CN109593827A - A kind of molecule chromatography PCR detection method - Google Patents
A kind of molecule chromatography PCR detection method Download PDFInfo
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- CN109593827A CN109593827A CN201910113649.7A CN201910113649A CN109593827A CN 109593827 A CN109593827 A CN 109593827A CN 201910113649 A CN201910113649 A CN 201910113649A CN 109593827 A CN109593827 A CN 109593827A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6804—Nucleic acid analysis using immunogens
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses a kind of molecules to chromatograph PCR detection method, chromatographs detection cartridge using a kind of multilayer down flow type and is detected, comprising: sample to be tested is carried out PCR reaction, obtains PCR product by the first step；The PCR product that sample to be tested marks is placed on the conjugate pad of the detection cartridge and reacts by second step, and purple red precipitate band occur in detection line or point；Excessive sample colloidal gold composite will continue to penetrate into the nature controlling line being coated on conjugate pad or point, in conjunction with the Biotin- bovine serum albumin(BSA) on nature controlling line or point, also there is purple red precipitate band, if purple red precipitate band do not occur in nature controlling line or point, indicate that the detection is invalid；Third step, activation detection cartridge switch, checks result.The present invention can eliminate sample liquid reflux to preventing false positive test results, influence detection by sample property, complex matrices and external aerosol, easy to operate, testing result is accurate.
Description
Technical field
The present invention relates to molecular Biological Detection technology, especially microorganism, virus and animal derived and kinds to examine skill
Art is specifically related to a kind of molecule chromatography PCR detection method.
Background technique
Colloidal gold immunity chromatography (Colloidal gold immunochromato graphy) is that the nineties are risen
A kind of quick diagnosis technology, principle is that special antibody is first fixed on to a zone of nitrocellulose membrane, when dry
After nitrocellulose membrane end thereof contacts sample liquid, due to capillarity, sample liquid will be along film PARALLEL FLOW forward, when flowing to
When being fixed with the region of antibody, corresponding antigen is specifically bound with antibody in sample liquid, and the region can be made to show centainly
Color, to realize the immunodiagnosis of specificity.On the basis of colloidal gold immunity chromatography, colloidal gold nucleic acid is chromatographed also gradually
Grow up, passes through biotin (Biotin)-biotin antibody, fluorescein (FITC)-anti-fluorescein antibody, digoxin (DIG)-ground
The systems such as digoxin antibody combine PCR and colloidal gold immunochromatographimethod.
Currently, there is no mature to grow up for colloidal gold nucleic acid chromatography, existing nucleic acid chromatographic test paper has following lack
Point:
1. test paper appearance is still traditional elongated paper slip, the direct engaged test paper slip of operator, there are sample pollutions
The case where；
2. sample application zone and detection zone are in two regions of same level, examined by way of through capillary lateral flow
The case where survey, there are sample liquid reflux；
3. lateral flow causes detection time too long, detection sensitivity is reduced.Sample liquid reflux leads to false positive results.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of facilitate to operate and significantly improve accuracy in detection and sensitivity
Molecule chromatograph PCR detection method, solve the above problem and defect possessed by existing colloidal gold nucleic acid chromatographic test paper.
In order to solve the above technical problem, the present invention provides a kind of molecules to chromatograph PCR detection method, is hung down using a kind of multilayer
Direct current ejector half chromatography detection cartridge, the detection cartridge have integrated housing member, the first shell including being located at cartridge top
Body component and second shell component positioned at cartridge lower part, the first shell component are equipped with sample-adding window, and sample-adding window is divided into
Set Test paper, the Test paper from top to bottom include: by filtration liner and the first filtration module at conjugate pad, by reinforcing
The conjugate pad of pad and the second filtering film layer composition, one to several layers water absorption pad；The Test paper is by sample application zone and detection zone knot
It is combined, forms a kind of down flow type chromatography structure；In the second shell component of the cartridge there is a stress to support structure
Part and the contraction members to link together with conjugate pad, stress supporting member squeeze conjugate pad, conjugate pad and suction
Water cushion is allowed to fit closely together with sample-adding window；Contraction members are touched, the conjugate to link together with contraction members is removed
Pad, is separated from each other conjugate pad with conjugate pad, so that the detection zone being located on conjugate pad is exposed to sample-adding window, to make to tie
Fruit visualization；
The detection method includes the following steps:
Sample to be tested is carried out PCR reaction, obtains PCR product by the first step；
The PCR product that sample to be tested marks is placed on the conjugate pad of the detection cartridge, sample to be tested by second step
The PCR product of label can dissolve the crosslinked on conjugate pad, acted on by the permeation filtration of the first filter membrane, liquid is seeped
Thoroughly to the segment to be checked on the conjugate pad for being coated with antibody, being coated on conjugate pad in the antibody and sample of detection line or point
And colloidal gold forms the immune complex of a double-antibody sandwich, purple red precipitate band occurs；Excessive sample colloidal gold is compound
Object will continue to penetrate into the nature controlling line being coated on conjugate pad or point, with the Biotin- bovine serum albumin on nature controlling line or point
Also there is purple red precipitate band in white combination, if purple red precipitate band do not occur in nature controlling line or point, indicates that the detection is invalid
's；
Third step, activation detection cartridge switch, checks result.
Preferably, in the first step, the sample to be tested increases following steps before carrying out PCR reaction:
(1) sample to be tested 36 DEG C Zengjing Granule 18-24 hours in enriched medium；
(2) liquid after above-mentioned increasing bacterium is taken, is added in the EP pipe containing buffer, 100 DEG C are boiled 10min；
(3) EP pipe is cooled to room temperature, and takes part liquid in pipe, is added in the PCR pipe containing reaction reagent.
Preferably, in the first step, PCR pipe is put into thermal cycler, reacts 90min；PCR pipe is taken out, 4 drop bufferings are added dropwise
Liquid.
Preferably, in second step, all liq in PCR pipe is added in the detection cartridge after reacting 2min, then be added dropwise 4
Buffer is dripped, 1min is reacted.
Preferably, the multilayer down flow type chromatography detection cartridge chromatographs traditional immune colloid gold lateral flow level
It is optimized to vertical current multilayer chromatographic, removes the setting of traditional test strip both ends water absorption pad, sample application zone and detection zone is organically whole
It is combined, guarantees that detection sample liquid all touches detection zone, to improve detection sensitivity.The detection chromatography cartridge can
To be in flat or three-dimensional column.
Preferably, it is one to fit together that the conjugate pad, which is by flexible or rigid filter liner and the first filter membrane,
Body structure；The conjugate pad of the detection card includes that the colloidal gold crosslinked containing marker or biotin or fluorescein or ground are high
The crosslinked of pungent antibody etc. can be used directly to combine PCR product or lossless DNA or antigen to generate signal.The conjugate
Pad is the integral structure to fit together by reinforcement patches and the second filter membrane, and the position of face sample-adding window, which is provided with, among reinforcement patches leads
Flow window；Position on second filter membrane positioned at face water conservancy diversion window includes the detection of capturing agent or antibody containing detection signal
Line or point and nature controlling line or point, that is, constitute detection zone.Whether develop the color on observation nature controlling line or point, so as to judge the cartridge
Detect whether effectively.
Preferably, two layers of filter membrane, the water absorption pad are set in the test paper of the multilayer down flow type chromatography detection card
It is connected with each other with reinforcement patches by the second filter membrane, the filtration liner is connected with reinforcement patches by the first filter membrane.It is described
Second filter membrane of conjugate pad is equipped with the annular diversion trench of the recess vertically opposite with sample-adding window edge, for dredging liquid
It flows downward, so that sample liquid be allowed to concentrate on detection zone.
Preferably, the detection zone of the Test paper and the sample-adding window position are perpendicular corresponding, and sample liquid flow direction is
From sample-adding window to the water absorption pad direction perpendicular flow positioned at bottom；The sample-adding window and the conjugate pad close contact, institute
Stating sample-adding window includes lantern ring or o-ring shape window frame, and conjugate pad does not contact directly with first shell component, but in support stress
Pass through under the extruding of component sample-adding window compress conjugate pad, make around do not stay a gap, also play in this way shielding interference and
The effect of pollution.Sample liquid is added drop-wise to sample-adding window, then the PCR product in the sample solution to be tested or lossless DNA or antigen contact and is permeated
Conjugate pad reaches the second filter membrane of conjugate bedding course；Extra liquid directly penetrates into water absorption pad or along annular water conservancy diversion
Slot imports water absorption pad.The PCR product or antibody for there was only the liquid in the sample solution to be tested and its carrying during this can permeate and sew
Object pad and reinforcement patches are closed, the molecule in other impurities and air can not pass through conjugate pad.
Preferably, in third step, the activation detection cartridge switch is specially to touch the contraction members；The detection card
Box is that mechanical touch is designed, and one end is equipped with sample-adding window on the first shell component, and the other end is equipped with sliding groove or aperture；It is described
The switch of contraction members in second shell component is stretched out from the sliding groove of first shell component, and the switch of contraction members is pulled to make
Contraction members are slided along sliding groove to the other end, remove the conjugate to link together with contraction members by sliding to shrink
Pad, so that conjugate pad is separated from each other with conjugate pad, thus release stress, after contraction members are touched, the conjugation positioned at upper layer
Object pad is retracted to sliding groove location, and the detection zone on the conjugate pad of lower layer is exposed in sample-adding window, can to show
Depending on reaction result；Or it pulls or rotates with cotton rope or adhesive tape or bolt by set aperture on first shell component and shrink structure
Part removes conjugate pad, so that the detection zone being located on conjugate pad is exposed to sample-adding window, to make result visualization；Testing result
It reads not by outside contamination and influences, the entire cartridge that detects is integrally formed, and is easy to use.The contraction members are to other end water
Horizontal drawing is dynamic or horizontally rotates or the mode of vertical pressing is pulled loose, rotated or activated by movement, contraction or activation.
Preferably, the stress supporting member is used to support water absorption pad, conjugate pad and conjugate pad and applies upward
Pressure；The stress surface size of stress supporting member and sample-adding window are in the same size, the area at least not less than sample-adding window, stress branch
Support component is placed in second shell component in a manner of piston-like；Stress supporting member uses column or mushroom-shaped or goblet
Type shape；Stress supporting member is single entirety or is made from multiple components.
Preferably, the detection line or point and nature controlling line or point are being set to the annular diversion trench intermediate vertical of conjugate pad just
To on the rotary island of sample-adding window, the detection zone of test paper is constituted.Since detection zone is located at immediately below sample application zone, so being coated on rotary island
On nature controlling line or point and detection line or point on the amount of capturing agent be reduced, to be dropped while guaranteeing accuracy in detection
Low cost.
Preferably, the first shell component and second shell component are integral, the first shell member parallel in
The edge of second shell component, which adjoins one another, closely to be snapped together；Or first shell component and second shell component can pass through
The attachmentes such as spring, hinge are attached or hydrant connection, and the connection type of first shell component and second shell component is unrestricted
System.Various shape, such as cuboid, square, gengon, cylinder etc. can be used in the detection cartridge.
Preferably, the material of the Test paper has permeability and filtration；The filtration liner using flexible or
The filtering material medium of rigidity, including polypropylene fibre (polypropylene fibre), polypropylene, metal or nonmetallic filter medium, metallic fiber
Sintering is glued, sintered powder material, more empty ceramics, sintering cellular plastics are sintered Lyu's oxide or glass filter；The filtering
It pads for effectively shielding the substances such as impurity and possible potential aerosol in sample liquid；First filter membrane is fine using acetic acid
Tieing up plain film or glass fibre membrane or PA membrane or polyethylene film or nitrocellulose filter etc. has the material of filtration；It is described
First filter membrane is coated with chemical colour reaction or luminescent substance for filtering fine impurities in sample liquid, evenly dispersed liquid；The reinforcing
Pad is using native cellulose, polyethylene (PE), polyvinyl chloride (PVC), polypropylene (PP), polystyrene (PS) etc., for reinforcing
Detection zone (including detection line or point & nature controlling line or point), constitutes the independent detection zones with certain space on second filter membrane
Domain；Second filter membrane is using cellulose acetate membrane or glass fibre membrane or PA membrane or polyethylene film or nitrocellulose
Film etc. has the material of filtration；(PCR product is lossless for test substance for being coated in capture sample for second filter membrane
DNA or antigen) with Quality Controls substances such as the immune substance and biotin, the seralbumins that generate signal.The upper layer of conjugate pad
It can effectively be shielded in sample liquid using flexible or rigidity the filtration liner of filtering material medium and the first filter membrane of lower layer
The substances such as impurity and possible potential aerosol, effectively filter fine impurities in sample liquid, evenly dispersed liquid is coated with chemical colour reaction
Or luminescent substance, a possibility that effectivelying prevent false positive, therefore have without setting up negative, positive and Matrix controls, reduce at
This advantages of.
Preferably, the water absorption pad is made of that one have spongy material to several layers, be located at below conjugate pad and
Above stress supporting member, to absorb the extra sample liquid for flowing through conjugate pad and conjugate pad, anti-backflow.
Compared to the prior art, the invention has the following advantages: the detection cartridge uses vertical current chromatographic technique, by
Power supporting member, which applies pressure, makes conjugate pad combine closely with conjugate pad, and centre is equipped with diversion trench, thus when detecting, no
There can be the phenomenon that sample liquid reflux and influence testing result (sample liquid reflux can be eliminated to prevent false positive test results),
And under the filtering of filtration liner, reinforcement patches and filter membrane, the interfering substance in sample liquid can be filtered, make detection not by shadow
It rings and (detection can be made not influenced by complex matrices in sample liquid)；And directly contacted with the marker on the conjugate pad, fastly
Fast accurate response observes by the naked eye the display of sample application zone to determine result.The detection cartridge is by traditional immune colloid gold
Level chromatography is optimized to multilayer and vertically chromatographs, and reduces the setting of both ends water absorption pad, sample application zone and detection zone are organically incorporated into
One part guarantees that test sample all touches detection zone, to improve detection sensitivity, has without setting up yin-yang pair
According to the advantages of reducing cost.Compared with the conventional method, the present invention has easy to operate, applicable sample extensive, prevents pollution, spirit
The advantages that sensitivity is high, result judgement is intuitive, save trouble and labor can be used for the detection of a variety of samples such as clinic, food, and testing result is quasi-
Really.
Detailed description of the invention
Referring to schematic figures, the feature and advantage of multilayer vertical detection cartridge of the invention will be apparent from, in which:
Fig. 1 is the front view of the embodiment of the present invention 1 (using cuboid shape)；
Fig. 2 is the side perspective exploded view of Fig. 1；
Fig. 3 is the planar structure decomposition diagram of Fig. 1；
Fig. 4 is the bottom view of the representative device of the embodiment of the present invention 1；
Fig. 5 is some components of the representative device of the embodiment of the present invention 1；Wherein, Fig. 5 a represents conjugate pad (using just
It is rectangular), Fig. 5 b represents conjugate pad (using round), and Fig. 5 c represents conjugate pad；
Fig. 6 is the front view of the embodiment of the present invention 2 (using round)；
Fig. 7 is the side structure decomposition diagram of Fig. 6；
Fig. 8 is the positive structure schematic of the embodiment of the present invention 2 (using round)；
Fig. 9 is the bottom view of the representative device of the embodiment of the present invention 2；
Figure 10 is some components of the representative device of Example 1 and Example 2 of the present invention；Wherein, Figure 10 a, which is represented, shrinks
Component (uses rectangle appearance), and Figure 10 b represents stress supporting member, and Figure 10 c represents contraction members (using circular appearance).
Description of symbols: 1- first shell component；2- second shell component；3- filtration liner；The first filter membrane of 4-；5-
Reinforcement patches；The first water absorption pad of 6-；The second water absorption pad of 7-；8- stress supporting member；The rectangular contraction members of 9-；10- is loaded window；11-
Second filter membrane；12-O type ring；13- sliding groove；14- diversion trench；15- conjugate pad；16- conjugate pad；51- nature controlling line or
Point；52- detection line or point；91- rounded constrictions component.
Specific embodiment
Multilayer vertical-type chromatography detection cartridge provided by the invention, including Test paper and plastic card sets, below in conjunction with attached
Figure describes the manufacturing process and specific structure of the detection cartridge.
Embodiment 1 is using cuboid shape multilayer vertical-type chromatography detection cartridge
As shown in Fig. 1-Fig. 5, Figure 10, a kind of multilayer vertical-type chromatography detection cartridge of the present invention, including Test paper and card
Box, the detection cartridge have an integrated housing member, including being located at the first shell component 1 of cartridge upper end and being located at
The second shell component 2 of cartridge lower end, there are gaps between first shell component 1 and second shell component 2, facilitate installation
(see Fig. 2).The detection sample-adding plot structure of the Test paper has at least two water absorption pads (the first water absorption pad 6 and the second water suction
Pad 7), one end is equipped with sample-adding window 10 on first shell component 1, and the other end is equipped with sliding groove 13 or aperture, and sample-adding window 10 is lower to be arranged
Test paper, the Test paper from top to bottom include: the conjugate pad 15 being made of filtration liner 3 and the first filter membrane 4, by adding
Gu conjugate pad 16, the first water absorption pad 6 and the second water absorption pad 7 that pad 5 and the second filtering film layer 11 form；The Test paper will add
Sample area is combined together with detection zone, forms a kind of down flow type chromatography structure.
There are a stress supporting member 8 and rectangular contraction members 9 in the second shell component 2 of the cartridge, it can be by first
Water absorption pad 6, the second water absorption pad 7, conjugate pad 16, conjugate pad 15 are closely joined together；
1 surface of first shell component is equipped with sample-adding window 10 and sliding groove 13, the detection sample application zone of institute's Test paper with
10 position of sample-adding window is corresponding, and sample flow direction is perpendicular flow.Rectangular contraction members 9 opens in second shell component 2
It closes and is stretched out from sliding groove 13.Rectangular contraction members 9 are connected with conjugate pad 15, and rectangular contraction members 9 pass through sliding to the other end
Releasable pressure is moved, so that conjugate pad 15 is separated from each other with conjugate pad 16, is exposed to detection zone in sample-adding window, to be in
Reveal visual reaction result；Testing result reads not by outside contamination and influences；Entire detection cartridge is integrally formed, and is conveniently made
With；Rectangular contraction members 9 be move horizontally, rotate horizontally or the mode of vertical pressing pulled loose, rotated or activated be pulled, turn
Dynamic or activation.
The conjugate pad 15 is the filtration liner 3 and the first filter membrane using flexible or rigidity filtering material medium
4 integral structures being combined into.
The filtration liner 3 of the conjugate pad 15 is using flexible or rigidity material medium, including polypropylene fibre (third
Synthetic fibre), polypropylene, metal or nonmetallic filter medium, metallic fiber sintered viscous, sintered powder material, more empty ceramics, sintering it is mostly empty
Sintering of plastics Lyu oxide or glass filter；Filtration liner 3 is used for the impurity in effectively shielding sample liquid and may be potentially
The substances such as aerosol；First filter membrane 4 is using cellulose acetate membrane or glass fibre membrane or PA membrane or polyethylene film or nitre
Acid cellulose film etc. has the material of filtration；First filter membrane 4 is for filtering fine impurities in sample liquid, uniform dispersion
Body is coated with chemical colour reaction or luminescent substance；Conjugate pad 15 includes the colloidal gold crosslinked containing marker, is used to combine to be checked
It surveys object and generates signal.
The conjugate pad 16 is reinforcement patches 5 and the integral structure that the second filter membrane 11 is combined into, including contains detection letter
Number capturing agent, the second filter membrane 11 of the conjugate pad 16 is equipped with the ring of the recess opposite with sample-adding 10 edge-perpendicular of window
Shape diversion trench 14, for dredging downward liquid flow, so that sample liquid be allowed to concentrate on detection zone.Reinforcement patches 5 use natural fiber
Element, polyethylene (PE), polyvinyl chloride (PVC), polypropylene (PP), polystyrene (PS) etc. are examined for reinforcing on the second filter membrane
Region (comprising detection line or point nature controlling line or point) is surveyed, the independent detection region with certain space is constituted；Second filter membrane 11
There is filtration using cellulose acetate membrane or glass fibre membrane or PA membrane or polyethylene film or nitrocellulose filter etc.
Material；Second filter membrane 11 is for test substance (PCR product or lossless DNA or antigen) in coating capture sample to generate letter
Number immune substance and the Quality Controls substance such as biotin, seralbumin.
The rectangular contraction members 9 connect conjugate pad 15, and sample application zone is separated with detection zone, testing result reading not by
Outside contamination and influence.
The rectangular contraction members 9 are to move horizontally, rotate horizontally or the mode of vertical pressing is pulled loose, rotated or activated
It is pulled, rotates or activates.
The detection line or point 52 and nature controlling line or point 51 be set on the rotary island that diversion trench impales on the second filter membrane 11,
In window in reinforcement patches 5, since detection zone is located at immediately below sample-adding window, so being coated on the antibody on nature controlling line or point 51
Or the amount of antigen reduces (see Fig. 5 a).
Embodiment 2 detects cartridge using the multilayer vertical-type chromatography of cylindrical outer shape
As shown in Fig. 6-Figure 10, a kind of multilayer vertical-type chromatography detection cartridge of the present invention, including Test paper and cartridge, it should
Detecting cartridge has an integrated housing member, including being located at the first shell component 1 of cartridge upper end and being located at cartridge
The detection sample-adding plot structure of the second shell component 2 of lower end, the Test paper has at least two water absorption pads (the first water absorption pad
6 and second water absorption pad 7), one end is equipped with sample-adding window 10 on first shell component 1, and the other end is equipped with sliding groove 13 or aperture, sample-adding
Window 10 is lower to be arranged Test paper, which from top to bottom includes: the conjugation being made of filtration liner 3 and the first filter membrane 4
Object pad 15, conjugate pad 16, the first water absorption pad 6 and the second water absorption pad 7 being made of reinforcement patches 5 and the second filtering film layer 11；It should
Sample application zone and detection zone are combined together by Test paper, form a kind of down flow type chromatography structure.
There is a stress supporting member 8 and rounded constrictions component 91 in the second shell component 2 of the cartridge, it can be by first
Water absorption pad 6, the second water absorption pad 7, the second filter membrane 11, reinforcement patches 5, the first filter membrane 4 and filtration liner 3 are close-coupled at one
It rises；
1 surface of first shell component is equipped with sample-adding window 10, the detection sample application zone of institute's Test paper and the sample-adding window 10
Position is corresponding, and sample flow direction is perpendicular flow.
The conjugate pad 15 is by the filtration liner 3 and the first filter membrane using flexible or rigidity filtering material medium
4 integral structures to fit together.The filtration liner 3 of conjugate pad 15 is using flexible or rigidity material medium, including poly- third
Alkene fiber (polypropylene fibre), polypropylene, metal or nonmetallic filter medium, metallic fiber sintered viscous, sintered powder material, more empty potteries
Porcelain, sintering cellular plastics sintering Lyu's oxide or glass filter；Filtration liner 3 is for effectively shielding the impurity in sample liquid
With the possible potentially substances such as aerosol；First filter membrane 4 using cellulose acetate membrane or glass fibre membrane or PA membrane or
Polyethylene film or nitrocellulose filter etc. have the material of filtration；First filter membrane 4 is used to filter fine impurities in sample liquid,
Evenly dispersed liquid is coated with chemical colour reaction or luminescent substance；Conjugate pad 15 includes the colloidal gold crosslinked containing marker, is used
To combine object to be detected to generate signal.The conjugate pad 15 includes the colloidal gold crosslinked containing marker, is used to combine anti-
Product or antigen is answered to generate signal.
The conjugate pad 16 is the integral structure to fit together by reinforcement patches 5 and the second filter membrane 11, including is contained
Detect the capturing agent of signal.Second filter membrane 11 of conjugate pad 16 is equipped with the recess opposite with sample-adding 10 edge-perpendicular of window
Annular diversion trench 14, for dredging downward liquid flow, so that sample liquid be allowed to concentrate on detection zone.Reinforcement patches 5 are using natural fine
Element, polyethylene (PE), polyvinyl chloride (PVC), polypropylene (PP), polystyrene (PS) etc. are tieed up, for reinforcing the second filter membrane
Detection zone (includes detection line or point nature controlling line or point), constitutes the independent detection region with certain space；Second filter membrane
11 using cellulose acetate membrane or glass fibre membrane or PA membrane or polyethylene film or nitrocellulose filter etc. there is filtering to make
Material；Second filter membrane 11 is for test substance (PCR product or lossless DNA or antigen) in coating capture sample to generate
The Quality Controls substance such as the immune substance and biotin of signal, seralbumin.
The rounded constrictions component 91 connects conjugate pad 15, and sample application zone is separated with detection zone, and testing result is read not
By outside contamination and influence.
The rounded constrictions component 91 is to move horizontally, rotate horizontally or the mode of vertical pressing is pulled loose, rotated or swashed
It is living.
The detection line or point 52 and nature controlling line or point 51 are set on the second filter membrane 11, since detection zone is located at sample-adding
Immediately below area, so the amount for being coated on nature controlling line or antibody or antigen on point 51 reduces (see Fig. 5 b).
Embodiment 3
By taking a kind of preparation method of the detection card of detection food-borne pathogens as an example, introduce detection of the present invention block and
The structure of its Test paper is as follows:
Signal generation unit subregion is equably coated on the first filter membrane 4 by step 1, is fitted in filtration liner 3
Conjugate pad 15 is obtained together, and raw material used in the present embodiment includes Streptavidin, colloidal gold and bovine serum albumin
It is white.
1) 0.2M K is used2CO3A certain amount of 30nm colloidal gold solution PH is adjusted to 7.0,6 μ l 10mg/ml strepto-s are added
Avidin mixes, and is closed with 10% (W/V) bovine serum albumin(BSA).
2) closed for second time is carried out with 10% (W/V) bovine serum albumin(BSA), 9000rpm is centrifuged 30min, will with gold mark dilution
Centrifugation is redissolved, and is coated on the first filter membrane 4 with Biodot point film instrument, rectangular or circular coating face is formed, with filtering
Liner 3 fits together, to form conjugate pad 15, as shown in Figure 5 c；
Step 2, the antibody coating of conjugate pad
1) NHS-Biotin and bovine serum albumin(BSA) (BSA) are coupled overnight, pH to 7.4 are adjusted, with the second filter membrane 11
As reaction film, above-mentioned mixed liquor is coated in nature controlling line or point 51 on the second filter membrane 11, mouse anti-FITC is coated in the
52,37 DEG C of drying of detection line or point on two filter membranes 11 save.As shown in Fig. 5 a, 5b, by the nature controlling line or point 51, detection
Line or point 52 are preferably configured to fillet or dot form, are located on the second filter membrane 11 on rectangular or border circular areas.
The assembling of step 3, detection chromatography cartridge
Above-mentioned conjugate pad 15 after treatment is placed in rectangular contraction members 9, and by conjugate pad 16, first
Water absorption pad 6 and the second water absorption pad 7 (material is water-absorbing material, including but not limited to blotting paper) are sequentially overlapped together, are placed
It is combined on second shell component 2, then by first shell component 1 and second shell component 2, it is spare, as shown in Figure 2.
By Fig. 2 and Fig. 3 it is found that multilayer vertical-type provided by the invention chromatography detects cartridge, including Test paper and first
Housing member 1, second shell component 2, in which:
The chromatography, which detects cartridge, has first shell component 1, second shell component 2, inside the second shell component 2,
It has been sequentially overlapped the first water absorption pad 6, the second water absorption pad 7 and conjugate pad 16, which has at least two coated films,
It is connected with each other between every two coated film by the second filter membrane 11, at least two coated film and filtration liner 3 are also by the
One filter membrane 4 is connected；It is coated with marker on the filtration liner 3, which is Streptavidin-colloidal gold, is not limited to
Fixed combination can also mark secondary antibody equimagnetic particle.
By Fig. 1, Fig. 6 it is found that the corresponding conjugate pad 15 of the first shell component 1 offers a sample-adding window 10, the sample-adding
Window 10 is also the observation window of observation testing result.
As shown in Figure 8, rounded constrictions component 91 is connected with conjugate pad 15, and rounded constrictions component 91 is by the other end
Releasable pressure is slided, conjugate pad 15 can be removed under the action of rounded constrictions component 91, be exposed to detection zone and added
In sample window 10, to show visual reaction result, and see being shown on conjugate pad 16 as a result, the sample-adding window 10
Shape can be round, square and polygon.Testing result reads not by outside contamination and influences；Entire detection cartridge one
It is body formed, it is easy to use；
As shown in Figure 4, the Starting mode of multilayer vertical-type chromatography detection cartridge provided by the invention can pass through slide
(sliding rectangular contraction members 9) discharges stress supporting member 8, and rectangular contraction members 9 are by sliding releasable pressure to the other end
Power, so that conjugate pad 15 is separated from each other with conjugate pad 16, after rectangular contraction members 9 are touched, conjugate pad 15 is reduced to sliding
13 position of slot, starting detection.
As shown in Figure 9, the Starting mode of multilayer vertical-type chromatography detection cartridge provided by the invention can be round by rotation
Contraction members 91 discharge stress supporting member 8, starting detection.
As shown in Figure 10, in multilayer vertical-type chromatography detection cartridge provided by the invention, rectangular 9 corresponding diagram 1 of contraction members,
Chromatography detection cartridge in Fig. 2 and Fig. 3 constitutes single chromatography detection cartridge；91 corresponding diagram 6 of rounded constrictions component, Fig. 7 and Fig. 8 institute
Show chromatography detection cartridge, constitutes multiple chromatography and detect cartridge.
In above-described embodiment, be using the second filter membrane 11 as water absorption pad on and conjugate pad 16 coating medium and company
It connects, coating medium and connection of first filter membrane 4 as conjugate pad 15 and conjugate pad 16, the second filter membrane 11 and the first mistake
Filter membrane 4 can be nitrocellulose filter, and can virtually completely use cellulose acetate membrane, glass fibre membrane, PA membrane, poly- second
Alkene film etc. substitutes above-mentioned nitrocellulose filter, and it is simple equivalent that this is those skilled in the art combine teachings herein that can make
Replacement, it will not be described here for detailed process.
In addition, in above-described embodiment, chromatography detection cartridge includes stress supporting member 8, and stress supporting member 8 can be
See in Figure 10, these embodiments are merely exemplary for the purpose of the present invention, and not restrictive, other forms also can be used
Stress supporting member.In some embodiments, stress supporting member 8 can be made of any material, be not limited to plastics.Fig. 3 institute
Show, stress supporting member 8 is used for water absorption pad 6, water absorption pad 7, conjugate pad 16 and conjugation in these non-limiting embodiments
Object pad 15 applies pressure.Stress supporting member 8 is placed on second shell component 2 in a manner of piston-like.As shown in fig. lob,
In some embodiments, stress supporting member 8 can have the shapes such as mushroom-shaped, goblet type, be made of head and bar, and head
Portion is wider than bar, and stress supporting member 8 can be single entirety and be also possible to multiple components compositions, and stress supporting member 8 is to guarantee
Chromatography detection cartridge is vertical current chromatography rather than lateral flow.
Stress supporting member 8 can apply the pressure perpendicular to conjugate pad 16, under pressure effect, conjugate pad 16 and conjugation
Object pad 15 is in close contact, and easing off the pressure can stop flowing, so that test sample rests on conjugate pad 16, convenient for improving inspection
It surveys sensitivity and prevents test sample reflux.
In above-described embodiment, chromatography detection cartridge includes contraction members (such as rectangular contraction members 9, rounded constrictions
Component 91), conjugate pad 15 is contacted, stress supporting member 8 compresses conjugate pad 15, conjugate pad from above and below respectively
16, water absorption pad 6, water absorption pad 7.As shown in Figure 2, rectangular contraction members 9 can apply pressure, so that stress supporting member 8 and knot
Close object pad 16, conjugate pad 15 constitutes 1 main body of first shell component；Contraction members are releasably pressed to detection cartridge rear slide
Power, so that conjugate pad 15 is separated from each other with conjugate pad 16, the reaction result of test sample and capturing agent exists only in combination
On object pad 16, contraction members can using move horizontally, rotate horizontally, vertical pressing and side sliding etc. modes operate.
In some embodiments, the sample-adding window 10 that conjugate pad 15 contacts may include lantern ring or o-ring 12 (see Fig. 3) etc.
Component, lantern ring or o-ring 12 can make sample liquid not flow to the place other than detection zone, directly contact detection zone；Relative to adding
10 edgewise compressive conjugate pad 15 of sample window, conjugate pad 15 does not contact directly with first shell component 1, but passes through sample-adding window
10, sample-adding window 10 is applied a sample to, then the sample solution to be tested contacts conjugate pad 15.
Moreover, chromatography detection cartridge corresponds to conjugate pad 15 equipped with a sample-adding window 10, reality in above-described embodiment
In, which is also the watch window that corresponding conjugate pads 16 locations of structures, is understood by an observation window all
Testing result.
First shell component 1 and second shell component 2 are integral, and in embodiment 1, first shell component 1 is parallel to
The edge of second shell component 2, which adjoins one another, to be snapped together.In some embodiments, first shell component 1 and second shell
Component 2 is attached by attachmentes such as spring, hinges or hydrant connection.The connection of first shell component 1 and second shell component 2
Mode is unrestricted.
After the manufacturing process for introducing above-mentioned detection chromatography card, then introduce above-mentioned detection card application method it is as follows:
Choosing meat, egg, milk is verification sample, and 28 parts in total, sample is divided into positive bacteria addition sample (22), nature
Sample (4) and negative sample (2) three classes, each positive bacteria addition sample add a kind of strain of corresponding detection project.It is natural
Sample is directly bought from market, and specially treated is not done.Negative sample is prepared food (being ensured to be object bacteria feminine gender).28 parts of samples are adopted
It is synchronous to be detected using GB4789.4-2010 detection method with the chromatography detection cartridge detection prepared in embodiment 1.As a result
As shown in table 1, it is shown that the chromatography, which detects cartridge, has good testing result.
Test example 1
In test example 1, when detecting salmonella, the segment of the conservative gene of salmonella is used to generate as template proprietary
PCR reaction, with FITC, biotin labeling PCR product, is then placed on conjugate pad 15, is coated on conjugate pad 15
Streptavidin-colloidal gold crosslinked, the PCR product of sample to be tested label, can dissolve the crosslinked on conjugate pad 15,
It is acted on by the permeation filtration of the first filter membrane 4, by Liquid Penetrant to the conjugate pad 16 for being coated with specific antibody, is coated with
Detection line or the rabbit anti-FITC antibody for putting 52 and segment to be checked and Streptavidin-colloidal gold in sample on conjugate pad 16
There is purple red precipitate band in the immune complex for forming a double-antibody sandwich.Excessive sample Streptavidin-colloidal gold is multiple
Closing object will continue to penetrate into the nature controlling line being coated on conjugate pad 16 or point 51, with the Biotin- ox on nature controlling line or point 51
Seralbumin combines, and purple red precipitate band also occurs, if nature controlling line or point 51 do not occur, indicates that the detection is invalid.
1. 28 parts of food samples chromatography detection cartridges of table and GB4789.4-2010 testing result
Testing result shows, by various concentration salmonella, the detection of different food products matrix, 24 parts of Salmeterol fluticasone propionate
Sample.Compared with national standard detection method result, determine that positive 18 met, negative 6 met are positive incongruent
0, negative incongruent 0.The testing result positive coincidence rate 100% of test example 1, negative match-rate 100%, chromatography detection
The result of cartridge is consistent with background, it is shown that the chromatography detection cartridge of this method preparation has preferable testing result.
Test example 2
In test example 2, when detecting Listeria Monocytogenes, with guarding for Listeria Monocytogenes
The segment of gene generates proprietary PCR reaction as template, with FITC, biotin labeling PCR product, is then placed on sewing
It closes on object pad 15, Streptavidin-colloidal gold crosslinked, the PCR product of sample to be tested label, meeting has been coated on conjugate pad 15
It by the crosslinked dissolution on conjugate pad 15, is acted on by the permeation filtration of the first filter membrane 4, by Liquid Penetrant to being coated with spy
On the conjugate pad 16 of heterogenetic antibody, it is coated on conjugate pad 16 in the rabbit anti-FITC antibody and sample of detection line or point 52
Segment to be checked and Streptavidin-colloidal gold form the immune complex of a double-antibody sandwich, purple red precipitate band occur.It crosses
Sample Streptavidin-colloidal gold composite of amount will continue to penetrate into the nature controlling line being coated on conjugate pad 16 or point 51,
In conjunction with the Biotin- bovine serum albumin(BSA) on nature controlling line or point 51, also there is purple red precipitate band, if nature controlling line or point 51 do not have
It occurs, indicates that the detection is invalid.
2. 28 parts of food samples chromatography detection cartridges of table and GB4789.30-2010 testing result
Testing result is shown, single by various concentration Listeria Monocytogenes, the detection of different food products matrix
Nucleus monocytogenes detect 28 parts of samples.Compared with national standard detection method result, positive 24 met are determined
Example, negative 4 met, positive incongruent 0, negative incongruent 0.The testing result positive coincidence rate of test example 2
100%, the result of negative match-rate 100%, chromatography detection cartridge is consistent with background, it is shown that the chromatography detection of this method preparation
Cartridge has preferable testing result.
Test example 3
In test example 3, when detecting escherichia coli O157:H7, with the conservative gene of escherichia coli O157:H7
Segment generates proprietary PCR reaction as template, with FITC, biotin labeling PCR product, is then placed on conjugate pad
On 15, Streptavidin-colloidal gold crosslinked is coated on conjugate pad 15, the PCR product of sample to be tested label can will be conjugated
Crosslinked dissolution on object pad 15, is acted on by the permeation filtration of the first filter membrane 4, and Liquid Penetrant is specific anti-to being coated with
On the conjugate pad 16 of body, it is coated on detection line or the rabbit anti-FITC antibody for putting 52 and to be checked in sample on conjugate pad 16
Section and Streptavidin-colloidal gold form the immune complex of a double-antibody sandwich, purple red precipitate band occur.Excessive sample
Product Streptavidin-colloidal gold composite will continue to penetrate into the nature controlling line being coated on conjugate pad 16 or point 51, with Quality Control
Biotin- bovine serum albumin(BSA) on line or point 51 combines, and purple red precipitate band also occurs, if nature controlling line or point 51 do not go out
It is existing, indicate that the detection is invalid.
3. 24 parts of food samples chromatography detection cartridges of table and GB4789.36-2008 testing result
Testing result is shown, passes through the detection of various concentration escherichia coli, different food products matrix, escherichia coli
Detect 24 parts of samples.Compared with national standard detection method result, determine that positive 16 met, negative 8 met are positive
Incongruent 0, negative incongruent 0.The testing result positive coincidence rate 100% of test example 3, negative match-rate 100%,
The result of chromatography detection cartridge is consistent with background, it is shown that the chromatography detection cartridge of this method preparation has preferable detection knot
Fruit.
Test example 4
In test example 4, when detecting salmonella, monoclonal antibody 8H10-B3 is marked into colloidal gold, and it is corresponding a certain amount of
Heat-inactivated salmonella be incubated for 5min in enzyme mark hole, after 0.1%PBST is washed three times, closed with 3% bovine serum albumin,
37 DEG C of closing 1h.Confining liquid is dried, after washing four times with 0.1%PBST, the original monoclonal antibody and its label glue of known concentration is added
Centrifuged supernatant after body gold, is sprayed on conjugate pad 15.Prepare secondary antibody, washs 5 times 5000 times of dilutions with 0.1%PBST
Sheep anti mouse ELIAS secondary antibody discards secondary antibody, and developing solution is added, and 37 DEG C of reaction 15min are added 2M sulfuric acid and terminate reaction.Using Bio-
The mixing monoclonal antibody diluted is sprayed in the detection line or point 52 of conjugate pad 16 by Dot three-dimensional point film instrument, the anti-mouse secondary antibody dilution of donkey
It for 1mg/mL, is sprayed on the nature controlling line or point 51 of conjugate pad 16, it is dry that conjugate pad is placed on 30 DEG C of vacuum ovens
More than 2.5h.Conjugate pad 15, conjugate pad 16, water absorption pad 6, water absorption pad 7 is good with position folded in a certain order.Pass through
The permeation filtration of one filter membrane 4 acts on, and by Liquid Penetrant to the conjugate pad 16 for being coated with specific antibody, is coated on combination
Sramana's antigen on object pad 16 in the monoclonal antibody and sample of detection line or point 52 forms the immune complex of a double-antibody sandwich, out
Existing purple red precipitate band.Excessive sample colloidal gold composite will continue to penetrate into the nature controlling line being coated on conjugate pad 16 or
Secondary antibody on point 51, with nature controlling line or point 51 reacts, and purple red precipitate band also occurs, if nature controlling line or point 51 do not go out
It is existing, indicate that the detection is invalid.
4. 24 parts of food samples chromatography detection cartridges of table and GB4789.36-2008 testing result
Testing result shows, by various concentration salmonella, the detection of different food products matrix, 20 parts of Salmeterol fluticasone propionate
Sample.Compared with national standard detection method result, determine that positive 16 met, negative 4 met are positive incongruent
0, negative incongruent 0.The testing result positive coincidence rate 100% of test example 4, negative match-rate 100%, chromatography detection
The result of cartridge is consistent with background, it is shown that the chromatography detection cartridge of this method preparation has preferable testing result.
From above-described embodiment and test example it is found that the invention has the following outstanding advantages:
1. the chromatography detection cartridge can be with the detection of the carry out index of rapid sensitive；
2. conjugate pad 15 and conjugate are padded 16 tighter integrations by the chromatography detection cartridge, exclusive vertical current is chromatographed,
Be conducive to improve detection efficiency, reduce sample consumption.
3. the chromatography detects cartridge and will test with inside setting as the result is shown and cartridge, the interference of external source sample and dirt are prevented
Dye.
Streptavidin-colloidal gold, bovine serum albumin(BSA), NHS-Biotin and mouse described in above embodiments are anti-
FITC is only several examples in a large amount of viable solutions of the invention, can also be coated with the objects such as primary antibody, secondary antibody
Matter, and the combination of antibody in conjugate pad 15, conjugate pad 16, Streptavidin, colloidal gold, fluorescein be not limited to
Upper embodiment, those skilled in the art can be adjusted without departing from the scope of the present invention.In the present invention, in order to
Statement is convenient, above-mentioned signal generation analytical unit is coated with to filtration liner 3, and connect conjugate pad by the first filter membrane 4
16, the integral structure which is made of filtration liner 3 and the first filter membrane 4 is also possible to the institute that bonds together
It is formed, as shown in Figure 3.
Above with reference to attached drawing and related data illustrated embodiment, invention is explained in detail, but this field
It is to be understood by the skilled artisans that the present invention is not limited to these specific embodiments, but can be without departing from the scope of the present invention
Or variations and modifications are carried out to it in the case where Spirit Essence.
Claims (10)
1. a kind of molecule chromatographs PCR detection method, it is characterised in that: cartridge is detected using a kind of multilayer down flow type chromatography,
The detection cartridge has integrated housing member, including being located at the first shell component (1) on cartridge top and being located at cartridge
The second shell component (2) of lower part, the first shell component (1) are equipped with sample-adding window (10), inspection are arranged under sample-adding window (10)
Test paper, the Test paper from top to bottom include: the conjugate pad (15) being made of filtration liner (3) and the first filter membrane (4),
By reinforcement patches (5) and second filtering film layer (11) form conjugate pad (16), one to several layers water absorption pad；The Test paper will
Sample application zone is combined together with detection zone, forms a kind of down flow type chromatography structure；In the second shell component (2) of the cartridge
With the contraction members (9) that a stress supporting member (8) and one link together with conjugate pad (15), stress supports structure
Part (8) squeezes conjugate pad (15), conjugate pad (16) and water absorption pad, is allowed to fit closely together with sample-adding window (10)；Touching
Dynamic contraction members (9) remove the conjugate pad (15) to link together with contraction members (9), make conjugate pad (15) and combine
Object pad (16) is separated from each other, and so that the detection zone being located on conjugate pad (16) is exposed to sample-adding window (10), to keep result visual
Change；
The detection method includes the following steps:
Sample to be tested is carried out PCR reaction, obtains PCR product by the first step；
The PCR product that sample to be tested marks is placed on the conjugate pad (15) of the detection cartridge, sample to be tested by second step
The PCR product of label can dissolve the crosslinked on conjugate pad (15), be acted on by the permeation filtration of the first filter membrane (4),
On Liquid Penetrant to the conjugate pad (16) for being coated with antibody, conjugate pad (16) on detection line or point (52) anti-will be coated on
Segment to be checked and colloidal gold in body and sample form the immune complex of a double-antibody sandwich, purple red precipitate band occur；
Excessive sample colloidal gold composite will continue to penetrate into the nature controlling line being coated on conjugate pad (16) or point (51), with Quality Control
Biotin- bovine serum albumin(BSA) on line or point (51) combines, and purple red precipitate band also occurs, nature controlling line or point (51) be not if having
There is purple red precipitate band, indicates that the detection is invalid；
Third step, activation detection cartridge switch, checks result.
2. detection method as described in claim 1, it is characterised in that: in the first step, the sample to be tested is carrying out PCR reaction
Before, increase following steps:
(1) sample to be tested 36 DEG C Zengjing Granule 18-24 hours in enriched medium；
(2) liquid after above-mentioned increasing bacterium is taken, is added in the EP pipe containing buffer, 100 DEG C are boiled 10min；
(3) EP pipe is cooled to room temperature, and takes part liquid in pipe, is added in the PCR pipe containing reaction reagent.
3. detection method as claimed in claim 2, it is characterised in that: in the first step, PCR pipe is put into thermal cycler, instead
Answer 90min；PCR pipe is taken out, 4 drop buffers are added dropwise.
4. detection method as claimed in claim 3, it is characterised in that: in second step, all liq in PCR pipe is added to described
It detects after reacting 2min in cartridge, then 4 drop buffers is added dropwise, react 1min.
5. detection method according to claim 1, it is characterised in that: the water absorption pad and reinforcement patches (5) pass through the second mistake
Filter membrane (11) is connected with each other, and the filtration liner (3) is connected with reinforcement patches (5) by the first filter membrane (4)；The conjugate
The second filter membrane (11) for padding (16) is equipped with the annular diversion trench (14) of the recess opposite with sample-adding window (10) edge-perpendicular, uses
In allowing sample liquid to concentrate on detection zone.
6. detection method according to claim 1, it is characterised in that: the detection zone of the Test paper and the sample-adding window
(10) position vertically corresponds to, and sample liquid flow direction is from sample-adding window (10) to the water absorption pad direction vertical current for being located at test paper bottom
It is dynamic；The sample-adding window (10) contacts with the conjugate pad (15), and the sample-adding window (10) includes lantern ring or o-ring shape window frame,
Contact conjugate pad (15) directly with first shell component (1), but by sample-adding window (10) in stress supporting member (8)
Extruding under compress conjugate pad (15), make be loaded window (10) around leave no gaps；By sample liquid be added drop-wise to sample-adding window (10), then to
Sample measuring liquid contacts and vertically penetrates the second filter membrane (11) that conjugate pad (15) reach conjugate pad (16) bottom；Extra liquid
Body directly penetrates into water absorption pad or imports water absorption pad along annular diversion trench (14).
7. detection method as described in claim 1, which is characterized in that in third step, the activation detection cartridge switch is specific
To touch the contraction members (9)；One end is equipped with sample-adding window (10) on the first shell component (1), and the other end is equipped with sliding
Slot (13) or aperture；Sliding of the switch of contraction members (9) in the second shell component (2) from first shell component (1)
Slot (13) stretches out, and pulls the switch of contraction members (9) to slide contraction members (9) to the other end along sliding groove (13), passes through
The conjugate pad (15) removed and linked together with contraction members (9) is shunk in sliding, makes conjugate pad (15) and conjugate pad
(16) it is separated from each other, releases stress, be exposed to the detection zone on conjugate pad (16) in sample-adding window (10), it can to show
Depending on reaction result；Or it pulls or rotates with cotton rope or adhesive tape or bolt by set aperture on first shell component (1) and receive
Contracting component (9) removes conjugate pad (15), so that the detection zone being located on conjugate pad (16) is exposed to sample-adding window (10), to make
Result visualization；The contraction members be by level pull, horizontally rotate or vertical pressing in a manner of pulled loose, rotated or swashed
It is living.
8. detection method according to claim 1, which is characterized in that the stress supporting member (8) is used to support water suction
Pad, conjugate pad (16) and conjugate pad (15) simultaneously apply pressure；The stress surface size of stress supporting member (8) is greater than or waits
In the area of sample-adding window (10)；Stress supporting member (8) is placed in second shell component (2) in a manner of piston-like；Stress branch
It supports component (8) and uses column or mushroom-shaped or goblet type shape, be single entirety or be made from multiple components.
9. detection method as claimed in any of claims 1 to 8 in one of claims, which is characterized in that the detection line or point (52) and
Nature controlling line or point (51) are set to the rotary island of annular diversion trench (14) intermediate vertical face sample-adding window (10) of conjugate pad (16)
On, constitute the detection zone of test paper；Since detection zone is located at immediately below sample application zone, so the nature controlling line or point that are coated on rotary island
(51) be reduced with detection line or the amount of the capturing agent on point (52), thus reduced on the basis of guaranteeing accuracy in detection at
This.
10. detection method according to claim 1, it is characterised in that: the first shell component (1) and second shell structure
Part (2) is integral or the first shell component (1) is parallel to the edge of second shell component (2) and adjoins one another and be fastened on
Together or first shell component (1) is attached with second shell component (2) by spring, hinge or hydrant connects；The mistake
Filter liner (3) is using flexible or rigidity filtering material medium, including polypropylene fibre, polypropylene, metal or nonmetallic filtering
Medium, metallic fiber sintered viscous, sintered powder material, more empty ceramics, sintering cellular plastics sintering Lyu's oxide or glass filtration
Medium；The filtration liner (3) is for effectively shielding impurity and possible potential aerosol matter in sample liquid；First mistake
Filter membrane (4) was had using cellulose acetate membrane or glass fibre membrane or PA membrane or polyethylene film or nitrocellulose filter etc.
The material of filter effect；It is aobvious to be coated with chemistry for filtering fine impurities in sample liquid, evenly dispersed liquid for first filter membrane (4)
Color or luminescent substance；The reinforcement patches (5) use native cellulose, polyethylene, polyvinyl chloride, polypropylene or polystyrene, use
In reinforcing detection zone on the second filter membrane (11), the independent detection region with certain space is constituted；Second filter membrane
(11) using cellulose acetate membrane or glass fibre membrane or PA membrane or polyethylene film or nitrocellulose filter there is filtering to make
Material；Second filter membrane (11), which is used to be coated with, captures in sample test substance to generate immune substance and the life of signal
Object element, seralbumin Quality Control substance.
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| CN201910113649.7A CN109593827A (en) | 2019-02-14 | 2019-02-14 | A kind of molecule chromatography PCR detection method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910113649.7A CN109593827A (en) | 2019-02-14 | 2019-02-14 | A kind of molecule chromatography PCR detection method |
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Cited By (1)
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| CN113311175A (en) * | 2021-05-28 | 2021-08-27 | 四川省畜牧科学研究院 | Colloidal gold detection test strip for Sichuan Huaniu cattle, preparation method and application |
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Application publication date: 20190409 |