CN106319041B - A kind of preparation method of nanoparticle polychrome multiple detection test paper - Google Patents
A kind of preparation method of nanoparticle polychrome multiple detection test paper Download PDFInfo
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- CN106319041B CN106319041B CN201610634638.XA CN201610634638A CN106319041B CN 106319041 B CN106319041 B CN 106319041B CN 201610634638 A CN201610634638 A CN 201610634638A CN 106319041 B CN106319041 B CN 106319041B
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- probe
- nucleic acid
- capture probe
- instruction
- acid sequence
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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
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
Abstract
The present invention provides a kind of preparation methods of nanoparticle polychrome multiple detection test paper, specifically, the present invention provides a kind of preparation methods of nanoparticle Test paper for detection of nucleic acids, instruction probe is provided first, instruction probe can be specifically bound with target nucleic acid sequence, it indicates that 5 ' ends of probe are combined with colour developing nanosphere and combine, indicates that 3 ' ends of probe are combined with biotin；Then capture probe is provided, and capture probe and Streptavidin are fixed on Test paper.The Test paper of method preparation of the invention, it is not only easy to operate, quick, cheap, but also can realize that multiple nucleic acid detects.
Description
Technical field
The invention belongs to field of biotechnology, specifically, the present invention relates to a kind of examinations of nanoparticle polychrome Multiple detection
The preparation method of paper.
Background technique
Nucleic acid detection technique can be used to detect pathogen nucleic acid, including DNA and RNA.Compared with conventional Serologic detection,
Detection of nucleic acids is the DNA or ribonucleic acid of direct detection pathogen, has the characteristics that high specificity, high sensitivity,
Pathogen recall rate can be greatly improved, shorten the detection window phase, greatly improve monitoring efficiency.
Present nucleic acid detection technique is based on PCR principle mostly, and that such as common includes quantitative fluorescent PCR (Realtime
FluorescencequantitativePCR, RTFQPCR) technology, rely on the amplification technique of nucleic acid sequence
(NucleicAcidSequence-basedAmplification, NASBA), rolling circle amplification
(RollingCircleAmplification, RCA), loop-mediated isothermal amplification technique (Loop-
MediatedIsothermalAmplification, LAMP), the amplification technique of transcriptive intermediate
(TranscriptionMediatedAmplifi-cation, TMA) etc..Either quantitative fluorescent PCR or other PCR amplifications
Technology, will be by high-end, expensive instrument and equipment when detecting nucleic acid, and complicated for operation, monitoring condition requires height, and is difficult
Realize the Multiple detection of pathogen.Therefore, those skilled in the art are dedicated to developing a kind of easy to operate, quick, cheap,
The technology of multiple nucleic acid detection can be achieved.
Summary of the invention
The purpose of the present invention is to provide a kind of nanoparticle polychrome multiple detection test papers and preparation method thereof.
The first aspect of the present invention provides a kind of preparation method of nanoparticle Test paper for detection of nucleic acids,
Comprising steps of
(1) instruction probe is provided
The instruction probe is arranged for specifically binding with the first complementary combined area of target nucleic acid sequence, the finger
Show that 5 ' ends of probe are combined with colour developing nanosphere and combine, 3 ' ends of the instruction probe are combined with biotin；
(2) capture probe is provided
The capture probe is arranged for specifically binding with the second complementary combined area of target nucleic acid sequence, and institute
It states the first complementary combined area and the second complementary combined area and is at least spaced 3 nucleotide；
(3) preparation of test strips
The test strips include loading area, detection zone and the suction zones successively arranged；The detection zone is close to the loading
Area side includes detection line, and the detection line is fixed with the capture probe, and the detection zone is wrapped close to the suction zones side
Nature controlling line is included, the nature controlling line is fixed with Streptavidin.
Preferably, target nucleic acid sequence number >=2；The instruction probe corresponds respectively to each target nucleic acid sequence
Column, and respectively instruction probe 5 ' holds the colour developing nanosphere for being combined with different colours；The capture probe corresponds respectively to each mesh
Nucleic acid sequence is marked, and each capture probe is once individually fixed in the different location of the detection zone.
Preferably, target nucleic acid sequence number >=3, more preferably described target nucleic acid sequence number >=5.
Preferably, the instruction probe includes 10~50 nucleotide.
Preferably, the capture probe includes 10~50 nucleotide.
Preferably, in the step (3), the capture probe is fixed on nitrocellulose filter close to the loading area
Side forms the detection line, Streptavidin is fixed on nitrocellulose filter formed close to the suction zones side it is described
Nature controlling line.
Preferably, in the step (3), CaCl is first used2Solution is crossed on nitrocellulose filter, between two lines
Distance >=1.5mm, 78 DEG C~82 DEG C baking 15min~20min after drying；It is drawn after capture probe and Streptavidin are dissolved in water
In CaCl2In solution scribing line, 60 DEG C~65 DEG C baking 1h~2h after drying.
Preferably, in the step (3), CaCl2Liquid quality fraction is 5%~15%.
Preferably, in the step (3), CaCl2Solution scribe widths are 1mm.
Preferably, in the step (3), CaCl2Liquid quality fraction is 10%.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 shows the structure of nanoparticle polychrome multiple detection test paper of the invention.
Fig. 2 shows the testing principle of nanoparticle polychrome multiple detection test paper of the invention.
Specific embodiment
The single-stranded DNA sequence (capture probe) that target gene (DNA, RNA) can be captured is fixed on nitric acid fibre by the present invention
On the specific position for tieing up plain film, the fixed Streptavidin in quality control area.Furthermore it is possible to other sites with the target gene
The single stranded DNA of sequence complementation is used as DNA probe, and two sections of the ssDNA probe are received with biotin and particular color respectively
Meter Wei Qiu phase, which is coupled, becomes instruction probe.When target gene, instruction probe exist simultaneously the capture simultaneously and on nitrocellulose filter
Probe interaction, takes the position that colored nanosphere is enriched to capture probe, to observe specific position color
Variation.And the specific position color in the absence of target gene where capture probe does not change.
Different location on a nitrocellulose filter places different capture probes simultaneously, and corresponding instruction
Probe carries the nanosphere of different colours, these instruction probes combine in the same system with target gene, then simultaneously
It is acted on the nitrocellulose filter for being fixed with capture probe, then the target gene is the positive, Quality Control at capture probe colour developing
Region is presented specific color and (according to number of probes and carries color and different, such as due to capturing unbonded instruction probe
Black).
The scope of application and advantage:
Pcr amplification product analysis, alternative PCR product agarose gel electrophoresis analysis, has easy to operate, detection time
The advantages that short, visual result can analyze the close product of multiple sizes simultaneously, and as a result the holding time is long；Nucleic acid hybridization analysis is carried out,
There are the advantages such as resolution ratio is higher, result is more intuitive compared with standard hybridization analysis；For nucleic acid signal amplification system, high-resolution point
Signal is calculated in analysis, avoids the occurrence of analytical error, has the advantages such as detection cycle is short, technical requirements are low, error is small.
Nanoparticle polychrome multiple detection test paper according to the present invention is designed according to following principle:
1. probe designs
Capture probe: it complementary with the specific region of target nucleic acid sequence specificity can combine.
Indicate probe: 5 ' hold in conjunction with the nanoparticle of polychrome, 3 ' ends are in conjunction with biotin, sequence and target nucleic acid sequence
Specific region specificity is complementary to combine (being different from capture probe region).
2. develop the color film design
Bar shaped: " loading area ", " test paper area ", " suction zones " are from left to right followed successively by.Test paper area is from left to right successively with spy
Different capture probe scribing line, hot roasting mode is fixed after dry, and right end uses Streptavidin scribing line as nature controlling line.
3. indicating that probe captures target gene
Single-stranded target gene directly reacts at room temperature 20 minutes.
90 DEG C of double stranded target gene are reacted 5 minutes, are cooled to room temperature rapidly.
4. develop the color test paper reaction
Bar shaped: the reaction system after instruction probe capture is added to loading area, and each detection line and matter are observed after 1-10 minutes
Control the colour developing situation of line.
5. interpretation of result
Nature controlling line (color is determined according to the quantity of polychrome microballoon) reaction is as the believable basis of reaction.It examines on this basis
Survey line or test point show that correct color is the positive, and not developing the color or developing the color is not all negative with design colours or mistake is examined
It surveys.
Above-mentioned probe can be used artificial synthesized method to be prepared, and can be visited instruction using the known technology of this field
The combination of needle and colour developing nanoparticle (microballoon), and be combined with biotin.The synthesis of probe and probe and marker
Combination, generally entrust commercialized biotechnology company to complete.
Combined with specific embodiments below, further statement is of the invention.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.The experimental method of detailed conditions is not specified in the following example, usually according to conventional strip
Part such as U.S. Sambrook.J etc. writes " Molecular Cloning: A Laboratory room guide " (Huang Peitang etc. is translated, Beijing: Science Press, 2002)
Described in condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number be by weight
It calculates.Experimental material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel.
1 test strips of embodiment analyze target gene
(1) artificial synthesized following target gene segment
A、5’ATCTAGGTACTCTAGGACTAATAGCCAACCGCTACT 3’(SEQ ID NO.1)
B、5’CCTAAGTCATGGCTTGAATCTGAGTCCTGAATCT 3’(SEQ ID NO.2)
C、5’AGAGTTCGAATCGTACAAGTTACTACGTATATCGT 3’(SEQ ID NO.3)
D、5’TGAACGTCCAGTGCAGTACTGCCTCGAGTCGACTT 3’(SEQ ID NO.4)
E、5’TCGACTGCTAGTCAGACACAGGATCAATCCAATGCA 3’(SEQ ID NO.5)
It is all artificial sequence above, is single-stranded DNA sequence, is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.By these
The mixing that various combination is carried out after sequence dissolution, mixed various combination is marked.
(2) probe is indicated
Artificial synthesized following instruction probe a, b, c, d, e, respectively with target gene segment A, B, C, D, E in step (1)
5 ' end underscore partial complementarities pairing:
A, 5 ' CTAGAGTACCTAGAT 3 ' (SEQ ID NO.6) (green microspheres)
B, 5 ' AAGCCATGACTTAGG 3 ' (SEQ ID NO.7) (red microsphere)
C, 5 ' TACGATTCGAACTCT3 ' (SEQ ID NO.8) (blue microballoon)
D, 5 ' TGCACTGGACGTTCA 3 ' (SEQ ID NO.9) (purple microballoon)
E, 5 ' CTGACTAGCAGTCGA 3 ' (SEQ ID NO.10) (yellow microballoon)
The above artificial sequence 5 ' holds the colour developing nanosphere that different colours are connected in a manner of amino, carboxyl, and 3 ' ends are with life
Object element label, the synthesis of artificial sequence and label are completed by Shanghai Jierui Biology Engineering Co., Ltd.5 color micro-spheres are marked
Sequence mixing, be denoted as " instruction probe mixed liquor ".
(3) capture probe
Artificial synthesized following instruction probe a ', b ', c ', d ', e ', respectively in step (1) target gene segment A, B,
C, 3 ' the end underscore partial complementarity pairings of D, E:
a’、5’AGTAGCGGTTGGCTATTA 3’(SEQ ID NO.11)
b’、5’AGATTCAGGACTCAG 3’(SEQ ID NO.12)
c’、5’ACGATATACGTAGTA 3’(SEQ ID NO.13)
d’、5’AAGTCGACTCGAGGC 3’(SEQ ID NO.14)
e’、5’TGCATTGGATTGATC 3’(SEQ ID NO.15)
The above sequence is all artificial sequence, is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.
(4) prepared by test strips:
By CaCl2Then (mass fraction 10%) soluble in water uses CaCl2Solution is crossed on nitrocellulose filter,
The spacing distance of scribe widths 1mm, two lines are 2mm, and scribing position is the fixation position of subsequent captured probe, 80 after drying
DEG C baking 20min；After capture probe a ', b ', c ', d ', e ' and the Streptavidin of above-mentioned preparation are dissolved in water (50 μ g/ml),
It draws in CaCl2Solution scribing line is upper (scribe widths 1mm, the spacing distance of two lines are 2mm), toasts 2 hours for 60 DEG C after drying.?
Loading area is set on the left of nitrocellulose filter, and right side is connected with blotting paper.
The present inventor has found that short nucleotide sequence (being lower than 100bp) is difficult to be firmly fixed to nitric acid fibre under study for action
Tie up on plain film, capture probe it is fixed it is bad will lead to subsequent detection during, band disperse, or even can not show band, it is close and
There is the result of false negative.By repetition test, it has been found that first using CaCl2Solution is crossed on nitrocellulose filter, is dried
It is crossed in same position using capture probe after roasting, shorter nucleotide sequence firmly very can be fixed on nitric acid
Cellulose membrane.By optimization, it is determined that optimal CaCl2Solution concentration (mass fraction) is 5%~15%, CaCl2Solution scribing line
Baking condition is 78 DEG C~82 DEG C baking 15min~20min afterwards.
(5) target gene detects:
1, sequence mark: instruction 100 μ l of probe mixed liquor being added into the 100 μ l of mixture of target gene, is sufficiently mixed,
Room temperature reaction 20 minutes.
2, loading: test strips are laid flat, reaction solution be added to left side loading area, wait liquid gradually turn right blotting paper shifting
It is dynamic.
Result judgement: end of reaction, using rightmost side black stripe as system Quality Control mark, if rightmost side band is black
It carries out analyzing other bands.Other banded zone colors are consistent or colourless with design colours, can further analyze.Color development
With the colour developing band of design band solid colour, indicate that sample object band is positive.Band indicates sample object band without color
Feminine gender, as shown in Figure 2.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (9)
1. a kind of preparation method of the nanoparticle Test paper for detection of nucleic acids, comprising steps of
(1) instruction probe is provided
The instruction probe is arranged for specifically binding with the first complementary combined area of target nucleic acid sequence, and the instruction is visited
5 ' ends of needle are combined with colour developing nanosphere, and 3 ' ends of the instruction probe are combined with biotin；
(2) capture probe is provided
The capture probe is arranged for combined area complementary with the second of target nucleic acid sequence and specifically binds, and described the
One complementary combined area and the second complementary combined area are at least spaced 3 nucleotide；
(3) preparation of test strips
The test strips include loading area, detection zone and the suction zones successively arranged；The detection zone is close to the loading area one
Side includes detection line, and the detection line is fixed with the capture probe, and the detection zone includes matter close to the suction zones side
Line is controlled, the nature controlling line is fixed with Streptavidin；
Wherein, in the step (3), the capture probe is fixed on nitrocellulose filter close to loading area side shape
At the detection line, Streptavidin is fixed on nitrocellulose filter and forms the nature controlling line close to the suction zones side
Also, in the step (3), CaCl is first used2Solution is crossed on nitrocellulose filter, and the distance between two lines >=
1.5mm, 78 DEG C~82 DEG C baking 15min~20min after drying；Then capture probe and Streptavidin are dissolved in after water draw in
CaCl2In the scribing line of solution, 60 DEG C~65 DEG C baking 1h~2h after drying.
2. the method as described in claim 1, which is characterized in that target nucleic acid sequence number >=2；The instruction probe
Each target nucleic acid sequence is corresponded respectively to, and respectively instruction probe 5 ' holds the colour developing nanosphere for being combined with different colours；It is described
Capture probe corresponds respectively to each target nucleic acid sequence, and each capture probe is successively individually fixed in the difference of the detection zone
Position.
3. the method as described in claim 1, which is characterized in that target nucleic acid sequence number >=3.
4. the method as described in claim 1, which is characterized in that the instruction probe includes 10~50 nucleotide.
5. the method as described in claim 1, which is characterized in that the capture probe includes 10~50 nucleotide.
6. method as claimed in claim 3, which is characterized in that target nucleic acid sequence number >=5.
7. the method as described in claim 1, which is characterized in that in the step (3), CaCl2Liquid quality fraction is 10%.
8. the method as described in claim 1, which is characterized in that in the step (3), CaCl2Liquid quality fraction be 5%~
15%.
9. the method as described in claim 1, which is characterized in that in the step (3), CaCl2Solution scribe widths are 1mm.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1452662A (en) * | 2000-07-07 | 2003-10-29 | 海伦·李 | Improved capture and detection format versatility for dipstick assays |
| CN1522368A (en) * | 2002-04-05 | 2004-08-18 | ���µ�����ҵ��ʽ���� | Test strip for chromatography and process for producing the same |
| CN102154498A (en) * | 2011-03-21 | 2011-08-17 | 厦门大学 | Nucleic acid detecting method |
| CN102230032A (en) * | 2011-06-27 | 2011-11-02 | 广州弗赛生物科技有限公司 | Isothermal chain multiple detection card of pathogen nucleic acid |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100730054B1 (en) * | 2005-08-05 | 2007-06-20 | 가부시키가이샤 히타치플랜트테크놀로지 | Apparatus and method for separating oil from water |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1452662A (en) * | 2000-07-07 | 2003-10-29 | 海伦·李 | Improved capture and detection format versatility for dipstick assays |
| CN1522368A (en) * | 2002-04-05 | 2004-08-18 | ���µ�����ҵ��ʽ���� | Test strip for chromatography and process for producing the same |
| CN102154498A (en) * | 2011-03-21 | 2011-08-17 | 厦门大学 | Nucleic acid detecting method |
| CN102230032A (en) * | 2011-06-27 | 2011-11-02 | 广州弗赛生物科技有限公司 | Isothermal chain multiple detection card of pathogen nucleic acid |
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