CN104404137A - Closed portable test strip colourimetry nucleic acid detection apparatus, detection container and detection method - Google Patents
Closed portable test strip colourimetry nucleic acid detection apparatus, detection container and detection method Download PDFInfo
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Abstract
The invention discloses a closed portable test strip colourimetry nucleic acid detection apparatus, a detection container and a detection method The apparatus comprises a reaction tube and the detection container; and one end of the detection container is in sealed connection with the reaction tube, a test strip can be put into the detection container, the detection container is provided with an elongated cavity for accommodating the test strip, and the other end of the detection container is connected with a detection container cover. The method comprises the following steps: preparing an amplification reaction solution in the reaction tube, or adding the prepared amplification reaction solution into the reaction tube, connecting the detection container provided with the test strip with the reaction tube provided with the amplification reaction solution, carrying out an amplification reaction when the reaction tube is connected with the detection container, allowing the reaction solution to enter the detection container or allowing the test strip to enter the reaction tube after the amplification reaction ends, immersing the test strip in a liquid obtained after the amplification reaction, and carrying out nucleic acid detection according to the change of the color of the test stripe. The apparatus, the container and the method are mainly used for nucleic acid amplification and specificity detection, and have the advantages of simple design and reasonable structure.
Description
Technical field
The invention belongs to field of nucleic acid detection, be specifically related to reaction unit and method that one can be used for Common Polymerase Chain Reaction (PCR) and the detection of constant-temperature amplification product.
Background technology
Along with the development of science and technology, detection of nucleic acids is applied to the field such as medical diagnosis, food safety detection more and more.At present conventional nucleic acid amplification method has amplification technique based on classical Common Polymerase Chain Reaction (PCR) and emerging isothermal amplification technology, as cross primer isothermal amplification technology (CPA), loop-mediated isothermal amplification technology (LAMP), strand displacement amplification (SDA), relies on the amplification (HDA) etc. of helicase.Due to the highly sensitive of nucleic acid amplification technologies, the output of amplified production is very high, therefore faces Aerosol.
In addition, based on the detection method of amplified production, traditional real time fluorescent quantitative detection method needs integrated huge, expensive, accurate optical device, is unfavorable for that the miniaturization development and application of device is in Site Detection.Non-optical detection method emerging at present, as electrochemical method, HNB colorimetry, nephelometry, greatly simplifies the requirement of device, the operation but the needs that these methods have are uncapped, and as easy as rolling off a logly causes Aerosol Pollution, and some detection sensitivities are not high, and result not easily judges.Application number be 201410431991.9 Chinese invention patent application can make positive sample react after reaction solution from colourless become coloured, nucleic acid amplification is detected become naked eyes visible, compare with traditional colorimetric method, its colour developing is sensitiveer, but this technology relates to detection specificity not yet.Therefore, develop airtight, miniaturization, portable nucleic acid detection apparatus, it is combined with existing detection method and seems more and more important.
Test strip method is subject to extensive concern because of its detection specificity, highly sensitive and simply portable characteristic.But current business-like reagent paper detector design is complicated, is unfavorable for reducing costs.Therefore develop simple and easy to do, reagent paper detector with low cost, very necessary.
Summary of the invention
An object of the present invention is to provide a kind of colorimetry nucleic acid detection method, and nucleic acid reaction system can be made when not opening reaction vessel, carries out PCR reaction or carries out different types of isothermal amplification reactions and carry out specific detection by test strip.For this reason, the present invention is by the following technical solutions:
Colorimetry nucleic acid detection method, is characterized in that it comprises the following steps:
There is provided the detection receptacle that always can be connected with reaction tubes, and the connection between them is sealing, detection receptacle can either be connected with reaction tubes and can be placed into test strip, and has the long strip type cavity holding test strip;
Configure amplification reaction solution in reaction tubes after, or the amplification reaction solution configured is added in reaction tubes, the detection receptacle that test strip is housed is connected with the reaction tubes that amplification reaction solution is housed, tight amplified reaction under reaction tubes and detection receptacle attached state, after reaction to be amplified terminates, reaction solution is made to enter in detection receptacle, or make test strip enter in reaction tubes, test strip is made to be immersed in after amplified reaction in liquid, such as put upside down or the linker of tilt reaction tubes and detection receptacle, or promote test strip and progress into amplification reaction solution, detection of nucleic acids is carried out by the colour-change in test strip.
Further, it carries out wax oil sealing to the upper strata of the amplification reaction solution in reaction tubes.
Two of object of the present invention is to provide the device of a kind of stopping property portable test strip colorimetric determination nucleic acid amplification, nucleic acid reaction system can being made when not opening reaction vessel, carrying out PCR reaction or carrying out different types of isothermal amplification reactions and carry out specific detection by test strip.For this reason, the present invention is by the following technical solutions:
A kind of stopping property portable test strip colorimetry nucleic acid detection apparatus, is characterized in that it comprises reaction tubes, detection receptacle; Detection receptacle comprises the cavity placed for colloidal gold strip, and one end of described detection receptacle is the structure that can be connected with reaction tubes, and is sealing between this structure and reaction tubes, has the connected entrance communicated with described cavity at this end; The other end of described detection receptacle also has the mouth communicated with described cavity, and this end is connected with the lid of detection receptacle; The cavity placed for colloidal gold strip in detection receptacle communicates with reaction tubes through described connected entrance.
The mouth of the other end of described detection receptacle is the test strip filler straight-through with cavity.
Further, the structure that can be connected with reaction tubes is the structure inserted by reaction tubes.Described is the lid of reaction tubes by the structure that reaction tubes inserts.Further, the axes normal of described cavity and reaction tubes, in this structure, described detection receptacle can be cuboid.
Further, described detection receptacle also can be tubular, and its one end can be inserted by reaction tubes and seal.In this structure, the lid of described detection receptacle can adopt piston-type, and its medial extremity has the position that can connect test strip, and in this structure, place's test strip in the cavities can be connected on piston.
Three of object of the present invention is to provide a kind of detection receptacle of test strip colorimetric determination nucleic acid amplification, can be applied in nucleic acid detection apparatus, nucleic acid reaction system can being made when not opening reaction vessel, carrying out PCR reaction or carrying out different types of isothermal amplification reactions and carry out specific detection by test strip.For this reason, the present invention is by the following technical solutions:
A kind of detection receptacle of test strip colorimetric determination nucleic acid amplification, it is characterized in that it comprises the cavity placed for colloidal gold strip, one end of described detection receptacle is the structure that can be connected with reaction tubes, and be sealing between this structure and reaction tubes, there is at this end the connected entrance communicated with described cavity; The other end of described detection receptacle also has the mouth communicated with described cavity, and this end is connected with the lid of detection receptacle; The cavity placed for colloidal gold strip in detection receptacle communicates with reaction tubes through described connected entrance.
The mouth of the other end of described detection receptacle is the test strip filler straight-through with cavity.
Further, the structure that can be connected with reaction tubes is the structure inserted by reaction tubes.Described is the lid of reaction tubes by the structure that reaction tubes inserts.Further, the axes normal of described cavity and reaction tubes, described detection receptacle can also be in cuboid.
Further, described detection receptacle also can be tubular, and its one end can be inserted by reaction tubes and seal.In this structure, the lid of described detection receptacle can adopt piston-type, and in this structure, place's test strip in the cavities can be connected on piston.
Compared with prior art, the present invention has following beneficial effect:
1. reaction vessel separates with detection receptacle but is communicated with by the present invention, is interconnected, can be integrated in nucleic acid amplification reactor by test strip, just can realize color reaction, greatly reducing aerocolloidal pollution without the need to opening reaction vessel by communicating aperture;
2. apparatus of the present invention are simple, can carry out PCR reaction or carry out different types of isothermal amplification reactions, and do not affect follow-up colloidal gold strip detection, the detection realizing nucleic acid amplification product that just can be easy.
3. the present invention not only ensure that the rapidly and efficiently property of ELISA test strip, and with low cost, can complete the specific detection of whole nucleic acid amplification when covered.
Accompanying drawing explanation
Fig. 1 is the sectional view of a kind of embodiment in the embodiment of the present invention 1.
Fig. 2 is the exploded view of Fig. 1.
Fig. 3 is the sectional view of another kind of embodiment in the embodiment of the present invention 1.
Fig. 4 is the exploded view of Fig. 3.
Fig. 5 is the sectional view of the embodiment of the present invention 2.
Fig. 6 is the exploded view of Fig. 5.
Fig. 7 is the result figure of aperture non-oil envelope nucleic acid detection apparatus specific detection experiment in embodiment 6.
Wherein, A is the aperture non-oil sealing nucleic acid detection apparatus figure before amplified reaction, fills 50 μ L amplification reaction solutions in reaction tubes; B is negative control; C is positive control, and wherein on colloidal gold strip, C line is control line, and T line is detection line.
Fig. 8 is the result figure of macropore oil sealing nucleic acid detection apparatus specific detection experiment in embodiment 7.
Wherein, A is the macropore oil sealing nucleic acid detection apparatus figure before amplified reaction, fills 25 μ L amplification reaction solutions in reaction tubes, and reaction solution upper strata is 15 μ L paraffin oil sealings; B is negative control; C is positive control, and wherein on colloidal gold strip, C line is control line, and T line is detection line.
Fig. 9 is the result figure of syringe type nucleic acid detection apparatus specific detection experiment in embodiment 8.
Wherein, A is the syringe type nucleic acid detection apparatus figure before amplified reaction, fills 25 μ L amplification reaction solutions in reaction tubes, and reaction solution upper strata is 15 μ L paraffin oil sealings; B is negative control; C is positive control, and wherein on colloidal gold strip, C line is control line, and T line is detection line.
Figure 10 is colloidal gold strip colour developing schematic diagram.
The cellulose acetate film of colloidal gold strip is marked with in advance the antibody of colloid gold label.Due to positive DNA product being marked with vitamin H and fluorescein isothiocyanate FITC, therefore positive products can with the antibodies of colloid gold label, formed composite structure.This mixture, at the captured antibody capture of detection line (T line), forms sandwich structure, occurs red stripes at T line; Unconjugated product moves forward further under syphonic effect, and the mouse-anti IgG that the antibody of colloid gold label and control line (C line) mark in advance catches, and presents red stripes at C line, and therefore result is positive.When amplified production is negative, can not sandwich structure be formed, therefore not have red stripes to occur at T line, and the antibody of colloid gold label on cellulose acetate film moves forward under syphonic effect, combine with the mouse-anti IgG of the upper mark of control line (C line), C line presents red stripes, and therefore result is negative.
Embodiment
embodiment 1, with reference to Fig. 1,2,3,4:
Stopping property provided by the present invention portable test strip colorimetry nucleic acid detection apparatus, comprises reaction tubes 5, detection receptacle 2; Detection receptacle 2 comprises the long strip type cavity 4 placed for colloidal gold strip, one end of described detection receptacle is can insert the lid 20 of the reaction tubes be connected with reaction tubes 5, and is seal between this structure and reaction tubes, has communicate with described cavity at this end
The axes normal of described cavity 4 and reaction tubes, described detection receptacle can be cuboid.
Different according to the lid of reaction tubes and the mode of communicating of detection receptacle, this embodiment can be divided into two kinds again:
The first: the lid of reaction tubes and connected entrance 21 opening less (as shown in Figure 1, 2) of the detection receptacle for placing test strip be communicated with it.The shape of cross section of this connected entrance 21 is any, is preferably rectangle (comprising square) and circle, and be more preferably circular, opening diameter size is preferably less than or equal to 2mm, is more than or equal to 1mm, is more preferably 1.5mm.
The second: the lid of reaction tubes and the connected entrance 21 comparatively large (as shown in Figure 3,4) of the rectangular parallelepiped detection receptacle for placing test strip be communicated with it.Preferably the equitant part of wall of the lid of reaction tubes and the cavity of placement test strip is all removed, and makes both connected component cross-sectional areas reach maximum as far as possible.
Above-mentioned closed nucleic acid detection apparatus, the detection receptacle being integrated with the lid of reaction tubes is formed in one.
Namely the axes normal of the reaction tubes that described cavity and detection receptacle will connect, also described detection receptacle is horizontal detection receptacle or also to can be described as detection receptacle relative to reaction tubes be horizontal container relative to reaction tubes.The height of described reaction tubes preferably, is less than or equal to 20mm, is more than or equal to 10mm.
In the first embodiment above-mentioned, react the volume of working fluid preferably between 50 ~ 450 μ L, more preferably between 50 ~ 100 μ L.In above-mentioned the second embodiment, react the volume of working fluid preferably between 20 ~ 450 μ L, more preferably between 20 ~ 50 μ L.
During assembling, desirable above-mentioned detection receptacle, puts into the colloidal gold strip marked for material to be detected from mouth 22, and the mark end of colloidal gold strip is near connected entrance 21, mouth 22 near the mouth 22 of detection receptacle, then seals with lid 3 by the unmarked end of colloidal gold strip.
Nucleic acid detection apparatus of the present invention mainly for do nucleic acid amplification in enormous quantities detect time, the problem such as Aerosol Pollution detecting and formed of uncapping.According to the needs of real reaction, in detection receptacle, add the colloidal gold strip of the specific marker for different testing sample, when use, only need reaction solution be added in reaction tubes, carry out after being connected with detection receptacle reacting and detecting.
The reaction vessel (reaction tubes 5) of apparatus of the present invention is for holding nucleic acid amplification reaction liquid, and the material of reaction vessel can be high temperature resistant, do not affect nucleic acid amplification reaction and carry out.Reaction vessel has good stopping property, moisture evaporation does not occur in reaction process and scatters and disappears, can independently carry out PCR reaction or other isothermal amplification reactions.When covered, by operations such as rocking or shake, put upside down, the liquid in reaction vessel can enter detection receptacle by communicating aperture and carry out test strip colorimetric determination.
Utilize the nucleic acid detection method of above-mentioned nucleic acid detection apparatus, specifically comprise the steps:
(1) get the reaction tubes of the airtight nucleic acid detection apparatus of machine-shaping, put into pcr amplification reaction liquid (or the isothermal amplification reactions liquid such as CPA, LAMP).As detection receptacle adopts in above-mentioned the second embodiment, reaction solution upper strata paraffin oil seals.
It should be noted that for colloidal gold strip, the ratio of wax oil and amplification reaction solution.The volume ratio of paraffin oil and amplification reaction solution should be less than 2.5:1.Experiment proves, when the ratio of paraffin oil and amplification reaction solution is 2.5:1, colloidal gold strip does not have band occur; When the ratio of paraffin oil and amplification reaction solution is 2:1, colloidal gold strip there is once in a while band occur, but occur that the reaction times needed for band is longer.When the ratio of paraffin oil and amplification reaction solution is 1.5:1, colloidal gold strip has band occur, and speed of response is fast, band color is obvious.
Therefore the ratio of preferred paraffin oil and amplification reaction solution should be less than or equal to 1.5:1, will ensure that paraffin oil forms sealing on amplification reaction solution upper strata simultaneously.
(2) get one end airtight good detection receptacle with lid 3, be connected with the reaction tubes of liquid feeding.Then the nucleic acid amplification procedures under relevant temperature is carried out.As: in the enterprising performing PCR reaction of the accurate hot block of temperature control; Water-bath or hot block are carried out increase temperature lower than the isothermal amplification reactions of 90 DEG C, can be cross primer constant-temperature amplification (CPA), also can be loop-mediated isothermal amplification (LAMP).
It should be noted that and carry out amplified reaction, when especially carrying out water-bath amplified reaction, the mouth of pipe that make the reaction tubes of nucleic acid detection apparatus upward, to ensure that reaction can be carried out smoothly, in amplification process, reaction solution can not leak on colloidal gold strip in any form.
(3) after nucleic acid amplification reaction terminates, by the nucleic acid detection apparatus reaction tubes mouth of pipe down, be inverted whipping twice by airtight nucleic acid detection apparatus, enable reaction solution penetrate on colloidal gold strip by connected entrance, room temperature is placed, the colour developing result of observing colloid gold test paper strip.
The principle that colloidal gold strip detects is: be fixed on cellulose acetate film by specific antigen or antibody with ribbon, colloid gold label reagent (antibody or monoclonal antibody) is adsorbed on pad, after in the sample pad that sample to be checked is added to test strip one end, moved forward by wicking action, react to each other after dissolving the colloid gold label reagent on pad, when moving to the region of fixing antigen or antibody again, there is specific binding with it again and be trapped in the binding substances of thing to be checked and gold marked reagent, be gathered in detection zone, by being observed visually colour developing result.Bibliographical information is had about with the principle that nucleic acid amplification detects relevant colloidal gold strip, as " Rapid and sensitive detection of white spot syndrome virus by loop-mediated isothermal amplification combined with a lateral flow dipstick " (W. Jaroenrama, W. Kiatpathomchai and T. W. Flegel, Molecular and Cellular Probes, 2009, 23, 65-70.) with " Detection of Roundup Ready soybean by loop-mediated isothermal amplification combined with a lateral-flow dipstick " (X. Wang, D. Teng, Q. Guan, F. Tian and J. Wang, Food Control, 2013, 29, 213-220).
embodiment 2, with reference to Fig. 5,6.
Stopping property provided by the present invention portable test strip colorimetry nucleic acid detection apparatus, it comprises reaction tubes 5, detection receptacle 2; Comprise the long strip type cavity 4 placed for colloidal gold strip, one end of described detection receptacle is the structure that can be connected with reaction tubes, and is sealing between this structure and reaction tubes, has the connected entrance 21b communicated with described cavity at this end; The other end of described detection receptacle also has the mouth 22b communicated with described cavity, and this end is connected with the lid 3b of detection receptacle; The cavity 4 placed for colloidal gold strip in detection receptacle communicates with reaction tubes 5 through described connected entrance 21b.
Described detection receptacle 2 in tubular, the inner peripheral surface of its one end as the insertion detection receptacle at reaction tubes detection receptacle be enclosed within its outer time the structure be connected with reaction tubes.In this embodiment, the lid 3b of described detection receptacle can adopt piston-type, and in this structure, place's test strip in the cavities can be connected on piston, has more the protruding parts 30b connecting test strip on the inner mountain of piston.In the present embodiment, detection receptacle is in being similar to syringe shape, and after the completion of reaction, adopting the mode pushing piston test strip to be immersed in gradually in reaction solution, is that manufacturing installation or operating gear are all very convenient.
Further, in this this embodiment, reaction tubes bottom design becomes plane and non-tapered, and object facilitates test strip to be deep into reaction container bottom, carries out colloidal gold strip colorimetric detection.
Above-mentioned closed nucleic acid detection apparatus, the height of its reaction tubes preferably, is less than or equal to 20mm, is more than or equal to 10mm.
In said syringe formula nucleic acid detection apparatus, the volume of amplification reaction solution preferably between 20 ~ 450 μ L, more preferably between 20 ~ 50 μ L.
Utilize the nucleic acid detection method of above-mentioned nucleic acid detection apparatus, specifically comprise the steps:
(1) get the reaction tubes of the airtight nucleic acid detection apparatus of machine-shaping, put into pcr amplification reaction liquid (or the isothermal amplification reactions liquid such as CPA, LAMP); The colloidal gold strip marked for material to be detected is connected to the protruding parts 30b of piston, the mark end of colloidal gold strip is in colloidal gold strip that one end away from outstanding position, piston is inserted the far-end of detection receptacle, the end that the near-end position of detection receptacle is connected with reaction tubes.Reaction tubes is inserted detection receptacle, is connected and seals.
It should be noted that in reaction tubes after adding nucleic acid amplification liquid, before starting the reaction or during amplified reaction carries out, test strip end all can not go deep in reaction solution, and the position of test strip will remain on reaction solution upper end.
Nucleic acid detection apparatus of the present invention mainly for do nucleic acid amplification in enormous quantities detect time, the problem such as Aerosol Pollution detecting and formed of uncapping.According to the needs of real reaction, at the outstanding position of syringe piston, reaction solution, to the colloidal gold strip of the specific marker of different testing sample, when use, only need add in reaction tubes, carry out reacting and detecting by connecting needle.
The reaction vessel of apparatus of the present invention is used for holding nucleic acid amplification reaction liquid, and the material of reaction vessel can be high temperature resistant, does not affect nucleic acid amplification reaction and carries out.Reaction vessel has good stopping property, moisture evaporation does not occur in reaction process and scatters and disappears, can independently carry out PCR reaction or other isothermal amplification reactions.When covered, by pressing piston, test strip is immersed in reaction solution, carries out specific detection by the colour-change of colloidal gold strip.
In the present embodiment, wax oil sealing is carried out on the upper strata of the amplification reaction solution in reaction tubes.
embodiment 3:the non-oil sealing nucleic acid detection apparatus of aperture is applied to PCR and detects transgenosis terminator T-Nos gene.Its proofing unit adopts the first embodiment of embodiment 1.
Before reaction, reagent prepares: 50 μ L amplification reaction solutions (include primer, template, from He Gao bio tech ltd, Shanghai transgene component NOS terminator real-time fluorescence PCR detection kit, article No. GMO-NOS-100) and water.
Nucleic acid carries out pcr amplification reaction: 98 DEG C of reaction 2min; 40 circulations, each circulation comprises 98 DEG C of reaction 10s, 55 DEG C of reactions 30s and 72 DEG C of reaction 1min; 72 DEG C of reaction 10min.
Test strip colorimetric determination pcr amplification: after having reacted, reverses whipping twice by device, make reaction solution penetrate in test strip by communicating aperture, and room temperature places 1 ~ 2min, observes test strip colour developing result.Positive findings is that (C line and T line) appears in two red stripes, and negative findings occurs (C line) for only having a red stripes.
embodiment 4:macropore oil sealing nucleic acid detection apparatus is applied to PCR and detects transgenosis terminator T-Nos gene.
Proofing unit adopts embodiment 1 the second embodiment, and 25uL amplification reaction solution, adopts paraffin oil sealing above reaction solution, other are with embodiment 3.
embodiment 5:syringe type nucleic acid detection apparatus is applied to PCR and detects transgenosis terminator T-Nos gene.
Adopt the proofing unit of embodiment 2, nucleic acid amplification reaction is with embodiment 4.
Test strip colorimetric determination pcr amplification: pressing piston, make colloidal gold strip go deep into reaction solution, until be deep into bottom reaction tubes, reaction solution is risen in test strip by syphonic effect, and device room temperature is placed 1 ~ 2min, observes test strip colour developing result.The positive is that (C line and T line) appears in two red stripes, negative for only having a red stripes to occur (C line).
embodiment 6:the non-oil sealing nucleic acid detection apparatus of aperture is applied to cross primer constant-temperature amplification (CPA) and detects transgenosis terminator T-Nos gene.Its proofing unit adopts the first embodiment of embodiment 1.
Before reaction, reagent prepares: in the reaction tubes of embodiment 1.1, add 50 ul reaction amplification liquid (include primer, dNTP, Tris-HCl damping fluid, Mg
2+, template, from Hangzhou You Sida biotech company transgenosis NOS sequence constant-temperature amplification kit, article No. I016-01), and water.
Nucleic acid constant-temperature amplification reaction is carried out: said apparatus is placed in 63 ° of C heating in water bath 1h.
Test strip colorimetric determination CPA increases: with embodiment 3.
embodiment 7:macropore oil sealing nucleic acid detection apparatus is applied to PCR and detects transgenosis terminator T-Nos gene.
Proofing unit adopts embodiment 1 the second embodiment, and 25uL amplification reaction solution, adopts paraffin oil sealing above reaction solution.
All the other steps are with embodiment 6.
embodiment 8:syringe type nucleic acid detection apparatus is applied to cross primer constant-temperature amplification (CPA) and detects transgenosis terminator T-Nos gene.
Adopt the proofing unit of embodiment 2, nucleic acid amplification reaction is with embodiment 7.
Test strip colorimetric determination CPA increases: with embodiment 5.
embodiment 9:the non-oil sealing nucleic acid detection apparatus of aperture is applied to cross primer constant-temperature amplification (CPA) and detects transgenosis terminator T-Nos gene.Its proofing unit adopts the first embodiment of embodiment 1.
Nucleic acid constant-temperature amplification reaction is carried out: be placed in by said apparatus on 63 ° of C metal fever blocks and heat 1h.
All the other steps are with embodiment 6.
embodiment 10:macropore oil sealing nucleic acid detection apparatus method is applied to cross primer constant-temperature amplification (CPA) and detects transgenosis terminator T-Nos gene.
Proofing unit adopts embodiment 1 the second embodiment, and nucleic acid constant-temperature amplification reaction is carried out: be placed in by said apparatus on 63 ° of C metal fever blocks and heat 1h.
All the other steps are with embodiment 7.
embodiment 11:syringe type nucleic acid detection apparatus is applied to cross primer constant-temperature amplification (CPA) and detects transgenosis terminator T-Nos gene.
Adopt the proofing unit of embodiment 2, nucleic acid constant-temperature amplification reaction is carried out: be placed in by said apparatus on 63 ° of C metal fever blocks and heat 1h.
All the other steps are with embodiment 8.
embodiment 12:the non-oil sealing nucleic acid detection apparatus of aperture is applied to loop-mediated isothermal amplification (LAMP) and detects transgenosis terminator T-Nos gene.Its proofing unit adopts the first embodiment of embodiment 1.
Before reaction, reagent prepares: 50ul reaction amplification liquid (includes primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, template, from Guangzhou enlightening Australia NOS gene nucleic acid detection kit (constant temperature fluorescent method), article No. PA101S).All the other steps are with embodiment 6.
embodiment 13:macropore oil sealing nucleic acid detection apparatus is applied to loop-mediated isothermal amplification (LAMP) for test strip colorimetry and detects transgenosis terminator T-Nos gene.
Proofing unit adopts embodiment 1 the second embodiment, and before reaction, reagent prepares: 25uL amplification reaction solution, adopts oil sealing above reaction solution.
All the other steps are with embodiment 10.
embodiment 14:syringe type nucleic acid detection apparatus is applied to loop-mediated isothermal amplification (LAMP) for test strip colorimetry and detects transgenosis terminator T-Nos gene.
Adopt the proofing unit of embodiment 2, before reaction, reagent preparation and nucleic acid amplification reaction carry out same embodiment 5.
All the other steps are with embodiment 8.
embodiment 15:the non-oil sealing nucleic acid detection apparatus of aperture is applied to loop-mediated isothermal amplification (LAMP) and detects transgenosis terminator T-Nos gene.Its proofing unit adopts the first embodiment of embodiment 1.
Nucleic acid constant-temperature amplification reaction is carried out: be placed in by said apparatus on 63 ° of C metal fever blocks and heat 1h.
All the other steps are with embodiment 12.
embodiment 16:macropore oil sealing nucleic acid detection apparatus is applied to loop-mediated isothermal amplification (LAMP) and detects transgenosis terminator T-Nos gene.Proofing unit adopts embodiment 1 the second embodiment.
Nucleic acid constant-temperature amplification reaction is carried out: be placed in by said apparatus on 63 ° of C metal fever blocks and heat 1h.
All the other steps are with embodiment 13.
embodiment 17:syringe type nucleic acid detection apparatus is applied to loop-mediated isothermal amplification (LAMP) and detects transgenosis terminator T-Nos gene.Adopt the proofing unit of embodiment 2.
Nucleic acid constant-temperature amplification reaction is carried out: be placed in by said apparatus on 63 ° of C metal fever blocks and heat 1h.
All the other steps are both embodiment 14.
embodiment 18:the non-oil sealing nucleic acid detection apparatus of aperture is applied to loop-mediated isothermal amplification (LAMP) and detects streptococcus aureus.Its proofing unit adopts the first embodiment of embodiment 1.
Before reaction, reagent prepares: 50ul reaction amplification liquid (includes primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, template, from Guangzhou enlightening Australia streptococcus aureus kit for detecting nucleic acid (constant temperature fluorescent method), article No. FA109S).
Nucleic acid constant-temperature amplification reaction is carried out: be placed in by said apparatus on 63 ° of C metal fever blocks and heat 1h.
Detection of nucleic acids step: with embodiment 3.
embodiment 19:macropore oil sealing nucleic acid detection apparatus is applied to cross primer constant-temperature amplification (CPA) for test strip colorimetry and detects streptococcus aureus.Proofing unit adopts embodiment 1 the second embodiment.
25uL reaction amplification liquid (includes primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, template, from Yousida Biological Technology Co., Ltd., Hangzhou's streptococcus aureus constant-temperature amplification nucleic acid reagent box, article No. I011-01).Paraffin oil sealing is adopted above reaction solution.
Nucleic acid constant-temperature amplification reaction is carried out: said apparatus is placed in 63 ° of C heating in water bath 1h.
Detection of nucleic acids step is both embodiment 4.
embodiment 20:syringe type nucleic acid detection apparatus is applied to loop-mediated isothermal amplification (LAMP) for test strip colorimetry and detects Salmonellas.Adopt the proofing unit of embodiment 2.
Before reaction, reagent prepares: 25 ul reaction amplification liquid (include primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, template, from Guangzhou enlightening Australia Salmonellas kit for detecting nucleic acid (constant temperature fluorescent method), article No. FA105S), adopt paraffin oil sealing above reaction solution.
Nucleic acid constant-temperature amplification reaction is carried out: be placed in by said apparatus on 63 ° of C metal fever blocks and heat 1h.
Detection of nucleic acids step is both embodiment 5.
embodiment 21:the non-oil sealing nucleic acid detection apparatus of aperture is applied to cross primer constant-temperature amplification (CPA) for test strip colorimetry and detects Salmonellas.Its proofing unit adopts the first embodiment of embodiment 1.
Before reaction, reagent prepares: 50uL reaction amplification liquid (includes primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, template, from Yousida Biological Technology Co., Ltd., Hangzhou's salmonella constant temperature amplification of nucleic acid test kit, article No. I047-01).
Nucleic acid constant-temperature amplification reaction is carried out: said apparatus is placed in 63 ° of C heating in water bath 1h.
Detection of nucleic acids step is both embodiment 3.
embodiment 22:macropore oil sealing nucleic acid detection apparatus is applied to loop-mediated isothermal amplification (LAMP) for test strip colorimetry and detects Pseudomonas aeruginosa.Proofing unit adopts embodiment 1 the second embodiment.
25uL reaction amplification liquid (includes primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, template, from Guangzhou enlightening Australia pseudomonas aeruginosa kit for detecting nucleic acid (constant temperature fluorescent method), article No. FA112S).Paraffin oil sealing is adopted above reaction solution.
Nucleic acid constant-temperature amplification reaction is carried out: be placed in by said apparatus on 63 ° of C metal fever blocks and heat 1h.
Detection of nucleic acids step is both embodiment 4.
embodiment 23:syringe type nucleic acid detection apparatus is applied to cross primer constant-temperature amplification (CPA) for test strip colorimetry and detects pseudomonas aeruginosa.Adopt the proofing unit of embodiment 2.
25uL reaction amplification liquid (includes primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, template, from Guangzhou enlightening Australia pseudomonas aeruginosa kit for detecting nucleic acid (constant temperature fluorescent method), article No. FA112S/L).Paraffin oil sealing is adopted above reaction solution.
Nucleic acid constant-temperature amplification reaction is carried out: said apparatus is placed in 63 ° of C heating in water bath 1h.
Detection of nucleic acids step is with embodiment 5.
Claims (9)
1. colorimetry nucleic acid detection method, is characterized in that, it comprises the following steps:
There is provided the detection receptacle that can be connected with reaction tubes, and the connection between them is sealing, detection receptacle can either be connected with reaction tubes and can be placed into test strip, and has the long strip type cavity holding test strip;
Configure amplification reaction solution in reaction tubes after, or the amplification reaction solution configured is added in reaction tubes, the detection receptacle that test strip is housed is connected with the reaction tubes that amplification reaction solution is housed, tight amplified reaction under reaction tubes and detection receptacle attached state, after reaction to be amplified terminates, reaction solution is entered in detection receptacle, or make test strip enter in reaction tubes, make test strip be immersed in after amplified reaction in liquid, carry out detection of nucleic acids by the colour-change in test strip.
2. a kind of stopping property as claimed in claim 1 portable test strip colorimetry nucleic acid detection apparatus, it is characterized in that, it carries out wax oil sealing to the upper strata of the amplification reaction solution in reaction tubes.
3. a stopping property portable test strip colorimetry nucleic acid detection apparatus, is characterized in that, it comprises reaction tubes, detection receptacle; Detection receptacle comprises the cavity placed for colloidal gold strip, and one end of described detection receptacle is the structure that can be connected with reaction tubes, and is sealing between this structure and reaction tubes, has the connected entrance communicated with described cavity at this end; The other end of described detection receptacle also has the mouth communicated with described cavity, and this end is connected with the lid of detection receptacle; The cavity placed for colloidal gold strip in detection receptacle communicates with reaction tubes through described connected entrance.
4. a kind of stopping property as claimed in claim 3 portable test strip colorimetry nucleic acid detection apparatus, is characterized in that, the mouth of the other end of described detection receptacle is the test strip filler straight-through with cavity.
5. a kind of stopping property as claimed in claim 3 portable test strip colorimetry nucleic acid detection apparatus, is characterized in that, the structure that can be connected with reaction tubes is the structure inserted by reaction tubes.
6. a kind of stopping property as claimed in claim 5 portable test strip colorimetry nucleic acid detection apparatus, is characterized in that, described is the lid of reaction tubes by the structure that reaction tubes inserts.
7. the detection receptacle of a test strip colorimetric determination nucleic acid amplification, it is characterized in that it comprises the cavity placed for colloidal gold strip, one end of described detection receptacle is the structure that can be connected with reaction tubes, and be sealing between this structure and reaction tubes, there is at this end the connected entrance communicated with described cavity; The other end of described detection receptacle also has the mouth communicated with described cavity, and this end is connected with the lid of detection receptacle; The cavity placed for colloidal gold strip in detection receptacle communicates with reaction tubes through described connected entrance.
8. the detection receptacle of a kind of test strip colorimetric determination nucleic acid amplification as claimed in claim 7, is characterized in that, the mouth of the other end of described detection receptacle is the test strip filler straight-through with cavity.
9. the detection receptacle of a kind of test strip colorimetric determination nucleic acid amplification as claimed in claim 7, it is characterized in that, the structure that can be connected with reaction tubes is the structure inserted by reaction tubes.
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| CN105018341A (en) * | 2015-07-06 | 2015-11-04 | 西安交通大学 | Paper-based nucleic acid amplification and detection integrated detection device and detection method |
| CN105063180A (en) * | 2015-06-16 | 2015-11-18 | 浙江大学 | Sealed portable nucleic acid detection apparatus and method thereof |
| CN105273997A (en) * | 2015-10-21 | 2016-01-27 | 西安交通大学 | Paper-based nucleic acid extraction, amplification and detection integrated detection system and paper-based nucleic acid extraction, amplification and detection integrated detection method |
| CN106148181A (en) * | 2015-05-12 | 2016-11-23 | 厦门大学 | A kind of nucleic acid amplification reaction pipe of controllable liquid closed loop flow path |
| CN106497778A (en) * | 2016-11-04 | 2017-03-15 | 中国人民解放军军事医学科学院基础医学研究所 | A kind of device for the detection of constant temperature nucleic acid amplification quick visualization |
| CN106967790A (en) * | 2017-02-20 | 2017-07-21 | 浙江大学 | A kind of detection method of nucleic acid amplification |
| CN113607724A (en) * | 2021-06-29 | 2021-11-05 | 中国人民解放军东部战区疾病预防控制中心 | Integrated detection device and detection method for LAMP or RT-LAMP |
| WO2021237396A1 (en) * | 2020-05-25 | 2021-12-02 | 杭州梓晶生物有限公司 | Integrated self-service nucleic acid detection device and use method thereor |
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| CN106148181A (en) * | 2015-05-12 | 2016-11-23 | 厦门大学 | A kind of nucleic acid amplification reaction pipe of controllable liquid closed loop flow path |
| CN105063180A (en) * | 2015-06-16 | 2015-11-18 | 浙江大学 | Sealed portable nucleic acid detection apparatus and method thereof |
| CN105018341A (en) * | 2015-07-06 | 2015-11-04 | 西安交通大学 | Paper-based nucleic acid amplification and detection integrated detection device and detection method |
| CN105273997A (en) * | 2015-10-21 | 2016-01-27 | 西安交通大学 | Paper-based nucleic acid extraction, amplification and detection integrated detection system and paper-based nucleic acid extraction, amplification and detection integrated detection method |
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| CN106497778A (en) * | 2016-11-04 | 2017-03-15 | 中国人民解放军军事医学科学院基础医学研究所 | A kind of device for the detection of constant temperature nucleic acid amplification quick visualization |
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| CN106967790A (en) * | 2017-02-20 | 2017-07-21 | 浙江大学 | A kind of detection method of nucleic acid amplification |
| CN106967790B (en) * | 2017-02-20 | 2020-08-25 | 浙江大学 | Detection method for nucleic acid amplification |
| WO2021237396A1 (en) * | 2020-05-25 | 2021-12-02 | 杭州梓晶生物有限公司 | Integrated self-service nucleic acid detection device and use method thereor |
| CN113607724A (en) * | 2021-06-29 | 2021-11-05 | 中国人民解放军东部战区疾病预防控制中心 | Integrated detection device and detection method for LAMP or RT-LAMP |
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Application publication date: 20150311 |