CN101144814A - Method for detecting, identifying and/ or quantifying compound using adapter type reagent - Google Patents
Method for detecting, identifying and/ or quantifying compound using adapter type reagent Download PDFInfo
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- CN101144814A CN101144814A CNA200710163431XA CN200710163431A CN101144814A CN 101144814 A CN101144814 A CN 101144814A CN A200710163431X A CNA200710163431X A CN A200710163431XA CN 200710163431 A CN200710163431 A CN 200710163431A CN 101144814 A CN101144814 A CN 101144814A
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Abstract
The present invention relates to a method using adapter type reagent to detect, identify, and/or ration the compound. The present invention adopts the SELEX technology, uses various biologic molecules possessing the diagnosing value including the nucleotide sequence, the protein, the aminophenol, the polypeptide chain, the glucide, or the complete thalli as the target matters, and filters the corresponding high specificity adapter from which to obtain the target matters. The present invention has the advantages that the repeatability and the stability of the test result can be improved greatly, lots difference of the test can be reduced, and the reliability of the test result can be fully ensured. The present invention can be used to diagnose the clinic target molecular quickly and simply, and can provide favorable basis for the lab diagnosis. The present invention has short test time, low reagent cost, short research and development period, stable quality, etc.
Description
Technical field
The invention belongs to biomedical check field, the present invention relates to use the method for aptamer-based reagent detection, evaluation and/or quantification compound, this invention also relates to the preparation method of the aptamer-based detection kit that is used for this method.
Background technology
The reagent that is applied to clinical detection at present mostly is antibody class greatly, and majority is an import reagent.Because difference and stability problem in the antibody preparation process, every batch of reagent must be done typical curve when having determined quantitative measurement, and this makes complicated operating process loaded down with trivial details, and the material of or non-immunogenicity less to a part of immunogenicity can't detect.The present invention is directed to above problem, adopt adaptive sub-technology, screening process by the SELEX technology, obtain the special adaptive son of high-affinity of the target material of various tool clinical diagnosis meanings, be used for fast, accurately detecting corresponding target material, detect cost to improve the problem that existing clinical detection reagent exists, to reduce, research and develop the chemical diagnosis reagent of autonomous innovation.
The oligonucleotide aptamer technology is a kind of novel Protocols in Molecular Biology that development in recent years is got up, be that (systematic evolution of ligands byexponential enrichment, SELEX) screening obtains the phyletic evolution technology that adopts exponential enrichment part.The SELEX technology is a triage techniques of founding the beginning of the nineties in last century, be a kind of effective ways of studying nucleic acid structure and function, its ultimate principle is single stranded oligonucleotide library of external chemosynthesis, mix with the target material with it, form target material nucleic acid complexes, wash the nucleic acid that does not combine off, separate the nucleic acid molecules that combines with the target material with the target material, with this nucleic acid molecules is that template is carried out pcr amplification, carries out the screening process of next round again.By screening and the amplification that repeats, some do not combine with the target material or with the DNA of target material low-affinity, middle affinity or RNA molecule by flush away, and be referred to as " adaptive son " (aptamer) the DNA of high-affinity or RNA molecule are arranged from being separated the hangar with the target material, and purity is carried out and is increased with the SELEX process, from pmol to nmol, even, occupy the great majority in storehouse at last to μ mol.By the in-vitro screening and the amplification of many wheels, can from the random oligonucleotide library, filter out the adaptive son of specificity.A typical SELEX process may filter out adaptive son in several weeks to some months, and uses " SELEX technology automatically ", has not only reduced the interference of human factor, more can finish a SELEX process in a couple of days.This technology has big, the advantages such as the target molecule scope is wide, affinity is high, high specificity of storage capacity, be successfully applied to the screening of target substances of multiple types, except that being used for nucleotide sequence, protein, amino acid, also can be used for dyestuff, medicine micromolecule (as theophylline), growth molecule, peptide chain, steroids, carbohydrate, even can be used for complete cell, virus, spore and prion etc.Adaptive son has high degree of specificity, by reverse SELEX technology, even can filter out its corresponding adaptive son under the situation of not knowing target character.Aspect clinical detection, particularly to some unknown pathogenic bacterias or viral research, though do not know the epi-position of its inner structure, function and these materials, but with it as the target material, arrive its corresponding adaptive son by the SELEX technology screening, to detect the target material, become the research focus in this field.
Adaptive son has high-affinity to the identification of target molecule, and binding target molecule with high specificity.The adaptive subsolution of having sifted out at present generally at 0.05～50pmo1/L, is higher than the aglucon of other types from constant (kd).Adaptive son can be told fine distinction on the target molecule structure, can distinguish the difference of a methyl or a hydroxyl.Adaptive son can be used for the analogue that monoclonal antibody is difficult to distinguish or the antidiastole of cross-reacting antigen.Such as these 4 kinds of hormones of hTSH, hLH, hCG and hFSH, identical α chain is arranged, the structure of β chain is also quite similar, thereby will prepare with the specific monoclonal antibody of other three kinds of hormone no cross reactions very difficult.And adaptive son no cross reaction between these four kinds of hormones.Therefore, adaptive son can be applicable to the detection to certain target molecules.Need the certification mark thing is directly connected on the adaptive son in the technology of the adaptive son detection of existing application target molecule, not only influenced the dirigibility of detection method itself, and owing to of the influence of certification mark thing to adaptive sub-steric conformation, especially when the certification mark thing is relatively large, can directly cause the identification of adaptive son, the affinity of binding target molecule, specific change, thus accuracy and precision that influence detects.
The present inventor is through studying for a long period of time for this reason, proposed to contain single-side prolong-armed adaptive sub-fragment, and it is coated on the latex particle, when serum specimen adds reagent, target molecule to be measured in the serum forms aggegation with the latex particle that comprises adaptive sub-fragment, make the incident light scattering, scattered intensity is directly proportional with target material in the serum, can directly detect the concentration of respective target molecule by turbidimetry.This method not only provides the method for testing of the adaptive son of alternative utilization, and can avoid the direct influence that is connected with adaptive son adaptive son identification, binding ability of certification mark thing.Adopt aptamer-based molecule to do reagent molecule and can make the repeatability of detection better, reduce differences between batches, fully guarantee the reliability of measurement result.This detectable is applied to automatic clinical chemistry analyzer, can realizes the robotization and the mass that detect.This method also has short, advantages such as reagent cost is low, the R﹠D cycle is short, steady quality of test duration in addition.
Summary of the invention
The objective of the invention is to use the method for aptamer-based reagent detection, evaluation and/or quantification compound, this invention also relates to the preparation method and the application of the aptamer-based detection kit that is used for this method.
The phyletic evolution technology (SELEX technology) of the inventive method utilization index level enrichment part, to have the various biomolecule of diagnostic value, comprise nucleotide sequence, protein, amino acid, peptide chain, carbohydrate, or complete thalline is the target material, screening obtains the adaptive son of the corresponding high specific of these target materials, provides foundation for further utilizing adaptive sub-technology to carry out clinical diagnosis.Described adaptive son is the single stranded oligonucleotide that screening obtains, it is from a kind of strand at random library, described strand at random library can be that DNA also can be the RNA molecule, its two ends are that fixed sequence program is used to design primer, middle for random series be used to provide combine with various target materials enrich the nucleic acid aglucon.
The present invention utilizes the screening process of SELEX technology by repeatedly, obtain the special adaptive son of high-affinity of various target materials to be measured, by being modified, adaptive son strengthens its stability then, by single-side prolong-armed design and linking group labeled molecule such as fluorescein, biotin, radioactive isotope, collaurum or latex particle are connected on the adaptive sub-molecule again, be used for detecting corresponding target material from blood, urine, tissue specimen and various clinical samples, reach purpose quick, that accurately diagnose, may further comprise the steps:
1) the target material that seek from the various materials of tool clinical diagnosis meaning, preparation can be used for the SELEX technology screening comprises:
(1) protein of tool diagnostic significance, peptide chain are as hTSH, hLH, hCG, hFSH hormone; Human immunoglobulin(HIg) molecule etc.;
(2) nucleotide sequence of tool diagnostic significance is as hbv nucleic acid fragment, HIV viral nucleic acid fragment etc.;
(3) bacterium of tool diagnostic significance or virion are as Much's bacillus, Bacillus anthracis etc.
2) utilize the SELEX technology screening to obtain the adaptive son of various target materials, comprising:
(1) make up the suitable library of single stranded oligonucleotide at random, comprise single stranded DNA and/or RNA library, its two ends are that fixed sequence program is used to design primer, middle for random series be used to provide combine with various target materials enrich the nucleic acid aglucon;
(2) optimize the pcr amplification condition;
(3) asymmetric PCR method or biotin streptavidin paramagnetic particle method prepare single-stranded DNA banks;
(4) with nitrocellulose filter, affine resin or microwell plate be the target screening substances process of separating medium;
3) set up the clinical sample to be measured of adaptive sub-technology for detection, comprising:
(1) provides the adaptive son of high-affinity of target material, and it is modified, be primarily aimed at the adaptive son of RNA and modify;
(2) adaptive sub-molecule is carried out single-side prolong-armed modification;
(3) adopt linking group that labels such as fluorescein, biotin, radioactive isotope, collaurum or latex particle are marked at the extension arm end the adaptive son that obtains is carried out mark;
(4) mix the reagent molecule contain adaptive son and to be detected, evaluation and/or quantified sample;
(5) signal of detection bond changes.
Wherein single-side prolong-armed is nucleotide fragments; Adaptive son can be the adaptive son of microorganism, virus, the adaptive son of protein, peptide hormone or the adaptive son of nucleic acid molecules, the preferably adaptive son of each proteinoid, peptide hormone; The certification mark thing is one or more combinations in radioactive isotope, chemiluminescent groups, chemiluminescence group, latex particle, fluorescin, enzyme, antigen, antibody, part or the acceptor, preferably latex particle; The preparation method of aptamer-based detection molecules provided by the invention comprises single-side prolong-armed and cohesive process label.
The invention provides the application in clinical detection, drugs detection, food safety detection of described kit of the inventive method or aptamer-based detection molecules of the present invention.
The adaptive son of human immunoglobulin(HIg) E provided by the invention, its sequence are 5 '-GGGGACGTTTATCCGTCCCTCCTAGTGGCGTGCCCC-3 '.
At adaptive subsequence part nucleic acid fragment is strand, can prolong by modification.And single-side prolong-armed be the single-chain nucleic acid sequence that is connected with adaptive subsequence, and its nucleotide sequence end is connected with the certification mark thing, therefore can detect measured object by the single-side prolong-armed certification mark thing that has.In this article, nucleic acid is represented its sequence with symbol well-known to those skilled in the art, and what sequence was represented if no special instructions is the sequence order of holding to 3 ' extreme direction from 5 '.
Term herein " single-side prolong-armed " refers to one section continuous sequence in the end adding of adaptive subsequence, thereby labeled molecule and the nucleic acid fragment that contains adaptive son can be combined, do not influence simultaneously the steric conformation of adaptive son, its sequence length is decided on the sequence length of adaptive son usually, above-mentioned single-side prolong-armed suitable sequence length is preferably 5 to 50 bases, more preferably 5 to 30 bases, more preferably 8 to 15 bases.
Term herein " adaptive son " is well known to those skilled in the art, refer to can with the single-chain nucleic acid of specific target molecule specific bond, comprise DNA or RNA fragment.Can obtain the adaptive son of certain target molecules at short notice by the SELEX technology.At present there has been a large amount of adaptive subsequences to be disclosed, therefore adaptive son herein can be selected from the adaptive son of microorganism, virus, the adaptive son of protein, peptide hormone or the adaptive son of nucleic acid molecules, the adaptive son of protein, peptide hormone preferably is as the adaptive son of human immunoglobulin(HIg) E.Because adaptive son has the character that combines certain target molecules similar with antibody, therefore adaptive son can replace the effect of antibody in detection, and the inventor has designed the prolongation sequence of single-chain nucleic acid in the side of adaptive subsequence, obtained the method for first aspect of the present invention, make adaptive subsequence and prolong fragment can the incorporation of markings molecule, then the signal by the certification mark molecule change calculate target molecule existence whether and content, thereby detection, evaluation and/or quantifying target molecule.
The present invention can modify increase stability to it, with the degraded of opposing nucleic acid in vivo enzyme further by finding the structure of adaptive son; Adopt standard items to carry out doubling dilution and detect the typical curve of making determinand.The inventive method can significantly improve repeatability, the stability of testing result, reduce the differences between batches that detect, and fully guarantee the reliability of measurement result.Can be used for quick, the easy diagnosis of clinical target molecule, can provide favourable foundation for laboratory diagnosis, this method also has short, advantages such as reagent cost is low, the R﹠D cycle is short, steady quality of test duration.The inventive method will be opened new approach for clinical diagnosis, also provide new research thinking for the exploitation the detection kit.So the present invention has better detection specificity and repeatability than the clinical detection method of routine, but also has the following advantages: 1. detection time is short.After mixing adaptive son detection sequence and sample, usually as long as reaction just can detect in 5 minutes.2. production cost is low.Adaptive son and extension arm sequence thereof are as long as just can obtain by chemosynthesis and modification, and the DNA synthetic technology is very ripe at present, and cost is extremely low.And protein antibodies will obtain by animal immune and purifying, and cost is higher.3. the new-product development cycle is short.By the SELEX technology, can in 1 month, obtain the adaptive son that combines with the target molecule high special; And the research and development time of protein antibodies needs more than 6 months at least.4. constant product quality.Nucleic acid molecules is more stable than protein antibodies, is easy to long-time preservation, and this prolongs the term of validity of product greatly.
In this article, linking group is the chemical group that those skilled in the art know, and they can be connected labeled molecule on the adaptive daughter nucleus acid fragment by covalent bond.These technology extensively are present among the nucleic acid technique for fixing of nucleic acid microarray, nucleic acid chip.In order more stably to synthesize aptamer-based detectable, the preferred linking group of the present invention is antigen or antibody, part or acceptor, more preferably biotin or affinity element.Pass through biotin at extension arm sequence end---the combination of affinity element makes labeled molecule be connected to the complete aptamer-based detectable of formation on the nucleic acid fragment.In a specific embodiment of the present invention, preferred single-chain nucleic acid fragment contains adaptive subsequence and the single-side prolong-armed sequence (catenation sequence is preferably GCGCGCGC) of human immunoglobulin(HIg) E; Linking group is a biotin, is marked at the 5 ＇ end of single-side prolong-armed sequence.
In this article, the certification mark thing that single-side prolong-armed sequence connects be can be detected material, include but not limited to one or more combinations in radioactive isotope, chemiluminescent groups, chemiluminescence group, fluorescin, enzyme, colloid gold particle, the latex particle.Radioisotopic suitable example has
32P,
125I,
35S,
3H etc.Chemiluminescent groups has luminol etc.; The chemiluminescence group comprises rhodamine, FITC, TRITC etc.By the chemical synthesis process of routine, can be to adaptive daughter nucleus acid sequence with enzyme labelings such as horseradish peroxidase, alkaline phosphatases.And there have been a large amount of commercial certification mark thing and reaction kit thereof available.In the present invention, the certification mark thing of preferred adaptive subsequence connection is a latex particle.
Description of drawings
Fig. 1 is the process flow diagram of the adaptive son of SELEX process screening human immunoglobulin(HIg) E;
Fig. 2 is not in conjunction with the adaptive sub-compound of human immunoglobulin(HIg) E;
Fig. 3 is the typical curve that detects human immunoglobulin(HIg) E with the human immunoglobulin(HIg) E detection kit of the present invention's preparation.
Embodiment
Preparation method with the adaptive son of high-affinity DNA that can combine with human immunoglobulin(HIg) E is an example, carries out according to the following step:
1. prepare and purifying protein
The preparation of related human immunoglobulin(HIg) E and purifying be this area oneself know technology (commercially available), will obtain the pure product of human immunoglobulin(HIg) E as screening target material.
2. make up random single chain DNA (ssDNA) library and primer
(1) synthetic ssDNA library at random
Synthetic length is the library of ssDNA at random of 79bp, and two ends are fixed sequence program, middle 35 single stranded DNA storehouses that base is a random series, and its storage capacity is 10
14-15[5 ＇-CCC CTG TCA GAA GTG TTT CTCAA-(N35)-AGA GAT GAG CAG GCG GAT ACT-3 ＇], wherein N represents base A, G, C, any one among the T.Random single-stranded DNA banks is synthetic by Synbiotics AB.
(2) primer is synthetic
Primer is by Primer Design5.0 software design, and upstream primer and downstream primer correspond respectively to the fixed sequence program at two ends, random dna library, downstream primer 5 ＇ end mark biotin.
3. the preparation of the adaptive son of specificity:
Repeat to select and amplification at the constructed ssDNA library of testing molecule by the SELEX technology,, the adaptive son that is obtained is cloned, checked order, and check the adhesion of itself and target molecule through strict selection and the amplification of number wheel.The screening number of times is decided according to every strict degree of taking turns washing non-specific adsorption DNA or RNA and target Substance Properties, the screening wheel number is set at 12 takes turns.
The carbonic acid buffer bag of 10 μ g human immunoglobulin(HIg) E being used pH9.6 is by on elisa plate, and 37 ℃ act on 3 hours, set the blank hole simultaneously.Sample aperture and control wells were sealed 2 hours with 3% BSA.The single-stranded DNA banks 1ng of purifying is acted on 40 minutes with the blank hole earlier in SELEX binding buffer liquid, anti-sieve is removed the single stranded DNA that combines with BSA, transfer to human immunoglobulin(HIg) E bag then by hole and protein combination 40 minutes, with SELEX binding buffer liquid washing 6 times, add the SELEX eluent again in 80 ℃ of effects 10 minutes, the ssDNA that wash-out combines with human immunoglobulin(HIg) E down through the extracting of phenol chloroform, precipitation with alcohol, obtains the single stranded DNA of purifying.Repeat above-mentioned steps, 12 affinity of taking turns adaptive son in back and human immunoglobulin(HIg) E reach capacity, obtain can with the single-strand DNA aptamer of human immunoglobulin(HIg) E high-affinity.
Not modified adaptive son as adaptive son is carried out suitable modification, then can be avoided degraded easily by the degraded of the nucleotidase in the body fluid.Can strengthen of the opposing of adaptive son with the modification of phosphorus sulphur acetate (phosphrothiate) connector to nuclease.Nuclease in the body fluid is the pyrimidine specificity, and the pyrimidine that replaces 2 ＇ positions then can be avoided being degraded, and with preparing adaptive son again behind 2 ＇ amino or the fluorine-based substituted pyrimidines of 2 ＇, then is more suitable for diagnosis and treatment.
4. adaptive sub-affinity is measured
Every SELEX screening product of taking turns carries out pcr amplification with the primer of mark biotin, behind the extracting of phenol chloroform, precipitation with alcohol purifying, measures content, with the testing protein molecular mixing and add the horseradish peroxidase-labeled Avidin, chromogenic assay affinity.
5. the clone of adaptive son and order-checking
Last is taken turns the adaptive son usefulness of the ssDNA that filters out and is not carried out pcr amplification with the primer of biotin, product the pMD18-TSimple Vector kit that provides with TaKaRa company behind the purifying is provided is connected on the pMD18-T carrier, be transformed into bacillus coli DH 5 alpha, ammonia Bian resistance screening, the single growth bacterium colony of picking checks order.
6. with the pairing adaptive son biotin labeling of each single growth bacterium colony, amount is 0.1 μ g, with the human immunoglobulin(HIg) E of every hole 10 μ g bag quilt in SELEX binding buffer liquid 37 ℃ combine 40 minutes, with SELEX dcq buffer liquid washing 6 times, the horseradish peroxidase mark strepto-affinity element that adds 1: 1000,37 ℃ act on 30 minutes, with PBS damping fluid washing 4 times, flush away is not gone up the enzyme mark strepto-affinity element that the DNA biotin combines with human immunoglobulin(HIg) E, add tetramethyl benzidine then color development at room temperature effect 20 minutes, add the reaction of 2M concentrated sulphuric acid color development stopping, 450nm enzyme connection instrument is measured the OD value, choose the highest adaptive son of DNA of 0D value, this adaptive son be can with the adaptive son of DNA of human immunoglobulin(HIg) E specific bond, should adaptive son order-checking obtain its sequence, and carry out the secondary structure collection of illustrative plates (see figure 2) that structure analysis obtains this adaptive son.
The used SELEX binding buffer liquid of the present invention is: 8.1mM Na
2HPO
4, 1.1mM KH
2PO
4, 1mMMgCl
2, 2.7mM KC1,138mM NaCl, pH 7.4; SELEX dcq buffer liquid is: 5 mM Na
2HPO
4, 5mM KH
2PO
4, 2mM MgCl
2, 1% (v/v) Tween-20, pH7.4; The PBS damping fluid is 10 μ MPBS buffer, 0.2M NaCl, pH7.9.
The present invention tests used phenol chloroform extract: the phenol of 25ml, the chloroform of 24ml, the iso pentane alcohol mixture of 1ml.
To carry out single-side prolong-armed modification by the adaptive son that said process obtains, by the plain linking group of biotin one affinity fluorescein, biotin, radioactive isotope, collaurum or latex particle are marked at single-side prolong-armed end then, the adaptive son that obtains is converted into the adaptive son of report, just can from clinical blood, urine, tissue specimen and culture supernatant, detect corresponding target material, thereby reach purpose quick, that accurately detect.
The adaptive filial generation of the human immunoglobulin(HIg) E that obtains with the present invention is for antibody, carry out the detection of human immunoglobulin(HIg) E, have many superiority: because the affinity of adaptive son and antigen is better than the affinity between the antigen-antibody, thereby adaptive son combines with strong specificity between conjugated antigen, almost can avoid non-specific binding fully, even and the albumen of less immunogenic can obtain the adaptive son of its high specific too; The screening of adaptive son is shorter than the antibody screening time, is easy to preparation, good stability, and easy mark.Therefore, utilize the adaptive son of specificity of SELEX technology screening human immunoglobulin(HIg) E, a kind of new research thinking will be provided for the diagnosis of people's immunoglobulin E.
Claims (10)
1. method of using aptamer-based reagent detection, evaluation and/or quantification compound, it is characterized in that, utilize the SELEX technology, to have the various biomolecule of diagnostic value, comprise that nucleotide sequence, protein, amino acid, peptide chain, carbohydrate or complete thalline are the target material, screening obtains the adaptive son of the corresponding high specific of these target materials, may further comprise the steps:
1) the target material that seek from the various materials of tool clinical diagnosis meaning, preparation can be used for the SELEX technology screening comprises:
(1) protein of tool diagnostic significance, peptide chain are as hTSH, hLH, hCG, hFSH hormone; The human immunoglobulin(HIg) molecule;
(2) nucleotide sequence of tool diagnostic significance is as hbv nucleic acid fragment, HIV viral nucleic acid fragment;
(3) bacterium of tool diagnostic significance or virion are as Much's bacillus, Bacillus anthracis;
2) utilize the SELEX technology screening to obtain the adaptive son of various target materials, comprising:
(1) make up the suitable library of single stranded oligonucleotide at random, comprise single stranded DNA and/or RNA library, its two ends are that fixed sequence program is used to design primer, middlely combine the nucleic acid aglucon that enriches with various target materials for random series is used to provide;
(2) optimize the pcr amplification condition;
(3) asymmetric PCR method or biotin streptavidin paramagnetic particle method prepare single-stranded DNA banks;
(4) with nitrocellulose filter, affine resin or microwell plate be the target screening substances process of separating medium;
3) set up the clinical sample to be measured of adaptive sub-technology for detection, comprising:
(1) provides the adaptive son of high-affinity of target material, and it is modified;
(2) adaptive sub-molecule is carried out single-side prolong-armed modification;
(3) adopt linking group that the certification mark thing is carried out mark at the extension arm end to the adaptive son that obtains;
(4) mix the detectable contain adaptive son and to be detected, evaluation and/or quantified sample;
(5) signal of detection bond changes.
2. the method for the aptamer-based reagent detection of application according to claim 1, evaluation and/or quantification compound is characterized in that, described single-side prolong-armed be nucleotide fragments, its sequence length is 5 to 50 bases.
3. the method for the aptamer-based reagent detection of application according to claim 1, evaluation and/or quantification compound, it is characterized in that described adaptive son is adaptive son or protein, the adaptive son of peptide hormone or the adaptive son of nucleic acid molecules of microorganism, virus.
4. the method for the aptamer-based reagent detection of application according to claim 1, evaluation and/or quantification compound, it is characterized in that, described certification mark thing is one or more combinations in radioactive isotope, chemiluminescent groups, chemiluminescence group, latex particle, fluorescin, enzyme, antigen, antibody, part or the acceptor, preferably latex particle.
5. a kit of using aptamer-based reagent detection, evaluation and/or quantification compound is characterized in that, comprises the adaptive son that obtains by following steps:
1) the target material that seek from the various materials of tool clinical diagnosis meaning, preparation can be used for the SELEX technology screening comprises:
(1) protein of tool diagnostic significance, peptide chain are as hTSH, hLH, hCG, hFSH hormone; The human immunoglobulin(HIg) molecule;
(2) nucleotide sequence of tool diagnostic significance is as hbv nucleic acid fragment, HIV viral nucleic acid fragment;
(3) bacterium of tool diagnostic significance or virion are as Much's bacillus, Bacillus anthracis;
2) utilize the SELEX technology screening to obtain the adaptive son of various target materials, comprising:
(1) make up the suitable library of single stranded oligonucleotide at random, comprise single stranded DNA and/or RNA library, its two ends are that fixed sequence program is used to design primer, middle for random series be used to provide combine with various target materials enrich the nucleic acid aglucon;
(2) optimize the pcr amplification condition;
(3) asymmetric PCR method or biotin streptavidin paramagnetic particle method prepare single-stranded DNA banks;
(4) with nitrocellulose filter, affine resin or microwell plate be the target screening substances process of separating medium;
3) set up the clinical sample to be measured of adaptive sub-technology for detection, comprising:
(1) provides the adaptive son of high-affinity of target material, and it is modified;
(2) adaptive sub-molecule is carried out single-side prolong-armed modification;
(3) adopt linking group that the certification mark thing is connected the extension arm end the adaptive son that obtains is carried out mark;
(4) mix the detectable contain adaptive son and to be detected, evaluation and/or quantified sample;
(5) signal of detection bond changes.
6. kit according to claim 5 is characterized in that, described single-side prolong-armed be nucleotide fragments, its sequence length is 5 to 50 bases.
7. kit according to claim 5 is characterized in that, described adaptive son is adaptive son or protein, the adaptive son of peptide hormone or the adaptive son of nucleic acid molecules of microorganism, virus.
8. kit according to claim 5 is characterized in that, described linking group is antigen or antibody, part or acceptor, one or more combinations in biotin or the affinity element, preferably biotin-avidin group.
9. kit according to claim 5, it is characterized in that, described certification mark thing is one or more combinations in radioactive isotope, chemiluminescent groups, chemiluminescence group, latex particle, fluorescin, enzyme, antigen, antibody, part or the acceptor, preferably latex particle.
10. each described kit of claim 5-9 or each described method of claim 1-4 are applied in clinical detection, drugs detection, the food safety detection.
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- 2007-10-22 CN CNA200710163431XA patent/CN101144814A/en active Pending
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