CN105273997A - Paper-based nucleic acid extraction, amplification and detection integrated detection system and paper-based nucleic acid extraction, amplification and detection integrated detection method - Google Patents
Paper-based nucleic acid extraction, amplification and detection integrated detection system and paper-based nucleic acid extraction, amplification and detection integrated detection method Download PDFInfo
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Abstract
The invention discloses a paper-based nucleic acid extraction, amplification and detection integrated detection system and a paper-based nucleic acid extraction, amplification and detection integrated detection method and belongs to the technical field of biological test paper detection. According to the detection method, integrated operation of nucleic acid extraction, amplification and detection of any food, environment, clinic samples and the like is achieved, and particularly, paper-based nucleic acid extraction, LAMP (loop-mediated isothermal amplification) and test paper detection are achieved. The invention further provides a small-sized handheld detection device capable of achieving nucleic acid amplification temperature. Detection results can be observed easily by naked eyes, and quantifying is achieved by mobile phone software. Total amplification and detection time is about one hour. Compared with other existing methods, the detection method has the advantages that nucleic acid extraction, amplification and detection can be simplified, operation steps are decreased and rapid efficient onsite detection is achieved.
Description
Technical field
The invention belongs to biological detection paper technical field, be specifically related to a kind of paper substrate nucleic acid extraction, amplification and detect integrated proofing unit and detection method.
Background technology
Detection of nucleic acids is molecular biological study hotspot, has been widely used in clinical disease diagnosis and the field such as popular bacterium or Viral diagnosis.Flourish along with micro-fluidic technologies, the detection method of miniaturization, low cost, facilitation, as nucleic acid detection paper method day by day comes into one's own.Because of raw sample (blood, saliva, urine etc.) complicated component, nucleic acid concentration is on the low side, and nucleic acid extraction and amplification step are necessary before detection paper.Conventional nucleic acid extracts and amplification needs expensive equipment and takes time and effort.Comparatively speaking, paper substrate nucleic acid extraction, amplification and detection paper have that cost is low, simplicity of design, are easy to use and the advantage such as easy to carry.Current paper substrate detection of nucleic acids research, comprise and make on paper, realize HumanImmunodeficiencyVirus (HIV), Chlamydiatrachomatis and InfluenzaA detection, but paper substrate extraction, amplification and detection paper proceed step by step, do not realize the integrated of three, make integrated operation step complicated, cannot carry out efficiently, fast detecting.
Along with the development of biotechnology, low cost, miniaturization, portable integrated integrated detection method are day by day by the attention of people.Recently, there is correlative study to report, " paper machine " achieves nucleic acid extraction, amplification and detects integrated.But nucleic acid amplification need use external source or powerful large-scale heater heats, and step is various, also need extraneous UV-light acquisition testing signal, efficient Site Detection cannot be reached.
Summary of the invention
In order to overcome the defect that above-mentioned prior art exists, the object of the present invention is to provide a kind of paper substrate nucleic acid extraction, amplification and detecting integrated proofing unit and detection method, the design of this detection system is exquisite, and temperature-controllable is simple to operate; This detection method can simplify the operation steps of nucleic acid extraction, amplification and detection, reaches object that is efficient, rapid detection.
The present invention is achieved through the following technical solutions:
The invention also discloses a kind of paper substrate nucleic acid extraction, amplification and detect integrated detection system, being made up of detection casing and test strip;
Described detection box house is provided with heated for controlling temperature equipment, and offer cavity at detection casing top, be provided with heating chamber and test chamber in cavity, the degree of depth of heating chamber is greater than test chamber; Cover plate is provided with above test chamber;
Described test strip is lateral flow test strip, comprises and detects pad, chromatographic film, absorption pad and supporting pad, and detect one end that pad is arranged on chromatographic film, absorption pad is arranged on the other end of chromatographic film; Chromatographic film is provided with detection zone and check plot; Supporting pad is arranged on below chromatographic film, and the length of supporting pad is greater than the length of chromatographic film;
Described supporting pad is four layers, and top layer supporting pad is close to below chromatographic film; The front end place of second layer supporting pad is provided with amplification pad, and on this amplification pad, apply colour developing nm gold particles layer, forms amplification region; Third layer supporting pad is provided with FTA card; The front end place of base layer support pad is provided with bottom absorption pad;
Described detection system also comprises the gummed paper for blocking amplification pad and FTA card.
The volume of described detection casing is less than 7cm × 12cm × 11cm.
Heating chamber is rectangular parallelepiped, and volume is less than 5mm × 10mm × 25mm; Test chamber is right cylinder, and diameter is less than 5mm, and the degree of depth is less than 10mm.
Described heating chamber is made up of aluminium alloy, the outside coated insulation material layer of heating chamber.
Temperature control scope in heating chamber is ring temperature+5 DEG C ~ 100 DEG C, temperature-controlled precision reaches ± and 0.1 DEG C.
Described test chamber is made up of polyoxymethylene, and the cover plate above test chamber is made of plastics.
The top detecting casing is provided with temperature and regulates touching display screen, and the inside detecting casing is provided with rechargeable lithium ion batteries.
Chromatographic film is made up of nitrocellulose filter, for fixing thing to be checked and contrast;
Absorption pad is made up of cellulose membrane, for providing REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE power for liquid-flow in test strip;
Detect pad to be made up of glass fibre membrane or trevira film, for carrying colour developing nm gold particles with amplification pad;
Supporting pad is made up of polychloroethylene film, is used as the substrate supporting to detect pad, chromatographic film and absorption pad.
The invention also discloses and carry out based on above-mentioned paper substrate nucleic acid extraction, amplification and the integrated detection system of detection the method that detects, comprise the following steps:
1) be first that the sample drop to be detected of 10 μ L is added on FTA card by total amount, extract nucleic acid, with 40 μ LFTA purifying agents and 80 μ LTE damping fluids, FTA card is cleaned up after 15 minutes, then absorption pad is taken apart;
2) after 5 minutes, to increase pad and FTA engages, the amplifing reagent of 25 μ L and primer are dripped on amplification pad, form amplification region, with gummed paper, amplification region is hidden again, the amplification region of test paper and detection zone are separated, then test paper is placed in the heating chamber detecting casing and carries out nucleic acid amplification;
3) to be amplified complete after, gummed paper is taken apart, is combined by the amplification region of test paper and detection zone by one-sided adhesive, then the upper cover of test chamber is opened, in test chamber, drip 50 μ L damping fluids, the test paper that amplification region and detection zone combine is put into test chamber and carries out detection of nucleic acids;
4), after detection reaction completes, observe detection zone and check plot, if develop the color in detection zone and check plot simultaneously, show the positive; If do not develop the color in detection zone, developing the color in check plot, shows feminine gender; If all do not develop the color in detection zone and check plot, result is unreliable, needs duplicate detection.
Compared with prior art, the present invention has following useful technique effect:
The detection system that paper substrate nucleic acid extraction disclosed by the invention, amplification and detection are integrated, be made up of detection casing and test strip, detect box house and be provided with heated for controlling temperature equipment, testing chamber is offered at casing top, and testing chamber comprises the test chamber of a heating chamber and a normal temperature.The test strip of native system is lateral flow test strip, more existing test strip, devises three layers of extra supporting pad, can realize nucleic acid extraction, constant-temperature amplification and detection on paper substrate.Test strip of the present invention is first placed on room temperature and carries out nucleic acid extraction, nucleic acid amplification is carried out in heating chamber, to be amplified complete after transfer to again in test chamber and carry out detection of nucleic acids, complete integration operation very easily, native system can carry out the regulation and control of temperature according to different amplification temperature simultaneously, simple to operate, easy to use.
Nucleic acid extraction disclosed by the invention, amplification and detection method, first with gummed paper, the amplification region on test paper is hidden, the amplification region of test paper and detection zone are separated, to watch out for liquid evaporation, then test paper is placed in heating chamber and increases, to be amplified complete after, combined by test paper two regions by one-sided adhesive, and detect in test chamber after dripping damping fluid.Method provided by the invention, can nucleic acid extraction, any one nucleic acid amplification be carried out to any food, environment and clinical sample and detect integration operation, especially on paper substrate, realize nucleic acid amplification (LAMP) and detect integrated, the temperature range that paper substrate LAMP nucleic acid amplification is suitable for is 65 DEG C, and process only needs 45 minutes.About 1 hour of total extraction, amplification and detection time.
Accompanying drawing explanation
Fig. 1 is the structural representation of proofing unit of the present invention;
Fig. 2 is the structure vertical view of proofing unit of the present invention;
Fig. 3 is the structural representation of test strip of the present invention;
Wherein, 1 is casing; 2 is cavity; 3 is heating chamber; 4 is test chamber; 5 is temperature adjustment touching display screen; 6 for detecting pad; 7 is chromatographic film; 8 is absorption pad; 9 is support pad; 10 is amplification pad; 11 is detection zone; 12 is check plot; 13 is FTA card; 14 is bottom absorption pad; 15 is gummed paper.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail, and the explanation of the invention is not limited.
See Fig. 1 and 2, the proofing unit that a kind of paper substrate nucleic acid extraction disclosed by the invention, amplification and detection are integrated, this device is Portable integrated structure, comprise the detection casing 1 that inside is provided with heated for controlling temperature equipment, cavity 2 is offered at detection casing 1 top, be provided with heating chamber 3 and test chamber 4 in cavity 2, the degree of depth of heating chamber 3 is greater than test chamber 4; Cover plate is provided with above test chamber 4.Be provided with temperature at detection casing 1 top and regulate touching display screen 5, the digital code that can realize observed temperature and design temperature shows.
Preferably, proofing unit volume is no more than 7cm × 12cm × 11cm.Heating chamber 3 in cavity 2 is rectangular parallelepipeds, and volume is no more than 5mm × 10mm × 25mm.Test chamber 4 is a right cylinder or other cross-sectional shape, and the not super 5mm of diameter, the degree of depth is no more than 10mm, and test chamber does not heat, and is normal temperature environment.Test chamber 4 is made up of corrosion resistant polyoxymethylene, and its top is provided with upper plastic cover.
Heating chamber 3 is made up of, as aluminium alloy etc. the good corrosion resistant metallic substance of heat conductivility.Its bottom surface is heated by electric heating film, and outside has lagging material to be incubated, to reduce the temperature fluctuation in cavity.The temp scope of heating chamber 3 is ring temperature+5 DEG C ~ 100 DEG C, and temperature-controlled precision can reach the highest ± 0.1 DEG C.
The temperature range that paper substrate LAMP nucleic acid amplification is suitable for is 65 DEG C, process 45 minutes, and rear 95 DEG C of heating 30 seconds, to allow nucleic acid denaturation.
Heating chamber 3 in order to paper substrate isothermal nucleic acid amplification, as LAMP, RPA, NASBA, SDA or HDA etc.Before nucleic acid amplification, amplifing reagent, target nucleic acid and primer are dripped on the pad of test paper, then test strip is placed in heating chamber.
The built-in rechargeable lithium ion batteries of detection casing 1 of described detection system, charges by external charger.Charge power supply is alternating-current: voltage AC220V ± 10%; Frequency: 50Hz; Peak power is 50W.
Temperature control with fully-automatic intelligent PID unidirectional power temperature control method, temperature continuously adjustabe.When about 24 hours can be maintained without the need to operational lifetime when external source.
See Fig. 3, test paper of the present invention is lateral flow test strip, is made up of detection pad 6, chromatographic film 7, absorption pad 8 and support pad 9.Detect one end that pad 6 is arranged on chromatographic film 7, absorption pad 8 is arranged on the other end of chromatographic film 7; Chromatographic film 7 is provided with detection zone 11 and check plot 12; Supporting pad 9 is arranged on below chromatographic film 7, and the length of supporting pad 9 is greater than the length of chromatographic film 7; Described supporting pad 9 is four layers, and top layer supporting pad is combined with chromatographic film 7; The front end place of second layer supporting pad is provided with amplification pad 10, and on this amplification pad 10, apply colour developing nm gold particles layer, forms amplification region; Third layer supporting pad is FTA card 13; The front end place of base layer support pad is provided with bottom absorption pad 14; Also comprise the gummed paper 15 for blocking amplification pad 10 and FTA card 13.
Pad 6 material in test paper amplification region 10 and dropping nanometer gold district is glass fibre membrane, increases and carry colour developing nm gold particles for carrying out.Chromatographic film 7 material is nitrocellulose filter, and for the detection thing of fixing 2mg/ml, the contrast of Streptomycin sulphate and 100 μMs, contrast molecule, forms detection zone 11 and check plot 12.Absorbent pad material is Mierocrystalline cellulose, provides REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE power for giving solution flowing in test paper.Support pad material is polychloroethylene film, is used as the substrate supporting pad, chromatographic film, absorption pad.Instruction particle is nm gold particles, and granularity is 13nm.
For realizing nucleic acid extraction, amplification is integrated with detection, and test strip comprises amplification region and Liang Ge region, detection zone, but both pads are spaced in advance.After FTA card 13 realizes nucleic acid extraction, rinse with 40 μ LFTA card purifying agents and 80 μ L damping fluids, rear bottom absorption pad 14 to be torn.Be first that the amplifing reagent of 25 μ L and primer drip on the amplification region pad of test paper by total amount before amplification, with gummed paper 15, amplification region 10 and FTA card 13 are hidden, to watch out for liquid evaporation, then test strip is placed in heating chamber, lid is covered, then increases.To be amplified complete after, allow two regions combine by one-sided adhesive, and detect after dripping damping fluid.The pad material in the above two region can be glass fibre membrane or trevira film, all can load bearing grain.
Detection method disclosed by the invention, comprises the following steps:
1) be first that the sample drop to be detected of 10 μ L is added on FTA card 13 by total amount, extract nucleic acid, with 40 μ LFTA purifying agents and 80 μ LTE damping fluids, FTA card 13 is cleaned up after 15 minutes, then absorption pad 8 is taken apart;
2) after 5 minutes, amplification pad 10 and FTA card 13 are closed, the amplifing reagent of 25 μ L and primer are dripped on amplification pad 10, form amplification region, with gummed paper 15, amplification region is hidden again, the amplification region of test paper and detection zone are separated, then test paper is placed in the heating chamber 3 detecting casing 1 and carries out nucleic acid amplification;
3) to be amplified complete after, gummed paper 15 is taken apart, is combined by the amplification region of test paper and detection zone by one-sided adhesive, then the upper cover of test chamber 4 is opened, in test chamber 4, drip 50 μ L damping fluids, the test paper that amplification region and detection zone combine is put into test chamber 4 and carries out detection of nucleic acids;
4), after detection reaction completes, observe detection zone and check plot, if develop the color in detection zone and check plot simultaneously, show the positive; If do not develop the color in detection zone, developing the color in check plot, shows feminine gender; If all do not develop the color in detection zone and check plot, result is unreliable, needs duplicate detection.
The present invention utilizes Streptomycin sulphate on detection line, catch amplified material with vitamin H.Target compound can be combined with nm gold particles.Capture molecules can be combined with nm gold particles, also can with target compound specific binding.Contrast molecular energy and capture molecules specific binding.After detection reaction completes, observe detection zone 11 and check plot 12, detected result shows two-region and develops the color simultaneously, shows the positive; Do not develop the color in detection zone, developing the color in check plot, shows feminine gender; Do not develop the color in two-region, result is unreliable, needs duplicate detection.
In sum, the invention provides one and can carry out nucleic acid extraction to any food, environment and clinical equal samples, any one nucleic acid amplification and detection integration operation, especially on paper substrate, realize nucleic acid extraction, constant-temperature amplification (LAMP) and detection paper, provide a kind of small hand-held proofing unit realizing nucleic acid amplification temperature simultaneously.Detected result can detect by an unaided eye easily, quantitative with cell phone software.Only need total amplification and detection time about 1 hour.Compared with other method existing, method provided by the invention can simplify nucleic acid extraction, amplification and detection, reduces operation steps, realizes on-the-spot rapid detection efficiently.
Claims (9)
1. the detection system that paper substrate nucleic acid extraction, amplification and detection are integrated, is characterized in that, is made up of detection casing (1) and test strip;
Described detection casing (1) inside is provided with heated for controlling temperature equipment, cavity (2) is offered at detection casing (1) top, be provided with heating chamber (3) and test chamber (4) in cavity (2), the degree of depth of heating chamber (3) is greater than test chamber (4); Cover plate is provided with in test chamber (4) top;
Described test strip is lateral flow test strip, comprise and detect pad (6), chromatographic film (7), absorption pad (8) and supporting pad (9), detect one end that pad (6) is arranged on chromatographic film (7), absorption pad (8) is arranged on the other end of chromatographic film (7); Chromatographic film (7) is provided with detection zone (11) and check plot (12); Supporting pad (9) is arranged on chromatographic film (7) below, and the length of supporting pad (9) is greater than the length of chromatographic film (7);
Described supporting pad (9) is four layers, and top layer supporting pad is close to chromatographic film (7) below; The front end place of second layer supporting pad is provided with amplification pad (10), and at this amplification pad (10) upper coating colour developing nm gold particles layer, forms amplification region; Third layer supporting pad is provided with FTA card (13); The front end place of base layer support pad is provided with bottom absorption pad (14);
Described detection system also comprises the gummed paper (15) for blocking amplification pad (10) and FTA card (13).
2. the detection system that a kind of paper substrate nucleic acid extraction according to claim 1, amplification and detection are integrated, it is characterized in that, the volume of described detection casing (1) is less than 7cm × 12cm × 11cm.
3. the detection system that a kind of paper substrate nucleic acid extraction according to claim 1, amplification and detection are integrated, it is characterized in that, heating chamber (3) is rectangular parallelepiped, and volume is less than 5mm × 10mm × 25mm; Test chamber (4) is right cylinder, and diameter is less than 5mm, and the degree of depth is less than 10mm.
4. the detection system that a kind of paper substrate nucleic acid extraction according to claim 1, amplification and detection are integrated, it is characterized in that, described heating chamber (3) is made up of aluminium alloy, the outside coated insulation material layer of heating chamber (3).
5. a kind of paper substrate nucleic acid extraction according to claim 1, amplification and the integrated detection system of detection, it is characterized in that, the temperature control scope in heating chamber (3) is ring temperature+5 DEG C ~ 100 DEG C, temperature-controlled precision reaches ± and 0.1 DEG C.
6. the detection system that a kind of paper substrate nucleic acid extraction according to claim 1, amplification and detection are integrated, it is characterized in that, described test chamber (4) is made up of polyoxymethylene, and the cover plate of test chamber (4) top is made of plastics.
7. the detection system that a kind of paper substrate nucleic acid extraction according to claim 1, amplification and detection are integrated, it is characterized in that, the top detecting casing (1) is provided with temperature and regulates touching display screen (5), and the inside detecting casing (1) is provided with rechargeable lithium ion batteries.
8. the detection system that a kind of paper substrate nucleic acid extraction according to claim 1, amplification and detection are integrated, is characterized in that:
Chromatographic film (7) is made up of nitrocellulose filter, for fixing thing to be checked and contrast;
Absorption pad (8) is made up of cellulose membrane, for providing REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE power for liquid-flow in test strip;
Detection pad (6) and amplification pad (10) are made up of glass fibre membrane or trevira film, for carrying colour developing nm gold particles;
Supporting pad (9) is made up of polychloroethylene film, is used as the substrate supporting to detect pad (6), chromatographic film (7) and absorption pad (8).
9. carry out the method detected based on the detection system that paper substrate nucleic acid extraction according to claim 1, amplification and detection are integrated, it is characterized in that, comprise the following steps:
1) be first that the sample drop to be detected of 10 μ L is added on FTA card (13) by total amount, extract nucleic acid, with 40 μ LFTA purifying agents and 80 μ LTE damping fluids, FTA card (13) is cleaned up after 15 minutes, then absorption pad (8) is taken apart;
2) after 5 minutes, to increase pad (10) and FTA card (13) closes, the amplifing reagent of 25 μ L and primer are dripped in amplification pad (10), form amplification region, gummed paper (15) is used to be hidden amplification region again, the amplification region of test paper and detection zone are separated, then test paper is placed in the heating chamber (3) detecting casing (1) and carries out nucleic acid amplification;
3) to be amplified complete after, gummed paper (15) is taken apart, combined by the amplification region of test paper and detection zone by one-sided adhesive, then the upper cover of test chamber (4) is opened, in test chamber (4), drip 50 μ L damping fluids, the test paper that amplification region and detection zone combine is put into test chamber (4) and carries out detection of nucleic acids;
4), after detection reaction completes, observe detection zone and check plot, if develop the color in detection zone and check plot simultaneously, show the positive; If do not develop the color in detection zone, developing the color in check plot, shows feminine gender; If all do not develop the color in detection zone and check plot, result is unreliable, duplicate detection.
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| CN110591911A (en) * | 2019-09-30 | 2019-12-20 | 中国科学院长春光学精密机械与物理研究所 | Optical-mechanical-electrical integrated full-automatic digital PCR device |
| CN111733288A (en) * | 2020-06-22 | 2020-10-02 | 厦门大学 | Nucleic acid detection method and device and application in COVID-19 detection |
| CN115141745A (en) * | 2022-09-01 | 2022-10-04 | 苏州雅睿生物技术股份有限公司 | Multi-channel split type kit, nucleic acid detection device and detection method |
| CN115141745B (en) * | 2022-09-01 | 2022-11-25 | 苏州雅睿生物技术股份有限公司 | Multi-channel split type kit, nucleic acid detection device and detection method |
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