CN105063180A - Sealed portable nucleic acid detection apparatus and method thereof - Google Patents
Sealed portable nucleic acid detection apparatus and method thereof Download PDFInfo
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- CN105063180A CN105063180A CN201510332532.XA CN201510332532A CN105063180A CN 105063180 A CN105063180 A CN 105063180A CN 201510332532 A CN201510332532 A CN 201510332532A CN 105063180 A CN105063180 A CN 105063180A
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Abstract
The present invention provides a portable visualized nucleic acid detection method and a sealed portable nucleic acid detection apparatus. The nucleic acid detection apparatus comprises a visualized mixing container, wherein the visualized mixing container is provided with a plurality of connection ports, such that the visualized mixing container can be used as the total sealing body for connecting the nucleic acid amplification reaction tube on the visualized mixing container and the detection reaction liquid storage container on the visualized mixing container, or can be adopted as the total sealing body for connecting the nucleic acid amplification reaction tube on the visualized mixing container and the dilution storage container on the visualized mixing container. According to the present invention, the nucleic acid isothermal amplification reaction can be performed without interference in the case of achievement of no reaction tube opening, the subsequent detection can be normally performed according to the sequence, and the apparatus and the method can be suitable for chromogenic assay and test paper strip assay.
Description
Technical field
The invention belongs to field of nucleic acid detection, be specifically related to the device and method that one can be used for the detections such as Common Polymerase Chain Reaction (PCR), constant temperature nucleic acid amplification.
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 the amplification (RAA) etc. of cross primer isothermal amplification technology (CPA), loop-mediated isothermal amplification technology (LAMP), strand displacement amplification (SDA), the amplification (HDA) relying on helicase, restructuring polymeric enzymatic amplification (RPA), recombinase-mediated.Due to the highly sensitive of nucleic acid amplification technologies, the output of amplified production is very high, therefore faces Aerosol.
Although the method based on nucleic acid amplification has very high sensitivity, how a problem highly studied is judged to amplification.Method the most traditional is, after amplification terminates, product is carried out gel electrophoresis.But the Aerosol Pollution that this method needs manually to open reaction tubes and easily causes laboratory after electrophoresis terminates, and the time of gel electrophoresis at least wants 0.5-1h usually, and need the instrument of the huge costlinesses such as gel imaging system, therefore very unfavorable to field quick detection.In addition this method needs the operation of professional, is unfavorable for promoting.Method based on fluorescent quantitation can realize Real-Time Monitoring amplification procedure, but this method owing to wanting real-time collecting fluorescent signal in reaction process, therefore still needs the augmentation apparatus of specialty, is unfavorable for that the miniaturization development and application of device is in Site Detection.Non-fluorescence nucleic acid Fast Detection Technique emerging at present such as electrochemical method, development process, nephelometry and test strip method etc. greatly simplify the requirement of reaction kit, especially development process and test strip method, is easy to the advantages such as judgement owing to having easy and simple to handle, quick, result and is widely used.But these methods are equally owing to being need operation of uncapping after nucleic acid amplification, as easy as rolling off a logly cause Aerosol Pollution.For the problems referred to above, applicant also develops colorimetry nucleic acid detection apparatus, see the Chinese invention patent document of application number 201410431991.9, the feature of this patented technology is that reacting front is placed in the inner connecting leg be communicated with by nitrite ion and amplifing reagent, when reaction terminating, device is rocked can realize nitrite ion with amplification liquid mix, after positive sample is reacted, reaction solution becomes coloured from colourless, nucleic acid amplification is detected become naked eyes visible.But need to design for different reaction systems based on the device of this method and process different reaction tubess and lid, during production, the difficulty of die sinking is comparatively large, and whole device exists certain problem in stopping property simultaneously.
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.And for the amplified reaction such as amplification (RAA) of polymeric enzymatic amplification (RPA) of such as recombinating, recombinase-mediated, due to its reaction reagent comparatively thickness, be unfavorable for that amplified production is detected by syphonic effect in test strip; Or as cross primer amplification (CPA), polymerase chain reaction (PCR) although etc. its reagent be unlikely to thickness to affecting ELISA test strip result false negative, but ELISA test strip needs certain volume to ensure usually, as the liquor capacity of at least 50ul or 100ul, and larger amplification volume often means the increase of reaction cost, be therefore the means that investigator takes usually by carrying out effectively dilution to amplified production.If but amplified production is directly uncapped dilution, be easy to cause crossed contamination.
Summary of the invention
The present invention's first object is to provide a kind of visual portable nucleic acid detection method, can realize when not opening reaction tubes, nucleic acid constant-temperature amplification is reacted can carry out uninterruptedly, the detection carried out afterwards can normally be carried out according to the order of sequence, and is applicable to development process detection and the detection of test strip method.For this reason, the present invention is by the following technical solutions:
A kind of visual portable nucleic acid detection method, it is characterized in that, it comprises the following steps:
Multiple nucleic acid amplification reaction pipe is provided, and the visual mixing vessel that can be connected together with described multiple nucleic acid amplification reaction pipe is provided, and the connection between them is sealing, described visual mixing vessel, as total sealing member of these nucleic acid amplification reaction pipes, makes this visual mixing vessel and the assembling of these nucleic acid amplification reaction pipes be formed as obturator;
In multiple nucleic acid amplification reaction pipe one of them or be severally loaded into nucleic acid amplification reaction liquid before assembling, remaining part or all of nucleic acid amplification reaction pipe is loaded into detection reaction liquid before assembling, or remaining part or all of nucleic acid amplification reaction pipe is loaded into diluent before assembling;
After nucleic acid amplification reaction terminates, when not separated visual mixing vessel and nucleic acid amplification reaction pipe, the sealing member that the visual mixing vessel of direct inversion and multiple nucleic acid amplification reaction pipe are formed, carry out mixing operation, if development process detects, then visual mixing vessel carries out the reaction vessel of detection reaction as the liquid after nucleic acid reaction and detection reaction liquid, observes the colour-change after detection reaction to carry out the detection of nucleic acid reaction result; Or, if test strip method detects, then visual mixing vessel is as the mixing vessel of the liquid after nucleic acid reaction and diluent, observe test strip and the detection of nucleic acid reaction result is carried out to the colour-change of liquid after the nucleic acid reaction after diluted, further, test strip is placed on to be connected with described visual mixing vessel and in the ELISA test strip container communicated.
Described multiple nucleic acid amplification reaction pipe adopts the form of orifice plate, and described visual mixing vessel is as total sealing member of each hole pipe in orifice plate; One of them hole pipe in orifice plate or several holes pipe load nucleic acid amplification reaction liquid, remaining hole pipe dress detection reaction liquid or diluent.
Another technical problem to be solved of the present invention is to provide the portable detection of nucleic acids device of a kind of stopping property, can realize when not opening reaction tubes, nucleic acid constant-temperature amplification is reacted can carry out uninterruptedly, the detection carried out afterwards can normally be carried out according to the order of sequence, and is applicable to development process detection and the detection of test strip method.For this reason, the present invention is by the following technical solutions:
The portable detection of nucleic acids device of a kind of stopping property, it is characterized in that, comprise visual mixing vessel, described visual mixing vessel has multiple Link Port, wherein one or more Link Ports have the structure that can be connected with nucleic acid amplification reaction pipe, and this Link Port is used for being connected with nucleic acid amplification reaction pipe, remaining Link Port also all has syndeton, these Link Ports can be connected with diluent storage container or detection reaction liquid storage container, make described visual mixing vessel can as total sealing member of the nucleic acid amplification reaction pipe of connection on it and detection reaction liquid storage container, and make nucleic acid amplification reaction pipe and detection reaction liquid storage container all be in the same side of visual mixing vessel, the encloses container can putting upside down use is formed together with detection reaction liquid storage container with nucleic acid amplification reaction pipe, or, as total sealing member of the nucleic acid amplification reaction pipe connected on it and diluent storage container, and make nucleic acid amplification reaction pipe and diluent storage container all be in the same side of visual mixing vessel, the encloses container can putting upside down use is formed together with diluent storage container with nucleic acid amplification reaction pipe.
Described detection reaction liquid storage container and diluent storage container adopt the container identical with nucleic acid amplification reaction interface tube or adopt the container identical with nucleic acid amplification reaction pipe.
Described detection reaction liquid storage container adopts the pipe identical with nucleic acid amplification reaction interface tube with diluent storage container.
When the sealing member of total of described visual mixing vessel as the nucleic acid amplification reaction pipe connected on it and diluent storage container, its be connected with communicate with it for the ELISA test strip container placing test strip.
Described visual mixing vessel self has oral area and has the stopper be tightly connected with oral area.
Described visual mixing vessel adopts elongated vessel, and described multiple Link Port is on the sidewall of visual mixing vessel.
The present invention can directly utilize existing PCR pipe or eight connecting legs or 96 hole PCR plate etc. as reaction tubes, in reaction process the present invention can directly as pipe lid by PCR pipe or the sealing such as eight connecting legs or 96 hole PCR plate, do not need the special reaction tubes of process mating in addition again, the layout of visual mixing vessel Link Port, size and commercially available PCR pipe or eight connecting legs or 96 hole PCR plate consistent, whole device versatility is good, and testing cost is low.
Proofing unit provided by the present invention forms the space that airtight inside is connected in visual mixing vessel; Only need proofing unit of the present invention be connected with multiple existing PCR reaction tubes or eight connecting legs or 96 orifice plates etc. and seal when nucleic acid amplification reaction, nucleic acid amplification reaction liquid and Visual retrieval reagent, diluent are directly positioned in the reaction tubess such as existing PCR pipe, one of them pipe is used for carrying out nucleic acid constant-temperature amplification reaction, and other reaction tubes all or part of holds reagent for nucleic acid constant-temperature amplification reaction product Visual retrieval or dilution reagent.After amplified reaction terminates, be inverted, by the mixing vessel in apparatus of the present invention, dress nucleic acid amplification reaction liquid be communicated with the pipe of Visual retrieval reagent, just can realize the detection of visual nucleic acid amplification without the need to opening reaction tubes, greatly reducing aerocolloidal pollution.
Proofing unit provided by the present invention is a kind of miniaturization, portable test set, can low cost, easy to operate the nucleic acid amplification that realizes detect.
Adopt color developing detection time, proofing unit provided by the present invention both ensure that differential responses pipe independent, separate store liquid, before the reaction separate, be contained in pipe without interfering with each other, ensure reaction normally carry out; Again can when covered, by operations such as acutely rocking or shake, put upside down, the detection reaction liquid of differential responses pipe pre-stored is mixed for Visual retrieval by mixing space device is communicated with between differential responses pipe.
When adopting test strip method to detect, realize when not opening lid too, nucleic acid amplification reaction can carry out uninterruptedly, afterwards by the dilution reagent of different ratios and nucleic acid reaction Reagent evaluation are mixed, can be according to actual needs, nucleic acid amplification product is diluted, suitable nucleic acid concentration and viscosity can be reached, carry out ELISA test strip.
When adopting color developing detection, the quantity of described Link Port, the quantity of also namely supporting with the present invention reaction tubes, is preferably 2-5, is more preferably 2-4, but is not limited only to this.The pipe that described device connects can be the multiple reaction tubess such as PCR pipe, eight connecting legs, 96 hole PCR plate, small-sized centrifuge tube, is not limited in a certain or two kinds.The nucleic acid amplification reaction pipe that proofing unit of the present invention connects both can arrangement on laterally equidirectional, also can longitudinal, oblique etc. multi-direction on cross arrangement, be even linked to be cellular or reticulated structure etc.The position that proofing unit of the present invention and PCR pipe or eight connecting legs or 96 orifice plates are connected both can seal from the inwall of pipe and pipe lid, can be entangled firm by pipe lid again from the outer wall of pipe.
When adopting test strip method to detect, the quantity of described Link Port, also namely supporting with the present invention reaction tubes quantity, is preferably 2-3, is more preferably 2, but is not limited only to this.The pipe that described device connects can be the multiple reaction tubess such as PCR pipe, eight connecting legs, 96 orifice plates, small-sized centrifuge tube, is not limited in a certain or two kinds.The pipe that described device connects both can in laterally equidirectional arrangement, also can be longitudinal, oblique etc. multi-direction upper cross arrangement, even be linked to be cellular or reticulated structure etc., as long as in the same side of visual mixing vessel, container and other liquid of making to hold nucleic acid amplification reaction liquid can not at amplified reaction forward slip value.
ELISA test strip container can arrange along the parallel arrangement of reaction tubes, also can arrange at any directions such as horizontal vertical direction or vertical vertical direction with reaction tubes.
Accompanying drawing explanation
Fig. 1 is the detection of nucleic acids device schematic diagram in the embodiment of the present invention 1, and wherein 1 is the tube wall cavity be connected with PCR pipe, and 2 is mixing vessel, and 3 is the stopper of mixing vessel.
Schematic diagram when Fig. 2 is dilution type reagent paper detector (the small-sized mixing vessel) of detection device of the present invention employing embodiment 2.1, wherein 1 is the tube wall cavity be connected with PCR pipe, 2 is mixing vessel, 3 is rectangular parallelepiped visual stick form test paper bar detection receptacle, and 4 is the lid of ELISA test strip container.
Fig. 3 is the schematic diagram of detection device of the present invention when adopting dilution type reagent paper detector (the large-scale mixing vessel, have uptake zone) of embodiment 2.2, wherein 1 is the tube wall cavity be connected with PCR pipe, 2 is mixing vessel, 3 is rectangular parallelepiped visual stick form test paper bar detection receptacle, 4 is uptake zone, 5 is the oral area of mixing vessel, and 6 is stopper.
Fig. 4 is the schematic diagram of detection device of the present invention when adopting dilution type reagent paper detector (the large-scale mixing vessel, without uptake zone) of embodiment 2.3, wherein 1 is the tube wall cavity be connected with PCR pipe, 2 is mixing vessel, 3 is rectangular parallelepiped visual stick form test paper bar detection receptacle, 4 is the oral area of mixing vessel, and 5 is stopper.
The different embodiment schematic diagram that Fig. 5, Fig. 6, Fig. 7 are respectively detection device of the present invention when being dilution type reagent paper detector.
Embodiment
embodiment 1, with reference to Fig. 1.
A kind of can directly as the PCR pipe of nucleic acid amplification or the lid of eight connecting leg lids or 96 hole PCR plate and the Portable visual proofing unit detected for visual nucleic acid amplification, this device comprises visual mixing vessel 2, mixing vessel 2 side have multiple with PCR pipe or the tube wall cavity 1 that is connected of eight connecting legs or 96 orifice plates as Link Port, described visual mixing vessel self has oral area and has the stopper 3 be tightly connected with oral area.
Visual detection device of the present invention can connect two simultaneously, three, four ... even more manage, adjust as required; Visual detection device of the present invention also simultaneously from the multi-direction extension such as horizontal and vertical, even can cover monoblock 96 hole PCR plate or 384 hole PCR plate etc., adjusts as required.The position be connected with PCR pipe or eight connecting legs or 96 hole PCR plate both can seal from the inwall of pipe and pipe lid, can be entangled firm by pipe lid again from the outer wall of pipe.The capacity of visual mixing vessel and the summation adaptation of nucleic acid amplification reaction liquid and detection reaction liquid.
The using method of above-mentioned visual detection device, specifically comprises the steps:
(1) get PCR pipe or eight connecting legs or 96 orifice plates etc., add pcr amplification reaction liquid (or the isothermal amplification reactions liquid such as CPA, LAMP, RPA, RAA), Visual retrieval reagent etc. respectively at different reaction tubess.According to the demand of temperature of reaction, on nucleic acid amplification reaction liquid, layer-selective takes oil sealing.
(2) seal with the reaction tubes of above-mentioned visual detection device to liquid feeding, then carried out the nucleic acid amplification procedures under relevant temperature.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, it can be cross primer constant-temperature amplification (CPA), also can be loop-mediated isothermal amplification (LAMP), or in pocket, carry out the amplification (RAA) of restructuring polymeric enzymatic amplification (RPA) or recombinase-mediated.
It should be noted that and carry out amplified reaction, when especially carrying out water-bath amplified reaction, upward, to ensure that reaction can be carried out smoothly, in amplification process, reaction solution can not mix the mouth of pipe that make reaction tubes in any form.
(3) after nucleic acid amplification reaction terminates, by nucleic acid amplification reaction pipe mouth of pipe whipping twice down, amplification reaction solution and visual colouring reagents are flowed out from reaction tubes and enters mixing vessel, room temperature is placed, and observes reagent colour-change.
embodiment 2.1, with reference to Fig. 2
A kind of can directly as the portable dilution type reagent paper detector for test strip color developing detection nucleic acid amplification of PCR pipe lid or eight connecting leg lids or 96 orifice plate lids, this device can connect simultaneously and seal multiple PCR pipe or eight connecting legs or 96 hole PCR plate etc., and to be communicated with by reaction tubes by visual mixing vessel 2 and to place test strip.
Described visual mixing vessel 2 side have multiple with PCR pipe or the tube wall cavity 1 that is connected of eight connecting legs or 96 orifice plates as Link Port, test strip is placed on and is connected with visual mixing vessel 2 and in the ELISA test strip container 3 communicated.
The end of described ELISA test strip container 3 has entrance and with lid 4.Test strip can be put into from this inlet portion, also can put into from Link Port (tube wall cavity 1).
For preventing test strip to be shifted in device whipping process after amplification and premature contact reaction reagent or dilution reagent, the height trying visual mixing vessel 2 is preferably less than or equal to 2mm, is more than or equal to 1mm.
embodiment 2.2, with reference to Fig. 3
In embodiment 2.1, the cross section of ELISA test strip container 3 is substantially identical with visual mixing vessel 2.And in the present embodiment, the higher primary school of the ELISA test strip container 3 in strip is in the height of mixing vessel.For the convenience of die sinking, now test strip can from just filling in the oral area 5 of the mixing vessel 2 self of the ELISA test strip container 3 in strip, and oral area 5 is by stopper 6 shutoff.For ensureing that test strip fully contacts with reaction reagent, the absorption pad of test strip preferably suitably can enter into mixing vessel, in the setting area, position of ELISA test strip container 3, the uptake zone of described Link Port 1 is not had at mixing vessel, the cross section size of uptake zone, between mixing vessel 2 and ELISA test strip container 3, makes test strip not block the reaction tubes mouth of pipe and allows latex not enter mixing vessel.The height of above-mentioned mixing vessel is preferably greater than or equal to 2.5mm, is less than or equal to 8mm.The height of ELISA test strip container is preferably less than or equal to 2mm, is more than or equal to 1mm.
embodiment 2.3, with reference to Fig. 4
In the present embodiment, for put test strip in strip ELISA test strip container 3 higher primary school in mixing vessel height and be directly communicated with mixing vessel.For the convenience of die sinking, now test strip can from just filling in the oral area 4 of the mixing vessel 2 self of the ELISA test strip container 3 in strip, and oral area 4 is by stopper 5 shutoff.For ensureing that test strip fully contacts with reaction reagent, the absorption pad of test strip preferably suitably can enter into mixing vessel, and does not more preferably block the reaction tubes mouth of pipe for test strip and allow latex not enter mixing vessel.The height of above-mentioned mixing vessel is preferably greater than or equal to 2.5mm, is less than or equal to 8mm.The height of ELISA test strip container is preferably less than or equal to 2mm, is more than or equal to 1mm.
In above-mentioned 2.12.22.3 embodiment, mixing vessel also can adopt rectangular parallelepiped or bar shaped, and the volume of diluent is preferably less than or equal to 250 μ L, is more preferably less than or equal to 200 μ L.The ELISA test strip container 2 in strip for placing test strip can in the parallel direction of mixing vessel, also can be crisscross arbitrarily in the vertical direction of mixing vessel (as Fig. 5, Fig. 6, Fig. 7) etc., only otherwise affecting it is connected with reaction tubes.Mixing vessel also can simultaneously in the multi-direction connection two such as horizontal and vertical, three, four ... even more manage, adjust as required.The tube wall be connected with PCR pipe or eight connecting legs or 96 orifice plates both can seal from the inwall of pipe and pipe lid, can be entangled firm by pipe lid again from the outer wall of pipe.
The preparation method of above-mentioned airtight nucleic acid detection apparatus, comprises the steps:
(1) test section of the dilution type reagent paper detector of machine-shaping is got, this test section is by being communicated with the mixing vessel of reaction tubes and forming for the rectangular parallelepiped ELISA test strip container two portions placing test strip of being communicated with mixing vessel, and two portions are formed in one; Or have uptake zone between them.
(2) in rectangular parallelepiped ELISA test strip container for placing the test strip marked for material to be detected, the mark end of test strip near mixing vessel, unmarked end at rectangular parallelepiped ELISA test strip container away from mixing vessel one end.
(3) test strip of this ELISA test strip container fills in mouth supporting lid or plug seal.
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, to in the rectangular parallelepiped ELISA test strip container be connected with mixing vessel, add the colloidal gold strip of the specific marker for different testing sample, when use, only need reaction solution and diluent etc. be added in reaction tubes, carry out reacting and detecting.
Apparatus of the present invention can, directly as amplified reaction pipe pipe lid, not need to process reaction tubes in addition again.Only need the nucleic acid amplification reaction reagent prepared be joined in PCR pipe or eight connecting legs before reaction, diluent (water or phosphate buffered saline buffer etc.) is joined in other pipe, with this device reaction solution and diluent be built and seal.For amplifications such as PCR or LAMP needing high temperature incubation, when Wu Regai, oil sealing can be taked to prevent reaction solution from evaporating; The lower amplification of temperature of reaction, as RPA and RAA etc., can not need oil sealing process.When covered, by operations such as rocking or shake, put upside down, be divided in reaction solution in differential responses pipe and diluent can be mixed by mixing vessel, then by the ELISA test strip in rectangular parallelepiped ELISA test strip container.
The using method of above-mentioned dilution type reagent paper detector, specifically comprises the steps:
(1) get PCR pipe or eight connecting legs or 96 orifice plates etc., add pcr amplification reaction liquid (or the isothermal amplification reactions liquid such as CPA, LAMP, RPA, RAA), Visual retrieval reagent etc. respectively at different reaction tubess.According to the demand of temperature of reaction, on nucleic acid amplification reaction liquid, layer-selective takes oil sealing.
(2) seal with the above-mentioned dilution type reagent paper detector of the machine-shaping reaction tubes to liquid feeding, then carried out the nucleic acid amplification procedures under relevant temperature.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, it can be cross primer constant-temperature amplification (CPA), also can be loop-mediated isothermal amplification (LAMP), or in pocket, carry out the amplification (RAA) of restructuring polymeric enzymatic amplification (RPA) or recombinase-mediated.
It should be noted that and carry out amplified reaction, when especially carrying out water-bath amplified reaction, upward, to ensure that reaction can be carried out smoothly, in amplification process, reaction solution can not mix the mouth of pipe that make reaction tubes in any form.
(3) after nucleic acid amplification reaction terminates, by nucleic acid amplification reaction pipe mouth of pipe whipping twice down, amplification reaction solution and diluent are flowed out from reaction tubes and enters mixing vessel, room temperature is placed, and observes reagent colour-change.
embodiment 3.1:the detection application of installation of embodiment 1 is in detecting transgenosis terminator T-Nos gene based on molybdenum blue method-ring mediated isothermal amplification (LAMP).
Before reaction, reagent prepares: in PCR eight connecting leg pipe 1, be added with 19ul reaction amplification liquid (include primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, Bst polysaccharase, template, from Guangzhou enlightening Australia NOS gene nucleic acid detection kit (constant temperature fluorescent method), article No. PA101S), and the heat resistant inorganic Pyrophosphate phosphohydrolase of 1ul0.1U/ul (from NewEnglandBiolabs, article No. M0296s), oil sealing; Ascorbic acid solution and the 68ul water of 2ul10% is added in PCR eight connecting leg pipe 2; 4ul ammonium molybdate-soluble tartrate antimony solution (comprising 21mM ammonium molybdate, 2mM soluble tartrate antimony, 5M sulfuric acid) and 106ul water is added in PCR eight connecting leg pipe 3.And get a Visual retrieval evenly mixing device by three seals of tube.
Nucleic acid constant-temperature amplification reaction is carried out: said apparatus is placed in 63 ° of C heating in water bath 1h.
Visual retrieval constant-temperature amplification: after having reacted, inverted device, makes the liquid in three reaction tubess fully mix, judged result after 10min: the positive is blue, and feminine gender is colourless.
embodiment 3.2: the detection application of installation of embodiment 2.1 is in detecting transgenosis terminator T-Nos gene based on ring mediated isothermal amplification (LAMP).
Before reaction, reagent prepares: in PCR eight connecting leg pipe 1, be added with 20ul reaction amplification liquid (include labeled primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, Bst polysaccharase template), oil sealing; 200ul water is added in PCR eight connecting leg pipe 2; And the detection device of Example 2.1 is by two seals of tube.
Nucleic acid constant-temperature amplification reaction is carried out: said apparatus is placed in 63 ° of C heating in water bath 1h.
Test strip color developing detection constant-temperature amplification: after having reacted, inverted device, makes the reaction solution in the first reaction tubes mix with the diluent in the second reaction tubes, and penetrates in test strip, and room temperature places 1 ~ 2min, observes the result of test strip colour developing.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 3.3: the detection application of installation of embodiment 2.3 is in detecting transgenosis terminator T-Nos gene based on ring mediated isothermal amplification (LAMP).
Adopt the sealing of the detection device of embodiment 2.3 that the PCR pipe of amplification reaction solution and diluent is housed.Other are with embodiment 3.2.
embodiment 3.4: the detection application of installation of embodiment 2.2 is in detecting transgenosis terminator T-Nos gene based on ring mediated isothermal amplification (LAMP).
The PCR pipe of amplification reaction solution and diluent is equipped with in the detection device device sealing of embodiment 2.2.Other are with embodiment 3.2.
embodiment 3.5:the detection application of installation of embodiment 1 is in detecting transgenosis terminator T-Nos gene based on molybdenum blue method-cross primer amplification (CPA).
Before reaction, reagent prepares: the a-quadrant of reactor in PCR eight connecting leg pipe 1, adds 19ul reaction amplification liquid and (includes primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, Gspf polysaccharase, template, from Hangzhou You Sida biotech company transgenosis NOS sequence constant-temperature amplification kit, article No. I016-01), and the heat resistant inorganic Pyrophosphate phosphohydrolase of 1ul0.1U/ul is (from NewEnglandBiolabs, article No. M0296s), oil sealing; Ascorbic acid solution and the 68ul water of 2ul10% is added in PCR eight connecting leg pipe 2; 4ul ammonium molybdate-soluble tartrate antimony solution (comprising 21mM ammonium molybdate, 2mM soluble tartrate antimony, 5M sulfuric acid) and 106ul water is added in PCR eight connecting leg pipe 3.
Nucleic acid constant-temperature amplification reaction is carried out: said apparatus is placed in 63 ° of C heating in water bath 1h.
Visual retrieval constant-temperature amplification: after having reacted, inverted device, makes the liquid in three reaction tubess fully mix, judged result after 10min.The positive is blue, and feminine gender is colourless.
embodiment 3.6:the detection application of installation of embodiment 2.1 detects transgenosis terminator T-Nos gene in cross primer amplification (CPA).
Before reaction, reagent prepares: in PCR eight connecting leg pipe 1, be added with 20ul reaction amplification liquid (include labeled primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, Gspf polysaccharase, template, from Hangzhou You Sida biotech company transgenosis NOS sequence constant-temperature amplification kit, article No. I016-01), oil sealing; 200ul water is added in PCR eight connecting leg pipe 2; And the detection device of Example 2.1 is by two seals of tube.
Other are with embodiment 3.2.
embodiment 7:the detection application of installation of embodiment 2.3 detects transgenosis terminator T-Nos gene in cross primer amplification (CPA).
Adopt the sealing of the detection device of embodiment 2.3 that the PCR pipe of amplification reaction solution and diluent is housed.Other are with embodiment 3.2.
embodiment 8:the detection application of installation of embodiment 2.2 detects transgenosis terminator T-Nos gene in cross primer amplification (CPA).
Adopt the sealing of the detection device of embodiment 2.2 that the PCR pipe of amplification reaction solution and diluent is housed.Other are with embodiment 3.2.
embodiment 9:the detection application of installation of embodiment 2.1 detects transgenosis terminator T-Nos gene in restructuring polymeric enzymatic amplification (RPA).
Before reaction, reagent prepares: in PCR eight connecting leg pipe 1, be added with 20ul reaction amplification liquid (include labeled primer, dNTP, Tris-HCl damping fluid, Mg
2+, SSA associated proteins, Bsu polysaccharase, RecA albumen, template etc., from Zhejiang Taijing Biotechnology Co., Ltd.); 200ul water is added in PCR eight connecting leg pipe 2; And the detection device of Example 2.1 is by two seals of tube.
Other are with embodiment 3.2.
embodiment 10:the detection application of installation of embodiment 2.3 detects transgenosis terminator T-Nos gene in restructuring polymeric enzymatic amplification (RPA).
Adopt the sealing of the detection device of embodiment 2.1 that the PCR pipe of amplification reaction solution and diluent is housed.Other are with embodiment 3.2.
embodiment 11:the detection application of installation of embodiment 2.2 detects transgenosis terminator T-Nos gene in restructuring polymeric enzymatic amplification (RPA).
Adopt the sealing of the detection device of embodiment 2.2 that the PCR pipe of amplification reaction solution and diluent is housed.Other are with embodiment 3.2.
embodiment 12:the detection application of installation of embodiment 2.1 detects transgenosis terminator T-Nos gene in polymerase chain reaction (PCR).
Before reaction, reagent prepares: in PCR eight connecting leg pipe 1, add 20 μ L amplification reaction solutions (include labeled primer, dNTP, Tris-HCl damping fluid, Mg
2+, TaqDNA nucleic acid polymerase template, from Biorad, article No. 172-5120, template), oil sealing; 200ul water is added in PCR eight connecting leg pipe 2; And the detection device of Example 2.1 is by two seals of tube.
Other are with embodiment 3.2.
embodiment 13:the detection application of installation of embodiment 2.3 detects transgenosis terminator T-Nos gene in polymerase chain reaction (PCR).
Adopt the sealing of the detection device of embodiment 2.3 that the PCR pipe of amplification reaction solution and diluent is housed.Other are with embodiment 3.2.
embodiment 14:the detection application of installation of embodiment 2.2 detects transgenosis terminator T-Nos gene in polymerase chain reaction (PCR).
Adopt the sealing of the detection device of embodiment 2.2 that the PCR pipe of amplification reaction solution and diluent is housed.Other are with embodiment 3.2.
embodiment 15:the detection application of installation of embodiment 1 is in detecting transgene promoter CaMV35S gene based on molybdenum blue method-ring mediated isothermal amplification (LAMP).
Before reaction, reagent prepares: in PCR eight connecting leg pipe 1, be added with 19ul reaction amplification liquid (include primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, Bst polysaccharase, template, from Guangzhou enlightening Australia CaMV35S gene nucleic acid detection kit (constant temperature fluorescent method), article No. PA102S/L), and the heat resistant inorganic Pyrophosphate phosphohydrolase of 1ul0.1U/ul (from NewEnglandBiolabs, article No. M0296s), oil sealing.Other are with embodiment 3.1.
embodiment 16:the detection application of installation of embodiment 1 is in detecting transgene promoter CaMV35S gene based on molybdenum blue method-ring mediated isothermal amplification (LAMP).
Before reaction, reagent prepares: in PCR eight connecting leg pipe 1, be added with 19ul reaction amplification liquid (include primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, Bst polysaccharase, template, from Guangzhou enlightening Australia CaMV35S gene nucleic acid detection kit (constant temperature fluorescent method), article No. PA102S/L), and the heat resistant inorganic Pyrophosphate phosphohydrolase of 1ul0.1U/ul (from NewEnglandBiolabs, article No. M0296s), oil sealing.Other are with embodiment 3.1.
embodiment 17: the detection application of installation of embodiment 2.1 is in the native gene detecting ox based on ring mediated isothermal amplification (LAMP).
Before reaction, reagent prepares: in PCR eight connecting leg pipe 1, be added with 20ul reaction amplification liquid (include labeled primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, Bst polysaccharase template), oil sealing; 200ul water is added in PCR eight connecting leg pipe 2; And the detection device of Example 2.1 is by two seals of tube.
Before reaction, reagent prepares: in PCR eight connecting leg pipe 1, be added with 20ul reaction amplification liquid (include primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, Bst polysaccharase, template, from Guangzhou enlightening Australia ox native gene kit for detecting nucleic acid (constant temperature fluorescent method), article No. M103S), oil sealing.Other are with embodiment 3.2.
embodiment 18: the detection application of installation of embodiment 2.3 detects golden yellow grape bacillus in cross primer amplification (CPA).
Before reaction, reagent prepares: in PCR eight connecting leg pipe 1, add 25uL reaction amplification liquid (include 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), oil sealing.Other are with embodiment 3.2.
embodiment 19: the detection application of installation of embodiment 2.2 detects transgene promoter CaMV35S gene in restructuring polymeric enzymatic amplification (RPA).
Before reaction, reagent prepares: in PCR eight connecting leg pipe 1, be added with 20ul reaction amplification liquid (include labeled primer, dNTP, Tris-HCl damping fluid, Mg
2+, SSA associated proteins, Bsu polysaccharase, RecA albumen, template etc., from Zhejiang Taijing Biotechnology Co., Ltd.); 200ul water is added in PCR eight connecting leg pipe 2; And the detection device of Example 2.2 is by two seals of tube.
Claims (9)
1. a visual portable nucleic acid detection method, it is characterized in that, it comprises the following steps:
Multiple nucleic acid amplification reaction pipe is provided, and the visual mixing vessel that can be connected together with described multiple nucleic acid amplification reaction pipe is provided, and the connection between them is sealing, described visual mixing vessel, as total sealing member of these nucleic acid amplification reaction pipes, makes this visual mixing vessel and the assembling of these nucleic acid amplification reaction pipes be formed as obturator;
In multiple nucleic acid amplification reaction pipe one of them or be severally loaded into nucleic acid amplification reaction liquid before assembling, remaining part or all of nucleic acid amplification reaction pipe is loaded into detection reaction liquid before assembling, or remaining part or all of nucleic acid amplification reaction pipe is loaded into diluent before assembling;
After nucleic acid amplification reaction terminates, when not separated visual mixing vessel and nucleic acid amplification reaction pipe, the sealing member that the visual mixing vessel of direct inversion and multiple nucleic acid amplification reaction pipe are formed, carry out mixing operation, if development process detects, then visual mixing vessel carries out the reaction vessel of detection reaction as the liquid after nucleic acid reaction and detection reaction liquid, observes the colour-change after detection reaction to carry out the detection of nucleic acid reaction result; Or, if test strip method detects, then visual mixing vessel is as the mixing vessel of the liquid after nucleic acid reaction and diluent, observe test strip and the detection of nucleic acid reaction result is carried out to the colour-change of liquid after the nucleic acid reaction after diluted, further, test strip is placed on to be connected with described visual mixing vessel and in the ELISA test strip container communicated.
2. a kind of visual portable nucleic acid detection method as claimed in claim 1, it is characterized in that, described multiple nucleic acid amplification reaction pipe adopts the form of orifice plate, and described visual mixing vessel is as total sealing member of each hole pipe in orifice plate; One of them hole pipe in orifice plate or several holes pipe load nucleic acid amplification reaction liquid, remaining hole pipe dress detection reaction liquid or diluent.
3. the portable detection of nucleic acids device of stopping property, it is characterized in that, comprise visual mixing vessel, described visual mixing vessel has multiple Link Port, wherein one or more Link Ports have the structure that can be connected with nucleic acid amplification reaction pipe, and this Link Port is used for being connected with nucleic acid amplification reaction pipe, remaining Link Port also all has syndeton, these Link Ports can be connected with diluent storage container or detection reaction liquid storage container, make described visual mixing vessel can as total sealing member of the nucleic acid amplification reaction pipe of connection on it and detection reaction liquid storage container, and make nucleic acid amplification reaction pipe and detection reaction liquid storage container all be in the same side of visual mixing vessel, the encloses container can putting upside down use is formed together with detection reaction liquid storage container with nucleic acid amplification reaction pipe, or, as total sealing member of the nucleic acid amplification reaction pipe connected on it and diluent storage container, and make nucleic acid amplification reaction pipe and diluent storage container all be in the same side of visual mixing vessel, the encloses container can putting upside down use is formed together with diluent storage container with nucleic acid amplification reaction pipe.
4. the portable detection of nucleic acids device of a kind of stopping property as claimed in claim 3, it is characterized in that, described detection reaction liquid storage container and diluent storage container adopt the container identical with nucleic acid amplification reaction interface tube or adopt the container identical with nucleic acid amplification reaction pipe.
5. the portable detection of nucleic acids device of a kind of stopping property as claimed in claim 3, is characterized in that, described detection reaction liquid storage container adopts the pipe identical with nucleic acid amplification reaction interface tube with diluent storage container.
6. the portable detection of nucleic acids device of a kind of stopping property as claimed in claim 3, it is characterized in that, when the sealing member of total of described visual mixing vessel as the nucleic acid amplification reaction pipe connected on it and diluent storage container, its be connected with communicate with it for the ELISA test strip container placing test strip.
7. the portable detection of nucleic acids device of a kind of stopping property as claimed in claim 3, is characterized in that, described visual mixing vessel self has oral area and has the stopper be tightly connected with oral area.
8. the portable detection of nucleic acids device of a kind of stopping property as claimed in claim 3, is characterized in that, described visual mixing vessel adopts elongated vessel, and described multiple Link Port is on the sidewall of visual mixing vessel.
9. the portable detection of nucleic acids device of a kind of stopping property as described in claim 3,4,5,6,7 or 8, is characterized in that, the layout of described Link Port and size consistent with eight connecting legs or 96 hole PCR plate.
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Application publication date: 20151118 |