CN113755559A

CN113755559A - Nucleic acid detection method, nucleic acid detection system and kit

Info

Publication number: CN113755559A
Application number: CN202110945613.2A
Authority: CN
Inventors: 王丹丹; 甘明哲; 崔金辉
Original assignee: Suzhou Institute of Nano Tech and Nano Bionics of CAS
Current assignee: Suzhou Institute of Nano Tech and Nano Bionics of CAS
Priority date: 2021-08-17
Filing date: 2021-08-17
Publication date: 2021-12-07

Abstract

The invention discloses a nucleic acid detection method, a nucleic acid detection system and a kit. The nucleic acid detection method comprises the following steps: providing a pretreatment chip for nucleic acid detection, which comprises a sample loading pad, a nucleic acid binding card, a water absorption pad and the like; applying a liquid sample to be detected on the sample loading pad, enriching target nucleic acid contained in the liquid sample on the nucleic acid binding card through the capillary action of the water absorption pad, and drying the nucleic acid binding card; and placing the dried nucleic acid binding card into a nucleic acid amplification module, adding a nucleic acid amplification assembly to form a nucleic acid amplification reaction system, carrying out nucleic acid amplification reaction, and detecting by using a fluorescence detection module. The invention can conveniently, quickly, accurately and low-cost detect the target nucleic acid without using large or precise instruments, has high detection sensitivity and good detection stability, can detect various nucleic acid samples at any time and any place, and well meets the detection requirements of various scenes such as outdoors and the like.

Description

Nucleic acid detection method, nucleic acid detection system and kit

Technical Field

The invention belongs to the technical field of nucleic acid detection, and particularly relates to a nucleic acid detection method, a nucleic acid detection system and a kit.

Background

The nucleic acid detection has important significance in life science, is widely applied to clinical diagnosis, agricultural monitoring and food safety, and particularly becomes the gold standard for diagnosis and judgment of diseases when large-scale infectious diseases occur. The rapid and high-quality extraction of nucleic acid in a sample is a prerequisite for various rapid detection application technologies of nucleic acid, but the existing kit extraction method is still complicated in steps and needs expensive or precise instruments and equipment such as a low-temperature centrifuge, a pipettor, a vortex instrument and the like, and meanwhile, the content of target nucleic acid is usually very low when an actual sample is detected.

For example, CN110982876A provides a pretreatment method for viral nucleic acid detection, which directly mixes a pretreatment solution containing a sample with a nucleic acid releasing agent and a qPCR amplification reagent, wherein the sample pretreatment solution used comprises: Tris-HCl, EDTA-2Na, sodium chloride, ribonuclease inhibitor and antibiotic, the pH value is 6.5-8.0, and qPCR can be directly carried out. Although the method shortens the detection time to a certain extent, the complexity of the sample source and the addition of the nucleic acid releasing agent cause certain interference on the subsequent qPCR, and the detection sensitivity is reduced. CN106318865A proposes a portable device for nucleic acid extraction and gene amplification, which comprises a nucleic acid extraction and pretreatment module and a gene amplification module. The device adds the lysate with complicated nucleic acid preliminary treatment in-process, and the filter membrane filters, and the required instrument of processes such as adding buffer solution and these in-process is realized through a multi-functional hot lid, has portablely, quick advantage. Although the device does not need equipment matched with the device to complete the nucleic acid extraction process, the device has a more complex structure and contains various joints and valves, and liquid is easy to leak, so the requirement on tightness is strict, and the manufacturing cost is higher; meanwhile, the extracted nucleic acid can be amplified in the next step only by further elution, so that the utilization rate of the nucleic acid is reduced. In recent years, researchers have proposed a method for detecting viruses in serum using a paper chip. The paper chip utilizes a loading pad to load and crack viruses, then utilizes a D.W. solution to transfer the cracked viruses to a binding pad loaded with chitosan, simultaneously utilizes the change of pH value to release the viruses loaded on the binding pad to a reaction cavity, utilizes RT-Lamp to carry out nucleic acid amplification, and utilizes Chemi-Doc XRS + imaging system to carry out signal detection after reacting for 60min at 65 ℃. The method provides a paper chip integrating virus extraction and amplification, and has the advantages of portability, low price and the like, but a pipettor and the like are needed for sample application, a constant-temperature 65 ℃ hot stage is needed for heating for reaction, the energy consumption is high, and particularly, the method needs to be matched with expensive detection equipment for detecting amplified signals, and cannot completely meet the requirement of outdoor detection anytime and anywhere.

Disclosure of Invention

The invention mainly aims to provide a nucleic acid detection method, a nucleic acid detection system and a kit, so as to overcome the defects in the prior art.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

one aspect of the present invention provides a nucleic acid detection method, comprising:

providing a pretreatment chip for nucleic acid detection, which comprises a sample loading pad, a nucleic acid binding card and a water absorption pad which are sequentially connected along a set direction, wherein the nucleic acid binding card can capture target nucleic acid;

applying a liquid sample to be detected on the sample loading pad, enriching target nucleic acid contained in the liquid sample on the nucleic acid binding card through the capillary action of the water absorption pad, and drying the nucleic acid binding card;

and placing the dried nucleic acid binding card into a nucleic acid amplification module, adding a nucleic acid amplification assembly to form a nucleic acid amplification reaction system, carrying out nucleic acid amplification reaction, and detecting by using a fluorescence detection module.

Another aspect of the present invention provides a nucleic acid detecting system comprising:

the pretreatment chip for nucleic acid detection comprises a sample loading pad, a nucleic acid binding card and a water absorption pad which are sequentially connected along a set direction, wherein the nucleic acid binding card can capture target nucleic acid;

the nucleic acid amplification module is used for accommodating a nucleic acid amplification reaction system formed by matching a nucleic acid binding card which is dried and captures target nucleic acid with a nucleic acid amplification component;

a portable fluorescence detector, comprising:

a heating element for heating the nucleic acid amplification reaction system to a temperature sufficient to initiate the nucleic acid amplification reaction,

a light source for providing excitation light to irradiate the nucleic acid amplification reaction system to generate a fluorescent signal; and

and the fluorescence detection module is used for taking a fluorescence signal generated by the nucleic acid amplification reaction system under the irradiation of the excitation light so as to realize the detection of the target nucleic acid.

In yet another aspect of the present invention, there is provided a kit comprising:

the nucleic acid amplification chip is used for accommodating a nucleic acid amplification reaction system formed by matching a nucleic acid binding card which is dried and captures target nucleic acid with a nucleic acid amplification component; and

the capillary is pre-stored with a nucleic acid amplification assembly, and each component of the nucleic acid amplification assembly is pre-stored in a plurality of different storage sections of the lumen of the capillary respectively, wherein the plurality of storage sections are distributed along the length direction of the capillary.

Compared with the prior art, the technical scheme provided by the invention at least has the following advantages:

(1) the provided pretreatment chip for nucleic acid detection has the advantages of simple structure, small and compact size, low price and convenient use, can quickly finish the extraction and enrichment of target nucleic acid without using a large or precise instrument, can be conveniently separated from a nucleic acid binding card enriched with the target nucleic acid, can be directly applied to subsequent detection experiments without elution, is not only convenient and quick, but also is beneficial to greatly improving the detection sensitivity and the detection stability.

(2) Each link in the provided nucleic acid detection method only needs simple operation, has low energy consumption, can fully meet the requirement of enabling a user to carry out detection outdoors or on site at any time, and has high detection sensitivity, stability and accuracy.

(3) The provided nucleic acid detection system has the advantages of simple structure, complete functions, portability, easy operation and low energy consumption, and can realize detection at any time and any place and for many times.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a schematic top view of a pre-processing chip in example 1 of the present application.

FIG. 2 is a schematic cross-sectional view of the pretreatment chip of FIG. 1 taken along the direction A-A.

FIG. 3 is a schematic diagram of another chip pretreatment according to embodiment 1 of the present application.

FIG. 4 is a schematic flow chart of a nucleic acid detection method in example 1 of the present invention.

FIG. 5 is a schematic diagram of a nucleic acid amplification chip in example 1 of the present invention.

FIG. 6 is a schematic view of a portable fluorescence detector according to embodiment 1 of the present invention.

FIG. 7 is a bar graph of the nucleic acid concentration and the detection signal in the sample of example 1 of the present invention (p < 0.05, N.S.: no significant difference, NC: blank, PC: internal standard).

FIG. 8 is a graph showing signals for detecting nucleic acids in the case of steel bead addition/non-steel bead addition in example 1 of the present invention.

FIG. 9 is a graph showing a comparison of the detection abilities of the nucleic acid detecting method using the pretreatment chip and the commercial nucleic acid detecting method in example 2 of the present invention.

FIG. 10 is a graph showing the effect of different drying methods on sample extraction and detection in example 3 of the present invention.

FIG. 11 is a graph showing the effect of different times of ethanol drying treatment on extraction and detection in example 3 of the present invention;

FIG. 12 is a schematic view showing the configuration in which the respective components of the nucleic acid amplification kit are prestored in different sites of the lumen of the capillary in example 1 of the present invention.

Description of reference numerals: 110. the chip comprises a pretreatment chip, 1101, a cover plate fixing layer, 1102, a nucleic acid extraction and enrichment layer, 1103, a substrate supporting layer, 11021, a sample loading pad, 11022, a nucleic acid binding card, 11023, a water absorption pad, 11011, a loading port, 11012, a nucleic acid binding card extraction port, 111, a nucleic acid amplification chip, 11112, a black matrix bottom layer, 11122, an amplification reaction layer, 11132, a cover plate layer, 111212, an amplification reaction chamber, 111222 and steel balls.

Detailed Description

As described above, in view of the defects of the prior art, the inventors of the present invention have made extensive studies and extensive practices to propose a technical solution of the present invention. The technical solution, its implementation and principles, etc. will be further explained as follows.

The nucleic acid detection method provided by the embodiment of the invention comprises the following steps:

In some embodiments, 0< the distance between the sample loading pad and the absorbent pad ≦ the length of the nucleic acid binding card in the set direction.

Further, referring to fig. 1 and 2, in a more specific embodiment of the present invention, a pretreatment chip 110 for nucleic acid detection includes a base support layer 1103 and a cover plate fixing layer 1101 disposed on the base support layer 1103, a nucleic acid extraction and enrichment layer 1102 is disposed between the base support layer 1103 and the cover plate fixing layer 1101, and the nucleic acid extraction and enrichment layer 1102 includes a sample loading pad 11021, a nucleic acid binding card 11022, and a water absorption pad 11023 sequentially connected along a set direction.

Wherein the nucleic acid binding card 11022 is capable of capturing and enriching target nucleic acids in a liquid sample applied on the sample application pad 11021. Obviously, to achieve this function, a capture object capable of specifically capturing the target nucleic acid, such as a nucleotide sequence complementary to the target nucleic acid, or a protein or other chemical substance, may be provided on the nucleic acid binding card 11022 in advance according to a known manner by those skilled in the art.

The cover plate fixing layer 1101 is also provided with a sample loading port 11011 and a nucleic acid binding card extraction port 11012 in regions corresponding to the sample loading pad 11021 and the nucleic acid binding card 11022, respectively. The nucleic acid binding card 11022 can be easily put into or taken out of the pretreatment chip 110 through the nucleic acid binding card take-out port 11012.

Further, it is also possible to form a groove-like structure on the top end face of the base support layer 1103 and to dispose the nucleic acid extracting and concentrating layer 1102 inside the groove-like structure.

Alternatively, as shown in FIG. 3, the nucleic acid extracting and concentrating layer 1102 may be disposed directly inside the base support layer 1103 with the remaining structure unchanged.

Further, the nucleic acid binding card 11022 may be a membrane or paper based card that can bind nucleic acid, such as, but not limited to, FTA card.

The materials of the sample pad 11021 and the absorbent pad 11023 are known in the art.

The base support layer 1103 and the cover fixing layer 1101 may be made of inorganic or organic materials such as glass and high molecular polymer. Preferably, the cover plate fixing layer 1101 may be formed of a hydrophobic material such as Polydimethylsiloxane (PDMS), so that a loss of the liquid sample and the target nucleic acid contained therein due to adhesion to the surface or inner wall of the cover plate fixing layer 1101 can be reduced.

Further, after the target nucleic acid contained in the liquid sample is enriched on the nucleic acid binding card 11022, the interfering substance on the nucleic acid binding card 11022 can be washed away by directly dropping a washing reagent or the like to the nucleic acid binding card 11022. Alternatively, it is preferable that the interfering substances on the nucleic acid binding card 11022 be washed away by applying a washing reagent to the sample loading pad 11021 and then flowing the washing reagent through the nucleic acid binding card 11022. Advantages of the latter cleaning method include: 1) the amount of the cleaning reagent is determined, and the cleaning effect is stable; 2) the cleaning solution completely flows through the nucleic acid binding card, the cleaning effect is good, the required cleaning solution is less, and the cleaning time is faster. The washing reagent may be water, buffer, etc.

In some embodiments, the nucleic acid detection method comprises: the drying treatment is performed on the nucleic acid-binding card by means of heat drying or natural drying.

Preferably, the drying treatment comprises: applying a volatile organic solvent to the nucleic acid binding card, and then allowing the nucleic acid binding card to dry naturally. Wherein, the volatile organic solvent is preferably ethanol. The drying treatment mode can accelerate the drying speed, shorten the drying time, does not need auxiliary operations such as heating and the like, does not cause the loss of target nucleic acid, and is particularly suitable for the detection requirements of environments such as the outdoor environment and the like.

In the present invention, the capture and enrichment of the target nucleic acid by the nucleic acid binding card can be generally completed within 10 min.

In some embodiments, the nucleic acid detection method specifically comprises:

a nucleic acid amplification chip is adopted as the nucleic acid amplification module;

and adding the dried nucleic acid binding card and the nucleic acid amplification assembly into the nucleic acid amplification chip to form the nucleic acid amplification reaction system, and then putting the nucleic acid amplification chip into a portable fluorescence detector to carry out the nucleic acid amplification reaction.

Further, the nucleic acid amplification chip may include:

a first amplification chamber for housing the nucleic acid amplification reaction system;

a second amplification chamber for containing a liquid sample for use as a blank set and/or a third amplification chamber for containing a liquid sample for use as an internal standard.

Preferably, the nucleic acid amplification chip may simultaneously comprise one or more first amplification chambers, one or more second amplification chambers, and one or more third amplification chambers, and particularly, the nucleic acid amplification chip may comprise a plurality of first amplification chambers to realize a single-time detection of a plurality of samples.

The nucleic acid amplification chip can be made of polymethyl methacrylate (PMMA), and the cost is low.

In order to avoid the interference of the nucleic acid amplification reaction caused by the material of the nucleic acid amplification chip itself, the inner wall of each amplification chamber may be passivated in advance, for example, by forming a coating on the inner wall of each amplification chamber using PDMS or the like.

Further, the portable fluorescence detector may include:

a heating element for heating the nucleic acid amplification reaction system to a temperature sufficient to initiate the nucleic acid amplification reaction;

a light source for providing excitation light to irradiate the nucleic acid amplification reaction system to generate a fluorescent signal.

Wherein, the heating element can adopt various electric heating elements and the like.

The fixed position of the nucleic acid amplification chip in the portable fluorescence detector is within the range of the heating region of the heating element and within the irradiation range of the exciting light emitted by the light source, so that the nucleic acid amplification chip can be directly detected without adjusting the position of the nucleic acid amplification chip after the amplification reaction is finished.

For example, the heating element may include upper and lower heating plates, and the nucleic acid amplification chip may be held between the upper and lower heating plates for heating, and the heating may be performed uniformly and at a high heating rate.

Wherein, the light source preferably adopts a laser light source.

More preferably, the excitation light is linear laser, such as a linear laser, and the linear laser irradiates the supernatant of the nucleic acid amplification reaction system, so as to avoid the interference of strong fluorescence and reflection generated by a nucleic acid binding card, a stirrer (such as a steel ball) and the like in the amplification reaction system on detection.

In addition, the portable fluorescence detector may further include components such as a temperature control element, a power source (e.g., a dry battery, a rechargeable battery, etc.), a power switch, a light source control element, a display module, etc., which may cooperate with the heating element, the light source, etc. to form a complete operating circuit in a manner known in the art, and will not be described in detail herein.

Furthermore, the portable fluorescence detector may further include a housing, the heating element, the light source, the temperature control element, the power source, and the like are integrally disposed in the housing, and the housing may further be cooperatively disposed with one or more openings and corresponding covers, so as to facilitate the insertion and removal of the nucleic acid amplification chip and the like into and from the portable fluorescence detector. When the portable fluorescence detector works, the whole portable fluorescence detector is closed, so that the interference of external factors on the amplification reaction process and the detection process is avoided.

The portable fluorescence detector has the advantages of small number of components, convenience for integrated arrangement, compact structure, small size, high utilization rate of internal space, convenience for carrying and easiness in use.

In some embodiments, the nucleic acid detection method specifically comprises:

pre-storing components of the nucleic acid amplification assembly in a plurality of different storage sections of a capillary lumen, wherein the plurality of storage sections are distributed along the length direction of the capillary;

applying pressure to the lumen of the capillary, thereby injecting and mixing the components of the nucleic acid amplification assembly into the nucleic acid amplification module and cooperating with the nucleic acid binding card to form the nucleic acid amplification reaction system.

Further, the storage sections are spaced apart from one another, for example, air sections may be provided between adjacent storage sections to prevent them from interfering with one another and mixing prior to use.

By adopting the pre-storage and use mode of the nucleic acid amplification component, the quantitative preparation of the nucleic acid amplification component can be realized, the possible adverse factors caused by the early mixing of the components can be avoided, the nucleic acid amplification component can be injected into a nucleic acid amplification chip by an injector and the like at one time during use, a pipettor and the like are not needed, the operation is convenient and fast, the use amount is accurate, and the kit is particularly suitable for field detection.

Wherein the nucleic acid amplification module may be of a type known in the art, which mainly comprises reaction buffer, primer mixture, probe, reverse transcriptase, RNA inhibitor, water, and amplification agent, etc. The nucleic acid amplification module may be commercially available or may be self-prepared according to the protocols described in textbooks and kits in the art. For example, the amplification agent may include magnesium acetate and RPA, a detection reagent manufactured by twist dx corporation of uk, and the like, without being limited thereto.

Furthermore, the nucleic acid amplification reaction system comprises a recombinase-mediated isothermal nucleic acid amplification reaction system or a rolling circle amplification reaction system, preferably the recombinase-mediated isothermal nucleic acid amplification reaction system, the corresponding nucleic acid amplification reaction temperature is low (37-42 ℃), the time is short (15-30min), the energy consumption is low, and the method is suitable for being used outdoors or in mobile environments. For example, in the case of a recombinase-mediated isothermal amplification reaction system, if the portable fluorescent detector uses a dry cell as a power source, detection can be repeated for 8 to 9 hours with only two cells, and detection can be performed outdoors at any time and place.

In some embodiments, the nucleic acid detection method may further comprise: paramagnetic or ferromagnetic stirrers are added into the nucleic acid amplification reaction system to continuously or intermittently stir the nucleic acid amplification reaction system under the action of a magnetic field, so that on one hand, the operation is favorable for fully contacting a nucleic acid amplification component with target nucleic acid on paper, on the other hand, the operation is favorable for uniformly mixing all components in the reaction system under the conditions of poor fluidity of the reaction system and the like, the progress of amplification reaction can be accelerated, the energy consumption is further reduced, and the result can be obtained as soon as possible (generally, the detection result can be obtained within 20 min).

Wherein, the stirrer can be a steel ball and the like, and can form the magnetic field through an external magnetic rod and the like, and the steel ball moves by moving the magnetic rod, so as to stir the nucleic acid amplification reaction system.

In some embodiments, the nucleic acid detection method may further comprise: the portable intelligent terminal is adopted as the fluorescence detection module, and the portable intelligent terminal absorbs the fluorescence signal generated by the nucleic acid amplification reaction system under the irradiation of the excitation light, so that the detection of the target nucleic acid is realized, and the detection at any time and any place outdoors is facilitated.

Specifically, the portable intelligent terminal may compare the taken fluorescence signal intensity of the nucleic acid amplification reaction system with a set reference fluorescence signal intensity, thereby implementing detection of the target nucleic acid.

The portable intelligent terminal comprises a smart phone, and can be a notebook computer, a tablet computer and the like with a camera, and is not limited thereto.

In a more specific embodiment of the present invention, taking the sample to be detected as the target nucleic acid wrapped by the biological membrane as an example, a corresponding nucleic acid detection method comprises:

extracting a sample to be detected, and cracking the sample by using a cracking solution to obtain a liquid sample;

dropping a liquid sample on the sample loading pad through the sample loading port, and capturing and enriching the target nucleic acid on the nucleic acid binding card through the capillary action of the water absorption pad;

drying the nucleic acid binding card enriched with the target nucleic acid, transferring the nucleic acid binding card to a nucleic acid amplification chip, adding a nucleic acid amplification component to form a nucleic acid amplification reaction system, and simultaneously adding steel balls into the nucleic acid amplification reaction system;

then starting the portable fluorescence detector to heat the nucleic acid amplification reaction system for nucleic acid amplification reaction, and simultaneously driving the steel balls to disturb the nucleic acid amplification reaction system for a plurality of times by adopting a magnetic rod so as to accelerate the progress of the nucleic acid amplification reaction;

in the process of or after the nucleic acid amplification reaction is carried out, particularly after the nucleic acid amplification reaction is finished, a smart phone is used for taking a fluorescent signal of the nucleic acid amplification reaction system for detection, so that a detection result is obtained.

The nucleic acid detection method can finish the detection of the liquid sample within about 25-40min, is convenient and quick, does not need complex sample pretreatment, has simple reaction system required by the detection, does not need the participation of criprpr related enzyme and the like, and has good detection precision (the detection limit can be as low as 10)⁰copies/. mu.L) with high accuracy and low energy consumption.

The embodiment of the invention also provides a nucleic acid detection system, which comprises:

a portable fluorescence detector, comprising:

Furthermore, two ends of the nucleic acid combination card are respectively lapped with the sample loading pad and the water absorption pad.

Wherein the nucleic acid amplification module comprises a nucleic acid amplification chip.

Furthermore, the materials and structures of the nucleic acid binding card and the nucleic acid amplification chip can be as described above, and are not described herein again.

Wherein, the structure of the portable fluorescence detector is as described above.

Further, the portable fluorescence detector has a space for accommodating the nucleic acid amplification chip.

Furthermore, the nucleic acid detection system also comprises a capillary tube in which the nucleic acid amplification components are prestored, wherein the components of the nucleic acid amplification components are prestored in a plurality of different storage sections of the lumen of the capillary tube respectively, and the plurality of storage sections are distributed along the length direction of the capillary tube. The capillary tube may be made of glass, polymer, or the like, and is preferably made of polymer, and can be bent to facilitate storage.

Further, the nucleic acid detection system further comprises a paramagnetic or ferromagnetic stirrer for continuously or intermittently stirring the nucleic acid amplification reaction system under the action of a magnetic field. Wherein the stirrer includes steel balls, etc., and is not limited thereto.

Furthermore, the fluorescence detection module adopts a portable intelligent terminal.

The embodiment of the invention also provides a kit, which comprises:

In some embodiments, the kit further comprises a paramagnetic or ferromagnetic stirrer for continuously or intermittently agitating the nucleic acid amplification reaction system under the action of a magnetic field.

In some embodiments, the kit further comprises a volatile organic solvent, preferably ethanol.

In some embodiments, the kit further comprises a washing reagent for washing at least the interferents on the nucleic acid binding card.

Further, the materials and structures of the nucleic acid binding card and the nucleic acid amplification chip can be as described above, and are not described herein again.

In some embodiments, the kit further comprises various reagents required for pretreating a sample to be detected to obtain the liquid sample, such as a lysis solution and the like. The lysis solution may include one or more of Trizol lysis solution, NP40 lysis solution, SDS lysis solution, Triton X-100 lysis solution, Tween 20 lysis solution, RIPA lysis solution, etc., but is not limited thereto. Further, the lysis solution may further include a ribonuclease inhibitor such as guanidine isothiocyanate, 8-hydroxyquinoline, β -mercaptoethanol, vanadyl riboside complex, a protein inhibitor of rnase, diethyl pyrocarbonate, and the like, without being limited thereto.

In some embodiments, the kit may further include other auxiliary devices, such as a dropper, a reagent tube, and the like, without being limited thereto.

In a word, the nucleic acid detection method, the nucleic acid detection system and the kit provided by the invention can be used for conveniently, quickly, accurately and inexpensively detecting the target nucleic acid without using a large or precise instrument, have high detection sensitivity and good detection stability, can be used for detecting various nucleic acid samples at any time and any place, and can well meet the detection requirements of various scenes such as outdoors and the like.

The technical scheme of the invention is further explained by combining the drawings and the embodiment.

The following examples use a nucleic acid detecting system including a pretreatment chip for nucleic acid detection, a nucleic acid amplification chip, a portable fluorescence detector, a fluorescence detection module, and the like, each of which has the structure as described above. Specifically, the pretreatment chip has the results shown in FIGS. 1 to 2 or the structure shown in FIG. 3, wherein the nucleic acid binding card is an FTA card. The nucleic acid amplification chip has 3 to 4 amplification chambers independent of each other, and is commercially available. The portable fluorescence detector comprises an upper heating plate, a lower heating plate, a linear laser light source, a temperature control module, a display module (digital display thermometer) and other components which are integrally arranged in a shell, the components are connected with a power supply, the power supply adopts two dry batteries and is also arranged in the shell, and the heating plate is connected with the temperature control module and the display module, as shown in figure 6. In addition, the portable fluorescence detector can also comprise a control module and the like so as to regulate and control the working states of at least one line laser light source, the temperature control module, the display module and the like.

Example 1 taking a sample to be detected as a target nucleic acid sample wrapped by a biological membrane as an example, referring to fig. 4, a method for detecting by using the nucleic acid detection system may include the following steps:

(1) sample pretreatment:

respectively adding a plurality of target nucleic acid standard samples wrapped by biological membranes into 200 mu L of lysis solution Trizol, and additionally adding ribonuclease inhibitor with the final concentration of 1U/mu L into the lysis solution to form a series of different dilution concentrations of 10³、10²、10¹、 10⁰And 10^-1copies/. mu.L of standard liquid sample.

The standard liquid samples are respectively added into a sample loading pad 11021 of the pretreatment chip through a loading port 11011, and nucleic acid is captured and enriched on a nucleic acid binding card 11022 through the capillary action of a water absorption pad 11023; after enrichment, 100 μ L of the wash reagent DEPC water was added to the sample loading pad to wash out the interferents on the nucleic acid binding card 11022. After the washing is finished, dipping the nucleic acid conjugate card 11022 in ethanol by using a disposable liquid adding device needle, and drying for 1 minute; each nucleic acid binding card 11022 is then transferred through the nucleic acid binding card removal port 11012 into one amplification chamber of the RT-RPA nucleic acid amplification chip.

(2) RT-RPA nucleic acid amplification: in this example, nucleic acid amplification can be performed using TwistDx RT-RPAkit. The sequence of the target nucleic acid amplified in the process is shown as SEQ ID NO.1, and the sequence of the internal standard nucleic acid is shown as SEQ ID NO.2 (the quality of a detected sample directly influences the accuracy of a diagnosis result, so RNase P ribonuclease universally existing in cells of various tissues and organs of a human body is selected as the internal standard to monitor the accuracy of the whole experimental process). Specifically, 43.75. mu.L of a reaction premix (29.5. mu.L of a reaction buffer, 4.2. mu.L of a 10uM primer mixture (see Table 1), 0.6. mu.L of a 10. mu.M probe (see Table 1) and 9.45. mu.L of DEPC water), 2.5. mu.L of 200U/. mu.L of a reverse transcriptase and 1.25. mu.L of 40U/. mu.L of an RNA inhibitor were first added to a detection reagent produced by TwistDx in Britain, mixed uniformly by pipetting with a 25. mu.L pipette, and then 2.5. mu.L of 280mM magnesium acetate was added to mix uniformly to form a nucleic acid amplification module, which was then pipetted into an amplification chamber of a nucleic acid amplification chip, into which a nucleic acid binding card 11022 enriched with a target nucleic acid had been added. As shown in FIG. 5, steel balls (with a diameter of 0.4-0.6 mm) can also be added into the RT-RPA reaction system as an experimental group. Further, a blank sample (only the nucleic acid amplification module) and an internal standard sample (the nucleic acid amplification module and the nucleic acid binding card are added, and the sequences of the primers and the probes are shown in Table 2) can be added into other amplification chambers of the nucleic acid amplification chip. Then the nucleic acid amplification chip is placed into a portable fluorescence detector and heated to 40 ℃ for amplification, after 5 minutes, a magnetic rod is used for driving steel balls outside to stir an RT-RPA reaction system in the nucleic acid amplification chip, and then the amplification is carried out for 15 minutes, thus carrying out the fluorescence detection. Wherein, the steel balls are added for stirring and mixing, which is beneficial to accelerating the progress of the amplification reaction and obtaining the experimental result earlier.

Preferably, as shown in FIG. 12, the components of the nucleic acid amplification kit, i.e., the primer mixture, the probe, the reverse transcriptase, the RNA inhibitor, the DEPC water, the detection reagent, the magnesium acetate, etc., may be pre-stored at different sites of a capillary lumen. When in use, the components are injected into the amplification chamber of the nucleic acid amplification chip by a syringe and the like and are uniformly mixed.

TABLE 1

TABLE 2

(3) And (3) detection: specifically, the detected fluorescence signals were compared with the fluorescence signals of the blank group (NC) by the mobile phone, and if the former and the latter are significantly different (the former is higher than the latter), the result can be determined to be positive, as shown in fig. 7. As can be seen, the nucleic acid detecting system and method of the present example can rapidly detect the concentration as low as 10 in 25-40min⁰copies/. mu.L of liquid sample.

Also, referring to FIG. 8, it can be seen that the concentration is 10⁰Whether the samples of the standard liquid of copies/mu L are added into the steel ball to stir and mix has a significant influence on the detection result. Adding steel balls to stir and mix, which is favorable for accelerating the progress of the amplification reaction and detecting the concentration of 10 earlier⁰copies/. mu.L of target sample.

Example 2 in this example, the effect of the sample collection pattern on the assay results was also identified. Wherein the method of example 1 was used for a concentration of 10¹Samples of copies/. mu.L of standard liquid were tested (defined as examples).

At the same time, the concentration of 10 was measured by the following method¹Samples of copies/. mu.L of standard liquid were tested (defined as comparative examples) and specifically: soaking FTA card in 200 μ L10¹In a sample of copies/. mu.L standard liquid, FTA cards having a diameter of 1.5mm were sufficiently dried for 1 hour to capture and enrich the target nucleic acid therein.Then, 200. mu.L of a purification reagent (GE Life science, USA) was added to the mixture through a disposable pipette and soaked for 5 minutes, the supernatant was discarded, and the procedure was repeated 2 times; then adding 200 mu LTE buffer solution (10mM Tris-HCl, 0.1mM EDTA, pH 8.0), soaking for 5 minutes, discarding the supernatant, and repeating the step for 3 times; after washing, the FTA card is dried at room temperature for at least 15 minutes; and finally, referring to the RT-RPA nucleic acid amplification process, adding the dried FTA card into an RT-RPA system to amplify the nucleic acid amplification chip, and then detecting. The results of both measurements are shown in FIG. 9. Obviously, compared with the nucleic acid extraction method in the comparative example, the method for extracting nucleic acid and detecting nucleic acid by using the pretreatment chip in the embodiment has simple experimental steps and quick operation, can greatly shorten the detection time, and simultaneously improves the detection sensitivity.

Example 3 in this example, the influence of the drying method of the nucleic acid binding card after enrichment of the target nucleic acid on the detection result was also identified, that is, the method of example 1 was referred to, but the nucleic acid binding card after enrichment of the target nucleic acid was treated by drying after washing with water and ethanol, respectively. At a concentration of 10¹Taking samples of copies/muL standard liquid as an example, referring to fig. 10, the final detection results of different drying modes are consistent, but the drying time is greatly different, after the nucleic acid binding card is enriched, the dipping water needs 30-60min at 25 ℃ for full drying, and a heating plate (the heating temperature interval is 45-60 ℃) needs 15-30min for full drying; however, after the nucleic acid binding card is enriched, the ethanol is placed on a heating plate (the heating temperature is 45-60 ℃) and is dried for 1-5min, as shown in FIG. 11. Wherein the negative control is a blank nucleic acid binding card treated with nuclease-free water instead of the standard liquid sample.

While the invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.

Sequence listing

<110> Suzhou nanotechnology and nano-bionic institute of Chinese academy of sciences

<120> nucleic acid detection method, nucleic acid detection system and kit

<160> 2

<170> SIPOSequenceListing 1.0

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<211> 156

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

acaccggaag ccaatatgga tcaagaatcc tttggtggtg catcgtgttg tctgtactgc 60

cgttgccaca tagatcatcc aaatcctaaa ggattttgtg acttaaaagg taagtatgta 120

caaataccta caacttgtgc taatgaccct gtgggt 156

<210> 2

<211> 118

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

agccactgac tcggatccgc aacaactcag ccatccacat ccgagtcttc agggtcacac 60

ccaagtaatt gaaaagacac tcctccactt atcccctccg tgatatggct cttcgcat 118

Claims

1. A method for detecting a nucleic acid, comprising:

providing a pretreatment chip for nucleic acid detection, which comprises a sample loading pad (11021), a nucleic acid binding card (11022) and a water absorption pad (11023) which are sequentially connected along a set direction, wherein the nucleic acid binding card (11022) can capture target nucleic acid;

applying a liquid sample to be detected on the sample loading pad (11021), enriching target nucleic acid contained in the liquid sample on a nucleic acid binding card (11022) through the capillary action of a water absorption pad (11023), and drying the nucleic acid binding card (11022);

and (2) placing the dried nucleic acid binding card (11022) into a nucleic acid amplification module, adding a nucleic acid amplification component to form a nucleic acid amplification reaction system, carrying out nucleic acid amplification reaction, and detecting by using a fluorescence detection module.

2. The method for detecting a nucleic acid according to claim 1, wherein: 0< the distance between the sample loading pad (11021) and the water absorption pad (11023) < the length of the nucleic acid binding card in a set direction.

3. The method for detecting a nucleic acid according to claim 1, comprising: the drying treatment is carried out on the nucleic acid binding card (11022) by adopting a heating drying or natural drying mode;

preferably, the drying treatment comprises: applying a volatile organic solvent on the nucleic acid binding card (11022), followed by allowing the nucleic acid binding card (11022) to dry naturally; more preferably, the volatile organic solvent comprises ethanol.

4. The method for detecting a nucleic acid according to claim 1, comprising: a nucleic acid amplification chip is adopted as the nucleic acid amplification module;

5. The method for detecting nucleic acid according to claim 4, wherein the portable fluorescence detector comprises:

a light source for providing excitation light to irradiate the nucleic acid amplification reaction system to generate a fluorescent signal;

preferably, the excitation light is a linear laser, and the range irradiated by the linear laser is a supernatant part of the nucleic acid amplification reaction system.

6. The method for detecting nucleic acid according to claim 1 or 4, wherein the nucleic acid amplification chip comprises:

7. The method for detecting a nucleic acid according to claim 1, comprising:

8. The method for detecting a nucleic acid according to claim 1, wherein: the nucleic acid amplification reaction system comprises a recombinase-mediated nucleic acid isothermal amplification reaction system or a rolling circle amplification reaction system, and preferably the recombinase-mediated nucleic acid isothermal amplification reaction system.

9. The method for detecting a nucleic acid according to claim 1, which specifically comprises: adding a paramagnetic or ferromagnetic stirrer into the nucleic acid amplification reaction system, and continuously or intermittently stirring the nucleic acid amplification reaction system under the action of a magnetic field.

10. The method for detecting a nucleic acid according to claim 1, which specifically comprises: a portable intelligent terminal is adopted as the fluorescence detection module, and the portable intelligent terminal is used for taking a fluorescence signal generated by the nucleic acid amplification reaction system under the irradiation of excitation light, so that the detection of target nucleic acid is realized;

preferably, the portable intelligent terminal is used for comparing the acquired fluorescence signal intensity of the nucleic acid amplification reaction system with a set reference fluorescence signal intensity, so as to realize the detection of the target nucleic acid;

preferably, the portable intelligent terminal comprises a smart phone.

11. A nucleic acid detecting system characterized by comprising:

the pretreatment chip for nucleic acid detection comprises a sample loading pad (11021), a nucleic acid binding card (11022) and a water absorption pad (11023) which are sequentially connected along a set direction, wherein the nucleic acid binding card (11022) can capture target nucleic acid;

the nucleic acid amplification module is used for accommodating a nucleic acid amplification reaction system formed by matching a dried nucleic acid binding card (11022) which captures target nucleic acid with a nucleic acid amplification component;

a portable fluorescence detector, comprising:

12. The nucleic acid detection system according to claim 11, wherein: the two ends of the nucleic acid combination card (11022) are respectively lapped with a sample loading pad (11021) and a water absorption pad (11023);

and/or the pretreatment chip comprises a base supporting layer (1103) and a cover plate fixing layer (1101) arranged on the base supporting layer (1103), wherein a nucleic acid extraction and enrichment layer (1102) is arranged between the base supporting layer (1103) and the cover plate fixing layer (1101), the nucleic acid extraction and enrichment layer (1102) comprises a sample loading pad (11021), a nucleic acid binding card (11022) and a water absorption pad (11023), wherein the areas, corresponding to the sample loading pad (11021) and the nucleic acid binding card (11022), on the cover plate fixing layer (1101) are also respectively provided with a sample loading port (11011) and a nucleic acid binding card extraction port (11012), preferably, a groove-shaped structure is formed on the top end face of the base supporting layer (1103), and the nucleic acid extraction and enrichment layer (1102) is arranged in the groove-shaped structure;

and/or, the nucleic acid binding card (11022) adopts a membrane or paper-based card capable of binding nucleic acid, preferably, the nucleic acid binding card (11022) adopts FTA card;

and/or the exciting light is linear laser, and the irradiation range of the linear laser is the supernatant part of the nucleic acid amplification reaction system;

and/or the portable fluorescence detector is provided with a space for accommodating the nucleic acid amplification chip;

and/or, the nucleic acid amplification module comprises a nucleic acid amplification chip comprising:

a first amplification chamber for housing the nucleic acid amplification reaction system,

a second amplification chamber for containing a liquid sample for use as a blank set and/or a third amplification chamber for containing a liquid sample for use as an internal standard;

and/or, the nucleic acid detection system further comprises a capillary tube in which a nucleic acid amplification component is prestored, wherein each component of the nucleic acid amplification component is prestored in a plurality of different storage sections of the lumen of the capillary tube respectively, and the plurality of storage sections are distributed along the length direction of the capillary tube; preferably, adjacent storage sections are air isolated;

and/or, the nucleic acid detection system further comprises a paramagnetic or ferromagnetic stirrer used for continuously or intermittently stirring the nucleic acid amplification reaction system under the action of a magnetic field, preferably, the stirrer comprises steel balls;

and/or the fluorescence detection module adopts a portable intelligent terminal, preferably a smart phone.

13. A kit, characterized by comprising:

the nucleic acid amplification chip is used for accommodating a nucleic acid amplification reaction system formed by matching a nucleic acid binding card (11022) which is dried and captures target nucleic acid with a nucleic acid amplification component; and

14. The kit of claim 13, further comprising:

a paramagnetic or ferromagnetic stirrer for continuously or intermittently stirring the nucleic acid amplification reaction system under the action of a magnetic field, preferably, the stirrer comprises steel balls;

and/or a volatile organic solvent, preferably ethanol;

and/or, washing reagents at least for washing away interferents on the nucleic acid binding card (11022);

and/or two ends of the nucleic acid binding card (11022) are respectively lapped with a sample loading pad (11021) and a water absorption pad (11023);

and/or, the nucleic acid amplification chip comprises: