include beta-galactosidase, horseradish peroxidase, alkaline phosphatase, urease, glucose oxidase. In addition, in some preferred embodiments, the secondary antibody may be in the form of a porous nanomaterial loaded with a plurality of detection labels, and the antibodies specifically binding to the primary antibody are labeled therein. In this case, the detection signal can be further amplified by the plurality of detection marks of the load, and the detection sensitivity can be improved.
In some embodiments, the magnetic beads used in the assay have a diameter of 0.5 to 2 μm and the droplets have a diameter of 1 to 50 μm.
The present application will be described below with reference to specific examples.
Example 1
This example provides a nucleic acid detection kit, wherein the microfluidic chip is as described above. The kit also comprises magnetic beads, primary antibodies, secondary antibodies and substrates. The magnetic beads are 20 mu L of streptavidin magnetic beads with the concentration of 10mg/mL and the particle size of 1 mu m, and HPV mRNA probes corresponding to nucleic acid fragments to be detected are coupled on the magnetic beads. 10 μ L of Anti-DNA at a concentration of 1 μ g/mL: RNA hybrid antibody (S9.6 antibody). The secondary antibody is 10 mu L of beta-galactosidase marked sheep anti-mouse antibody with the concentration of 1 mu g/mL. The substrate was FDG (fluorescein 2-. beta. -D-galactopyranoside). The volume of the sample containing the nucleic acid fragment to be detected was 1 mL.
The present embodiment also provides a method for detecting nucleic acid, comprising the steps of:
(1) and (3) mixing and incubating the solution of the magnetic beads coupled with the probes, the sample to be tested and a hybridization buffer (50mM sodium citrate, 750mM NaCl, pH 7.2) for 5min to obtain a solution containing magnetic bead-DNA: a solution of RNA complexes.
(2) And (3) mixing and incubating the reaction product in the step (1) with a primary antibody and a secondary antibody for 5min to form magnetic bead-DNA: a solution of magnetic bead complexes of RNA-primary anti-secondary antibody.
(3) Injecting the mixed solution into the microfluidic chip from the first liquid inlet, simultaneously injecting the substrate solution into the microfluidic chip from the third liquid inlet, injecting fluorine oil HFE7500 (fluorocarbon surfactant FSA, 1 wt%) into the microfluidic chip from the second liquid inlet, and mixing and reacting the first liquid inlet, the second liquid inlet and the third liquid inlet at flow rates of 0.1mL/h, 0.4mL/h and 0.1mL/h to form liquid drops.
(4) The droplet was imaged by fluorescence microscopy 20 minutes after the beginning of the entry into the detection chamber.
In the above detection method, the volume of the finally formed droplet is controlled to be 1 × 10-14About L.
In the above detection method, a standard curve is formed using purified HPV DNA as a standard. The concentration of HPV DNA is from 1X 106copy/mL was started and 10-fold gradient dilutions were performed. Microscopic images were recorded and analyzed to quantify the concentration of HPV DNA. Since the droplets are filled in the imaging area, the fraction of fluorescent bright droplets to the total amount of droplets is used as an output signal for calculation of HPV DNA concentration.
The results are shown in FIGS. 6 and 7, in which FIG. 6 is a fluorescence image of samples of different concentrations in example 1, and a to f are 10, respectively3、104、105、106、107、108The concentration gradient of copies/mL, FIG. 7 is a standard curve plotted for different concentrations of standard. With reference to FIG. 7, the detection range is 106copy/mL to 103copy/mL, linear relationship is Y5.829 × log (x) -18.59, R20.964. Thus, the detection method provided by this embodiment can detect a minimum of 103An order of magnitude of a nucleic acid molecule.
Compared with the commercial product Hybrid Capture 2, the method for detecting HPV provided by the embodiment has lower LOD and less time and reagent consumption. Compared with PCR-based methods, the detection method avoids the problems of high cost, complicated processing procedures and aerosol contamination. The method is simple, versatile, and has excellent quantification capability.
It can be seen from the above embodiments that, in the scheme of the present application, by compressing the conventional biochemical reaction in the femto-droplet reactor of the femto-upgrading, the micro biochemical signal can be concentrated, so that the signal which is not easy to be detected can be more easily detected. Modification of the probe with magnetic beads, after capture of the target nucleic acid molecule, DNA: the hybridizing duplex of RNA is separated by Anti-DNA: RNA hybrid antibody capture. The Anti-DNA of the mouse source and the RNA hybrid antibody are marked by an enzyme-labeled goat Anti-mouse antibody to form a final immune complex. The immune complex and the fluorogenic substrate thereof generate hundreds of thousands of liquid drops with the size of about ten microns through a liquid drop microfluidic chip. When the immune complex exists in the liquid drop, the enzyme in the liquid drop can catalyze the fluorescent substrate to generate a fluorescent signal, and the fluorescent signal is photographed and analyzed. According to the proportion of the fluorescent liquid drops in all the liquid drops, the absolute quantitative result of the nucleic acid molecules can be calculated.
In conclusion, the droplet-based nucleic acid hybridization method can realize amplification-free high-sensitivity nucleic acid detection, on one hand, the high-sensitivity detection in a complex sample is realized by utilizing the specificity of antigen-antibody combination, on the other hand, the method has the advantage of short detection time, and the whole detection process can be controlled within 30 minutes, so that the rapid diagnosis is facilitated. In addition, a plurality of microfluidic channels can be designed, so that detection and analysis of a plurality of targets can be realized.
Example 2
This example provides a nucleic acid detection kit, which is different from example 1 in that the secondary antibody is a horseradish peroxidase-labeled goat anti-mouse antibody, and the substrate is TMB (3,3 ', 5, 5' -tetramethyllbenzidine). The method in example 1 is adopted for detection, and analysis of the detection result shows that the detection range is similar to that of example 1 and the LOD is lower.
The present application has been described in detail with reference to the embodiments, but the present application is not limited to the embodiments described above, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present application. Furthermore, the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
Claims (10)
1. A method for detecting a nucleic acid, comprising the steps of:
providing a sample to be detected, a magnetic bead, a primary antibody and a secondary antibody, wherein the magnetic bead is marked with a nucleotide chain, the nucleotide chain is used for complementary pairing with a nucleic acid fragment to be detected in the sample to be detected to form a hybridization duplex, the primary antibody is used for specific binding with the hybridization duplex, the secondary antibody is used for specific binding with the primary antibody, and the secondary antibody is provided with a detection mark;
mixing and reacting the sample to be detected, the magnetic beads, the primary antibody and the secondary antibody to form a magnetic bead complex;
and detecting according to the detection label of the magnetic bead complex to obtain a detection result.
2. The method for detecting a nucleic acid according to claim 1, wherein the detection is carried out in a manner that: packaging the magnetic bead complex to form a liquid drop, and detecting according to the detection mark in the liquid drop to obtain a detection result;
preferably, the volume of the liquid drop is (1-100) multiplied by 10-15L。
3. The method for detecting nucleic acid according to claim 2, wherein the packaging method comprises the steps of:
providing an aqueous solution of an oil phase and the magnetic bead complex;
and (3) the oil phase is taken as a mobile phase, the aqueous solution is taken as a dispersed phase, and the water-in-oil droplets are formed by encapsulation under the action of pressure and/or shearing force.
4. The method for detecting nucleic acid according to claim 3, wherein the oil phase further comprises a surfactant.
5. The method for detecting a nucleic acid according to claim 2, wherein the detection is performed by: and analyzing to obtain the content of the nucleic acid fragment to be detected in the sample to be detected according to the quantity of the liquid drops encapsulated with the magnetic bead complex.
6. The method for detecting a nucleic acid according to any one of claims 1 to 5, wherein the detection marker is an enzyme;
preferably, the enzyme is selected from at least one of beta-galactosidase, horseradish peroxidase, alkaline phosphatase, urease and glucose oxidase.
7. The method of any one of claims 1 to 5, wherein the secondary antibody is a porous nanomaterial immobilized with an antibody, the porous nanomaterial carrying a plurality of the detection labels, and the antibody is configured to specifically bind to the primary antibody.
8. The method of detecting a nucleic acid according to any one of claims 1 to 5, wherein the diameter of the magnetic bead is 0.5 to 2 μm.
9. A nucleic acid detection kit, comprising:
the magnetic bead is marked with a nucleotide chain, and the nucleotide chain is used for complementary pairing with a nucleic acid fragment to be detected to form a hybrid duplex;
a primary antibody for specific binding to the hybridization duplex;
a secondary antibody for specific binding to the primary antibody, the secondary antibody further having a detection label;
the micro-fluidic chip is provided with a reaction chamber and a detection chamber which are communicated with each other, the reaction chamber is used for allowing the detection mark to react to generate a detection signal, and the detection chamber is used for detecting the detection signal.
10. The nucleic acid detection kit of claim 9, wherein the microfluidic chip further comprises a droplet formation region, the droplet formation region is provided with an oil phase inlet for injecting an oil phase, a water phase inlet for injecting the aqueous solution of the magnetic beads, and an outlet for forming droplets.
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