CN104293897A - Method for rapid identification and quantification of single-stranded small-molecular nucleic acid combination - Google Patents
Method for rapid identification and quantification of single-stranded small-molecular nucleic acid combination Download PDFInfo
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Abstract
The invention relates to a method for rapid identification and quantification of a single-stranded small-molecular nucleic acid combination. Specifically, the invention provides a method for rapid identification and semi-quantification of at least one single-stranded small-molecular nucleic acid with a specific sequence or the combination of various single-stranded small-molecular nucleic acids with different sequences by using a nucleic acid sequence hybridization technology and through a specific antigen-antibody reaction on a chromatography medium. The method is especially suitable for application in qualitation and semi-quantification determination aiming at small-molecular ribonucleic acid (miRNA) changes related to various diseases.
Description
Technical field
The present invention relates to field of nucleic acid detection, particularly, the invention provides a kind of method of Rapid identification and quantitative strand small molecules Nucleic acid combinations, effluent sheet and test kit.
Background technology
Found by INVENTIONModern cell and molecular biological research, intracellular genetic expression is subject to the strict regulation and control of many factors on a molecular scale.Regulate and control and regulation and control and messenger RNA(mRNA) post-translational control before messenger RNA(mRNA) translation after regulation and control and genetic transcription before regulation and control are divided into genetic transcription.Wherein important regulatory mechanism is a complementary sequence for the 3' end being combined in messenger RNA(mRNA) by non-coding short chain Yeast Nucleic Acid specificity, suppresses the protein translation process of messenger RNA(mRNA).The generation of inhibition is introduce nuclease binding site after combining on the one hand, cause this messenger RNA(mRNA) by nuclease degradation, be combine the local duplex structure produced on the other hand, prevent rrna to the protein translation process of messenger RNA(mRNA).This noncoding short chain Yeast Nucleic Acid is commonly referred to as small molecule RNA (miRNA).
MiRNA is the Yeast Nucleic Acid of small molecule, length by the data base querying of the miRNA had been found that generally between 15-30 Nucleotide.After miRNA is found, cause the concern of numerous science researcher, by the research of molecular biology and clinical medicine aspect during the nearly last ten years, find that there is multiple miRNA and cancer has direct dependency, the content of some miRNA raises in cancer patients, the then reduction had, causes reduction and the rising of the expression amount of the related protein by they regulation and control, helps growth and the diffusion of cancer cells.Some miRNA also changes in immunne response and cardiovascular disorder in addition.
Along with the going deep into of research of miRNA, increasing miRNA is found, and the dependency of increasing miRNA and various diseases is identified.MiRNA demonstrates increasing importance in the Diagnosis and Treat of disease.MiRNA detection method known at present has: the real-time chain amplification method of reverse transcription (RT-Realtime PCR), the cDNA microarray (microarray) of application Hybridization principle, and traditional hybrid method.
According to technology known at present, all for the single-minded miRNA molecule of a kind of sequence when detection miRNA, because the miRNA comparision contents in the sample to which of single kind is few, in order to the sensitivity improving detection generally will apply comparatively sensitive multiple spot fluorescent labelling techniques in hybrid method, or nucleic acid linkage flag fluorophor technology, the application of this technology often can not use the full nucleotide sequence of miRNA to hybridize, although sensitivity is improved, but its hybridization specificity just declines to some extent, causes accuracy to reduce.
Another method is the sensitivity being improved detection by the real-time chain amplification technology of Yeast Nucleic Acid reverse transcription, but the accuracy due to nucleic acid synthesizing enzyme is not 100%, the Nucleotide that introducing one is wrong in amplification procedure, will cause the decline of detection accuracy.And the cost that above-mentioned method detects is all higher, the fluorescence detection device all needing price comparison high also carries out comparatively complicated labeled reactant, may cause inaccurate result and the artificial error imported like this.
In sum, this area is high in the urgent need to a kind of accuracy, and detection efficiency is high, the miRNA detection method of low cost.
Summary of the invention
The object of this invention is to provide a kind of accuracy high, detection efficiency is high, the miRNA detection method of low cost.
A first aspect of the present invention, provide a kind of method detecting strand small molecules nucleic acid in sample, described method comprises step:
I testing sample mixes with detection solution by (), formed and detect mixture;
Wherein, described detection solution contains detection probes, capture probe and reporter probe, and described detection probes has: the capture probe land and the reporter probe land that lay respectively at flank, and the target nucleic acid molecules land between described capture probe land and described reporter probe land;
(ii) make described detection mixture carry out hybridization, thus formed containing " target nucleic acid molecules-capture probe-reporter probe-detection probes " tetraplex, hybridization after detection mixture;
(iii) the detection mixture after described hybridization is carried out chromatography on chromatography media, wherein said chromatography media is provided with one or more mixture trapping region, thus described tetraplex is trapped in described mixture trapping region, form one or more chromatographic band; And
(iv) whether and/or quantity, thus whether and/or quantity the existence determining described sample small molecular nucleic acid in the existence detecting tetraplex described in described chromatographic band.
In another preference, described capture probe land, reporter probe land and miRNA land are next-door neighbours.(the base number of transcribed spacer is 0 or 1).
In another preference, described detection method is quantitative or half-quantitative detection.
In another preference, in described sample, comprise the strand small molecules nucleic acid (miRNA) that at least one sequence is single-minded.
In another preference, in described sample, comprise the strand small molecules Nucleic acid combinations that multiple sequence is different.
In another preference, described chromatography media is cellulose membrane.
In another preference, described capture probe, detection probes and reporter probe are thymus nucleic acid oligonucleotide, and the sequence of described each probe is exogenous sequence.
In another preference, the quantity of described trapping region is 1-20, preferably 2-10, more preferably 2-5, is provided with 2-4 trapping region best.
In another preference, described capture probe with or be connected with conjugated group, and described mixture trapping region is fixed with and catches group, and described conjugated group is caught or be incorporated into described group specificity of catching.
In another preference, the conjugated group of described capture probe is marked at 5' end or 3' end.
In another preference, described conjugated group is selected from lower group: vitamin H (biotin), fluorescein, Cyanine dyestuff, antigen, aglucon.
In another preference, described in catch group and be selected from lower group: avidin, anti-fluorescein antibody, Cyanine dyestuff antibody, antibody, part.
In another preference, described conjugated group is selected from lower group: vitamin H (biotin), fluorescein (fluorescein), fluorescence dye Cy3 (C
31h
37kN
2o
8s
2), or its combination.
In another preference, described conjugated group passes through chemically modified covalent cross-linking in capture probe.
In another preference, the conjugated group of capture probe forms specificity with the 5' terminal nucleotide of capture probe and is combined, and produces color reaction by the reporter group of reporter probe wherein.
In another preference, the conjugated group of the 5' end of described capture probe combines with the associated proteins on chromatography media specific position or antibody, thus makes chromatography media to produce visible colour band.
In another preference, described reporter probe with or be connected with reporter group or detectable label.
In another preference, described reporter probe is connected by covalent cross-linking mode with described reporter group or detectable label.
In another preference, described reporter group is the group that can produce color reaction, is preferably selected from lower group: nm gold particles, alkaline phosphatase, or its combination.
In another preference, described reporter group can be marked at 3' end or 5' end.
In another preference, when reporter group is marked at 3' end, described hybridization check reaction is with the use of the capture probe being marked with conjugated group at 5' end, and the complementary sequence of reporter probe is held at 5', the detection probes that sequence capture probe is held at 3'.
In another preference, when reporter group is marked at 5' end, described hybridization check reaction is with the use of the capture probe being marked with conjugated group at 3' end, and the complementary sequence of reporter probe is held at 3', the detection probes that sequence capture probe is held at 5'.
In another preference, described detection probes has the one or more features being selected from lower group:
Described capture probe land is the complementary sequence of capture probe;
Described reporter probe land is the complementary sequence of reporter probe; With
Described target nucleic acid molecules land is the complementary sequence of target nucleic acid molecules to be detected.
In another preference, described detection probes has the complementary sequence of the capture probe of the 3' end be positioned at, be positioned at the complementary sequence of reporter probe of 5' end, and the complementary sequence of target nucleic acid molecules between capture probe complementary sequence and reporter probe complementary sequence.
In another preference, described detection probes has the complementary sequence of the capture probe being positioned at 5' end, be positioned at the complementary sequence of reporter probe of 3' end, and the complementary sequence of target nucleic acid molecules between capture probe complementary sequence and reporter probe complementary sequence.
In another preference, when described detection probes is above-mentioned probe, described capture probe is the capture probe that 3' end is marked with conjugated group; And described reporter probe is the reporter probe that 5' end is marked with reporter group.
In another preference, described method has following one or more features:
Length N1=10-30 Nucleotide of capture probe land; And/or
Length N2=10-30 Nucleotide of reporter probe land; And/or
Length N3=10-30 Nucleotide of miRNA land; And/or
A total length N4=32-90 Nucleotide of detection probes sequence.
In another preference, described mixture trapping region is connected with the associated proteins for detecting small molecular core acid molecule or antibody.
In another preference, described is selected from lower group for the associated proteins or antibody detecting small molecular core acid molecule: biotin-binding protein, anti-fluorescein antibody, anti-fluorescence dye Cy3 antibody, or its combination.
In another preference, described associated proteins or antibody are the antibody of the conjugated group of mark capturing probe.
In another preference, described associated proteins or antibody are used in conjunction with sequence capture probe.
In another preference, described chromatography media is also provided with positive control area, described positive control area is connected with the associated proteins for direct-detection reporter group or antibody.
In another preference, associated proteins or the antibody of described direct-detection reporter group are selected from lower group: nm gold particles associated proteins, anti-nanometer gold antibody, alkaline phosphatase associated proteins, alkali-resistivity phosphoesterase antibody, or its combination.
In another preference, described associated proteins or antibody are combined with reporter group, thus make chromatography media to produce positive control band.
A second aspect of the present invention, provide a kind of effluent sheet, described effluent sheet comprises chromatography media, be positioned at the sample application zone of chromatography media one end and be positioned at the optional water absorbent sheet of the chromatography media the other end, and for the clamping of fixing chromatography media and immobilization material, it is characterized in that
Described chromatography media is provided with and is one or morely connected with the trapping region of catching group, for catching conjugated group;
Described chromatography media is provided with the positive control area being connected with direct-detection reporter group, in conjunction with reporter group; With
Described chromatography media is also provided with sample application zone, for adding testing sample.
In another preference, described in catch the antibody that group is anti-binding group.
In another preference, described direct-detection reporter group is the antibody of anti-reporter group.
In another preference, the quantity of described trapping region is 1-20, preferably 2-10, more preferably 2-5, is provided with 2-4 trapping region best.
A third aspect of the present invention, provides the test kit that a kind of detection comprises the sample of strand small molecules nucleic acid, it is characterized in that, comprising:
Detect solution, in described detection solution, comprise capture probe, detection probes and reporter probe;
One or more container, for holding detection solution;
Effluent sheet as described in respect of the second aspect of the invention;
Optional sample adding device; With
Specification sheets.
In another preference, described test kit is also containing sample adding device; Preferably, sample adding device is dropper or kapillary.
In another preference, described chromatography media is cellulose membrane.
In another preference, described test kit detects strand small molecules nucleic acid by method as described in the first aspect of the invention.
A fourth aspect of the present invention, provides effluent sheet as described in the first aspect of the invention or test kit as described in respect of the second aspect of the invention for detecting the purposes of the strand small molecules nucleic acid in testing sample.
In another preference, described testing sample is biological sample, is preferably selected from lower group: whole blood sample, serum sample, humoral sample, saliva sample, urine sample.
A fifth aspect of the present invention, provides the pre-checking device of a kind of disease, and described device comprises the effluent sheet described in second aspect present invention, and for the test set (or detector) of the detection signal that detects described effluent sheet.
In another preference, whether and/or quantity, thus whether and/or quantity the existence determining described sample small molecular nucleic acid in the existence that described test set detects tetraplex described in described chromatographic band.
In another preference, the chromatography media of described effluent sheet is provided with 1-5 trapping region, is preferably provided with 2-4 trapping region, and the chromatography media of described effluent sheet is also provided with positive control area.
In another preference, described device can be used for detecting the disease relevant to small molecules nucleic acid.
In another preference, described device can be used for detecting the disease being selected from lower group: cancer, cardiovascular disorder, exogenous microbial infect.
Sixth aspect present invention, provide a kind of method that detection comprises the sample of strand small molecules nucleic acid, described method comprises: use capture probe, detection probes and reporter probe, chromatography media carrys out Rapid identification strand small molecular core acid content by narrow spectrum antigen antibody reaction and/or protein with the combination of the chemical group of its specificity combination.
A seventh aspect of the present invention, provides a kind of previewing method of disease, and described method comprises use effluent sheet as described in respect of the second aspect of the invention or the test kit as described in third aspect present invention detects.
In another preference, described disease is selected from lower group: cancer, cardiovascular disorder, exogenous microbial infect.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
Fig. 1 shows the inventive method probe used and target nucleic acid molecules, wherein, conjugated group (as conjugated group A, B and C) can select vitamin H (biotin), fluorescein (fluorescein) or fluorochrome Cy3 (C
31h
37kN
2o
8s
2; Ex:548nm, Em:562nm); Reporter group can select nm gold particles, fluorophor or enzyme labelling (as alkaline phosphatase).
Fig. 2 shows detection method schematic diagram of the present invention, wherein, after capture probe is combined with conjugated group, Nucleotide is formed with miRNA sequence to be detected, the reporter probe that the other end and one end of miRNA sequence are connected with reporter group is connected, form the nucleotide sequence with detection probes complementation, and after hybridizing, form stable duplex structure with detection probes, and described conjugated group can be caught by the group of catching in respective strap.
Fig. 3 shows the actual Detection results figure in the embodiment of the present invention 2.
Fig. 4 shows the actual Detection results figure in the embodiment of the present invention 3.
Embodiment
The present inventor is through long-term and deep research, be surprised to find that, detection probes, end modified capture probe and end-labelled reporter probe and sample is adopted to hybridize under suitable conditions, and carry out catching and developing the color on chromatography media, can very efficient and the qualitative or strand small molecules nucleic acid that detects quantitatively in sample easily.Based on above-mentioned discovery, contriver completes the present invention.
Term
As used herein, term " exogenous sequence " refers to the engineer's sequence in targeted tissue not containing identical sequence.
Term " conjugated group " refers to the group for mark capturing probe, described group be positioned at combining for the associated proteins or antibody detecting small molecular core acid molecule thus the nucleic acid molecule of hybridization is fixed on chromatography media on chromatography media.Described conjugated group can be (but being not limited to) vitamin H (biotin), antigen or aglucon etc. can with the group of special groups specific binding.
Term " is caught group " and is referred to that for the group with conjugated group specific binding, described group is arranged on chromatography media, is combined with the associated proteins be marked on capture probe or antibodies specific, thus is fixed on chromatography media by the nucleic acid molecule of hybridization.Described catch group can be (but being not limited to) biotin-binding protein, antigen or part etc. can with the group of special groups specific binding.
Capture probe
As shown in Figure 1, capture probe is a thymus nucleic acid oligonucleotide chain be made up of 10-30 Nucleotide.Its one end is marked with conjugated group, is preferably 5' end.This conjugated group can be vitamin H (biotin), fluorescein (fluorescein) or fluorochrome Cy3 (C
31h
37kN
2o
8s
2; Ex:548nm, Em:562nm) wherein any one or its combination.
Described conjugated group for combining with the group of catching on chromatography media, thus makes " target nucleic acid molecules-capture probe-reporter probe-detection probes ", and tetraplex is caught by the specific trapping region on chromatography media.
Catching group is the group be combined with capture probe specificity be fixed on chromatography media.That catches group chooses the kind depending on capture probe, usually biotin-binding protein (streptavidin can be selected, avidin or neutravidin), the antibody of anti-fluorescein antibody or anti-fluorescence dye Cy3, they are by the specific site of cross-linking at chromatography media, and by catching the capture probe through this site in conjunction with the conjugated group on capture probe.
When 4 kinds of nucleic acid molecule reach finite concentration by the mixture that base pairing is formed, corresponding colour band just produces color reaction, and the height of concentration can carry out certainly quantitative analysis (as shown in Figure 4) by the weight of the band that develops the color and width in certain scope.
In detecting for different target nucleic acid molecules, need to arrange different sequence capture probes respectively on chromatography media.Sequence capture probe is all the sequence of the exogenous engineer do not contained in targeted tissue, and by ordinary method, the Blast program as the gene database of NIH (U.S. National Institutes) is tested.
Reporter probe
As shown in Figure 1, reporter probe is a thymus nucleic acid oligonucleotide chain be made up of 10-30 Nucleotide.In detecting for different target nucleic acid molecules, the sequence of reporter probe generally remains unchanged, its sequence is the sequence of the exogenous engineer that does not contain in targeted tissue, by the Blast routine check (Fig. 1) of the gene database of NIH (U.S. National Institutes).
One end of reporter probe is marked with reporter group, is preferably 3' end.When reporter group is aggregated to a certain degree, the color that naked eyes can be observed can be produced.Reporter group can be nm gold particles, also can be fluorophor or enzyme labelling, such as alkaline phosphatase.
Detection probes
As shown in Figure 1, detection probes is a thymus nucleic acid oligonucleotide chain be made up of 32-90 Nucleotide.Its one end flank has reporter probe land, and described reporter probe land is the complementary sequence of reporter probe, is preferably positioned at 5' end; Its other end flank has capture probe land, described capture probe land is the complementary sequence of capture probe, preferably be positioned at 3' end, is target nucleic acid molecules land between these two sequences, and described target nucleic acid molecules land is the complementary sequence of target nucleic acid molecules (as miRNA).
Detection probes of the present invention is used for and sequence capture probe, and target miRNA sequence and reporter probe sequence hybridization form duplex structure tetraplex.
Target nucleic acid molecules
As shown in Figure 1, target nucleic acid molecules is a shorter nucleic acid molecule, and length is between 12-30 Nucleotide.It can be thymus nucleic acid, also can be Yeast Nucleic Acid.It can be natural or organism nucleic acid molecule freely, also can be the nucleic acid molecule by chemical process or biological engineering method synthetic.In preference of the present invention, target nucleic acid molecules is miRNA.
Chromatography media
Chromatography media refers to a kind of medium, it can be the chromatographic material of different pore size, as cellulose membrane or through certain chemically treated cellulose membrane, the water absorbent sheet of draw solution is had at the other end of medium, when the well of sample solution in one end of medium adds, sample solution spreads through the other end of siphonage to medium.
As shown in Figure 4, in one embodiment of the invention, on chromatography media, set gradually one or more test strip (target nucleic acid molecules trapping region) during use, and a positive control band is set after each bar test strip.
Wherein, described test strip contains or is fixed with antibody or the associated proteins of the respective capture group be combined with the capture probe of sequence to be detected respectively, thus combines with corresponding group of catching specifically, and the band corresponding to sequence to be detected is developed the color.
Described positive control band is directly in conjunction with associated proteins or the antibody of reporter group, as nm gold particles associated proteins, anti-nanometer gold antibody, alkaline phosphatase associated proteins or alkali-resistivity phosphoesterase antibody.Described associated proteins or antibody combine with reporter group, thus positive control band is developed the color.
The hybridization of nucleotide sequence
Term " hybridization " refers to base pairing in the singlestranded RNA of the Yeast Nucleic Acid of strand or thymus nucleic acid and another or another one section of complete complementary wherein or thymus nucleic acid, forms the process of hydrogen bond between its base.
Described base pairing can be that traditional Hua Sheng-Ke Like matches, and also can be to swing pairing.One typically swings the pairing that pairing is guanine and uridylic, forms 2 pairs of hydrogen bonds between them.
Hybridization carries out under certain reaction conditions, influential because have the temperature that hybridization uses, the salt concn in hybridization solution to the accuracy of hybridization, in concentration and other chemical substances of mixing in hybridization solution of hybridization solution middle probe molecule, as PVOH 6000, Formamid etc.The condition of hybridization adjusts according to the length of target nucleic acid molecules that will detect and the content of its all kinds of Nucleotide, to reach single-minded, and sensitive requirement.In the present invention, described crossover process at 65-80 DEG C, or is carried out at 40 ~ 65 DEG C.
In another preference, described in the inventive method, the process of nucleic acid hybridization comprises:
The 3' terminal nucleotide of capture probe and the 5' terminal nucleotide of target miRNA form adjacent Nucleotide;
The 3' terminal nucleotide of target miRNA and the 5' terminal nucleotide of reporter probe form adjacent Nucleotide; With
Detection probes and above-mentioned three single stranded nucleic acid molecules carry out hybridization and match.
The detection of strand small molecules nucleic acid
The method of strand small molecules nucleic acid in detection sample provided by the invention, comprising:
I testing sample mixes with detection solution by (), formed and detect mixture, wherein said detection solution contains detection probes, capture probe and reporter probe, and described detection probes has the capture probe land and reporter probe land that lay respectively at flank and the target nucleic acid molecules land between described capture probe land and described reporter probe land;
(ii) carry out hybridization, thus form the detection mixture containing " target nucleic acid molecules-capture probe-reporter probe-detection probes " tetraplex;
(iii) the detection mixture after hybridization is carried out chromatography on chromatography media, wherein said chromatography media is provided with one or more mixture trapping region, thus described tetraplex is trapped in described mixture trapping region, form one or more chromatographic band;
(iv) whether and/or quantity, thus whether and/or quantity the existence determining described sample small molecular nucleic acid in the existence detecting tetraplex in described chromatographic band.
Detection method can be used as qualitative detection, also can be used as quantitatively or half-quantitative detection.
As shown in Figure 2, the trapping region (antibody band) be combined with reporter group for the narrow spectrum associated proteins of the conjugated group of one or more capture probe or immune antibody band and an energy is had the water absorbent sheet from the application of sample end of medium to the other end successively respectively.In diffusion process, described probe is hybridized with the small molecules nucleic acid detected in sample, make reporter group produce color reaction, and combined group is caught, and makes chromatography media demonstrates band, thus detects target nucleic acid molecules.
When capture probe, detection probes, reporter probe or target ribonucleic acid lack, the stable tetraplex be made up of 4 nucleic acid molecule just can not be formed.Especially when target nucleic acid molecules lacks, can not be formed and only have capture probe, the mixture of 3 nucleic acid molecule of reporter probe and detection probes composition.When this solution containing the duplex structure tetraplex formed by 4 nucleic acid molecule is added on a chromatography media, spread to certain direction.Chromatography media there is the conjugated group of one or more difference energy and capture probe form the band of specificity combination, when the duplex structure tetraplex molecule formed by 4 nucleic acid molecule is through the band of energy with the wherein conjugated group specificity combination of capture probe, just be combined on this band, when this quasi-molecule enrichment, reporter group just by reporter probe wherein produces color reaction, by the power of the color reaction of these three bands, just can with the naked eye or instrument semidefinite form calculate the content of contained target nucleic acid molecules.
When detecting multiple miRNA molecule, according to the colour developing degree of strength of each band, also half-quantitative detection can be carried out to sequence to be detected.
Disease preliminary examination
Relevant miRNA for a kind of disease is classified, is divided into the miRNA that (a) content rises, the miRNA that (b) content declines and (c) miRNA that content is constant or substantially constant in contrast.The miRNA risen for content uses same capture probe, the miRNA constant for content uses same capture probe, the miRNA reduced for content uses same capture probe, by with the constant miRNA of content as contrast, the miRNA that sxemiquantitative content rises or declines, and contrast with healthy group (or normal population), can be quick, whether judgement sample suffers from this type of disease exactly, the need of doing more deep inspection or long-term observation.The metrical error that single miRNA content is too low and occur can be solved like this.
Use detection method of the present invention, preliminary examination can be carried out to various dissimilar disease.Representational disease comprises (but being not limited to): cancer, cardiovascular disorder, exogenous microbial infection etc.
Major advantage of the present invention:
(1) can be quicker, sxemiquantitative, detect the miRNA in testing sample more accurately, experiment condition is gentle, and accuracy is high.
(2) can effectively for the preliminary examination of some diseases relevant to miRNA content, as cancer, cardiovascular disorder, exogenous microbial infect.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
Material
The preparation of embodiment 1 effluent sheet
Configuration is containing biotin-binding protein (Neutravidin) respectively, anti-fluorescein antibody, the antibody of anti-fluorescence dye Cy3 antibody and sheep anti mouse, the antibody-solutions of antibody protein concentration between 0.1 milligram every milliliter to 2 milligrams every milliliter, the antibody-solutions prepared is added on nitrocellulose filter in a linear fashion with commercially available Hamilton micro sample adding appliance (2 microlitre) respectively, totally 4 band, dry after application of sample immediately.Close with the solution containing glucose and glycerine after drying, after confining liquid adds, dry immediately.
Embodiment 2 is for the detection of people source hsa-miR-21-5p
By the has-miR-21-5p of synthetic and the capture probe 1 (can be combined with the specificity detection probe for hsa-miR-21-5p) with vitamin H, with the capture probe 2 (can be combined with the specificity detection probe for hsa-miR-16-5p) of fluorescein, with the capture probe 3 (can be combined with the specificity detection probe for hsa-miR-92a-5p) of fluorescence dye Cy3, for the specificity detection probe of hsa-miR-21-5p, for the specificity detection probe of hsa-miR-16-5p, for the specificity detection probe of hsa-miR-92a-5p, the nm gold particles being combined with reporter probe and mouse source antibody mixes in hybridization solution, and (hybridization solution is 20mM Tris-Cl pH7.5, 5mM MgCl2, 1mM EDTA, 150mM NaCl, 0.02%Tween20, 1mg/ml BSA).The solution of mixing, 65 DEG C of insulations 20 minutes, detects with effluent sheet prepared by embodiment 1.By sample joining side flow well, after waiting sample to spread completely, biotin-binding protein Neutravidin site is had to occur the band of nanometer gold crosslinked.On positive control band, also there is the band of a nanometer gold simultaneously.And other sites do not have band, see Fig. 3.
Embodiment 3 for people source hsa-miR-21-5p, the detection of hsa-miR-16-5p, hsa-miR-92a-5p
By the hsa-miR-21-5p of synthetic, hsa-miR-16-5p, hsa-miR-92a-5p and the capture probe 1 (can be combined with the specificity detection probe for hsa-miR-21-5p) with vitamin H, with the capture probe 2 (can be combined with the specificity detection probe for hsa-miR-16-5p) of fluorescein, with the capture probe 3 (meeting and the specificity detection probe for hsa-miR-92a-5p) of fluorescence dye Cy3, for the specificity detection probe of hsa-miR-21-5p, for the specificity detection probe of hsa-miR-16-5p, for the specificity detection probe of hsa-miR-92a-5p, the nm gold particles being combined with reporter probe and mouse source antibody mixes in hybridization solution, and (hybridization solution is 20mM Tris-Cl pH7.5, 5mM MgCl
2, 1mM EDTA, 150mM NaCl, 0.02%Tween20,1mg/ml BSA).The solution of mixing is incubated 20 minutes at 65 ° of C, detects with effluent sheet prepared by embodiment 1.By sample joining side flow well, after waiting sample to spread completely, all there is the band of nanometer gold at 3 binding sites.On positive control band, also there is the band of a nanometer gold simultaneously, see Fig. 4.
Result shows, and the small molecule RNA of three-type-person's work synthesis is all detected, and creates the band of positive result on a corresponding position.Experiment shows that the method may be used for detecting the different small molecule RNA of base sequence, and specificity is high, easy to detect, without the need to the instrument of special expensive, to testing staff without very strong professional requirement, applied widely.
Discuss
By the research of molecular biology and clinical medicine aspect during the nearly last ten years, find that there is multiple miRNA and cancer has direct dependency, the content of some miRNA raises in cancer patients, the then reduction had, cause reduction and the rising of the expression amount of the related protein by they regulation and control, help growth and the diffusion of cancer cells.Some miRNA also changes in immunne response and cardiovascular disorder in addition.Utilize effluent sheet of the present invention or test kit, can be quick, semidefinite form ground detects multiple miRNA.By selecting the content miRNA relevant to specified disease to contrast miRNA with suitable, disease early diagnosis and therapy can be used for as the preliminary examination means of disease.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. detect a method for strand small molecules nucleic acid in sample, it is characterized in that, comprise step:
I testing sample mixes with detection solution by (), formed and detect mixture;
Wherein, described detection solution contains detection probes, capture probe and reporter probe, and described detection probes has: the capture probe land and the reporter probe land that lay respectively at flank, and the target nucleic acid molecules land between described capture probe land and described reporter probe land;
(ii) make described detection mixture carry out hybridization, thus formed containing " target nucleic acid molecules-capture probe-reporter probe-detection probes " tetraplex, hybridization after detection mixture;
(iii) the detection mixture after described hybridization is carried out chromatography on chromatography media, wherein said chromatography media is provided with one or more mixture trapping region, thus described tetraplex is trapped in described mixture trapping region, form one or more chromatographic band; And
(iv) whether and/or quantity, thus whether and/or quantity the existence determining described sample small molecular nucleic acid in the existence detecting tetraplex described in described chromatographic band.
2. the method for claim 1, is characterized in that, described capture probe with or be connected with conjugated group, and described mixture trapping region is fixed with and catches group, and described conjugated group is caught or be incorporated into described group specificity of catching.
3. the method for claim 1, is characterized in that, described reporter probe with or be connected with reporter group or detectable label.
4. the method for claim 1, is characterized in that, described detection probes has the one or more features being selected from lower group:
Described capture probe land is the complementary sequence of capture probe;
Described reporter probe land is the complementary sequence of reporter probe; With
Described target nucleic acid molecules land is the complementary sequence of target nucleic acid molecules to be detected.
5. the method for claim 1, is characterized in that, described mixture trapping region is connected with the associated proteins for detecting small molecular core acid molecule or antibody; And/or
Described chromatography media is also provided with positive control area, described positive control area is connected with the associated proteins for direct-detection reporter group or antibody.
6. an effluent sheet, described effluent sheet comprises chromatography media, be positioned at the sample application zone of chromatography media one end and be positioned at the optional water absorbent sheet of the chromatography media the other end, and for the clamping of fixing chromatography media and immobilization material, it is characterized in that,
Described chromatography media is provided with and is one or morely connected with the trapping region of catching group, for catching conjugated group;
Described chromatography media is provided with the positive control area being connected with direct-detection reporter group, in conjunction with reporter group; With
Described chromatography media is also provided with sample application zone, for adding testing sample.
7. detection comprises a test kit for the sample of strand small molecules nucleic acid, it is characterized in that, comprising:
Detect solution, in described detection solution, comprise capture probe, detection probes and reporter probe;
One or more container, for holding detection solution;
Effluent sheet as claimed in claim 6;
Optional sample adding device; With
Specification sheets.
8. the purposes of effluent sheet as claimed in claim 6 or test kit as claimed in claim 7, is characterized in that, for detecting the strand small molecules nucleic acid in testing sample.
9. the pre-checking device of disease, is characterized in that, described device comprises effluent sheet according to claim 6, and the test set (or detector) of the optional detection signal for detecting described effluent sheet.
10. a detection comprises the method for the sample of strand small molecules nucleic acid, it is characterized in that, use capture probe, detection probes and reporter probe, chromatography media carrys out Rapid identification strand small molecular core acid content by narrow spectrum antigen antibody reaction and/or protein with the combination of the chemical group of its specificity combination.
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