CN102296119B - Detection method and kit for milk globulin mRNA in circulatory blood - Google Patents
Detection method and kit for milk globulin mRNA in circulatory blood Download PDFInfo
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Abstract
The invention provides a specific detection method for a trace amount of breast adenocarcinoma cells in circulatory blood volume, which comprises: mixing a first adaptor coupled with a magnetic particle and a second adaptor coupled with nano gold particles with a sample to be mixed separately or at the same time; and detecting, identifying and/or quantifying target molecules. In addition, the invention also relates to a reagent for treating the blood sample to be treated, an intermediate, a detection kit and the like, which are used in the method. The method is simple and convenient for use and has high sensitivity, high specificity, high repeatability, quick result delivery, no need of complex instruments, and no special skills. Particularly, even if the sample to be detected is detected directly without accurate purification, the method can still keep practical sensitivity and specificity; and thus, the detection operation is facilitated greatly, the consumption of a reagent is saved, the detection process is accelerated, and the method and the kit can be promoted for actual industrial application.
Description
Technical field
The present invention relates to the nucleic acid detection method technical field, particularly, the present invention relates to detect, identify and/or quantitative the circulate method for detecting specificity of micro-breast cancer cell in the blood of people.In addition, the invention still further relates to test kit for this method, intermediate product with and application etc.
Background technology
Mammary cancer is one of modal lethality cancer of women, excision is the first-selection in the mammary cancer comprehensive treatment, but recurrence rate height, rate of transform height after the mammary cancer excision: cancer cells is easy to enter peripheral blood generation metastases from primary tumor, and metastases is the major cause of breast cancer treatment failure.A large amount of experiments have confirmed to circulate, and (Circulating Tumor Cells CTC) detects and will help early diagnosing mammary cancer, relapse and metastasis monitoring, judges patient's prognosis, instructs postoperative adjuvant therapy etc. tumour cell in the blood.
Human galactophore globin (Mammaglobin, MAM) be a kind of mammary tissue specific proteins mark of discovered in recent years, (Watson et al. specific expressed in former of mammary gland, transfer and peripheral blood cancer cells thereof, 1999, Watson and Fleming, 1996, Carter et al., 2002, Zuo et al., 1998).Utilize anti-MAM antibody patented to the specific detection of human galactophore globin in the world, as US6566072 (Mark A.Watson, Timothy P.Fleming, Washington University, 2003).The present domestic specific detection patent that also has at human galactophore globin: Chinese patent (application) CN03117222.9 discloses a kind of people MAM montage variant protein, utilizes this protein Preparation monoclonal antibody and for detection of MAM; Chinese patent (application) CN200510106613.4 discloses method and the test kit thereof that the latex particle that utilizes antibody labeling detects MAM.These methods are all utilized antibody to carry out MAM and are detected, therefore the accuracy that detects requires very high to the specificity of wherein antibody, otherwise a large amount of false-positive results can appear, because the production cost height of antibody reagent own, antibody must be preserved under suitable condition, transportation and use to prevent the antibody titer step-down, this has increased the uncertainty that detects cost and detected result greatly in addition.Watson etc. at first set up the method (Watson et al., 1999) that detects people MAM mRNA with RT-PCR; Chinese patent CN200710303872.5 sets up and utilizes RT-PCR to detect method and test kit thereof that people MAM mRNA expresses.But, this method need be utilized expensive RT-PCR equipment, the process of finishing RT-PCR in addition is very consuming time, the susceptibility that RT-PCR detects and specificity are very big to quality, testing environment purity (the no DNA crossed contamination) dependence of detection reagent, descend or false positive results otherwise be prone to detection sensitivity.These all most basic hospitals be difficult to overcome.
The beginning of the nineties in last century, Tuerk and Gold have founded index concentration formula aglucon systematize screening (Systematic Evolution of Ligands by Exponential Enrichment, abbreviate SELEX as) technology, external selection and amplification by many wheels, can from the random oligonucleotide library, filter out specificity aptamers (aptamer), referring to (Tuerk and Gold, 1990).At present, it has been found that the specificity aptamers of many molecules, aptamers as microorganism, biomacromolecule (as, vascular endothelial growth factor, the somatomedin in thrombocyte source, Prostatropin, L-selects plain, keratinocyte growth factor, IFN-, cytokines such as tumour necrosis factor) aptamers, micromolecular aptamers, can the specific combination corresponding microorganism, biomacromolecule, target molecules such as small molecules, referring to CN1490054A, CN1550501A, CN1563401A, CN101148666A, CN101835904A, the biotechnology journal, 20(4): 627-632, etc.The advantage of aptamers is that the identification of target molecule is had very high avidity, its dissociation constant even can reach 50pmol/L～10nmol/L.In addition, because the chemical structure of aptamers itself is single-chain nucleic acid, the combination of aptamers and target molecule can be subjected to the interference of impurity molecule, especially when target molecule and impurity molecule all be single-chain nucleic acid (as, scDNA, mRNA) situation under, because there is the character of pairing dimerization in nucleic acid chains itself, take place increasing situation about disturbing.These unfavorable factors have influenced accuracy and the precision of aptamers detection, make it be difficult to popularization and carry out the commercialization use, therefore still come into operation in practice based on the ELISA detection of antibody or PCR detection consuming time etc. at present.
Summary of the invention
First problem to be solved by this invention is that the specific detection that overcomes existing human galactophore globin requires height to the environment purity, the antagonist specificity requires problem very high and that false positive rate is higher, the detection method of mammaglobin mRNA in a kind of blood that circulates is provided, this method is easy to use, sensitive, high specificity, good reproducibility, it is quick to go out the result, need not complex instrument and special skill, and can utilize the testing sample of purifying without precision directly to detect, the situation that aptamers and target molecule (MAM mRNA) and other non-specific nucleic acid squences interfere with each other can not appear.
Another technical problem to be solved by this invention is to provide the specific detection test kit of micro-breast cancer cell in a kind of blood that circulates.
The specific detection test kit that another technical problem to be solved by this invention is to provide micro-breast cancer cell in a kind of blood that circulates is for the preparation of the application in the goods of the present invention's method aspect first.
Another technical problem to be solved by this invention is to provide the intermediate product binding substances in the specific detection of micro-breast cancer cell in a kind of blood that circulates.
In order to solve these problems of the prior art, aspect first, technical scheme provided by the invention is: the invention provides the detection method of mammaglobin mRNA in a kind of blood that circulates, may further comprise the steps,
(1) from mammary gland cell, comprises and extract mammaglobin mRNA in cancer cells and the normal cell;
(2) prepare with first aptamers of magnetic-particle coupling and with second aptamers of nm gold particles coupling, wherein first aptamers and second aptamers be respectively with the aptamers of the different sites specific combination of mammaglobin mRNA, and first aptamers does not have the complementary nucleic acid sequence with second aptamers and does not have the potential possibility of mutual cross combination mutually;
(3) with the aptamers of coupling with to be detected, evaluation and/or quantified sample is mixed and carry out magnetic resolution,
(4) detect the magnetic resolution product.
Preferably, in the method for first aspect of the present invention, wherein step (3) is, will with second aptamers of first aptamers of magnetic-particle coupling, nm gold particles coupling simultaneously with to be detected, evaluation and/or quantified sample is mixed and carry out magnetic resolution,
Preferably, in the method for first aspect of the present invention, wherein step (3) is:
1. first aptamers of magnetic-particle coupling is mixed with to be detected, evaluation and/or quantified sample;
2. carry out magnetic resolution, collect the product of magnetic force absorption gained; With
3. mix magnetic force absorption gained product and with second aptamers of nm gold particles coupling, carry out magnetic resolution then.
Preferably, in the method for first aspect of the present invention, wherein mammaglobin is human galactophore globin, and preferably its mRNA is shown in SEQ ID No.1.
Preferably, in the method for first aspect of the present invention, wherein the combining site of one of first aptamers and second aptamers is positioned on the 1-30 position of human galactophore globin mRNA, and another combining site of first aptamers and second aptamers is positioned on the 470-490 position of human galactophore globin mRNA.More preferably, wherein the identity of first aptamers and mammaglobin mRNA is less than 30%, and the identity of second aptamers and mammaglobin mRNA is less than 30%, most preferably first aptamers is shown in SEQID No.2, be 5 '-aaggaagccgctgtc-3 ', and second aptamers shown in SEQ ID No.3, be 5 '-aataaacatgatagc-3 '.Especially as the described method of the specific embodiment of the invention preparation with first aptamers magnetic-particle coupling and with second aptamers of nm gold particles coupling.
Preferably, in the method for first aspect of the present invention, second aptamers certification mark substance markers wherein.Wherein, the certification mark thing is one or more combinations in vitamin H Biotin, radio isotope, metal mark thing, chemiluminescent groups, chemiluminescence group, fluorescin, enzyme, antigen or antibody, part or the acceptor, most preferably is vitamin H Biotin.
Preferably, in the method for first aspect of the present invention, wherein second aptamers is the nm gold particles coupling by avidin and suis avidin-horseradish peroxidase bag quilt.
Preferably, in the method for first aspect of the present invention, wherein the magnetic resolution product is the product of magnetic force absorption gained.
Preferably, in the method for first aspect of the present invention, wherein the magnetic resolution product is through washing.
Preferably, in the method for first aspect of the present invention, the temperature of wherein carrying out step (3) is 20-42 ℃, is preferably 37 ℃.
Preferably, in the method for first aspect of the present invention, wherein the damping fluid of step (3) is Tris-HCl, PBS or the PBS that contains the Mg ion, is preferably the PBS that contains the Mg ion.
Aspect second, the invention provides the test kit of the method for detecting specificity of micro-breast cancer cell in a kind of blood that circulates, it comprise with first aptamers of magnetic-particle coupling and with second aptamers of nm gold particles coupling, wherein first aptamers and second aptamers be respectively with the aptamers of the different sites specific combination of mammaglobin mRNA, and first aptamers does not have the complementary nucleic acid sequence and does not have the potential that the phase mutual cross is combined with second aptamers.
Preferably, in the test kit of second aspect of the present invention, wherein the combining site of one of first aptamers and second aptamers is positioned on the 1-30 position of human galactophore globin mRNA, and another combining site of first aptamers and second aptamers is positioned on the 470-490 position of human galactophore globin mRNA.
Preferably, in the test kit of second aspect of the present invention, wherein the identity of first aptamers and mammaglobin mRNA is less than 30%, and the identity of second aptamers and mammaglobin mRNA is less than 30%, most preferably first aptamers is shown in SEQ ID No.2, be 5 '-aaggaagccgctgtc-3 ', and second aptamers is 5 '-aataaacatgatagc-3 ' shown in SEQ ID No.3.
Preferably, in the test kit of second aspect of the present invention, second aptamers certification mark substance markers wherein.Wherein, the certification mark thing is one or more combinations in vitamin H, radio isotope, metal mark thing, chemiluminescent groups, chemiluminescence group, fluorescin, enzyme, antigen or antibody, part or the acceptor, most preferably is vitamin H.
Preferably, in the test kit of second aspect of the present invention, wherein second aptamers is the nm gold particles coupling by vitamin H and suis avidin-horseradish peroxidase bag quilt.
Preferably, in the test kit of second aspect of the present invention, also comprise the reagent that can detect the certification mark thing, as the horseradish peroxidase chromogenic substrate.
Preferably, in the test kit of second aspect of the present invention, it also comprises damping fluid, and preferred buffer is Tris-HCl, PBS or the PBS that contains the Mg ion, more preferably contains the PBS of Mg ion.
Preferably, in the test kit of second aspect of the present invention, it also comprises the magnetic resolution device.
Aspect the 3rd, the test kit that the invention provides second aspect of the present invention is for the preparation of the application in the goods of the method for the present invention aspect first.
Aspect the 4th, the invention provides a kind of binding substances, by with first aptamers of magnetic-particle coupling, with second aptamers and the mammaglobin mRNA be combined into of certification mark thing coupling, wherein first aptamers and second aptamers be respectively with the aptamers of the different sites specific combination of mammaglobin mRNA, and first aptamers does not have the complementary nucleic acid sequence and does not have the potential that the phase mutual cross is combined with second aptamers.Preferably wherein, mammaglobin is human galactophore globin, and more preferably its mRNA is shown in SEQ ID NO:1.
Preferably, in the binding substances of the 4th aspect of the present invention, wherein the combining site of one of first aptamers and second aptamers is positioned on the 1-30 position of human galactophore globin mRNA, and another combining site of first aptamers and second aptamers is positioned on the 470-490 position of human galactophore globin mRNA.
In the binding substances of preferred the 4th aspect of the present invention, wherein the identity of first aptamers and mammaglobin mRNA is less than 30%, and the identity of second aptamers and mammaglobin mRNA is less than 30%, most preferably first aptamers is shown in SEQ ID No.2, be 5 '-aaggaagccgctgtc-3 ', and second aptamers shown in SEQ ID No.3, be 5 '-aataaacatgatagc-3 '.
Preferably, in the binding substances of the 4th aspect of the present invention, second aptamers certification mark substance markers wherein.
Preferably, in the binding substances of the 4th aspect of the present invention, wherein the certification mark thing is one or more combinations in vitamin H, radio isotope, metal mark thing, chemiluminescent groups, chemiluminescence group, fluorescin, enzyme, antigen or antibody, part or the acceptor, most preferably is vitamin H.
Preferably, in the binding substances of the 4th aspect of the present invention, wherein second aptamers is the nm gold particles coupling by vitamin H and suis avidin-horseradish peroxidase bag quilt.
Preferably, in the binding substances of the 4th aspect of the present invention, it can be at 20-42 ℃, is preferably combination under 37 ℃ the temperature.
Aspect the 5th, the invention provides mammaglobin mRNA aptamers, it is selected from 5 '-aaggaagccgctgtc-3 ' of SEQ ID No.2 and the 5 '-aataaacatgatagc-3 ' shown in the SEQ ID No.3.
In this article, term " detection " has implication well-known to those skilled in the art, comprises the existence of identifying target product and the quantity of quantitative objective product.The resulting net result of detection method of the present invention is existence or the quantity of mammaglobin mRNA.Owing to also exist a small amount of mammaglobin mRNA to see (Watson and Fleming, 1996) in the mammary gland of minority healthy individuals, therefore identify that mammaglobin mRNA can't directly draw the result who whether suffers from breast tumor or the breast tumor occurence tendency is arranged; Because implementer of the present invention is the clinicist not necessarily, therefore and do not know that patient is being got " sample " before whether through degree for the treatment of and treatment, therefore the quantity of the mammaglobin mRNA that quantitatively obtains can't directly draw and whether suffers from breast tumor or the breast tumor recurrence is arranged, shift the result of occurence tendency.The method of first aspect of the present invention only can be regarded an intermediate steps in the diagnostic procedure as, that obtain is mammaglobin mRNA result in " sample ", itself can not directly draw whether to suffer from breast tumor or the breast tumor recurrence is arranged, shift diagnostic results such as occurence tendency.In addition, because individual hereditary difference, cause the mammaglobin mRNA level of each individual patient that certain difference is arranged, age of patient in addition, the menstrual physiology state also can cause the difference of mammaglobin mRNA level in the blood, if directly judge whether to suffer from breast tumor or the breast tumor recurrence arranged, shift diagnostic results such as occurence tendency, the result that the method for first aspect of the present invention must be obtained and corresponding normal healthy controls or individual before treatment, after blood in mammaglobin mRNA level compare, and judge by experienced doctor whether corresponding individuality is enough to deduct idiogenetics, age, factors such as menstrual physiology state could attempt obtaining diagnostic result to the systematic effects of mammaglobin mRNA.Therefore, the method for first aspect of the present invention does not comprise the process that compares with corresponding normal healthy controls and individual treatment front and back.Even according to the specific embodiment of the present invention, the mammaglobin mRNA level of the different breast cancer cell lines of different individual patient also exists greatly difference, some in addition similar to background values, can be used as negative control, can't directly draw diagnostic result thus.
In this article, term " sample " refers to the potential stripped circulation blood sample that may contain mammaglobin mRNA, preferably from people's sample.Although the purification of samples with the hemorrhage total RNA of extracting also can use in the present invention,, sample of the present invention is preferably without the cracking liquid sample of purifying, contain the total RNA of blood.Those skilled in the art know the nucleated blood cell cracking of solid or extracting, purifying and the cellular elements biology techniques that keeps mRNA composition wherein not to be degraded.Sample of the present invention can be treated sample, as dilution process, hemocyte cracking processing and pcr amplification etc., also can be undressed sample.Treated sample can be further purified, with enrichment mRNA.Because sampling need have the doctor of qualification to decide according to individual particular case to human body, so the method for first aspect of the present invention does not comprise the process of the sample preparation of taking a sample and choosing wantonly, owing to do not know the sample degree whether process is handled and handled, therefore can't directly draw and whether suffer from breast tumor or diagnostic results such as breast tumor recurrence, transfer occurence tendency are arranged.The method of first aspect of the present invention does not comprise the process of the sample preparation of taking a sample and choosing wantonly.
In present patent application, mammaglobin (abbreviating MAM as) is human galactophore globin preferably, is more preferably the human galactophore globin cDNA sequence of mRNA shown in SEQ ID NO:1.Mammaglobin is only expressed in mammary gland and breast cancer cell, detects therefore whether mammaglobin mRNA can exist mammary gland and breast cancer cell for the sample that judgement detects in the circulation blood.
In present patent application, be used for the label of the different aptamers of difference, namely term such as " first ", " second " only is used for the different chemical entities of representative, does not limit the constitutional features of these chemical entities.The chemical entities that these terms are distinguished in this article mainly is aptamers, as DNA aptamers, RNA aptamers or have aptamers of certification mark thing and/or magnetic-particle etc.In this article, DNA and RNA represent its sequence with symbol well-known to those skilled in the art, and if no special instructions, what sequence was represented is the sequence order of holding to 3 ' extreme direction from 5 '.Be preferably 0.5～5:1 with first aptamers of magnetic-particle coupling with the mol ratio of second aptamers that detects the nm gold particles coupling, most preferably be 1:1.Because used two aptamers among the present invention, these two aptamers must be attached on the same target molecule, just can obtain detected result, thereby detected result are very special; If first aptamers of capture probe or magnetic-particle coupling and the combination of non-specific target molecule, but this non-specific target molecule possibility of combination is very little simultaneously with second aptamers that detects the nm gold particles coupling, thereby still can eliminate the influence of non-specific combination when detecting.Such aptamers double insurance makes method of the present invention greatly reduce false-positive result when detecting mammaglobin mRNA, has improved accuracy.Owing to used among the present invention and second aptamers that detects the nm gold particles coupling, wherein nm gold particles is coated with a plurality of suis avidins-horseradish peroxidase molecule, warp and horseradish peroxidase substrate color reaction can strengthen the detection sensitivity to mammaglobin mRNA.
Term in the present patent application " aptamers " is well-known to those skilled in the art, refer to can with the single-chain nucleic acid of specific target molecule (mammaglobin mRNA) combination, comprise DNA or RNA.Can obtain the aptamers of certain target molecules by SELEX.Owing to used two aptamers in the method for first aspect present invention, do not disturb mutually in order to make the aptamers that obtains, the inventor discovers, first aptamers does not have the complementary nucleic acid sequence and does not have the potential that the phase mutual cross is combined with second aptamers, for example the identity of first aptamers and second aptamers is less than 50%, preferably less than 40%, be more preferably less than 30%.Because the chemical classification of target molecule (mammaglobin mRNA) also belongs to nucleic acid, for fear of the interference of target molecule itself, the inventor discovers that the identity of first aptamers and mammaglobin mRNA is less than 30%, preferably less than 20%, be more preferably less than 10%; The identity of second aptamers and mammaglobin mRNA preferably less than 20%, is more preferably less than 10% less than 30%.Wherein, can adopt the BLAST algorithm of acquiescence to come identity between the definite kernel acid sequence, this knows to those skilled in the art.In the specific embodiment of the present invention, first aptamers is 5 '-aaggaagccgctgtc-3 ', and second aptamers is 5 '-aataaacatgatagc-3 '.
In present patent application, " the potential possibility of no phase mutual cross combination " in the method steps refers to first, second aptamers nucleotide sequence of detailed analysis, show that no tangible nucleic acid complementary sequence exists between the two, can not hybridize the stable aptamers combination of formation after the mixing, can not influence the combination of aptamers and target molecule.
In present patent application, magnetic-particle is magnetic nanoparticle (abbreviating MNP as) preferably, i.e. nano level size particles, and it has magnetic, can separate by equipment for magnetic separation.Magnetic-particle comprises nano particle, quantum dot or the nano dot etc. of magneticmetal.Described particle is superparamagnetism preferably, comprises the particle of records such as US6057107B, US6268222B, US6524793B.Preferably select for use and can obtain product by the commercial channel.In the present invention, preferred first aptamers and magnetic-particle are by the chemical reaction coupling.
In present patent application, with the marker on second aptamers that detects the nm gold particles coupling be the material that can detect, for example can excite by laser, radioactivity, chemoluminescence (comprising fluorescence), electroluminescence, electrochemiluminescence, enzyme catalysis colour developing or any method well known by persons skilled in the art realize.Exemplary survey marker includes but not limited to one or more combinations in vitamin H, radio isotope, chemiluminescent groups, chemiluminescence group, fluorescin, enzyme, antigen or antibody, part or the acceptor.Radioisotopic suitable example has
32P,
125I,
35S,
3H etc.By ordinary methods such as chemosynthesis, PCR, nick translations, can prepare radiolabeled second single-chain nucleic acid.Chemiluminescent groups has luminol,3-aminophthalic acid cyclic hydrazide etc.; The chemiluminescence group comprises rhodamine, FITC, TRITC etc.Be that chemical synthesis process by routine carries out to second single-chain nucleic acid with vitamin H (Biotin) mark, the above-mentioned single-chain nucleic acid that has a biotin labeling thing and preparation thereof and detection method are known, can summarize referring to (Tabor and Boyle, 2001).By chemical synthesis process, suis avidin-horseradish peroxidase molecule can be coated on the nm gold particles, its method can be referring to documents such as (Long and Keating, 2006, Mayer et al., 2000, Tang et al., 2008).The nm gold particles of the above-mentioned single-chain nucleic acid that has a biotin labeling thing and suis avidin-horseradish peroxidase bag quilt can special, high-affinity ground combination, and forms the second aptamers mixture with measuring ability.At present there has been a large amount of commercial horseradish peroxidase detection kit available, as the Streptavidin-Peroxidase that Sigma-Aldrich company sells, Supersignal West Pico Horseradish Peroxidase (HRP) the Detection test kit that Pierces (USA) company sells etc.In the specific embodiment of the present invention, utilize the nm gold particles energy in conjunction with the character than the multienzyme molecule, the certification mark thing adopts the coupled product of nm gold particles and horseradish peroxidase, has strengthened the detection sensitivity to mammaglobin mRNA greatly.
In present patent application, the magnetic resolution product is the product of magnetic force absorption gained, also can be the product that is not adsorbed by magnetic force, but the product of magnetic force absorption gained preferably.Can be adsorbed by magnetic force and be detected the product that obtains must be the product that combines first and second aptamers.By equipment for magnetic separation, can be attached on the test tube wall by the adsorbed material of magnetic force, then still do not resided in the solution by the adsorbed material of magnetic force, after draw solution, these two kinds of materials just can be separated, thereby reach the purpose of separation.In theory, in the product of magnetic force absorption gained and the product that is not adsorbed by magnetic force the sum total of certification mark thing equal to mix initial adding capture probe or with the amount of first aptamers of magnetic-particle coupling, both can convert according to the amount of the certification mark thing that mixes initial adding.Preferably in the step of magnetic resolution, wash the magnetic resolution product at last, namely the magnetic resolution product is through washing.
Binding substances of the present invention can be hatched under 20-42 ℃ temperature in conjunction with and do not separated, and preferably hatches the hybridization temperature that this temperature normally adopts for fear of aptamers and non-specific target molecular hybridization under 37 ℃ temperature; Be the carrying out of handled easily, allow that the temperature of testing circumstance has 1-2 ℃ deviation, but must under less than 45 ℃ temperature, carry out, preferably under 37 ℃ temperature, carry out.As surpassing this temperature, as 50 ℃, may cause aptamers to be combined with the specific target molecule, thereby reduce the sensitivity that detects.In this article, as do not add explanation, mix the action that refers to mixing and mix the process that (hatching) placed in the back.
The method operating time of the present invention is short.Wherein account for most time of placing in the mixing step of time less than 60 minutes, preferred 30-45 minute, as 30,35,40 minutes.
Than solution of the prior art, the invention has the beneficial effects as follows:
1, simple to operate, detection time is short.Because the sample that method of the present invention in fact only need detect reagent and band simply mix and place the back with magnetic separator absorption and detect, remove the detection step, other steps are simple, convenient, weak point consuming time, especially detect MAM mRNA, time much shorter with respect to conventional RT-PCR.Need not complex instrument and special skill, especially this method can be utilized the direct detection of testing sample of purifying without precision to be very easy to like this and detect operation, has saved the reagent use and has accelerated detection procedure, can promote the industry of carrying out reality and use.
2, high specificity, good reproducibility, accuracy rate height have effectively reduced false positive results.Owing to the present invention is with two non-interfering aptamers, adopt double insurance, thereby can keep the correct of result, greatly reduce the probability that false positive results occurs, make the suitable reality of the present invention promote.Method of the present invention can be utilized the testing sample of purifying without precision directly to detect and still can keep practical sensitivity and specificity, the situation that aptamers and target molecule (MAM mRNA) and other non-specific nucleic acid squences interfere with each other can not occur.
3, higher detection sensitivity relatively, suitable with the RT-PCR detection MAM mRNA of routine.
4, commercialization is easy, and it is low to detect cost.Because reagent itself can prepare by business-like method, and required instrument also can be bought and obtains in detecting, therefore can be fast in large, medium and small type hospital even clinic popularization and application; Wherein employed instrument comprises equipment for magnetic separation, the PCR instrument considerably cheaper of use in detecting than conventional RT-PCR, thereby easily commercialization is promoted.
5, constant product quality, condition of storage is less demanding.Aptamers is more stable than protein antibodies, and volatility is not preserved easily for a long time, and this causes the validity period of product to prolong greatly.
6, cost is low, because aptamers chemosynthesis in a large number, thereby the products production cost can significantly reduce than antibody detection method or RT-PCR method.
Description of drawings
Below in conjunction with drawings and Examples the present invention is further described:
Fig. 1 has shown different concns and the influence of first aptamers magnetic-particle coupling to color reaction intensity.
Fig. 2 has shown different add-ons and the influence of second aptamers nm gold particles coupling to color reaction intensity.
Fig. 3 has shown the influence of different experiments incubation temperature to color reaction intensity.
Fig. 4 has shown the influence of different experiments damping fluid to color reaction intensity.
Fig. 5 has shown that method of the present invention detects the result of the MAM mRNA in the MDA-MB361/231 cell total rna that dilutes in proportion.
Embodiment
For the ease of understanding, below will describe in detail the present invention by specific embodiment.It needs to be noted that these descriptions only are exemplary descriptions, do not constitute limitation of the scope of the invention.According to the argumentation of this specification sheets, many variations of the present invention, change all are apparent concerning one of ordinary skill in the art.
Below will describe with the form of giving an example, if any not detailed part, can be referring to laboratory manual commonly used, as the manufacturers instruction of " molecular cloning laboratory manual " and agents useful for same and instrument.Wherein, all chemical reagent all adopt AG, and experimental water filters through Milli-XQ, and each reagent all can obtain by commercial channel with material, particularly: (1.5 μ M are 20mg/mL) available from Polysciences company (U.S.) to have the magnetic corpusculum of C-terminal; 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (abbreviating EDC as) is available from Alfa Aesar company (U.S.); The gold nano grain (particle diameter is 10nm and 40nm) of nm gold particles (particle diameter is 5nm, 10nm, 30nm and 50nm), suis avidin (streptavidin) bag quilt is all available from the BB International(U.S.); Bovin serum albumin, suis avidin-horseradish peroxidase (S2438) are available from the Sigma-Aldrich(U.S.); Horseradish peroxidase substrate reagent box is available from the Millipore Corporation(U.S.); Nucleic acid primer, first aptamers and biotinylation second aptamers entrust Sigma-Aldrich company (USA) synthetic, and its sequence is as follows: 5 '-aaggaagccgctgtc-3 ' and 5 '-aataaacatgatagc-3 '; MCF-7 MDA-MB361(MAM mRNA expresses positive) and MDA-MB231(MAM mRNA expression feminine gender), all can be available from the U.S. representative microbial preservation center (ATCC), the substratum of cultivating it is substratum (the DMEM substratum of the Dulbecco improvement Eagle of the penicillin that adds 10% foetal calf serum bovine serum (Gibco company), L-L-glutamic acid (Gibco company), 50U/ml and 5 mcg/ml Streptomycin sulphates (Gibco company); Sigma-Aldrich company); It is TRIzol reagent that cell total rna extracts reagent, and RT-PCR reaction related reagent is available from (Gibco company, the U.S.).
The preparation that embodiment 1 detects with MAM mRNA sample and contrast thereof
Vitro culture MDA-MB361 cell, attached cell makes cell suspension through trysinization, and the centrifugal nutrient solution that goes adds physiological saline again, and the preparation single cell suspension is got a certain amount of cell (10 through cell counting
7) experimentize.According to manufacturer's explanation, extract reagent with cell total rna and extract the MDA-MB361 cell with cell total rna (MAM mRNA expresses positive), measure 260 and 280 nanometers optical density(OD) (OD value) with the amount of quantitatively total RNA.Then total RNA is carried out gradient dilution with TE damping fluid (pH7.2), comprise respectively from being equivalent to 10 in each dilution solution equal-volume
5(A group), 10
4(B group), 10
3(C group), 10
2(D group) and 10
1Total RNA amount of (E group) individual MDA-MB361 breast cancer cell.
According to aforesaid method, extract total RNA that MAM mRNA expresses negative MDA-MB231 cell, after diluting, comprise respectively from being equivalent to 10 in each dilution solution equal-volume
5(A
0Group), 10
4(B
0Group), 10
3(C
0Group), 10
2(D
0Organize) and 10
1(E
0Group) total RNA amount of individual MDA-MB231 breast cancer cell.
Synthetic and the preparation of embodiment 2 aptamers
Get the little liquid solution of magnetic that 12 microlitres have C-terminal and place the 1.5ml centrifuge tube, (add 150 microlitre 0.1M imidazole buffers (pH7.0) mixes back centrifugal at every turn with 0.1M imidazole buffer washing three times, abandon supernatant), add 300 microlitres and contain the 0.1M imidazole buffer (pH7.0) of 0.03M EDC, slowly shook centrifuge tube 20 minutes, and then first aptamers of 12pmol (5 '-aaggaagccgctgtc-3 ') added, mix 37 ℃ and hatched 1 hour, during slowly shake centrifuge tube continuously.Centrifuge tube placed apply magnetic force on the magnetic separator, blot supernatant liquor and (add 300 microlitre washing solns (the PBS solution that contains 100mM NaCl three times with the washing soln washing at every turn, pH7.2) and blot), in order to remove the unconjugated first free aptamers, add 240 microlitre lysate (20mM Tris-HCl then, pH8.0,0.5M NaCl) with its dissolving.Make first aptamers with the magnetic-particle coupling thus.
Get the nm gold particles solution (pH8.2 of 40nm particle diameter, 9x1010 particle/ml, the about 1.5pmol of 8 microlitre nano-Au solutions) with 3 microlitre suis avidin-horseradish peroxidase (1mg/ml) mixing, add water to 100 microlitres, after room temperature leaves standstill 2 hours, add 22 microlitre 5%(g/v) PEG20000, through centrifugal 45 minutes nm gold particles precipitations with suis avidin-horseradish peroxidase mark of 9000x g, use 1ml PBS(pH7.2) resuspending, the washing after, centrifugation again, three times repeatedly, be melted among the 10 microlitre PBS.Get capacity (10 microlitres, biotin labeled second aptamers 2pmol) (5 '-aataaacatgatagc-3 ') mix with it, hatched 24 hours for 40 ℃, add 1ml PBS then, with 2800x g centrifugal 45 minutes, blot supernatant liquor to remove the unconjugated second free aptamers, add 600 microlitre solution (45mmol NaCl, 30% sucrose, 0.25%Tween-20,0.25%SDS) then with the resolution of precipitate of redness, make second aptamers with the nm gold particles coupling thus.
Determining of embodiment 3 first aptamers and the second aptamers concentration
In the series reaction test tube, add the known MDA-MB361 cell total rna (1 microgram) that contains target molecule (MAM mRNA), with according to embodiment 2 preparation with first aptamers (the 2-20 microlitre magnetic-particle coupling, 1-10pmol) mix, add PBS(0.1M, pH7.2) to 100 microlitres, hatched 60 minutes in 37 ° of C; Then test tube is placed and apply magnetic force on the magnetic separator, blot supernatant liquor and (add 300 microlitre washing solns (the PBS solution that contains 100mMNaCl three times with the washing soln washing at every turn, pH7.2) and blot), add 95 microlitre PBS damping fluids, mixing, add again 5 microlitres according to embodiment 2 preparation with second aptamers nm gold particles coupling, hatched 60 minutes in 37 ° of C temperature, then test tube is placed and apply magnetic force on the magnetic separator, blot supernatant liquor, and with washing three times as above-mentioned washing soln.According to manufacturer's explanation, add the reaction substrate solution of horseradish peroxidase substrate reagent box and measure color reaction intensity (measuring 450nm place absorption spectrum) then.The result as shown in Figure 1, when with (end) of first aptamers of magnetic-particle coupling when concentration is got 1pmol, color reaction intensity is the highest, determines that therefore the suitableeest (end) concentration with first aptamers of magnetic-particle coupling is 1pmol.
Equally, in the series reaction test tube, add the known MDA-MB361 cell total rna (1 microgram) that contains target molecule (MAM mRNA), with 2 microlitres according to embodiment 2 preparation with first aptamers (1pmol) the magnetic-particle coupling, add PBS(0.1M, pH7.2) 100 microlitres, add respectively more different amounts according to embodiment 2 preparation with the second aptamers 1-9 microlitre nm gold particles coupling, hatched 60 minutes in 37 ℃, then test tube is placed and apply magnetic force on the magnetic separator, blot supernatant liquor.According to manufacturer's explanation, add the reaction substrate solution of horseradish peroxidase substrate reagent box and measure color reaction intensity (measuring 450nm place absorption spectrum) then.The result as shown in Figure 2, therefore when getting 5 microlitres with second aptamers of nm gold particles coupling, color reaction intensity is the highest, determines that the suitableeest adding dosage with second aptamers of nm gold particles coupling is 5 microlitres.
Determining of embodiment 4 experimental temperatures and buffering system
According to the concentration that embodiment 3 determines, the temperature of experiment is tested.Experiment is to carry out according to the methods that embodiment 3 determines, unique different be that 52 ℃ of 22 –, carry out for 5 ℃ at every interval.Experimental result shows that (as shown in Figure 3) under the situation of target molecule arranged, and the color reaction intensity level increases gradually between 37 ℃ of 22 –, reaches maximum until being warmed to 37 ℃, enters plateau subsequently (slightly descending), begins rapid decline when being warmed to 42 ℃; Do not having (result does not show) under the situation of target molecule, the association reaction intensity level maintains always that low-level almost do not have can detected peak value between 42 ℃ of 22 –.Thereby definite experimental temperature is optimum temps with 37 ° of C.
Get concentration and the above-mentioned temperature of reaction determined according to embodiment 3, the buffer system of experiment is tested, uniquely different be, respectively with three kinds of damping fluids experimentize test: TE (Tris-HCl10mM, EDTA1mM, pH7.5), PBS(0.1M, pH7.2) and contain 0.001M MgCl
2PBS.The result uses to contain MgCl as shown in Figure 4
2PBS the time, therefore color reaction intensity is the highest, determines that the buffer system of experiment is to contain 0.001M Mg
2+PBS be optimized buffer liquid.
The concentration of mammaglobin mRNA in embodiment 5 test sample
Condition according to embodiment 3-4 determines contains the MDA-MB361 cell total rna (A, B, C, D, E organize diluent) of target molecule (MAM mRNA) and expresses the negative MDA-MB231 cell total rna (A of target molecule (MAM mRNA) the known of embodiment 1 preparation
0, B
0, C
0, D
0, E
0The group diluent), carry out above-mentioned experiment respectively and detect (namely total RNA(1 microgram of above-mentioned experiment) and replace with these diluents), judge the existence of target RNA molecule according to the power of color reaction.
The result as shown in Figure 5, the detected result of expressing the negative MDA-MB231 cell total rna of target molecule (MAM mRNA) very at the bottom of the utmost point, can be used as negative control and uses, with its detection line as the standard baseline (being higher than this line is the detected result positive) that detects; And in three duplicate detection to MDA-MB361 serial dilution RNA sample, even minimum dilution sample detection value also significantly greater than the result of the negative control of high dilution, namely all can be at 1:10
5And detecting MAM mRNA in the above diluent, its sensitivity can reach other extent of dilution of 100,000/level at least.We have tested 1:10 separately
6Diluent is found difficult to distinguish.
Comparative example 1 with detect mammaglobin (mammaglobin, MAM) comparison of the method for mRNA with RT-PCR
Carry out according to conventional RT-PCR detection method, it comprises: get total RNA, 3 μ g Yeast Nucleic Acid random hexamer and DEPC water, cumulative volume is 20 μ l, and 72 ° of C heated 10 minutes; Add reverse transcription mixture (5 * cDNA reverse transcription damping fluid-250mM Tris – HCl pH value 8.3 that contains 4 μ l, Repone K 375mmol, 15mmol magnesium chloride, the dNTPs of 10pmol, the RNAase inhibitor of 20U and 200U reversed transcriptive enzyme (Gibco company).Last mixture after 37 ° of C are hatched 1.5h, 95 ° of deactivations in C5 minute.Carry out the nest-type PRC reaction then, this PCR reaction is carried out in two steps.The first step: the reaction cumulative volume is 25 microlitres, contains 2.5 μ lcDNA, 5 μ L1 * PCR damping fluid (200mM Tris – HCl pH value 8.4, Repone K 500mM, 2mmol magnesium chloride, 600nM primer, 200uMdNTPs, Taq enzyme 1U(Gibco company).The PCR reaction conditions arranges as follows: 94 ° C1 minute; 25 cycles amplification (94 ° of C45 seconds, 58 ℃ of 60 seconds and 72 ° of C30 seconds) was 72 ° of C extensions 10 minutes; MAM outer primer sequence is: 5 '-cagcggcttccttgatccttg-3 ', 5 '-tagcaggtttcaacaattgtc-3 '; Second step: get 5 microlitre PCR products and make template, quantitative PCR is again through as above same PCR reaction 25 cycles of amplification, its inner primer sequence is 5 '-agcactgctacgcaggctct-3 ', 5 '-ataagaaagagaaggtgtgg-3 ', in all quantitative PCR reactions, all there is corresponding house-keeping gene (GAPDH) to increase simultaneously and assesses with the quality to cDNA.After the PCR reaction was finished, through the agarose gel electrophoresis confirmation of ethidium bromide staining, the specificity product of pcr amplification was 202bp, can be used for detecting MAM mRNA.The quantitative fluorescent signal of RT-PCR of A, B, C, D, these samples of E, through the method for three repeated experiments RT-PCR can 100% ground at 1:10
5Detecting MAM mRNA in the diluent, its sensitivity also reached 100,000/; But we are to 1:10
6Diluent has carried out repeated experiments, but wherein has only 50% RT-PCR experiment can detect MAM mRNA, also is difficult to accurately differentiate the MAM mRNA in the 1:106 diluent.
This shows that the RT-PCR that method of the present invention can substitute expensive and complicated operation effectively detects, and detection sensitivity is suitable substantially.
Above-mentioned example only is explanation technical conceive of the present invention and characteristics, and its purpose is to allow the people who is familiar with this technology can understand content of the present invention and enforcement according to this, can not limit protection scope of the present invention with this.All equivalent transformations that spirit is done according to the present invention or modification all should be encompassed within protection scope of the present invention.
Claims (32)
1. the specific detection test kit of micro-breast cancer cell in the circulation blood, it comprise with first aptamers of magnetic-particle coupling and with second aptamers of nm gold particles coupling, wherein first aptamers and second aptamers be respectively with the aptamers of the different sites specific combination of mammaglobin mRNA, and first aptamers does not have the complementary nucleic acid sequence and does not have the potential that the phase mutual cross is combined with second aptamers;
Wherein the identity of first aptamers and mammaglobin mRNA is less than 30%, and the identity of second aptamers and mammaglobin mRNA is less than 30%, first aptamers is shown in SEQ ID No.2, be 5 '-aaggaagccgctgtc-3 ', and second aptamers shown in SEQ ID No.3, be 5 '-aataaacatgatagc-3 '.
2. test kit according to claim 1, wherein the combining site of one of first aptamers and second aptamers is positioned on the 1-30 position of human galactophore globin mRNA, and another combining site of first aptamers and second aptamers is positioned on the 470-490 position of human galactophore globin mRNA.
3. test kit according to claim 1, wherein second aptamers certification mark substance markers.
4. test kit according to claim 3, wherein the certification mark thing is one or more combinations in vitamin H, radio isotope, metal mark thing, chemiluminescent groups, chemiluminescence group, fluorescin, enzyme, antigen or antibody, part or the acceptor.
5. test kit according to claim 3, wherein second aptamers is the nm gold particles coupling by vitamin H and suis avidin-horseradish peroxidase bag quilt.
6. test kit according to claim 3, it also comprises the reagent that can detect the certification mark thing.
7. test kit according to claim 3, it also comprises damping fluid.
8. test kit according to claim 7, wherein damping fluid is Tris-HCl or PBS.
9. test kit according to claim 7, wherein damping fluid is the PBS that contains the Mg ion.
10. test kit according to claim 1, it also comprises the magnetic resolution device.
11. according to the application in the goods of arbitrary described test kit method for detecting specificity of micro-breast cancer cell in for the preparation of circulation blood of claim 1-10, the step of the method for detecting specificity of micro-breast cancer cell comprises in the blood that wherein circulates,
(1) from mammary gland cell, extracts mammaglobin mRNA;
(2) prepare with first aptamers of magnetic-particle coupling and with second aptamers of nm gold particles coupling, wherein first aptamers and second aptamers be respectively with the aptamers of the different sites specific combination of mammaglobin mRNA, and first aptamers does not have the complementary nucleic acid sequence with second aptamers and does not have the potential possibility of mutual cross combination mutually;
(3) with the aptamers of coupling with to be detected, evaluation and/or quantified sample is mixed and carry out magnetic resolution,
(4) detect the magnetic resolution product.
12. application according to claim 11, wherein step (3) is: will with second aptamers of first aptamers of magnetic-particle coupling, nm gold particles coupling simultaneously with to be detected, evaluation and/or quantified sample is mixed and carry out magnetic resolution.
13. application according to claim 11, wherein step (3) is:
1. first aptamers of magnetic-particle coupling is mixed with to be detected, evaluation and/or quantified sample;
2. carry out magnetic resolution, collect the product of magnetic force absorption gained; With
3. mix magnetic force absorption gained product and with second aptamers of nm gold particles coupling, carry out magnetic resolution then.
14. application according to claim 11, wherein mammaglobin is human galactophore globin.
15. application according to claim 14, wherein human galactophore globin mRNA is shown in SEQ ID No.:1.
16. application according to claim 14, wherein the combining site of one of first aptamers and second aptamers is positioned on the 1-30 position of human galactophore globin mRNA, and another combining site of first aptamers and second aptamers is positioned on the 470-490 position of human galactophore globin mRNA.
17. application according to claim 11, wherein second aptamers certification mark substance markers.
18. application according to claim 17, wherein the certification mark thing is one or more combinations in vitamin H, radio isotope, metal mark thing, chemiluminescent groups, chemiluminescence group, fluorescin, enzyme, antigen or antibody, part or the acceptor.
19. application according to claim 18, wherein the certification mark thing is vitamin H.
20. application according to claim 19, wherein second aptamers is the nm gold particles coupling by vitamin H and suis avidin-horseradish peroxidase bag quilt.
21. application according to claim 11, wherein the magnetic resolution product is the product of magnetic force absorption gained.
22. application according to claim 11, wherein the magnetic resolution product is through washing.
23. application according to claim 11, the temperature of wherein carrying out step (3) is 20-42 ℃.
24. application according to claim 23, the temperature of wherein carrying out step (3) is 37 ℃.
25. binding substances, its by with first aptamers of magnetic-particle coupling, with second aptamers and the mammaglobin mRNA be combined into of certification mark thing coupling, wherein first aptamers and second aptamers be respectively with the aptamers of the different sites specific combination of mammaglobin mRNA, and first aptamers does not have the complementary nucleic acid sequence and does not have the potential that the phase mutual cross is combined with second aptamers;
Wherein the combining site of one of first aptamers and second aptamers is positioned on the 1-30 position of human galactophore globin mRNA, and another combining site of first aptamers and second aptamers is positioned on the 470-490 position of human galactophore globin mRNA; Wherein the identity of first aptamers and mammaglobin mRNA is less than 30%, and the identity of second aptamers and mammaglobin mRNA is less than 30%, first aptamers is shown in SEQ ID No.2, be 5 '-aaggaagccgctgtc-3 ', and second aptamers shown in SEQ ID No.3, be 5 '-aataaacatgatagc-3 '.
26. binding substances according to claim 25, wherein mammaglobin is human galactophore globin.
27. binding substances according to claim 26, wherein human galactophore globin mRNA is shown in SEQ ID NO:1.
28. binding substances according to claim 25, wherein second aptamers certification mark substance markers.
29. binding substances according to claim 28, wherein the certification mark thing is one or more combinations in vitamin H, radio isotope, metal mark thing, chemiluminescent groups, chemiluminescence group, fluorescin, enzyme, antigen or antibody, part or the acceptor.
30. binding substances according to claim 29, wherein second aptamers is the nm gold particles coupling by vitamin H and suis avidin-horseradish peroxidase bag quilt.
31. binding substances according to claim 25, it can combination under 20-42 ℃ temperature.
32. the aptamers of mammaglobin mRNA, it is 5 '-aaggaagccgctgtc-3 ' of SEQ ID No.2 and 5 '-aataaacatgatagc-3 ' of SEQ ID No.3.
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| CN104360077A (en) * | 2014-11-25 | 2015-02-18 | 重庆市科学技术研究院 | Aptamer nucleic acid probe kit for detecting doxycycline residue as well as preparation method and application thereof |
| CN104459155A (en) * | 2014-12-10 | 2015-03-25 | 临沂大学 | Kit for detecting human B lymphoma cells |
| CN110981956B (en) * | 2019-10-18 | 2020-08-25 | 中国人民解放军军事科学院军事医学研究院 | Monoclonal antibody for identifying cell membrane mammaglobin A and application |
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