CN111575354A - Nucleic acid detection equipment and use method thereof - Google Patents
Nucleic acid detection equipment and use method thereof Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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Abstract
The invention solves the problems that the existing nucleic acid detection equipment cannot be portable or has low integration level, complex detection process, long time consumption and high requirement on detection personnel, provides simple, portable, rapid and low-cost equipment for effective detection and a using method of the equipment, and is characterized by comprising the following steps: a first protective layer; the sample adding layer consists of a sample adding area and a channel connected with the sample adding area; the reaction layer comprises a sample detection area, a negative control area and a positive control area which are independent; the second protective layer is a hydrophobic transparent film; the first protective layer, the sample adding layer, the reaction layer and the second protective layer are integrated together in sequence, the sample adding region is positioned at the lower part of the sample adding hole, and the tail end of the channel is respectively connected with the sample detection region, the negative control region and the positive control region; a method for using nucleic acid detection equipment comprises the following specific steps: preparing a sample solution; sample adding; and (6) observing.
Description
Technical Field
The invention relates to the technical field of instant detection, in particular to nucleic acid detection equipment and a using method thereof.
Background
Epidemic diseases are particularly viral epidemic diseases such as Human Immunodeficiency Virus (HIV), Zika virus (ZIKV), influenza A (H1N 1), novel coronavirus (SARS-CoV-2) and the like, which threaten human health worldwide. The most effective prevention and treatment measures for epidemic diseases are early detection and early diagnosis.
Currently, pathogen detection for epidemic diseases in clinical use is mainly based on genetic diagnosis technology of nucleic acid amplification, such as Polymerase Chain Reaction (PCR), loop-mediated isothermal amplification (LAMP), Recombinase Polymerase Amplification (RPA), and the like. The main implementation procedures of these techniques mainly include nucleic acid extraction from a sample, nucleic acid purification, nucleic acid amplification, and signal detection, which often require special laboratories, skilled laboratory personnel, and expensive laboratory instruments, and are also generally time-consuming. In order to detect pathogens over a wider range, particularly in resource poor areas or areas with greater human traffic, the inventors believe that in order to provide more effective control of epidemic diseases, a less expensive and more compact assay device/method needs to be established.
The microfluidic chip technology is a new technology, and is gradually developed towards integration, and the application of the microfluidic chip technology to the nucleic acid detection technology brings important innovation. The paper-based microfluid chip is simple to process, low in cost, portable, biodegradable, and light, and the porous structure of the paper-based microfluid chip can perform spontaneous capillary siphoning without extra fluid driving force, and can be used as a good checking and detecting platform. However, there is no highly integrated device for the time being, nucleic acid is usually extracted and purified in advance, amplification reagents are mixed in advance or reaction reagents are added subsequently, amplification reaction is separated from signal detection, and these defects limit the use environment and have high requirements on the capability of users. The inventor establishes a highly integrated, portable and simple nucleic acid detection device based on the research experience and academic foundation of the microfluidic technology, and can realize faster and more convenient nucleic acid detection.
Disclosure of Invention
The invention solves the problems that the existing nucleic acid detection equipment cannot be portable or has low integration level, complex detection flow, long time consumption and high requirement on detection personnel, and provides simple, portable, rapid and low-cost equipment for effective detection and a using method of the equipment.
According to a first aspect of the present invention, there is provided a nucleic acid detecting apparatus characterized by comprising:
the first protective layer is a hydrophobic transparent film, and a sample adding hole is formed in the middle of the film;
the sample adding layer consists of sample adding regions and channels connected with the sample adding regions, and the number of the channels is at least three;
the reaction layer comprises a sample detection area, a negative control area and a positive control area which are independent, the number of the sample detection area, the negative control area and the positive control area is at least one, LAMP amplification reaction reagents, primers of target nucleic acids and freeze-dried powder of detection reagents are fixed on the sample detection area, the negative control area is different from the sample detection area in that the primers of the target nucleic acids are not contained, and the positive control area is different from the sample detection area in that a positive control substance added with the target nucleic acids is added;
the second protective layer is a hydrophobic transparent film;
the first protective layer, the sample adding layer, the reaction layer and the second protective layer are integrated together in sequence, the sample adding region is positioned at the lower part of the sample adding hole, and the tail end of the channel is respectively connected with the sample detection region, the negative control region and the positive control region.
Further, the material of the sample adding layer and the reaction layer is porous fiber material, the pore diameter of the fiber is 0.2-30 μm, and the porous fiber material comprises hydrophilic filter paper or a filter membrane consisting of one or more of the following materials: cellulose acetate, cellulose nitrate, mixed cellulose.
Further, the LAMP amplification reaction reagent comprises: DNA amplificase, RNA reverse transcriptase, dNTPs, MgSO4, Tris-HCl, KCl, (NH4)2SO4, Triton X-100, MnCl2, and the detection reagent comprises: fluorescent probe, pH sensitive indicator and calcein.
Further, the hydrophobic transparent film comprises: PDMS, PCL, PP, PET.
Further, the width of the channel is 1-8mm, and the size of the first protective layer and the second protective layer is 10-60 mm.
Furthermore, the sample adding hole is covered with transparent adhesive tape.
Further, the processing method of the sample adding layer structure and the independent sample detection area, the independent negative control area and the independent positive control area of the reaction layer comprises the following steps: and (6) cutting.
Based on the nucleic acid detecting apparatus of the first aspect of the present invention, the second aspect of the present invention provides a method for using a nucleic acid detecting apparatus, comprising the steps of:
(1) preparing a sample solution:
soaking/mixing a sample to be detected in enzyme-free water, normal saline and cell lysis solution to form a sample solution;
(2) sample adding:
adding the sample solution into the sample adding region through the sample adding hole, and placing the device after the liquid drops are completely absorbed
Heating at constant temperature in a heating device at 65 ℃;
(1) and (3) observation:
and observing and comparing the detection layer every 10 min until a comparison result is obtained.
Further, the sample to be tested comprises: a swab sample, a blood sample, a urine sample, said cell lysate comprising: triton, urea, SDS, formamide.
The invention has the following beneficial effects:
(1) the invention is a paper-based device, which can realize rapid high-flux detection;
(2) the equipment has simple structure, the traditional processing methods of paper-based microfluid equipment such as hot wax printing, ultraviolet lithography and the like are not needed in the processing, and the processing is simple and easy to implement;
(3) the device is a highly integrated device, and direct detection can be carried out only by once sample adding/reagent adding;
(4) the device does not need other nucleic acid extraction and purification devices, only needs to be matched with physiological saline or cell lysate, does not need operations such as excessive solution preparation and the like, does not need strict laboratory environment and extremely skilled operators, and avoids a complicated and time-consuming extraction process;
(5) all reaction reagents and detection reagents are integrated on the device, and reagents do not need to be configured during detection, so that reagent pollution can be avoided.
Drawings
To more clearly illustrate the embodiments or prior art solutions of the present invention, the following embodiments or prior art solutions will be described
While the drawings that are needed to be utilized in the description of the technology are briefly described, it should be apparent that only the illustrated drawings in the following description are of a type that is presently preferred
For those skilled in the art, the embodiments of the present invention can be implemented without creative efforts
The figures provided obtain further figures
FIG. 1 is a schematic view of a nucleic acid detecting apparatus according to example 2 of the present invention；
FIG. 2 is a schematic diagram showing the assembly of the nucleic acid detecting apparatus according to example 2 of the present invention.
Description of specific symbols:
1-a first protective layer; 2-a sample addition layer; 3-a reaction layer; 4-a second protective layer.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, shall fall within the scope of the present invention.
Example 1:
the nucleic acid detection device comprises a first protective layer and a second protective layer, wherein the first protective layer and the second protective layer are both made of hydrophobic transparent thin film materials, a sample adding hole is formed in the middle of the first protective layer, and the first protective layer and the second protective layer are fixed together during integration, so that an interlayer positioned in the middle of the first protective layer and the second protective layer can be fixed.
The middle of the sample adding layer is provided with two interlayers which are in specific shapes and are respectively a sample adding layer and a reaction layer. The sample adding layer is composed of a sample adding area and channels connected with the sample adding area, the number of the channels is at least three, the reaction layer is composed of at least three independent areas, wherein, at least one sample detection area, at least one negative control area and at least one positive control area are arranged, and each independent area of the reaction layer is connected with the other end of the channel of the sample adding layer; the sample detection area contains LAMP amplification reaction reagent, a primer of the target nucleic acid and freeze-dried powder of the detection reagent, compared with the sample detection area, the negative control area is not provided with the primer of the target nucleic acid, and the positive control area is added with a positive control substance of the target nucleic acid. During the equipment, the application of sample position on application of sample layer is located below first protective layer application of sample hole, and the channel end connection reaction layer's each independent block on application of sample layer, and the fixing of application of sample layer and reaction layer is reached through first protective layer and second protective layer.
In addition, the material of the sample adding layer and the reaction layer is a hydrophilic material of porous fiber, and the pore diameter of the fiber is 0.2-30 μm.
Through the use of porous fiber hydrophilic material and protective layer hydrophobic material, a channel for sample flow is constructed, the sample enters a sample adding area through a sample adding hole, the sample flows into a sample detection area, a negative control area and a positive control area of a reaction layer through a channel connected with the sample adding hole, detection reagents fixed in the areas are different, the negative/positive of the sample can be determined by comparing the results of the areas, the driving force for sample flow comes from the capillary siphoning effect of porous fiber, in addition, a purification step is not required to be designed in the nucleic acid detection process, the reason is the use of the porous fiber material, in the whole process of solution sample flow, impurities with larger sizes, such as cell fragments and the like, in a sample solution can be adsorbed and filtered by the fiber, and the effect of nucleic acid purification is achieved.
The porous fiber material is used as a hydrophilic material, a hydrophilic area and a hydrophobic area are manufactured through pattern design and processing, the hydrophobic area is used as a barrier to prevent liquid solution from flowing to an area outside the hydrophilic area, three layers of porous fiber materials are processed into specific patterns to be superposed and combined to construct a channel for flowing of a liquid sample, so that the sample can flow into a sample detection hole and a negative control hole respectively through a sample adding area, a sample transferring area and a channel, the detection hole and the control hole both contain an amplification reagent and an indicator, the difference is that the detection hole contains a primer, and finally the negative/positive can be determined by comparing detection results of the sample detection hole and the invisible control hole.
According to the knowledge and experience of the technicians in the field, all the porous fibers capable of achieving the effect can obtain a detection result; any material can be selected as long as it can produce hydrophobic regions on the porous fibrous material. The porous material can be selected from hydrophilic filter paper, cellulose acetate filter membrane, cellulose nitrate filter membrane and mixed cellulose filter membrane; the material of the hydrophobic region can be selected from paraffin and polyhydroxybutyrate. The choice of reagents and indicators for the amplification reaction may be suitably selected in view of the particular viral nucleic acid characteristics in combination with literature and experience.
Example 2:
the zikv virus is detected using a nucleic acid detection device.
(1) Manufacture and assembly of nucleic acid detection equipment
As shown in the attached figure 1, the equipment is divided into four layers, wherein the first layer is a first protective layer 1, the fourth layer is a second protective layer 2, a sample adding hole with the diameter of 6mm is formed in the center of the first protective layer, and transparent adhesive is covered on the sample adding hole; the second layer is application of sample layer 2, the third layer is reaction layer 3, application of sample layer 2 and reaction layer 3 are tailor with the filter membrane of cellulose acetate and form, wherein, the aperture of cellulose acetate filter membrane is 15 μm, specific tailor requirement is, the size of application of sample region and the diameter of the three independent blocks of reaction layer 3 of application of sample layer 2 are 5mm, the outside of application of sample region of application of sample layer 2 is tailor and is had three length and be 30mm, width 3 mm's passageway, the block of reaction layer 3 is located the end of application of sample layer 2 passageway during the equipment, further fix through first protective layer 1 and second protective layer 2.
The three blocks of the reaction layer are respectively a sample detection zone, a negative control zone and a positive control zone, different reaction reagents are fixed by a freeze-drying method respectively, and the reaction layer comprises the following specific components:
a sample detection zone: 2X colorimetric RT-LAMP premixed liquid freeze-dried powder and primer mixture
Negative control zone: 2X colorimetric RT-LAMP premixed liquid freeze-dried powder
Positive control zone: 2X colorimetric RT-LAMP premix freeze-dried powder, a primer mixture and a virus standard substance.
Wherein the primer mixture comprises:
f3 (forward outer primer): 5'-CGGATGGGATAGGCTCAAAAAA-3'
B3 (reverse outer primer): 5'-ATGGACCTCCCGTCCTTG-3' the flow of the air in the air conditioner,
FIP (forward inner primer):
5'- CCTGAGGGCATGTGCAAACCTAGAATGGCAGTCAGTGGAGAT-3'
BIP (reverse inner primer):
5'-ACCCTCAACTGGATGGGACAACTGGAGCTTGTTGAAGTGGTG-3
LoopF (forward loop primer): 5' -CATCAATTGGCTTCACAACGC-3
Loop primer of LoopB (reverse loop primer): 5'-GGGAAGAAGTTCCGTTTTGCTC-3' are provided.
(2) Method for detection using nucleic acid detection device
S1: mixing a zika RNA standard sample and a blood sample to be detected to prepare a sample solution;
s2, uncovering the transparent adhesive on the surface of the sample adding hole, dripping the sample solution into the sample adding area, and after the solution is completely absorbed, sticking the transparent adhesive again;
s3: after 15 minutes, the apparatus was placed on a hot plate set at 68 ℃ and the color change of the reaction zone was observed after heating for 30 minutes.
(3) Results of the experiment
The sample detection zone is yellow, the negative control zone is red, and the positive control zone is yellow, which indicates that the sample is positive.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (9)
1. A nucleic acid detecting apparatus characterized by comprising:
the first protective layer is a hydrophobic transparent film, and a sample adding hole is formed in the middle of the film;
the sample adding layer consists of sample adding regions and channels connected with the sample adding regions, and the number of the channels is at least three;
the reaction layer comprises a sample detection area, a negative control area and a positive control area which are independent, the number of the sample detection area, the negative control area and the positive control area is at least one, LAMP amplification reaction reagents, primers of target nucleic acids and freeze-dried powder of detection reagents are fixed on the sample detection area, the negative control area is different from the sample detection area in that the primers of the target nucleic acids are not contained, and the positive control area is different from the sample detection area in that a positive control substance added with the target nucleic acids is added;
the second protective layer is a hydrophobic transparent film;
the first protective layer, the sample adding layer, the reaction layer and the second protective layer are integrated together in sequence, the sample adding region is positioned at the lower part of the sample adding hole, and the tail end of the channel is respectively connected with the sample detection region, the negative control region and the positive control region.
2. The nucleic acid detecting apparatus according to claim 1, wherein the sample addition layer,
The reaction layer is made of porous fiber material with the fiber aperture of 0.2-30 μm, and the porous fiber material comprises hydrophilic filter paper or a filter membrane consisting of one or more of the following components: cellulose acetate, cellulose nitrate, mixed cellulose.
3. The nucleic acid detection device of claim 1 or 2, wherein the LAMP
The amplification reaction reagent comprises: DNA amplificase, RNA reverse transcriptase, dNTPs, MgSO4, Tris-HCl, KCl, (NH4)2SO4, Triton X-100, MnCl2, and the detection reagent comprises: fluorescent probe, pH sensitive indicator and calcein.
4. The nucleic acid detecting apparatus according to any one of claims 1 to 3, wherein the nucleic acid detecting apparatus is a nucleic acid detecting apparatus
The hydrophobic transparent film of (1) comprises: PDMS, PCL, PP, PET.
5. The nucleic acid detecting apparatus according to any one of claims 1 to 4, characterized in that the nucleic acid detecting apparatus
The width of the channel is 1-8mm, and the size of the first protective layer and the second protective layer is 10-60 mm.
6. The nucleic acid detecting apparatus according to any one of claims 1 to 6, characterized in that the nucleic acid detecting apparatus
The sample adding hole is covered with transparent adhesive.
7. The nucleic acid detecting apparatus according to any one of claims 1 to 6, characterized in that the nucleic acid detecting apparatus
The processing method of the sample adding layer structure and the independent sample detection area, the negative control area and the positive control area of the reaction layer comprises the following steps: and (6) cutting.
8. A method of using a nucleic acid detecting apparatus, characterized in that it is used in any of claims 1 to 7
The nucleic acid detecting apparatus specifically comprises the following steps:
(1) preparing a sample solution:
soaking/mixing a sample to be detected in enzyme-free water, normal saline and cell lysis solution to form a sample solution;
(2) sample adding:
adding the sample solution into the sample adding region through the sample adding hole, and placing the device after the liquid drops are completely absorbed
Heating at constant temperature in a heating device at 65 ℃;
(3) and (3) observation:
and observing and comparing the detection layer every 10 min until a comparison result is obtained.
9. The method of using the nucleic acid detecting apparatus according to claim 8, wherein
The sample to be detected comprises: a swab sample, a blood sample, a urine sample, said cell lysate comprising: triton, urea, SDS, formamide.
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