CN102134596A - Nucleic acid cross flow test strip-based method for detecting single nucleotide polymorphism - Google Patents
Nucleic acid cross flow test strip-based method for detecting single nucleotide polymorphism Download PDFInfo
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Abstract
The invention relates to a nucleic acid cross flow test strip-based method for detecting single nucleotide polymorphism, comprising the following steps: firstly, preparing the nucleic acid cross flow test strip; secondly, obtaining a sample to be tested, denaturing and annealing; obtaining water, a nano-gold probe solutions, a connecting probe, Taq DNA ligase buffer solutions and Taq DNA ligase, and blending uniformly to obtain a mixed solution; adding KIF-1 and KIF-2 or the mixed solution of the two to the mixed solution, and blending uniformly; adding the sample to be tested, carrying out hybrid connection, denaturing, and annealing; and finally dropping obtained solutions on the binding area of the nucleic acid cross flow test strip, immersing the immersion area of the test strip into the running buffer solutions, and observing. The method achieves easy operation and low cost, is characterized by specificity, fastness as well as high resolution and sensitivity, and can be applied to the detection on the single nucleotide polymorphism and genetype as well as the identification on different pathogenic microorganisms of genes in hereditary diseases, communicable diseases, tumour and angiocardiopathy in clinical medicines.
Description
Technical field
The invention belongs to the detection range of single nucleotide polymorphism, particularly relate to a kind of method that detects single nucleotide polymorphism based on nucleic acid transverse flow test strip.
Background technology
Cardiovascular disorder is modern's first killer.Now the whole world has nearly 1/4th populations to be threatened by cardiovascular and relative disease, and throughout one's life, nearly 1/3rd people's life is shrouded by the cardiovascular disorder shade, has 1/5th population to die from cardiovascular related diseases at last.In generation, diagnosis and the treatment of cardiovascular disorder (as coronary heart disease), KIF6 (kinesin-like protein 6) gene important influence.KIF6 (kinesin-like protein 6) albumen is a kind of and the interior transportation of cell proteins associated matter.The KIF6 genotype provides the important information beyond traditional risk factors, with high-risk patient that helps identification coronary heart disease and the treatment of carrying out personalization.The single nucleotide polymorphism (SNP) of KIF6 (719 Arg) has been proved the increase that can predict coronary heart disease (CHD) risk, and reduces the accident of statin (statins) treatment.This class KIF6 genotype carrier is contained one or two 719Arg allelotrope (e.g., Arg/Trp or Arg/Arg), but not carrier's genotype shortage 719Arg allelotrope (e.g., Trp/Trp).And women KIF6 (719Arg) allelotrope carrier heart stalk and coronary heart disease (CHD) danger are higher than non-carrier.
Present modal method for detecting single nucleotide polymorphism comprises: sequencing, single strand conformation polymorphism detect (SSCP), connection or primer extension (ligation or primer extension), based on the fluorescent energy resonance transfer method (molecular-beacon-based fluorescence resonance energy transfer) of molecular beacon, methods such as Electrochemical Detection (electrochemical detecting) and Two Colour Fluorescence polarization technology.But these method ubiquity complicated operating process are more time-consuming and need valuable shortcomings such as detecting instrument.Therefore set up simple, special, fast, target nucleic acid and method for detecting single nucleotide polymorphism the promoting the use of in medical diagnosis and Clinical Laboratory of can being more convenient for easily.
Nearest a lot of research all concentrates on the exploitation of new detection technique so that make PCR and other amplification techniques more adapt to the demand of modern molecular diagnosis.Provide fast way as the test strip detection technique for biochemistry detection and medical science rapid detection.Yet, the at present antibody or the streptavidin test strip of test strip technology amplified production that has been mark mainly or oligonucleotide probe.This test strip has certain advantage, but also because used proteic antigen antibody reaction, need more mark, and the right quantity limitation of the antibody/haptens of high-affinity the detection of the many targets of gene, increased cost and step, and more be not easy to preserve.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method that detects single nucleotide polymorphism based on nucleic acid transverse flow test strip, this method is simple to operate, cost is low, have special, quick, high resolution, highly sensitive characteristics, can be applicable to the evaluation of the single nucleotide polymorphism of gene in inherited disease in the clinical medicine, communicable disease, tumour and the cardiovascular disorder, genotypic detection and Different Kinds of Pathogens microorganism.
Of the present inventionly a kind ofly detect the method for single nucleotide polymorphism based on nucleic acid transverse flow test strip, detecting with the gene pleiomorphism of cardiovascular diseases genes involved KIF is that example is described in detail, and comprising:
(1) will contain film capillaceous (Membrane) and stick on the chipboard, and uptake zone (Absorbent Pad) will be placed on contain on the film capillaceous overlapping 1-2mm again; Again land (Conjugation Pad) is placed on and contains on the film capillaceous overlapping 1-2mm; Be placed on the land immersing district (Immersion Pad) at last, overlapping 1-2mm gets nucleic acid transverse flow test strip; Described two detection lines (detection line 1, detection line 2) and the nature controlling line that has on the film capillaceous with capture dna probe bag quilt that contain;
(2) single nucleotide polymorphism of KIF6 DNA detects
Get 1 μ L sample to be tested, 95 ℃ of sex change 4min in the PCR instrument put into-20 ℃ of ice chest annealing 3min again;
At first preparation detects the genotypic reaction system of KIF6, the hybridization that is used for target dna and detection probes be connected, 4 μ L, Nano-Au probe solution 2 μ L, linking probe Z-KIF-1 and Z-KIF-2 (concentration is 1 μ M/L) each 1 μ L, TaqDNA ligase enzyme damping fluid 1 μ L and Taq dna ligase (concentration is 1 unit) solution 1 μ L fetch water, beat with liquid-transfering gun even, mixed solution; In mixed solution, add KIF-1, the KIF-2 of 1 μ L (concentration is 1 μ M/L) or their mixed solution, beat even with liquid-transfering gun; Add the sample to be tested after the above-mentioned processing then, beat even with liquid-transfering gun, afterwards again in the PCR instrument 45 ℃ of hybridization connect 30 minutes, 95 ℃ of sex change 4min again, be put into the 3min that anneals in-20 ℃ of ice chests again, at last the gained drips of solution be added in the land of above-mentioned nucleic acid transverse flow test strip, rapidly test strip is immersed the district and immerse in the running buffer (RunningBuffer), when arriving the uptake zone, liquid keeps flat test strip, observe phenomena behind the 10min.
The composition that contains film capillaceous in the described step (1) is a nitrocellulose filter; The composition of land is a polyester fiber; The composition that immerses the district is a glass fibre; The composition of uptake zone is a thieving paper; The model of glass fibre is SB06, and the model of polyester fiber film is VL68, and the model of thieving paper is SX27.
What contain on the film capillaceous the bag quilt in the described step (1) is the capture dna probe, and its sequence is one or several universal sequence, utilizes on the hybrid capture detection probes of DNA and its complementary universal sequence; That the bag quilt is Quality Control probe Control Probe on the nature controlling line, and wrap respectively by different capture probes (ZW-T and ZM-T) on two detection lines, wherein the capture dna concentration and probe concentration is 20 μ M/L, the bag dna probe consumption that is hunted down is 1 μ L/mm, and capture probe is dissolved in the solution that contains 2% sucrose and 5% methyl alcohol with the immobilized capture probes and the activity of preserving capture probe.Bag by line after test strip at room temperature dry and preserve.
The composition of the running buffer in the described step (2) is the Tris-HCl of 50mM/L, the NaCl of 50mM/L, the Tween-20 of 2ml/L and the sodium lauryl sulphate of 0.5g/L (SDS).
Table 1.KIF6 gene DNA sequence and probe sequence
In generation, diagnosis and the treatment of cardiovascular disorder (as coronary heart disease), KIF6 (kinesin-like protein 6) gene important influence.KIF6 (kinesin-like protein 6) albumen is a kind of and the interior transportation of cell proteins associated matter.The KIF6 genotype provides the important information beyond traditional risk factors, with high-risk patient that helps identification coronary heart disease and the treatment of carrying out personalization.The single nucleotide polymorphism (SNP) of KIF6 (719 Arg) has been proved the increase that can predict coronary heart disease (CHD) risk, and reduces the accident of statin (statins) treatment.
According to detecting principle (Fig. 1,3) and signal probe sequences Design (table 1), wrap by three lines at film (Membrane) with different capture probes, that the bag quilt is Quality Control probe Control Probe on the nature controlling line, be used to catch the Nano-Au probe that has neither part nor lot in ligation, the validity of colour developing indication test strip; And on first detection line 1 and second detection line 2 respectively bag be used to catch different connections survey product and assemble colour developing by different capture probes (ZW-T and ZM-T), thereby judge the genotype of Target DNA.
The present invention adopts dna sequence dna design detection probes and the linking probe of two probe in detecting methods according to KIF6 gene 719Arg allelotrope (Arg/Trp or Arg/Arg).At first choose the one section sequence (30bp) that has comprised KIF6 gene 719Arg allelotrope mononucleotide polymorphism site, synthetic two Target DNA, called after KIF-1 and KIF-2 correspond respectively to allelotrope KIF6 (719Arg) and KIF6 (719Trp).Sequence with the mononucleotide polymorphism site upstream is a template, designs its complementary nanometer gold detection probes (KIF-P-PS), and 5 ' is terminal through mercapto-functionalized modification, so that combine with nm gold particles.Design two linking probes (27bp) again, first base of 3 ' end is the single nucleotide polymorphism detection site, and the polymorphic site corresponding with KIF6 (719Arg) is C, probe called after Z-KIF-1, and the polymorphic site corresponding with KIF6 (719Trp) is T, probe called after Z-KIF-2; In addition 15 bases of 3 ' end and Target DNA (KIF-1 and KIF-2) are in the sequence complementation in polymorphic site downstream; 5 ' end 12 bases be one section with test strip on capturing probe complementary universal sequence.Capture probe on the test strip is two general sequence ZW-T and ZM-T, respectively with linking probe Z-KIF-1 and Z-KIF-2 half section sequence complementary pairing at 5 ' end.
Test strip detects the genotypic detection principle of KIF6 such as Fig. 1, Fig. 2 and Fig. 3, and all dna sequence dnas of using see Table 2, and the synthetic of probe finished by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Table 2.KIF6 gene DNA sequence and probe sequence
The preparation of described Nano-Au probe solution comprises:
The preparation of nanometer gold colloidal
Configuration concentration is that 1mM/L HAuCl4 solution and concentration are the citric acid three sodium solution of 38.8mM/L.HAuCl4 solution is heated to 130 ℃, in heat-processed, fully stir, the citric acid three sodium solution that rapid adding newly prepares when spending about 20 minutes (note insulation in this process, do not allow the cooling of HAuCl4 solution) 25ml continues stirring until solution and is cooled to room temperature, promptly forms burgundy solution.With 0.22 μ m cellulose nitrate nylon membrane filtering solution, can obtain evengranular nano-Au solution;
The preparation of the Nano-Au probe of dna marker
Getting concentration is the 13nm nano-Au solution 1.0mL of 12nM/L, and centrifugal 1 hour of 9500rpm, 4 ℃ go supernatant, and be resuspended with 97 μ L aseptic deionized waters; It is 3 μ M/L that the dna probe (concentration is 100 μ M/L) that adds 3 μ L marking sulfhydryls makes its ultimate density, and room temperature is placed spend the night (〉=12 hours); Progressively adding 1M/LNaCl solution, 0.1M/L (PH7.2) PB damping fluid (divides 3 times, for the first time each 3 μ L, each 4 μ L for the second time, each 5.5 μ L for the third time, 1 hour at interval) be respectively 0.1M/L, 10mM/L to final concentration, abundant mixing, room temperature is placed more than 48 hours; Add 0.01M/L PB, 0.1M/LNaCl mixing solutions to 1.0mL, centrifugal 1 hour of 9500rpm, 4 ℃ remove supernatant liquor, repeat twice; 0.01M/LPB, 0.1M/L NaCl mixing solutions with 100 μ l are resuspended, and 4 ℃ store for future use.
The preparation of described sample to be tested: adopt KIF6 genomic dna in the cracking process extracting serum sample, then KIF6DNA is carried out pcr amplification, make the clinical sample to be tested of KIF6 DNA.Wherein the pcr amplification system is: 10 * damping fluid, 1.5 μ l, Taq enzyme (1 unit), 25mmol/L MgCl
20.8 μ l, 25mmol/L dNTP 1 μ l, each 0.5 μ l of upstream and downstream primer, the primer final concentration is 0.5pmol/L; Amplification condition: 94 ℃ of sex change 4 minutes; 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 30 seconds, 35 circulations, 72 ℃ were extended 4 minutes.
The detected result of KIF6 DNA clinical sample single nucleotide polymorphism is judged
Observe the color of detection line 1, detection line 2 and three linearity region of nature controlling line in step (2) test strip, and judge the single nucleotide polymorphism of KIF6DNA clinical sample in view of the above.
If 1. nature controlling line does not manifest red stripes, this KIF6 genotype detection test strip is invalid so;
If 2. nature controlling line manifests red stripes, but detection line 1 and detection line 2 all do not manifest red stripes, and detecting the genotypic reaction system of KIF6 so has problem or invalid.
If 3. nature controlling line and detection line 1 all manifest red stripes, but detection line 2 does not manifest red stripes, the genotype of this KIF6 DNA is KIF6 (719Arg/Arg) so;
If 4. nature controlling line and detection line 2 all manifest red stripes, but detection line 1 does not manifest red stripes, the genotype of this KIF6DNA is KIF6 (719Trp/Trp) so;
If 5. detection line 1, detection line 2 and nature controlling line all manifest red stripes, the genotype of this KIF6DNA is KIF6 (719Arg/Trp) so.
The present invention utilizes the specific recognition single nucleotide polymorphism of Taq dna ligase in the ligase enzyme detection reaction (LDR), and according to the genotype of KIF6DNA clinical sample Nano-Au probe and corresponding acquisition sequence is linked together.Signal probe sequences Design such as table 1 are represented near KIF6 allelotrope 719Arg and the 719Trp Target dna fragmentation mononucleotide polymorphism site respectively with KIF-1 and KIF-2.As shown in Figure 2, in ligase enzyme detection reaction LDR, Target DNAKIF6 (719Arg) and KIF6 (719Trp), linking probe Z-KIF-1 and Z-KIF-2 also have the KIF-P-PS Nano-Au probe can hybridize the formation duplex structure, but because the specificity of Taq dna ligase, has only KIF6 (719Arg), Z-KIF-1 and KIF-P-PS and KIF6 (719Trp), these two kinds of structures of Z-KIF-2 and KIF-P-PS just can be under the effect of Taq dna ligase connect into a chain with linking probe Z-KIF-1 and Nano-Au probe KIF-P-PS or linking probe Z-KIF-2 and Nano-Au probe KIF-P-PS, be referred to as Z-KIF-Au-W and Z-KIF-Au-M respectively, therefore Nano-Au probe is connected together with acquisition sequence.And simultaneously, Target DNAKIF6 (719Arg), linking probe Z-KIF-2 and Nano-Au probe KIF-P-PS, perhaps these two kinds of structures of Target DNA KIF6 (719Trp), linking probe Z-KIF-1 and Nano-Au probe KIF-P-PS also can form two strands, but a single base mismatch is arranged in the joint, can't under the effect of Taq ligase enzyme, connect, after the reaction product sex change, therefore Nano-Au probe will separate with acquisition sequence.
The present invention utilizes base complementrity paired specificity in the DNA hybridization, and identification and the corresponding ligase enzyme detection reaction of KIF6DNA clinical sample genotype product (LDR Production) are caught Nano-Au probe in corresponding region clustering colour developing.The structure of test strip such as Fig. 1, two detection lines on test strip have fixed the paired single stranded DNA (capture probe ZW-T, ZM-T) mutually with linking probe Z-KIF-1 and Z-KIF-2 respectively.As shown in Figure 3, with ligase enzyme detection reaction product (LDRProduction) drip in test strip land (CP), and rapidly test strip is immersed when distinguishing in (IP) immersion running buffer (Running Buffer) solution, ligase enzyme detection reaction product (LDR Production) will be by siphon and on, the capture probe ZW-T of two detection lines and ZM-T will be respectively with wherein be connected product Z-KIF-Au-W and Z-KIF-Au-M hybridization, therefore Nano-Au probe is hunted down, and shows red thereby assemble; And under the situation of single base mismatch, only having linking probe Z-KIF-1 and Z-KIF-2 to be hunted down, Nano-Au probe will move to nature controlling line and combine colour developing with Control Probe capture probe.Therefore after experiment finishes, contrast the shade of two detection lines, just can judge the genotype of this KIF6DNA clinical sample.
Beneficial effect
The present invention is by ligase enzyme detection reaction (LDR), bind nucleic acid transverse flow test strip technology, set up a kind of fast, easy, disposable dna single nucleotide polymorphisms transmitter, and developed a kind of novel method that single nucleotide polymorphism detects that can be used for based on test strip.Relatively with existing other method, the present invention is based on the detection technique on the nucleic acid test strip, oligonucleotide probe is fixed on the test strip, utilize the Nano-Au probe of dna marker to be used for the detection of KIF6 gene mononucleotide polymorphism in conjunction with ligase enzyme detection reaction (LDR), but finish the detection to the KIF6 gene within a short period of time, the result is accurate, and is simple to operate, sensitivity can be satisfied the requirement of clinical detection, has a good application prospect aspect medical diagnosis.
Test strip preparation process based on nucleic acid among the present invention is simple, convenient, long and good stability of shelf time; The reaction process of ligase enzyme detection reaction (LDR) is simple, for single nucleotide polymorphism detection specificity height, and can finish within a short period of time; Have advantages such as quick, easy and cheap, sensitivity can be satisfied the requirement of clinical detection, has a good application prospect aspect medical diagnosis.Therefore present method is a kind of novel method that is used for gene pleiomorphism and genotype detection clinically.
Description of drawings
Fig. 1 is the synoptic diagram of nucleic acid transverse flow test strip;
Immerse district (Immersion pad), land (Conjugation pad), film (Membrane) and uptake zone (Absorbent pad); Film (Membrane) bag is by three lines, and that the bag quilt is Quality Control probe Control Probe on the nature controlling line, and on detection line 1 and the detection line 2 respectively bag by different capture probes (ZW-T and ZM-T);
Fig. 2 is the principle schematic that detects the KIF6 single nucleotide polymorphism based on ligase enzyme detection reaction (LDR);
Ligase enzyme detection reaction (LDR) is utilized the specific recognition single nucleotide polymorphism of Taq dna ligase, and according to the genotype of KIF6DNA clinical sample Nano-Au probe and corresponding acquisition sequence is linked together;
Fig. 3 is the colour developing schematic diagram of single nucleotide polymorphism test strip
According to base complementrity paired specificity in the DNA hybridization, identification and the corresponding ligase enzyme detection reaction of KIF6DNA clinical sample genotype product (LDR Production) are caught Nano-Au probe in corresponding region clustering colour developing.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
Detect the foundation of KIF single nucleotide polymorphism method based on nucleic acid transverse flow test strip
(1) preparation of the oligonucleotide nano Au probe of marking sulfhydryl
1. nanometer gold colloidal preparation
Configuration concentration is that 1mM/L HAuCl4 solution and concentration are the citric acid three sodium solution of 38.8mM/L.HAuCl4 solution is heated to 130 ℃, in heat-processed, fully stir, the citric acid three sodium solution that rapid adding newly prepares when spending about 20 minutes (note insulation in this process, do not allow the cooling of HAuCl4 solution) 25mL continues stirring until solution and is cooled to room temperature, promptly forms burgundy solution.With 0.22 μ m cellulose nitrate nylon membrane filtering solution, can obtain evengranular nano-Au solution.
2. the preparation of the Nano-Au probe of dna marker
Getting concentration is the 13nm nano-Au solution 1.0mL of 12nM/L, and centrifugal 1 hour of 9500rpm, 4 ℃ go supernatant, and be resuspended with 97 μ L aseptic deionized waters; It is 3 μ M/L that the dna probe (concentration is 100 μ M/L) that adds 3 μ L marking sulfhydryls makes its ultimate density, and room temperature is placed spend the night (〉=12 hours); Progressively adding 1M/LNaCl solution, 0.1M/L (PH7.2) PB damping fluid (divides 3 times, for the first time each 3 μ L, each 4 μ L for the second time, each 5.5 μ L for the third time, 1 hour at interval) be respectively 0.1M/L, 10mM/L to final concentration, abundant mixing, room temperature is placed more than 48 hours; Add 0.01M/L PB, 0.1M/LNaCl mixing solutions to 1.0mL, centrifugal 1 hour of 9500rpm, 4 ℃ remove supernatant liquor, repeat twice; 0.01M/LPB, 0.1M/L NaCl mixing solutions with 100 μ l are resuspended, and 4 ℃ store for future use.
(2) design of KIF6 DNA detection probe and synthetic
The present invention adopts dna sequence dna design detection probes and the linking probe of two probe in detecting methods according to KIF6 gene 719Arg allelotrope (Arg/Trp or Arg/Arg).At first choose the one section sequence (30bp) that has comprised KIF6 gene 719Arg allelotrope mononucleotide polymorphism site, synthetic two Target DNA, called after KIF-1 and KIF-2 correspond respectively to allelotrope KIF6 (719Arg) and KIF6 (719Trp).Sequence with the mononucleotide polymorphism site upstream is a template, designs its complementary nanometer gold detection probes (KIF-P-PS), and 5 ' is terminal through mercapto-functionalized modification, so that combine with nm gold particles.Design two linking probes (27bp) again, first base of 3 ' end is the single nucleotide polymorphism detection site, and the polymorphic site corresponding with KIF6 (719Arg) is C, probe called after Z-KIF-1, and the polymorphic site corresponding with KIF6 (719Trp) is T, probe called after Z-KIF-2; In addition 15 bases of 3 ' end and Target DNA (KIF-1 and KIF-2) are in the sequence complementation in polymorphic site downstream; 5 ' end 12 bases be one section with test strip on capturing probe complementary universal sequence.Capture probe on the test strip is two general sequence ZW-T and ZM-T, respectively with linking probe Z-KIF-1 and Z-KIF-2 half section sequence complementary pairing at 5 ' end.
Test strip detects the genotypic detection principle of KIF6 such as Fig. 1, Fig. 2 and Fig. 3, and all dna sequence dnas of using see Table 1, and the synthetic of probe finished by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
(3) preparation of nucleic acid transverse flow test strip
Test strip is formed by sticking on four of plastics adhesive patch cardboard parts, and (composition of 3mm * 15mm) is a glass fibre, is used for immersing and absorption running buffer (Running Buffer) for Immersion Pad, IP to immerse the district; (composition of 3mm * 10mm) is a polyester fiber, is used for reacting with the detection drips of solution thereon for Conjugation Pad, CP in the land; Film (composition of 3mm * 25mm) is a nitrocellulose filter for Membrane, M, in kapillary is arranged, utilize its syphonic effect will detect solution and up inhale, final and some DNA hybridization thereon; (3mm * 15mm) composition is a thieving paper, is used for the syphonic effect of reinforcing membrane for Absorbent Pad, AP in the uptake zone.
Package program is as follows: as shown in Figure 1, at first (M 25mm) sticks on the chipboard with film; (AP 15mm) is placed on the film (M) overlapping 2mm the uptake zone again; (CP 10mm) is placed on the film (M) overlapping 2mm the land again; (IP 15mm) is placed on the land (CP) overlapping 2mm immersing the district at last.
According to detecting principle (Fig. 1,3) and signal probe sequences Design (table 1), wrap by three lines at film (Membrane) with different capture probes, that the bag quilt is dna probe Control Probe on the nature controlling line, be used to catch the Nano-Au probe that has neither part nor lot in ligation, the validity of colour developing indication test strip; And on detection line 1 and the detection line 2 respectively bag be used to catch different connections survey product and assemble colour developing by different capture probes (ZW-T and ZM-T), thereby judge the genotype of Target DNA.Concentration and probe concentration is 20 μ M/L, and bag is 1 μ L/mm by the line consumption.Be the immobilized capture probes and the activity of preserving capture probe, capture probe is dissolved in the solution that contains 2% sucrose and 5% methyl alcohol.Bag by line after test strip at room temperature dry and preserve.
(4) detection of the single nucleotide polymorphism of KIF6DNA and judgement
At first preparation detects the genotypic reaction system of KIF6, the hybridization that is used for target dna and detection probes be connected, get the centrifuge tube of 3 150 μ L, difference mark 1,2,3, respectively add water 4 μ L, Nano-Au probe solution 2 μ L, linking probe Z-KIF-1 and Z-KIF-2 (concentration is 1 μ M/L) each 1 μ L, Taq dna ligase damping fluid 1 μ L and Taq dna ligase (concentration is 1 unit) 1 μ L, beat evenly with liquid-transfering gun, 4 ℃ stand-by.
Get synthetic Target DNA (concentration is 1 μ M/L) in (2) then, the KIF-1 and the KIF-2 that add 1 μ L in the centrifuge tube 1,2 respectively, the KIF-1 and the KIF-2 that add 1 μ L in the centrifuge tube 3 simultaneously, beat even with liquid-transfering gun, 45 ℃ of hybridization connect 30 minutes in the PCR instrument then, 95 ℃ of sex change 4min are put into the 3min that anneals in-20 ℃ of ice chests more again; Get 3 test strip in (3), respectively mark 1,2,3; Again with the drips of solution of above-mentioned centrifuge tube in the land of corresponding test strip (CP), rapidly test strip being immersed district (IP) immerses in running buffer (Running Buffer) solution, when liquid arrives uptake zone (AP), take out test strip and keep flat observe phenomena behind the 10min.
(5) judgement of the single nucleotide polymorphism of KIF6DNA
Observe the color of detection line 1, detection line 2 and three linearity region of nature controlling line in the test strip in the above-mentioned steps, and judge the single nucleotide polymorphism of KIF6DNA clinical sample in view of the above.
1. the detected result of KIF6 (719Arg) type DNA is judged
Nature controlling line on the test strip 1 and detection line 1 all manifest red stripes, but detection line 2 does not manifest red stripes, are KIF6 (719Arg) so judge this KIF6DNA, and be consistent with predicting the outcome of experimental program;
2. the detected result of KIF6 (719Trp) type DNA is judged
Nature controlling line on the test strip 2 and detection line 2 all manifest red stripes, but detection line 1 does not manifest red stripes, are KIF6 (719Trp) so judge this KIF6DNA, and be consistent with predicting the outcome of experimental program;
3. the detected result of KIF6 (719Arg/Trp) type DNA is judged
Detection line 1 on the test strip 3, detection line 2 and nature controlling line all manifest red stripes, are KIF6 (719Arg/Trp) so judge this KIF6DNA, and be consistent with predicting the outcome of experimental program.
By above result as can be seen, the result of experimental verification is consistent with predicting the outcome of experimental program, has shown that this method is used for feasibility and accuracy that single nucleotide polymorphism detects.
Embodiment 2
The single nucleotide polymorphism of KIF6DNA clinical sample detects and judges
(1) preparation of the oligonucleotide nano Au probe of marking sulfhydryl
Method is as described in the embodiment 1/ (1);
(2) design of KIF6DNA detection probes and synthetic
The present invention adopts dna sequence dna design detection probes and the linking probe of two probe in detecting methods according to KIF6 gene 719Arg allelotrope (Arg/Trp or Arg/Arg).At first choose the one section sequence (30bp) that has comprised KIF6 gene 719Arg allelotrope mononucleotide polymorphism site and be Target DNA, comprise allelotrope KIF6 (719Arg) and KIF6 (719Trp).Sequence with the mononucleotide polymorphism site upstream is a template, designs its complementary nanometer gold detection probes (KIF-P-PS), and 5 ' is terminal through mercapto-functionalized modification, so that combine with nm gold particles.Design two linking probes (27bp) again, first base of 3 ' end is the single nucleotide polymorphism detection site, and the polymorphic site corresponding with KIF6 (719Arg) is C, probe called after Z-KIF-1, and the polymorphic site corresponding with KIF6 (719Trp) is T, probe called after Z-KIF-2; In addition 15 bases of 3 ' end and Target DNA (KIF-1 and KIF-2) are in the sequence complementation in polymorphic site downstream; 5 ' end 12 bases be one section with test strip on capturing probe complementary universal sequence.Capture probe on the test strip is two general sequence ZW-T and ZM-T, respectively with linking probe Z-KIF-1 and Z-KIF-2 half section sequence complementary pairing at 5 ' end.
Test strip detects the genotypic detection principle of KIF6 such as Fig. 1, Fig. 2 and Fig. 3, and all dna sequence dnas of using see Table 2, and the synthetic of probe and primer finished by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
(3) preparation of nucleic acid transverse flow test strip
Method is as described in the embodiment 1/ (3);
(4) preparation of KIF6DNA clinical sample;
Gathering 2 genotype respectively is the individual serum sample of KIF6 (719Arg/Arg), KIF6 (719Trp/Trp) and KIF6 (719Arg/Trp), adopt KIF6 genomic dna in the cracking process difference extracting serum sample again, then KIF6DNA is carried out pcr amplification, makes the clinical sample to be tested of KIF6DNA:
With the serum cracking centrifugal after, get 1.5 μ l, add distilled water to 15 μ l; PCR system: 10 * damping fluid, 1.5 μ l, Taq enzyme (1 unit), 25mmol/L MgCl
20.8 μ l, 25mmol/L dNTP 1 μ l, each 0.5 μ l of upstream and downstream primer, the primer final concentration is 0.5pmol/L; Amplification condition: 94 ℃ of sex change 4 minutes; 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 30 seconds, 35 circulations, 72 ℃ were extended 4 minutes.Finally obtain three kinds of each 2 PCR product samples (the about 265bp of amplified production) (being designated as sample 1～sample 6);
(5) detection of KIF6DNA clinical sample single nucleotide polymorphism
1. at first preparation detects the genotypic reaction system of KIF6, the hybridization that is used for target dna and detection probes be connected, get the centrifuge tube of 6 150 μ L, add water 4 μ L, Nano-Au probe solution 2 μ L, linking probe Z-KIF-1 and Z-KIF-2 (concentration is 1 μ M/L) each 1 μ L, Taq dna ligase damping fluid 1 μ L and Taq dna ligase (concentration is 1 unit) solution 1 μ L, beat evenly with liquid-transfering gun, 4 ℃ stand-by.
2. the KIF6DNA clinical sample from (4) is respectively got 10 μ L (being designated as sample 01～sample 06) then, and 95 ℃ of sex change 4min in the PCR instrument put into-20 ℃ of ice chest annealing 3min again.
3. get the mixed solution that 1 μ L adds corresponding centrifuge tube in (5)/1. respectively in the sample from (5)/2., beat evenly with liquid-transfering gun, 45 ℃ of hybridization connect 30 minutes in the PCR instrument then, and 95 ℃ of sex change 4min are put into the 3min that anneals in-20 ℃ of ice chests more again; Get 6 test strip (being designated as 1～6) in (3), solution in (5)/is 3. dropped in the land (CP) of corresponding test strip respectively, rapidly test strip being immersed district (IP) immerses in the running buffer (Running Buffer), when liquid arrives uptake zone (AP), take out test strip and keep flat observe phenomena behind the 10min.
(6) detected result of KIF6DNA clinical sample single nucleotide polymorphism is judged
Observe the color of detection line 1, detection line 2 and three linearity region of nature controlling line in the test strip in (5)/3. step, and judge the single nucleotide polymorphism of KIF6DNA clinical sample in view of the above.
Detection line 1 and nature controlling line on the test strip 1,2 manifest red stripes, but detection line 2 does not manifest red stripes, are KIF6 (Arg/Arg) so judge the genotype of this KIF6DNA clinical sample, and be consistent with predicting the outcome of experimental program;
Detection line 2 and nature controlling line on the test strip 3,4 manifest red stripes, and still 1 does not manifest red stripes, is KIF6 (Trp/Trp) so judge the genotype of this KIF6DNA clinical sample, consistent with predicting the outcome of experimental program.
Detection line 1 on the test strip 5,6, detection line 2 and nature controlling line all manifest red stripes, are KIF6 (Arg/Trp) so judge the genotype of this KIF6DNA clinical sample, and be consistent with predicting the outcome of experimental program.
By above result as can be seen, the result of experimental verification is consistent with predicting the outcome of experimental program, has shown that this method is used for feasibility and accuracy that single nucleotide polymorphism detects.
Claims (8)
1. one kind is detected the method for single nucleotide polymorphism based on nucleic acid transverse flow test strip, comprising:
(1) will contain film capillaceous and stick on the chipboard, and the uptake zone will be placed on contain on the film capillaceous overlapping 1-2mm again; Again the land is placed on and contains on the film capillaceous overlapping 1-2mm; Be placed on the land immersing the district at last, overlapping 1-2mm gets nucleic acid transverse flow test strip; Described two detection lines and the nature controlling line that has on the film capillaceous with capture dna probe bag quilt that contain;
(2) get 1 μ L sample to be tested, 95 ℃ of sex change 4min in the PCR instrument put into-20 ℃ of ice chest annealing 3min again;
Each 1 μ L, Taq dna ligase damping fluid 1 μ L of 4 μ L, Nano-Au probe solution 2 μ L, linking probe Z-KIF-1 and Z-KIF-2 and the concentration of fetching water is the Taq dna ligase 1 μ L of 1 unit, beat with liquid-transfering gun even, mixed solution; In mixed solution, add KIF-1, the KIF-2 of 1 μ L or their mixed solution, beat even with liquid-transfering gun; Add the sample to be tested after the above-mentioned processing then, beat even with liquid-transfering gun, afterwards again in the PCR instrument 45 ℃ of hybridization connect 30 minutes, 95 ℃ of sex change 4min again, be put into the 3min that anneals in-20 ℃ of ice chests again, at last the gained drips of solution be added in the land of above-mentioned nucleic acid transverse flow test strip, rapidly test strip is immersed the district and immerse in the running buffer, when arriving the uptake zone, liquid keeps flat test strip, observe phenomena behind the 10min.
2. a kind of method according to claim 1 based on nucleic acid transverse flow test strip detection single nucleotide polymorphism, it is characterized in that: the composition that contains film capillaceous in the described step (1) is a nitrocellulose filter; The composition of land is a polyester fiber; The composition that immerses the district is a glass fibre; The composition of uptake zone is a thieving paper.
3. a kind of method that detects single nucleotide polymorphism based on nucleic acid transverse flow test strip according to claim 1, it is characterized in that: what contain on the film capillaceous the bag quilt in the described step (1) is the capture dna probe, its sequence is one or several universal sequence, utilizes on the hybrid capture detection probes of DNA and its complementary universal sequence; That the bag quilt is Quality Control probe Control Probe on the nature controlling line, and wraps respectively by different capture probes: ZW-T and ZM-T on two detection lines; Wherein the capture dna concentration and probe concentration is 20 μ M/L, and wrapping the dna probe consumption that is hunted down is 1 μ L/mm, and capture probe is dissolved in the solution that contains 2% sucrose and 5% methyl alcohol.
4. a kind of method that detects single nucleotide polymorphism based on nucleic acid transverse flow test strip according to claim 3, it is characterized in that: the sequence of described capture probe ZW-T is 5 '-TGCGGGTAATCG-TTTTTTTTTTTTTTTT-3 ', the sequence of capture probe ZM-T is 5 '-CAGCATCGTGCG-TTTTTTTTTTTTTTTT-3 ', and the sequence of Quality Control probe Control Probe is 5 ' AGAGGAGTTGGGACC-AAAAAAAAAAAAAAA-3 '.
5. a kind of method that detects single nucleotide polymorphism based on nucleic acid transverse flow test strip according to claim 1, it is characterized in that: the preparation of sample to be tested in the described step (2): adopt KIF6 genomic dna in the cracking process extracting serum sample, then KIF6 DNA is carried out pcr amplification, promptly; Wherein the pcr amplification system is: 10 * damping fluid, 1.5 μ l, Taq enzyme, 25mmol/L MgCl
20.8 μ l, 25mmol/L dNTP 1 μ l, each 0.5 μ l of upstream and downstream primer, the primer final concentration is 0.5pmol/L; Amplification condition: 94 ℃ of sex change 4 minutes; 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 30 seconds, 35 circulations, 72 ℃ were extended 4 minutes.
6. a kind of method according to claim 1 based on nucleic acid transverse flow test strip detection single nucleotide polymorphism, it is characterized in that: the sequence of the Nano-Au probe in the described step (2) is 5 '-PO4-GGTCCCAACTCCTCT-TTTTTTTTTTTTTTT-(CH2) 3-SH-3 '; Linking probe Z-KIF-1 sequence is 5 '-CGATTACCCGCA ACTCCCAGCATGAAC-3 ', and the sequence of linking probe Z-KIF-2 is 5 '-CGCACGATGCTG ACTCCCAGCATGAAT-3 '; The sequence of Target DNA KIF-1 is 5 '-AGAGGAGTTGGGACCGTTCATGCTGGGAGT-3 ', the sequence of TargetDNA KIF-2 is 5 '-AGAGGAGTTGGGACCATTCATGCTGGGAGT-3 '.
7. a kind of method that detects single nucleotide polymorphism based on nucleic acid transverse flow test strip according to claim 1, it is characterized in that: the composition of the running buffer in the described step (2) is the Tris-HCl of 50mM/L, the NaCl of 50mM/L, the Tween-20 of 2ml/L and the sodium lauryl sulphate of 0.5g/L.
8. a kind of method that detects single nucleotide polymorphism based on nucleic acid transverse flow test strip according to claim 1, it is characterized in that: the Taq dna ligase damping fluid composition in the described step (2) is the Tris-HCl of 200mmol/L, the KAcO of 250mmol/L, the MgAcO of 100mmol/L, the NAD of 10mmol/L, 1% Triton X-100, pH
7.6@25 ℃
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