CN103045720B - Targeting molecule for detecting pathogenic cell and application thereof - Google Patents
Targeting molecule for detecting pathogenic cell and application thereof Download PDFInfo
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- CN103045720B CN103045720B CN201110315281.6A CN201110315281A CN103045720B CN 103045720 B CN103045720 B CN 103045720B CN 201110315281 A CN201110315281 A CN 201110315281A CN 103045720 B CN103045720 B CN 103045720B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
Abstract
The invention relates to a targeting molecule for detecting a pathogenic cell and an application thereof. According to a method provided by the invention, signal amplification is conducted on maker, namely oligonucleotide, so that the detection sensitivity of special molecules on the surface of the pathogenic cell can be improved; and various molecules to be detected can be encoded by different oligonucleotide sequences, so that multiple detections can be realized.
Description
Technical field
The invention belongs to biological technical field; More specifically, the present invention relates to targeting molecule of detecting in pathogeny cell and its preparation method and application.
Background technology
Numerous clinical datas and research show, to the detection by quantitative of pathogeny cell, are accurately to judge conditions of patients and give the direct evidence for the treatment of in time.For example, in blood, the quantity of circulating tumor cell is the important factor that determines patient's prognosis.Equally, to the plasmodial detection in blood, be also the important indicator of the judgement malaria state of an illness.
Traditional detection method is based on to the detection of pathogeny cell-surface antigens and quantitatively substantially.In these methods, first antibody be attached on a solid carrier, due to interaction antibody and the cell to be detected formation mixture of antibody-antigen.Same principle, antibody may first form mixture by antigen-antibody reaction and cell to be checked, and then is attached on a solid carrier.Thereafter, the cell of being caught by antibody is collected with mark to come quantitatively with this.Such as immunofluorescence technique detects circulating tumor cell.First with the antibody that is attached to magnetic bead or micro-fluid chip, by tumour cell, from blood, enrichment is out.Then use with the antibody labeling tumour cell of marker, tumor cell specific and carry out detection by quantitative.In general, marker comprises radioactive isotropic substance, dyestuff, fluorescein and enzyme (for the reaction of enzyme mark) etc.Although the detection method based on antigen capture is widely used clinically, these methods have many weak points:
(1) detection sensitivity is low.It is mainly by cell antigen are directly detected that antigen enzyme mark detects.The detection limit of these methods is subject to target cell surface antigen and counts quantitative limitation, and detectability is conventionally～10
-9m.Therefore (antigen concentration < 10 when cell quantity is rare
-12m), insufficient sensitivity.Therefore, this area is necessary to develop new detection reagent or method very much at present, to improve the sensitivity of detection.
(2) flux is low.The flux of these methods is limited to manually.Therefore, flux is lower, is not suitable for a large amount of examinations.
(3) troublesome poeration and personal errors are large.The operation steps of traditional method is complicated, and manual request is high.Therefore unavoidably there are a lot of artificial experimental errors of introducing, thereby cause the credibility of detected result to decline.
(4) Multiple detection.It is the important guiding index of clinical diagnosis and treatment somatotype that a plurality of antigens of sample are detected simultaneously.Due to the restriction of marker, traditional method detects the 3-4 of a pathogeny cell antigen at most in same detection sample simultaneously.Therefore, can not carry out large-scale scanning to the antigen of sample.
Now technology to the detection of certain cell often the diad theory based on an A-B: A represent that one for the ligand molecular (K of specific, the high-affinity of cell
d< 10
-6m), such as the polypeptide fragment of monoclonal antibody, high-affinity, high-affinity chemistry small molecules etc., for the surface antigen of combining target cell specifically; B represents one or more dye marker molecules, for quantitative detection of molecules A.A and B can be by the molecule of chemical covalent linkage coupling.For example, the antibody of anti-CD45 is by chemical bond CO-NH and fluorescent tag molecule fluorescein succinimide ester linkage.The antibody of the anti-CD45 of this fluorescein can be for specific fluorescent mark detection by quantitative white corpuscle.A and B can be also that molecule by non-bonding bonding is to (as hydrogen bond, Van der Waals force etc.).Such as, A represents the antibody of the anti-CD45 of vitamin H covalent linkage coupling, B represents the alkaline phosphatase of avidin covalent linkage coupling, for detection of with quantitatively.The CD45 antigen combination of A and leukocyte surface, thus and B reaches the object of detection by the hydrogen bonded of vitamin H and avidin.
Although above-mentioned these methods can detect target cell very easily, the detection limit of these methods is subject to target cell surface antigen and counts quantitative limitation, detectability is generally～and 10
-9m, sensitivity is not high.
Therefore, this area is necessary to develop new detection reagent or method very much at present, to improve the sensitivity of detection.
Summary of the invention
The object of the present invention is to provide oligonucleotide probe of targeting molecule coupling and preparation method thereof.
In a first aspect of the present invention, a kind of targeting molecule that detects pathogeny cell is provided, structure is as follows:
X-Y；
Wherein, X represents the binding molecule of selectively targeted pathogeny cell;
Y represents oligonucleotide probe;
"-" represents the relation of covalently bound (bonding), coupling or coupling between X and Y.
In a preference, X is selected from: the antibody of selectively targeted pathogeny cell, part, chemical small molecules or polypeptide.
In another preference, described X is less than 10 for the equilibrium dissociation constant of pathogeny cell
-6mol/L (M).
In another preference, described pathogeny cell is tumour cell, and X is folic acid or folic acid-cysteine conjugate.
In another preference, described tumour cell is circulating tumor cell (CTC).
In another preference, described oligonucleotide probe is the oligonucleotide probe through chemically modified, and described chemically modified strengthens the stability of row oligonucleotide probe.
In another preference, in the sequence of described oligonucleotide probe, on phosphate bond, there is thio-modification.
In another preference, described " on phosphate bond, having thio-modification " refers in the sequence of oligonucleotide probe, and P-O sulfo-deoxidation occurs and forms P-S.
In another preference, described " on phosphate bond, having thio-modification " occurs on part phosphate bond or occurs on all phosphate bonds.
In another preference, described oligonucleotide probe is strand or two strands.
In another preference, the length of described oligonucleotide probe is 10-150bp (two strands) or 10-150nt (strand); Preferably, the length of described oligonucleotide probe is 20-100bp (two strands) or 20-100nt (strand); More preferably, the length of described oligonucleotide probe is 20-80bp (two strands) or 20-80nt (strand).
In another aspect of this invention, provide a kind of method of preparing targeting molecule, described targeting molecular structure is as follows:
X-Y；
Wherein, X represents the binding molecule of selectively targeted pathogeny cell, and this binding molecule is folic acid-cysteine conjugate;
Y represents oligonucleotide probe;
"-" represents the relation of covalently bound (bonding), coupling or coupling between X and Y;
Described method comprises:
(a) oligonucleotide is mixed with succinimide-4-hexanaphthene-1-carbonic ether (SMCC) solution, amino on oligonucleotide and succinimide-4-hexanaphthene-1-carbonic ether, with C-N covalent bonding, are obtained to oligonucleotide-succinimide-4-hexanaphthene-1-carbonic ether bonding product;
(b) in oligonucleotide-succinimide-4-hexanaphthene-1-carbonic ether bonding product, add folic acid-cysteine conjugate, by the maleimide on the sulfenyl of folic acid-halfcystine and succinimide-4-hexanaphthene-1-carbonic ether with C-S covalent bonding; Obtain oligonucleotide-folic acid-halfcystine product.
In another aspect of this invention, the purposes of the targeting molecule described in providing, for the preparation of the reagent or the test kit that detect pathogeny cell.
In another aspect of this invention, provide a kind of test kit that detects pathogeny cell, comprising described targeting molecule.
In another preference, described test kit comprises 1-100 kind targeting molecule, and in each targeting molecule, X is identical or different, and the base sequence of Y is different; Thereby when targeting molecule is when a kind of, this test kit can be used for different specific molecular on different pathogeny cells or same pathogeny cell surface to carry out Multiple detection.
In another preference, in described test kit, also comprise: the primer of oligonucleotide probe in the targeting molecule described in specific amplification; And/or
Specificity is for the detection probes of oligonucleotide probe in described targeting molecule, and this detection probes is carried detectable label.
In another preference, described primer is for two terminal sequences of the oligonucleotide probe in described targeting molecule; The intermediate sequence of the oligonucleotide probe in described detection probes and described targeting molecule is complementary.
In another preference, described primer is for two terminal sequences of the oligonucleotide probe in described targeting molecule; In described detection probes and described oligonucleotide probe with the sequence location of primer sequence complementation outside intermediate sequence locations complementary.
In another preference, the length of described primer is 8-50bp; Be preferably 10-30bp.
In another preference, described detection probes is fluorescent probe, and its detectable label carrying is fluorescence molecule; More preferably, described detection probes is TaqMan probe.
In another aspect of this invention, provide a kind of method that detects pathogeny cell, comprising:
(1) by described targeting molecule and testing sample blend, hatch;
(2) hatch and finish rear collecting cell, the targeting molecule from cell described in wash-out (preferably, using the glycine buffer wash-out of pH2.5);
(3) take the targeting molecule of step (2) wash-out is template, carries out PCR quantitative analysis, and what according to quantitative analysis results, know pathogeny cell in testing sample exists situation and amount.
In another preference, described primer is for two terminal sequences of the oligonucleotide probe in described targeting molecule; The intermediate sequence of the oligonucleotide probe in described detection probes and described targeting molecule is complementary.
In another preference, described primer is for two terminal sequences of the oligonucleotide probe in described targeting molecule; In described detection probes and described oligonucleotide probe with the sequence location of primer sequence complementation outside intermediate sequence locations complementary.
In another preference, the length of described primer is 8-50bp; Be preferably 10-30bp.
In another preference, described detection probes is fluorescent probe, and its detectable label carrying is fluorescence molecule; More preferably, described detection probes is TaqMan probe.
In another preference, in step (3), with specificity, for primer and/or the specificity of oligonucleotide probe in (amplification) this targeting molecule, for the detection probes (this detection probes is carried detectable label) of oligonucleotide probe in described targeting molecule, carry out pcr amplification, obtain PCR quantitative analysis results; There is situation and amount in what according to quantitative analysis results, know pathogeny cell in testing sample.
In another preference, in step (3), control group and/or standard substance group are also set, there is situation and amount in what take this to analyze pathogeny cell in testing sample.
In another preference, the method for described detection pathogeny cell is non-diagnostic or curative.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
accompanying drawing explanation
Fig. 1, the product preparing is carried out to HPLC analysis (260nm).
The HPLC purity check collection of illustrative plates of A, folic acid-halfcystine-ssPO25;
The HPLC purity check collection of illustrative plates of B, folic acid-halfcystine-ssPO45;
The HPLC purity check collection of illustrative plates of C, folic acid-halfcystine-ssPO100;
The HPLC purity check collection of illustrative plates of D, folic acid-halfcystine-ssPS45;
The HPLC purity check collection of illustrative plates of E, folic acid-halfcystine-dsPO45.
Fig. 2, the product preparing is carried out to ultraviolet spectro-photometric analysis.
The ultraviolet spectro-photometric analysis of A, folic acid-cysteine conjugate;
B, the ultraviolet spectro-photometric analysis of the ssPO25 of coupling not;
The ultraviolet spectro-photometric analysis of C, folic acid-halfcystine-ssPO25 couplet;
The ultraviolet spectro-photometric analysis of D, folic acid-halfcystine-ssPO45 couplet;
The ultraviolet spectro-photometric analysis of E, folic acid-halfcystine-ssPS45 couplet;
The ultraviolet spectro-photometric analysis of F, folic acid-halfcystine-ssPO100 couplet.
Fig. 3, the avidity of folic acid-halfcystine-oligonucleotide probe and folacin receptor is detected, obtain the competition binding curve of folic acid-halfcystine-ssPO25.
The PCR of Fig. 4, folic acid-halfcystine-oligonucleotide probe detects, the amplification of different lengths probe.
Fig. 5, his-and-hers watches 4 data are carried out linear regression analysis.Using the cell quantity in standard substance as independent variable, the cell quantity on average detecting is as relying on variable, and result obtains vertical axis intercept 1.99, and slope is 1.159; And R
2=0.99.
Fig. 6, respectively available from the hydrothorax of 3 cases for sample folic acid-halfcystine-OG (OG is oregon green 488, it is a kind of fluorescence molecule, green, what dye is folacin receptor) and DAPI dyeing (blueness, what dye is nucleus) result, wherein left column is optical transmission imaging results, for the form of observation of cell; The coloration result of folic acid-halfcystine-OG and DAPI is classified on the right side as.What be singly blueness is white corpuscle; What blue, green dichromatism pair dyed is cancer cells.
Embodiment
The present invention has disclosed a kind of reagent and method of new detection pathogeny cell, and method of the present invention is by carrying out signal amplification to marker oligonucleotide, and (detection is limited to 10 to have improved the detection sensitivity of pathogeny cell surface specific molecular
-14m); And different oligonucleotide sequences can be encoded to multiple molecules detected, therefore can realize Multiple detection.
Term
As used herein, described " pathogeny cell " refers to the cell (be fixed on lesion tissue or discharged and dissociated by lesion tissue and be present in body fluid) that comes from lesion tissue, on this cell surface, have specific molecular (for example surface antigen or acceptor), this specific molecular is that this kind of pathogeny cell is peculiar, and can be by specific binding molecule (as antibody, part) combination, this specific binding molecule is the binding molecule of selectively targeted pathogeny cell.For example, folic acid can be identified and in conjunction with the folacin receptor (folate receptor) on cancer cells surface, the Her2 antigen on breast cancer cell surface can be identified and be incorporated into the monoclonal antibody of anti-Her2; LHRH polypeptide can be identified and in conjunction with the luteinizing hormone-releasing hormone (LRH) acceptor (LHRH receptor) on prostate cancer cell surface etc.
As used herein, described " binding molecule " be specific molecular (for example surface antigen or acceptor) on target pathogeny cell specifically, and it has high affinity, its for equilibrium dissociation constant be conventionally less than 10
-6.Described " binding molecule " is such as but not limited to antibody, part, chemical small molecules or polypeptide etc.
As used herein, described " specificity is for the primer of oligonucleotide probe in targeting molecule " refers to a primer or pair of primers, its at least part of sequence can with targeting molecule in oligonucleotide probe complementary, this primer can be by the increase sequence of oligonucleotide probe of round pcr.Described primer is primer pair normally, comprises and the forward of oligonucleotide probe two ends complementation and reverse sequence.
As used herein, described " cancer " or " tumour " have no particular limits, and preferably should " cancer " or " tumour " idiopathy rise and can in blood circulation, disseminate cancer cell or tumour cell.For example be selected from (but being not limited to): nasopharyngeal carcinoma, the esophageal carcinoma, cancer of the stomach, liver cancer, mammary cancer, large bowel cancer, prostate cancer, lung cancer, cervical cancer, leukemia, oral carcinoma, salivary gland tumor, nasal cavity and paranasal sinus malignant tumour, laryngocarcinoma, tumor of ear, ocular tumor, thyroid tumor, mediastinal tumor, the wall of the chest, pleural tumor, intestinal tumor, tumor of biliary tract, pancreas and ampulla Tumors, mesentery and retroperitoneal tumor, tumor of kidney, adrenal tumor, tumor of bladder, prostate cancer, tumor of testis, penile cancer, carcinoma of endometrium, malignant tumor of ovary, malignant trophoblastic tumor, carcinoma vulvae and carcinoma of vagina, malignant lymphoma, multiple myeloma, soft tissue neoplasm, bone tumor, skin and adnexal tumor, malignant melanoma, nervous system neoplasm, tumors in children.As the preferred embodiment of the present invention, described " cancer " or " tumour " are selected from: breast (gland) cancer, lung cancer.
As used herein, described " equilibrium dissociation constant (equilibrium dissociation constant, Kd) " refers to occupy on half cell the concentration of specific acceptor or required part, antibody, chemical small molecules or the polypeptide of surface molecular.Kd value and receptor affinity are inverse relationship, and Kd value is less, show that avidity is higher; Otherwise Kd value is larger, show that avidity is lower.Conventionally Kd is less than 10
-6m represent acceptor or surface molecular and part, antibody, chemical small molecules or polypeptide avidity high.
As used herein, it is upper that described " specificity " refers to that the specific molecular on circulating tumor cell surface (as surface antigen, acceptor) can be identified and/or be incorporated into antibody, part, polypeptide or chemical small molecules, but nonrecognition and be incorporated into other irrelevant molecule.
As used herein, described " TaqMan fluorescent probe " is a kind of oligonucleotide, and its two ends are a fluorescent emission group of mark (as detectable signal) and a fluorescent quenching group respectively.When probe is complete, the fluorescent signal of fluorescent emission group transmitting is quenched group and absorbs; During pcr amplification, 5 '-3 ' 5 prime excision enzyme activity of Taq enzyme is cut degraded by probe enzyme, make fluorescent emission group separated with fluorescent quenching group, thereby can receive fluorescent signal by fluorescence detecting system, it is DNA chain of every amplification, just there is fluorescence molecule to form, the Complete Synchronization that accumulative total of fluorescent signal and PCR product form, thus realize quantitative and qualitative analysis.
As used herein, described " circulating tumor cell (CTC) " refers to cancer (tumour) cell being present in blood (peripheral blood), the concentration of CTC can diagnosing cancer, to cancer carry out by stages, prognosis etc.
Ultimate principle
Method of the present invention is mainly based on binding molecule-widow (gathering) nucleotide probe, the specific molecular of the selectively targeted pathogeny cell surface of described binding molecule.Described oligonucleotide probe is for the amplification template as follow-up PCR quantitative analysis.Described binding molecule, by being combined with the specific molecular of pathogeny cell surface, carries out mark to pathogeny cell.Thereby reach cell to be checked is carried out to quantitative object and oligonucleotide is used for that by quantitative PCR technique part is carried out to detection by quantitative.
The technique effect of realizing based on above-mentioned principle is as follows: first, due to the amplification effect of PCR to oligonucleotide, detectability can be reduced greatly; Secondly, the technology of PCR can detect great amount of samples in 2 hours, and flux is high; Again, due to the diversity of oligonucleotide, can carry out Multiple detection to the not isoacceptor of different pathogeny cells or same pathogeny cell surface.Therefore, reagent of the present invention and method have solved the many indeterminable problems of traditional method.
Targeting molecule
Targeting molecule of the present invention is mainly based on following structure:
X-Y；
Wherein, X represents a specific high-affinity molecule for pathogeny cell (common Kd < 10
-6mol/L), such as the polypeptide fragment of antibody, high-affinity, high-affinity chemistry small molecules etc., for specifically in conjunction with the specific molecular on pathogeny cell; Y is for the amplification template as follow-up PCR quantitative analysis, for the detection by quantitative of target pathogeny cell.
X is the key molecule of performance guide effect, it is for identifying and/or in conjunction with pathogeny cell, therefore, it is established according to the type of the pathogeny cell of required detection conventionally, and for example folic acid can be identified and in conjunction with the folacin receptor (folate receptor) on lung carcinoma cell surface; The Her2 antigen on breast cancer cell surface can be identified and be incorporated into the monoclonal antibody of anti-Her2; LHRH polypeptide can be identified and in conjunction with the luteinizing hormone-releasing hormone (LRH) acceptor (LHRH receptor) on prostate cancer cell surface.
At present, for the specific molecular that is present in various pathogeny cell surfaces, there is deep research, also developed the various binding molecules for these surface moleculars, comprised antibody, part, polypeptide, chemical small molecules etc.The present invention has no particular limits binding molecule, and these binding molecules all can be applied in method of the present invention, thereby designs the various targeting molecules for pathogeny cell.
Y is the oligonucleotide molecules with certain length, thereby can design specific primer based on this oligonucleotide molecules, realizes pcr amplification, by amplification effect, reduces detectability, improves the susceptibility detecting.Described oligonucleotide molecules can be double-stranded or strand.There is no particular limitation for the sequence of described oligonucleotide, and preferably it is not complementary with the gene order of human body or animal body, and those skilled in the art know its design and synthetic method.The length of described oligonucleotide can be preferably 10-150bp (two strands) or 10-150nt (strand).
The present invention also comprises the modified oligonucleotide molecules that adopts as obtain based on means such as nucleic acid chains backbone modification technology, and described modification does not change oligonucleotide molecules binding characteristic substantially; Preferably those can improve the modification of oligonucleotide molecules stability.For example, the described thio-modification that is modified to, or carry out alkyl modified in 2 ' position of ribose.Should be understood that and anyly can keep the modification of described oligonucleotide molecules binding characteristic to be included in the present invention.
The backbone modification method of oligonucleotide has multiple, comprises sulfo-method, and the method is that the Sauerstoffatom on phosphate bond on DNA skeleton is substituted with sulphur atom, and described sulfo-can be sulfo-on whole phosphate bonds or the sulfo-on part phosphate bond.The modification of sulfo-can strengthen the stability of described oligonucleotide molecules greatly, thereby is conducive to obtain detected result accurately.
Preferably, between X and Y, the form with chemical covalent linkage connects.
As optimal way of the present invention, the inventor, after repeatedly studying, has developed the targeting molecule of detection circulating tumor cell (CTC).This is can identify and in conjunction with the folacin receptor (folate receptor) of tumor cell surface due to folic acid.In this targeting molecule, X is folic acid or folic acid-cysteine conjugate.Folic acid is compared with natural folic acid with the conjugate of halfcystine, has improved the avidity with folacin receptor; And, folic acid is connected with halfcystine, can increase significantly the solvability of folic acid, thereby improve the amount of the folic acid contacting with circulating tumor cell.The preparation of folic acid-cysteine conjugate can adopt technology known in the art to carry out.The number of the halfcystine being connected with folic acid can be one or more, and for example 1～3, the halfcystine within the scope of this can be folic acid good hydrophilic environment is provided.In this targeting molecule, the sequence of Y is diversified, and is sulfo-or non-sulfo-.
Test kit
One or more targeting molecules of the present invention's exploitation can be comprised in test kit, thereby are convenient to those skilled in the art's operation.
The primer supporting with described targeting molecule also can be comprised in test kit, this primer is primer pair normally, the sequence of that comprise forward and two terminal sequence complementations reverse and described targeting molecule, thereby the described targeting molecule of can take is template, carry out pcr amplification, realize the amplification of detected result.
Preferably, in described test kit, also comprise the fluorescent probe that fluorescent PCR is used, TaqMan probe for example, thus be convenient to the quantitative analysis of PCR product.
In described test kit, described targeting molecule and supporting primer or fluorescent probe thereof are loaded in suitable container independently, and more preferably described targeting molecule and supporting primer thereof or fluorescent probe are also formulated into suitable consumption or concentration.
In described test kit, also can comprise other reagent, for example, for the reagent (as archaeal dna polymerase, Realtime PCR Master Mix) of pcr amplification, damping fluid (as PBS), washings etc.
Described test kit also can comprise: test kit working instructions, and with guidance technology personnel's operation.
Application
The invention still further relates to the method for utilizing described targeting molecule to detect pathogeny cell, described method can be take and diagnosed the illness as object, for example, patient is carried out to prognosis, or judge whether doubtful crowd suffers from disease; Or take and non-ly diagnose the illness as object, for example, pure scientific research, or pure cell typing (as distinguished the classification of tumour cell).
The method of described detection pathogeny cell comprises:, by described targeting molecule and testing sample blend, hatch (1); (2) hatch and finish rear collecting cell, the targeting molecule from cell described in wash-out; (3) take the targeting molecule of step (2) wash-out is template, with specificity, for the primer of oligonucleotide probe in (amplification) this targeting molecule, carries out pcr amplification, obtains PCR quantitative analysis results; There is situation and amount in what according to this quantitative analysis results, know pathogeny cell in testing sample.
The preferred fluorescent quantitative PCR technique of described PCR therefore, preferably, also adds fluorescent probe in pcr amplification system, thereby can be by specific quantitative PCR instrument Realization analysis easily; More preferably, described fluorescent probe is TaqMan probe.
As optimal way of the present invention, control group and/or standard substance group are also set, it is the group of known folic acid-halfcystine-oligonucleotide probe quantity, the standard substance that a series of different amts are set conventionally form standard substance group, and what take this to analyze pathogeny cell in testing sample exists situation and amount.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, writes molecular cloning experiment guide, Science Press, the condition described in 2002, or the condition of advising according to manufacturer conventionally as J. Pehanorm Brooker etc. according to normal condition.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Unless otherwise defined, the same meaning that all specialties of using in literary composition and scientific words and one skilled in the art are familiar.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
Material
H-Cys (Trt)-2-Cl Trt resin, HBTU and HOBT are all purchased from Novabiochem (U.S.A.).Erythrocyte cracked liquid is purchased from Suo Laibao biological (Beijing, China).Amido modified single strain oligonucleotide (ssPO25, ssPO45, ssPO52, ssPO100), PBS, CD45 Dynabeads magnetic bead and PCR detection reagent are purchased from Invitrogen (Long Island, U.S.A.).SMCC (succinimide-4-hexanaphthene-1-carbonic ether) is purchased from Thermo Scientific (U.S.A.).Cell strain used is all purchased from Chinese Academy of Sciences's cell bank (Shanghai, China).Folic acid-halfcystine-OG (oregon green 488) molecule is prepared as follows.Unless otherwise indicated, embodiment folic acid used is γ folic acid.The primer of oligonucleotide and PCR is purchased from Invitrogen company.PD-10 post (Sephadex G-25M) is purchased from General Electrical Healthcare (U.S.).The reagent of quantitative PCR is purchased from TOYOBO company (Japan).Quantitative PCR instrument is the 7300realtime PCR system of Applied Biosystems.All the other reagent are all purchased from Sigma Aldrich.Folic acid antibody is purchased from Cortez Diagnostics (U.S.).
" folic acid-halfcystine-OG " preparation method is as follows:
First, synthesize folic acid-halfcystine conjugates.The γ folic acid of 441mg is slowly dissolved in to 20mL bis-sulphur MSMs, then add the dicyclohexylcarbodiimide of 1.2mmol and the N-hydroxy thiosuccinimide of 2mmol in 50 ℃ of reactions 6 hours.γ folic acid-N-hydroxy thiosuccinimide that this reaction obtains and the halfcystine of 10 times of equimolar amounts add 50 microlitre pyridines 25 ℃ of reactions 5 hours.20mL acetonitrile precipitation centrifugal collection for crude product (wherein comprising γ folic acid-halfcystine and alpha-isomer-halfcystine).With after product, with ether, wash three times.The product of collecting is finally used vacuum-drying, and weighs, and weight is 390mg.Microsorb carbon-18 preparative Reversed Phase High Performances (250mm * 21mm) separation and purification of U.S. Agilent Technologies for this crude product.The elution time of γ folic acid-halfcystine is respectively 7.6 minutes.Separated γ folic acid-halfcystine (this is the conjugates that fat key CO-NH connects, containing 2 halfcystines) is 58mg, and yield is 12%.Then, the 1.5mg conjugates after purifying is dissolved in the two sulphur MSMs of 100 μ L, and add Oregon Green 488 succinimide esters of 1.0mg and incubated at room 4 hours.Reacted product high-efficient liquid phase chromatogram purification, obtains the fluorescently-labeled folic acid probe of Oregon Green 488 (being called for short OG) and (is called: folic acid-halfcystine-OG).
The preparation of embodiment 1, double-stranded oligonucleotide probe
Two single strain oligonucleotides (wherein a strand is modified with amino (Amino)) that dissolve respectively sequence complementation with the deionized water through sterilizing, are mixed with 50 μ M single strain oligonucleotide solution.Annealing reaction system is set as follows:
Remove the water of nuclease: 40 μ l;
Annealing buffer (5X) 20 μ l (wherein 10mM Tris, pH 7.5-8.0,50mM NaCl, 1mM EDTA); Forward chain F-ssPO45 (50 μ M) 20 μ l; Reverse strand R-ssPO45 (50 μ M) 20 μ l; Cumulative volume 100 μ l.
According to said sequence, add successively all ingredients, mix.Then PCR instrument is set as follows and carries out annealing reaction:
| Step | Temperature | Time | Explanation |
| 1 | 95℃ | 2 minutes | Allow the abundant sex change of single strain oligonucleotide |
| 2 | Within every 8 seconds, decline 0.1 ℃, be down to 25 ℃ | Approximately 90 minutes | Annealing |
By the reaction product after annealing, with 37 ℃ of enzymes of exonuclease I, cut 1 hour, remove unreacted single strain oligonucleotide.Then with DNA, reclaim post recovery double chain nucleotide standby.
Oligonucleotide sequence is as follows:
F-ssPO45：5’Amino C6-GGTAG GCATG AACTT GA AGA ACCCT CAACT CGACT CCACG ACACC-3’(SEQ ID NO：1)；
R-ssPO45：5’GGTGT CGTGG AGTCG AGTTG AGGGT TCTTC AAGTT CATGC CTACC-3’(SEQ ID NO：2)。
The preparation of embodiment 2, folic acid-halfcystine-oligonucleotide probe
First prepare folic acid-cysteine conjugate.The H-Cys of 270mg (the Trt)-2-Cl Trt resin surface molecular number of resin (take be 1 equivalent) is dipped in 5mL dimethyl formamide to 30 minutes.Add the Fmoc-Glu-OtBu (purchased from Novabiochem, the U.S.) of 4 equivalents and the N of 4 equivalents, N diisopropylethylamine, and add the HOBT of 2.5 equivalents and 2.5 equivalent HBTU to react 1 hour.Afterwards, add 15 minutes removal blocking group Fmoc of pyridine of 20%, rear use 5mL dimethyl formamide washing resin 3 times.After this, add the pteroic acid of 4 equivalents and the N of 4 equivalents, the HOBT of N diisopropylethylamine and 2.5 equivalents and HBTU reaction are spent the night.Next day, with dimethyl formamide, methylene dichloride and methyl alcohol, wash resin 3 times respectively.With nitrogen drying resin 60 minutes.After this, the mixture reaction 2 hours that adds 10mL trifluoroacetic acid, water and tri isopropyl silane (volume ratio, 95: 2.5: 2.5) in resin.Collect liquid reactants in round-bottomed flask, and on Rotary Evaporators volatilised liq to about 1mL.The ether that adds the precooling of 50mL, then 4, the centrifugal collection solid crude product of 000g.The crude product of collecting is purified and is obtained with HPLC, the conjugate 52mg of the folic acid that purity is 91% (for γ folic acid)-halfcystine.Carry out HPLC analytical spectra and mass spectroscopy to confirm to obtain the conjugate with certain purity.
SMCC is dissolved in the mixture of 500uL water and dimethyl sulfoxide (DMSO) (volume ratio 1: 1), then adding concentration is in the strand of 1mg/mL or double chain oligonucleotide (sequence is as follows) and incubated at room 4 hours.This reaction is with C-N covalent bonding by the amino on oligonucleotide and SMCC.PD-10 post for reacted product (Sephadex G-25M) is removed excessive SMCC.Then in reaction system, adding final concentration is folic acid-cysteine conjugate of 1mg/mL.This reaction is with C-S covalent bonding with the sulfenyl of folic acid-halfcystine and the maleimide on SMCC.End product is purified and can be obtained folic acid-halfcystine-oligonucleotide probe with HPLC.
Single strain oligonucleotide can be following several one of them:
ssPO25：5’Amino C6-ACTTG AAGAA CCCTC AACTC GACTC 3’(SEQ ID NO：3)；
ssPO45：5’Amino C6-GGTAG GCATG AACTT GA AGA ACCCT CAACT CGACT CCACG ACACC-3’(SEQ ID NO：1)；
ssPS45：5’Amino C6-GGTAG GCATG AACTT GA AGA ACCCT CAACT CGACT CCACG ACACC-3’(SEQ ID NO：1)；
dsPO45：5’Amino C6-GGTAG GCATG AACTT GA AGA ACCCT CAACT CGACT CCACG ACACC-3’(SEQ ID NO：1)；
ssPO100：5’Amino C6-GGTAG GCATG AACTT GA AGA ACCCT CAACT CGACT CCACG ACACC TACAT GTAAG GCCGA TACGG ACCTT ATCGC GTCAT CGACT GGGCT AATCA AGCGT 3’(SEQ ID NO：4)；
Wherein, ss represents single strain oligonucleotide; Ds represents double-stranded oligonucleotide; PO represents oligodeoxynucleotide; PS represents sulfo-oligodeoxynucleotide; The length of digitized representation oligonucleotide below.Special needs to be pointed out is, sulfo-oligodeoxynucleotide is stable in the extreme, is the better selection for detection of reagent.
Embodiment 3, HPLC analyze
The above-mentioned product preparing is carried out respectively to HPLC analysis (260nm), and Figure 1A is shown in by the HPLC purity check collection of illustrative plates of folic acid-halfcystine-ssPO25.HPLC purity check shows that probe purity is 97.23%.
Figure 1B is shown in by the HPLC purity check collection of illustrative plates of folic acid-halfcystine-ssPO45.HPLC purity check shows that probe purity is 93.87%.
Fig. 1 C is shown in by the HPLC purity check collection of illustrative plates of folic acid-halfcystine-ssPO 100.HPLC purity check shows that probe purity is 100%.
Fig. 1 D is shown in by the HPLC purity check collection of illustrative plates of folic acid-halfcystine-ssPS45.HPLC purity check shows that probe purity is 100%.
Fig. 1 E is shown in by the HPLC purity check collection of illustrative plates of folic acid-halfcystine-dsPO45.HPLC purity check shows that probe purity is 100%.
Embodiment 4, ultraviolet spectro-photometric analysis
By the oligonucleotide of coupling not, a series of folic acid-halfcystines-oligonucleotide conjugate of folic acid-cysteine conjugate and preparation carries out respectively uv-absorbing spectrum analysis.According to being the charateristic avsorption band of folic acid near near the charateristic avsorption band 350nm for nucleic acid prior art 260nm.If the scanning result of sample UV spectrum has near the absorption peak about 260nm and 350nm simultaneously, it contains Nucleotide and folic acid simultaneously so.
Fig. 2 A is the ultraviolet spectro-photometric analysis of folic acid-cysteine conjugate, Fig. 2 B is the ultraviolet spectro-photometric analysis of the ssPO25 of not coupling, Fig. 2 C is the ultraviolet spectro-photometric analysis of folic acid-halfcystine-ssPO25 couplet, Fig. 2 D is the ultraviolet spectro-photometric analysis of folic acid-halfcystine-ssPO45 couplet, Fig. 2 E is the ultraviolet spectro-photometric analysis of folic acid-halfcystine-ssPS45 couplet, and Fig. 2 F is the ultraviolet spectro-photometric analysis of folic acid-halfcystine-ssPO100 couplet.
As seen from Figure 2, the oligonucleotide of coupling does not only have the absorption peak of 217nm and 258nm left and right, and the absorption peak of folic acid-cysteine conjugate is in about 217nm, 281nm and 346nm left and right.And folic acid-halfcystine-oligonucleotide conjugate has the absorption peak at about 217nm, 258nm and 346nm.
The above results proves, the inventor has successfully prepared the conjugate of folic acid-halfcystine-oligonucleotide probe.
Embodiment 5, enzyme-linked immunosorbent assay (ELISA) detect folic acid-halfcystine-oligonucleotide
First with diluted sample solution, trial-product is diluted to suitable concentration range.And make positive control by the folic acid reference material (Sigma#) of purifying.Get standard solution and each 0.1ml of need testing solution of having diluted, add respectively in micro plate, every hole adds folic acid antibody 0.05ml, is placed on shaking table incubated at room 60 minutes, adds washing soln 0.3ml and washes plate three times.The two anti-0.1ml that add horseradish peroxidase-labeled, are placed on shaking table incubated at room 60 minutes, add washing soln 0.3ml and wash plate three times.Add substrate solution 0.1ml, in room temperature lucifuge, hatch 20 minutes, add reacting terminating solution 0.1ml, mix and be placed in microplate reader, take 570nm as reference wavelength, at wavelength 450nm place, measure absorbancy, record measurement result.
The detected result of probe is in Table 1.Only have the oligonucleotide of coupling folic acid-halfcystine just can detect the signal of folic acid, the concentration of detected folate content and ultraviolet determination is close, and this shows that the mode of coupling is folic acid-halfcystine: oligonucleotide carries out according to the ratio of 1: 1.And unlabelled oligonucleotide no signal.
Table 1
| Sample title | Ultraviolet quantitative concentrations | ELISA measures concentration | Remarks |
| Folic acid | 100nM* | 95.1nM | * directly weigh gained |
| Folic acid-halfcystine-ssPO25 | 100nM | 93.2nM | |
| Folic acid-halfcystine-ssPO45 | 100nM | 97.5nM | |
| Folic acid-halfcystine-ssPO100 | 100nM | 89.3nM | |
| Folic acid-halfcystine-ssPS45 | 100nM | 128.3nM | |
| Folic acid-halfcystine-dsPO45 | 100nM | 103.4nM | |
| ssPO25 | 100nM | ＜1nM | |
| ssPO45 | 100nM | ＜1nM | |
| ssPO100 | 100nM | ＜1nM | |
| ssPS45 | 100nM | ＜1nM | |
| dsPO45 | 100nM | ＜1nM |
The avidity of embodiment 6, folic acid-halfcystine-oligonucleotide probe and folacin receptor detects
KB cell is the cell strain that folacin receptor expression amount is higher.Get 10
5individual KB cell, is resuspended in 100 μ LPBS, and the determinand that adds fluorescent probe folic acid-halfcystine-OG (6nM) and different concns is totally 100 μ L.Room temperature lucifuge is hatched 60min; After finishing, reaction adds 200 μ L PBS, the centrifugal 15min of 600g; Abandon supernatant, add 300 μ L PBS re-suspended cells, flow cytometer fluorescence intensity.In reaction with unlabelled folic acid as positive control.By the fluorescence intensity recording, calculate the percentage (specific bound%) of specific binding and corresponding concentration and probe concentration, with Prism5 (Graphpad software Inc.), map, (constant numerical value is lower, and avidity is larger for Ki with binding competitive-One site Fit Ki, to calculate the inhibition constant (that is: the equilibrium dissociation constant of competitive blocker) of conjugates probe; Otherwise constant numerical value is higher, and avidity is less).The competition binding curve of folic acid-halfcystine-ssPO25 is shown in Fig. 3.All probe avidity detected results are in Table 2.
Table 2
| Determinand | Affinity constant Ki |
| Folic acid | 6nM |
| Folic acid-halfcystine-ssPO25 | 30.1nM |
| Folic acid-halfcystine-ssPO45 | 392nM |
| Folic acid-halfcystine-ssPO100 | 850nM |
| Folic acid-halfcystine-ssPS45 | 252nM |
| Folic acid-halfcystine-dsPO45 | 670nM |
Result demonstration, the avidity of probe and DNA length have close relationship.DNA is longer, and the avidity of probe and acceptor is more weak.
In addition, result also shows, the modification of DNA and probe avidity relation are little.
The PCR of embodiment 6, folic acid-halfcystine-oligonucleotide probe detects
First with deionized water, folic acid-halfcystine-oligonucleotide probe is diluted to 1.0E-9mol/L, 1.0E-10mol/L, 1.0E-11mol/L, 1.0E-12mol/L, five extent of dilution of 1.0E-13mol/L as standard substance successively according to concentration gradient, blank is deionized water.When PCR measures, according to the form below preparation amplification reaction solution, adds standard substance or product to be tested 2.5 μ L to carry out PCR reaction afterwards.
| Component | Amount (μ l)/part |
| Realtime PCR Master Mix | 12.5 |
| ddH 2O | 10 |
| FP(10μM) | 0.75 |
| RP(10μM) | 0.75 |
| Taq Man MGB probe (10uM) | 0.25 |
Reaction tubes is put into quantitative real time PCR Instrument, and order put in record, by following program, carries out the pre-amplified reaction of PCR.Analytical data base line adopts default value.Fluorescence threshold is set as 0.3.The Log value of standard substance concentration of take is X-coordinate, and CT value is ordinate zou, production standard curve, and calculate the concentration of probe with this.
Relevant primer and TaqMan MGB probe (TaqMan MGB probe in sequences Design with Y probe in one section of sequence complementation) sequence is as follows:
FP：5’-GGTAG GCATG AACTT GA-3’(SEQ ID NO：5)；
RP：5’-GGTGT CGTGG AGTCG-3’(SEQ ID NO：6)；
TaqMan MGB probe: 5 '-(6-FAM) AGTTG AGGGT TCT (MGB)-3 ' (SEQ ID NO:7).
The amplification of different lengths probe is as Fig. 4, and result shows, length, DNA backbone modification or single double-stranded variation affect without significance the result of PCR.The inventor has carried out analyzing further to the data that gather, and data declaration probe can be at R
2under=0.99 linear conditions, detect 10
-13the solution of M concentration.Therefore the detectability that, this method detects traditional enzyme mark as above has reduced by 10
5doubly.
The mensuration of embodiment 7, positive tumor cell folacin receptor quantity
For the positive cell of selecting to detect for CTC, the inventor has measured HeLa, KB, the isocellular folacin receptor quantity of M109, A549.Measuring method is as follows, first gets 2 * 10
5individual cell, after PBS washing, then add folic acid-halfcystine-ssPO25 or folic acid-halfcystine-ssPO45 probe mark cell, the concentration of probe is 20nM (folic acid-halfcystine-ssPO25) or 200nM (folic acid-halfcystine-ssPO45), incubated at room 20 minutes.After hatching end, add 1mL PBS termination reaction; Centrifugal collecting cell.Then add 1mL PBS centrifuge washing cell three times, finally add the 0.1M glycinate acid buffer re-suspended cell of 120 μ L PH2.5, wash-out probe.Centrifugal supernatant liquor is proceeded in new centrifuge tube, add 12 μ L 0.5MNaOH neutralizations.
Then, thus according to preceding method, carry out the number of probes that combination is measured in PCR quantitative analysis, i.e. the folacin receptor quantity of cell.The total acceptor number recording can be obtained to the acceptor number of each cell divided by the cell count adding, the results are shown in Table 3.
Table 3
From detected result, can find out, in four kinds of cell strains, KB expression amount is the highest, and secondly, M109's HeLa takes second place, and A549 is minimum.This and existing document (G.B.Henderson, et al, J.Membr.Biol.101 (1988) 247-258.C.A.Luhrs, et al.Arch.Biochem.Biophys.250 (1986) 94-105.M.A.Kane, et al, J.Clin.Invest.81 (1988) 1398-1406) in report, the expression level of the mRNA of these acceptors conforms to.
The detected result of two kinds of probes is similar, without obviously difference.
The rate of recovery, linearity and detectability that embodiment 8, CTC detect
The inventor takes a blood sample from Healthy People, then all blood samples is put together to the testing sample that mixes and be evenly divided into 3mL.Then respectively by 40,200,1,000 KB cell, adds in the as above sample of 3mL, is mixed with cell concn gradient standard model.Other 3mL do not add KB cell as negative control product.
During sample preparation, first in blood sample, add 9mL erythrocyte cracked liquid, after the abundant cracking of red corpuscle, centrifugal collection remaining cell.Centrifugal rear use 10mL PBS+0.1%BSA washed cell once, then adds 200ul antiCD-45Dynabeads in cell suspension, in 4 ℃ of refrigerators, hatches 30min.After hatching end, add 1mL PBS+0.1%BSA, put upside down and mix, be placed in magnetic field 10min.Then supernatant is transferred in new centrifuge tube to centrifugal collecting cell.
In the cell of collecting, add folic acid-halfcystine-ssPO25 probe mark cell, the concentration of probe is 20nM, incubated at room 20min.After hatching end, add 1mL PBS termination reaction.Centrifugal collecting cell, then adds 1mL PBS centrifuge washing cell three times, removes unconjugated probe.The 0.1M glycinate acid buffer re-suspended cell that finally adds 120 μ LpH2.5, wash-out probe.Centrifugal supernatant elutriant is proceeded in new centrifuge tube, add 12 μ L 0.5M NaOH neutralizations.Then, these samples are carried out to the number of probes that combination is measured in PCR quantitative analysis, i.e. the folacin receptor quantity of cell.The acceptor quantity of removing each positive cell with detected folacin receptor number obtains cell count.Result is as table 4.
Table 4
| Cell quantity in standard substance | On average detect cell quantity | The rate of recovery |
| 1000 | 1165 | 114％ |
| 200 | 210 | 92％ |
| 40 | 65 | 88％ |
| 0 | 25 | -- |
Note: the cell quantity * 100% in the rate of recovery=(on average detecting cell quantity-negative control product cell quantity)/standard substance.
Result shows, the cell rate of recovery of method of the present invention is very high, reaches or has approached 100%.This has also surpassed the positive magnetic bead sieve method (30%-70%) of CTC.
The inventor has done linear regression analysis to above-mentioned data.Using the cell quantity in standard substance as independent variable, the cell quantity on average detecting is as relying on variable, and result obtains vertical axis intercept 1.99, and slope is 1.159; And R
2=0.99, as Fig. 5.Therefore, removed the artificial loss of cancer cells in separation and concentration, the sensing range that present method is described is 0 to 1000 every 3mL blood sample of cell.
Embodiment 9, the qualitative and quantitative analysis to tumour cell in tumour patient hydrothorax sample
The inventor respectively gets 3mL hydrothorax sample from some patients with lung cancer, then by method as described in Example 8, carries out separation and concentration processing, and with folic acid-halfcystine-ssPO25 probe, tumour cell in hydrothorax sample is carried out to mark.The separated cell through mark, wash-out folic acid-halfcystine-ssPO25 probe from cell (concrete as aforementioned).After target PCR quantitative analysis, result is as table 5.
Table 5
| Example | Classification | Tissue subtype | Pattern representation | Tumour cell detects number |
| 1 | Lung cancer | Gland cancer | Bloody pleural fluid, has more insolubles precipitation | 4200/mL |
| 2 | Lung cancer | Squama cancer | Faint yellow hydrothorax, without obvious sediment | 45/mL |
| 3 | Lung cancer | Gland cancer | Bloody pleural fluid, without obvious sediment | 121/mL |
Result as shown in Figure 6.Same hydrothorax for sample folic acid-halfcystine-OG and DAPI coloration result all positive, under light microscopic, staining cell is compared with normal plasma cell greatly, is tumour cell.
Embodiment 10, the quantitative analysis to tumour patient CTC
The inventor respectively gets 3mL blood sample from some breast cancers and patients with lung cancer, then by method as above, carries out separation and concentration.And with folic acid-halfcystine-ssPO25 probe, tumour cell in hydrothorax sample is carried out to mark.The separated cell through mark, wash-out folic acid-halfcystine-ssPO25 probe from cell (concrete as aforementioned).After target PCR quantitative analysis, result is as table 6.
Table 6
Visible, " detecting number (quantity of circulating tumor cell (CTC)) " of in table 6, obtaining described and conformed to its clinical pathology.Therefore, adopting method mensuration CTC quantity of the present invention is valuable for analysis and the prognosis of clinical disease.
Conclusion
As mentioned above, the inventor's data shows, method of the present invention can be used for the content of quantitative analysis pathogeny cell (as tumour circulating cells CTC etc.) in patient's body fluid, as hydrothorax, blood sample etc.Therefore, to clinical tumor, diagnosis has following application to method of the present invention: a) early diagnosis cancer, b) the dynamic monitoring state of an illness, c) monitoring recurrence, and d) for judging chemotherapeutic efficacy and direction of medication usage.
All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document, are quoted as a reference separately.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 (7)
1. a test kit for detection by quantitative pathogeny cell, comprising:
The targeting molecule that detects pathogeny cell, structure is as follows: X-Y;
Wherein, X represents the binding molecule of selectively targeted pathogeny cell, is folic acid or folic acid-cysteine conjugate; Y represents oligonucleotide probe; The length of described oligonucleotide probe is 20-80bp or 20-80nt; "-" represents covalently bound, coupling between X and Y or the relation of coupling; With
The primer of oligonucleotide probe in targeting molecule described in specific amplification;
Described pathogeny cell is the tumour cell in blood or hydrothorax.
2. test kit as claimed in claim 1, is characterized in that, described tumour cell is the cell of lung cancer.
3. test kit as claimed in claim 2, is characterized in that, described tumour cell is circulating tumor cell.
4. test kit as claimed in claim 1, is characterized in that, in the sequence of described oligonucleotide probe, has thio-modification on phosphate bond.
5. a method of preparing targeting molecule, described targeting molecular structure is as follows:
X-Y；
Wherein, X represents the binding molecule of selectively targeted pathogeny cell, and this binding molecule is folic acid-cysteine conjugate;
Y represents oligonucleotide probe;
"-" represents covalently bound, coupling between X and Y or the relation of coupling;
Described method comprises:
(a) oligonucleotide is mixed with succinimide-4-hexanaphthene-1-carbonate solution, amino on oligonucleotide and succinimide-4-hexanaphthene-1-carbonic ether, with C-N covalent bonding, are obtained to oligonucleotide-succinimide-4-hexanaphthene-1-carbonic ether bonding product;
(b) in oligonucleotide-succinimide-4-hexanaphthene-1-carbonic ether bonding product, add folic acid-cysteine conjugate, by the maleimide on the sulfenyl of folic acid-halfcystine and succinimide-4-hexanaphthene-1-carbonic ether with C-S covalent bonding; Obtain oligonucleotide-folic acid-halfcystine product.
6. detect the purposes of the primer of oligonucleotide probe in the targeting molecule of pathogeny cell and the targeting molecule described in specific amplification, for the preparation of reagent or the test kit of detection by quantitative pathogeny cell; Wherein, the structure of the targeting molecule of described detection pathogeny cell is as follows: X-Y;
Wherein, X represents the binding molecule of selectively targeted pathogeny cell, is folic acid or folic acid-cysteine conjugate; Y represents oligonucleotide probe; "-" represents covalently bound, coupling between X and Y or the relation of coupling.
7. a method for non-diagnostic detection by quantitative pathogeny cell, comprising:
(1) by targeting molecule and testing sample blend, hatch; The structure of described targeting molecule is as follows: X-Y; Wherein, X represents the binding molecule of selectively targeted pathogeny cell, is folic acid or folic acid-cysteine conjugate; Y represents oligonucleotide probe; "-" represents covalently bound, coupling between X and Y or the relation of coupling;
(2) hatch and finish rear collecting cell, the targeting molecule from cell described in wash-out;
(3) take the targeting molecule of step (2) wash-out is template, carries out PCR quantitative analysis, and what according to quantitative analysis results, know pathogeny cell in testing sample exists situation and amount.
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| CN105779599B (en) * | 2016-04-05 | 2020-09-11 | 上海美吉生物医药科技有限公司 | Kit for detecting metastatic castration resistant prostate cancer drug resistance |
| CN105823878B (en) * | 2016-04-05 | 2017-06-23 | 上海美吉生物医药科技有限公司 | A kind of kit for detecting circulating tumor cell phenotype |
| CN106381286A (en) * | 2016-08-31 | 2017-02-08 | 上海美吉生物医药科技有限公司 | Folic acid immunomagnetic beads and preparation method thereof |
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