CN108414760A - A method of utilizing the theophylline in RNA aptamers and nanogold detection serum - Google Patents
A method of utilizing the theophylline in RNA aptamers and nanogold detection serum Download PDFInfo
- Publication number
- CN108414760A CN108414760A CN201810194965.7A CN201810194965A CN108414760A CN 108414760 A CN108414760 A CN 108414760A CN 201810194965 A CN201810194965 A CN 201810194965A CN 108414760 A CN108414760 A CN 108414760A
- Authority
- CN
- China
- Prior art keywords
- rna
- probe
- theophylline
- elisa plate
- target mirna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- ZFXYFBGIUFBOJW-UHFFFAOYSA-N theophylline Chemical compound O=C1N(C)C(=O)N(C)C2=C1NC=N2 ZFXYFBGIUFBOJW-UHFFFAOYSA-N 0.000 title claims abstract description 46
- 238000001514 detection method Methods 0.000 title claims abstract description 29
- 229960000278 theophylline Drugs 0.000 title claims abstract description 23
- 210000002966 serum Anatomy 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 17
- 108091008103 RNA aptamers Proteins 0.000 title claims abstract description 15
- 239000002679 microRNA Substances 0.000 claims abstract description 64
- 108091070501 miRNA Proteins 0.000 claims abstract description 58
- 239000000523 sample Substances 0.000 claims abstract description 50
- 238000002965 ELISA Methods 0.000 claims abstract description 33
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 17
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 14
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 14
- 108010083644 Ribonucleases Proteins 0.000 claims abstract description 13
- 102000006382 Ribonucleases Human genes 0.000 claims abstract description 13
- 150000001875 compounds Chemical class 0.000 claims abstract description 7
- 238000002203 pretreatment Methods 0.000 claims abstract description 7
- 238000007689 inspection Methods 0.000 claims abstract description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 18
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 11
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 9
- 239000007853 buffer solution Substances 0.000 claims description 9
- 230000000295 complement effect Effects 0.000 claims description 9
- 201000001275 rectum cancer Diseases 0.000 claims description 9
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 6
- 150000001408 amides Chemical class 0.000 claims description 6
- 229960002685 biotin Drugs 0.000 claims description 6
- 235000020958 biotin Nutrition 0.000 claims description 6
- 239000011616 biotin Substances 0.000 claims description 6
- 125000003636 chemical group Chemical group 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 230000000593 degrading effect Effects 0.000 claims description 6
- 238000009396 hybridization Methods 0.000 claims description 6
- 229920000136 polysorbate Polymers 0.000 claims description 6
- 230000002255 enzymatic effect Effects 0.000 claims description 5
- 208000015634 Rectal Neoplasms Diseases 0.000 claims description 4
- 206010038038 rectal cancer Diseases 0.000 claims description 4
- 102000040650 (ribonucleotides)n+m Human genes 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 108010067770 Endopeptidase K Proteins 0.000 claims description 3
- 239000013504 Triton X-100 Substances 0.000 claims description 3
- 229920004890 Triton X-100 Polymers 0.000 claims description 3
- 150000008065 acid anhydrides Chemical class 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 230000007547 defect Effects 0.000 claims description 3
- 230000032050 esterification Effects 0.000 claims description 3
- 238000005886 esterification reaction Methods 0.000 claims description 3
- 239000011888 foil Substances 0.000 claims description 3
- 108091023663 let-7 stem-loop Proteins 0.000 claims description 3
- 108091063478 let-7-1 stem-loop Proteins 0.000 claims description 3
- 108091049777 let-7-2 stem-loop Proteins 0.000 claims description 3
- 108091091807 let-7a stem-loop Proteins 0.000 claims description 3
- 108091057746 let-7a-4 stem-loop Proteins 0.000 claims description 3
- 108091028376 let-7a-5 stem-loop Proteins 0.000 claims description 3
- 108091024393 let-7a-6 stem-loop Proteins 0.000 claims description 3
- 108091091174 let-7a-7 stem-loop Proteins 0.000 claims description 3
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims description 3
- 229920003023 plastic Polymers 0.000 claims description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- 239000012460 protein solution Substances 0.000 claims description 3
- 239000003161 ribonuclease inhibitor Substances 0.000 claims description 3
- 239000012488 sample solution Substances 0.000 claims description 3
- 239000004094 surface-active agent Substances 0.000 claims description 3
- 238000010792 warming Methods 0.000 claims description 3
- 108091069780 let-7h stem-loop Proteins 0.000 claims description 2
- 102100033195 DNA ligase 4 Human genes 0.000 claims 1
- 101000927810 Homo sapiens DNA ligase 4 Proteins 0.000 claims 1
- 241000218636 Thuja Species 0.000 claims 1
- 239000002253 acid Substances 0.000 claims 1
- 230000015556 catabolic process Effects 0.000 claims 1
- 238000006731 degradation reaction Methods 0.000 claims 1
- 238000006911 enzymatic reaction Methods 0.000 claims 1
- 108010079502 exoribonuclease T Proteins 0.000 claims 1
- 230000002194 synthesizing effect Effects 0.000 claims 1
- 210000004885 white matter Anatomy 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 14
- 102000004169 proteins and genes Human genes 0.000 abstract description 5
- 108090000623 proteins and genes Proteins 0.000 abstract description 5
- 238000000605 extraction Methods 0.000 abstract description 4
- 238000012545 processing Methods 0.000 abstract description 4
- 206010058467 Lung neoplasm malignant Diseases 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 3
- 239000004615 ingredient Substances 0.000 abstract description 3
- 201000005202 lung cancer Diseases 0.000 abstract description 3
- 208000020816 lung neoplasm Diseases 0.000 abstract description 3
- 201000011510 cancer Diseases 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000013461 design Methods 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/06—Gastro-intestinal diseases
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of methods using the theophylline in RNA aptamers and nanogold detection serum, include the following steps：Biology and the pre-treatment of clinical sample, chimeric probe hybridize with target miRNA, connect capture sequence be modified on ELISA Plate in surface, nucleic acid hybridize to form ELISA Plate chimeric probe miRNA compounds, RNase ONE ribalgilases carry out inspection and chemoluminescence method detects target miRNA amounts and theophylline amount；The present invention is according to the pre-treatment scheme in Direct round pcrs, chemistry and biology processing is carried out to biological cell or clinical lung cancer sample, decompose protein and DNA ingredients, release total serum IgE therein, without carrying out the extraction of RNA, this way can not only simplify experiment flow, shorten detection time, and can save precious clinical tumor sample.
Description
Technical field
The present invention relates to medical detection technologies, specially a kind of to detect blood using RNA aptamers and nanogold
The method of theophylline in clear.
Background technology
Currently, the carcinoma of the rectum has become the highest cancer of most common in the world and related mortality.According in National Cancer
Morbidity and Analysis of Died Cases report of the heart to Chinese malignant tumour in 2010, the annual carcinoma of the rectum new cases about 600,000 in China,
Death about 490,000, morbidity and the dead first place for occupying all malignant tumours.For the prevention of the carcinoma of the rectum, early detection,
Early diagnosis is crucial.It realizes i.e. before clinical symptoms occurs in patient or before infiltration occurs for cancer and makes a definite diagnosis, to make patient obtain
To timely treatment, and then improve prognosis, achievees the purpose that improve cure rate, reduces the death rate.Currently, the most of carcinoma of the rectum I,
II phase patient, illness are not obvious, it is difficult to are found and be effectively treated.In view of rectum cancer incidence trend increase and its it is early
Phase finds the difficulty with diagnosis, urgently develops at present a kind of more more acurrate than prior art means reliable again simple and practicable new simultaneously
Technology come carry out carcinoma of the rectum screening with early diagnosis.
So how to design a kind of method using the theophylline in RNA aptamers and nanogold detection serum, become
We will currently solve the problems, such as.
Invention content
The purpose of the present invention is to provide a kind of sides using the theophylline in RNA aptamers and nanogold detection serum
Method, to solve the problems mentioned in the above background technology.
To achieve the above object, the present invention provides the following technical solutions：It is a kind of to be examined using RNA aptamers and nanogold
The method for surveying the theophylline in serum, includes the following steps：
1) pre-treatment of biology and clinical sample：Rectum cancer cell Tris-HCl buffer solutions are diluted, Proteinase K is added
It with EDTA, digests 2 hours at 37 DEG C, is mixed 30 minutes after adding SDS and 20 surfactants of Tween, removal package nucleic acid
Protein discharges nucleic acid；It is added followed by DNA enzymatic I, 37 DEG C are reacted 1 hour, after degrading the DNA in genome, are remained in solution
Remaining is the total serum IgE for including target miRNA；
2) chimeric probe hybridizes with target miRNA：The different DNA-RNA that equivalent is added in the sample solution handled well are embedding
Close probe, the corresponding target miRNA complete complementaries in the parts RNA of probe and label Biotin, parts DNA of probe and will
Be connected to corresponding DNA on ELISA Plate and capture sequence complete complementary, after being thoroughly mixed, solution is warming up to 90 DEG C, then by
It is gradually down to room temperature, is annealed, the parts RNA of probe is made to hybridize with its target miRNA；
3) connection captures sequence and is modified on ELISA Plate in surface：Acid anhydrides or amide surface are modified 96/384 hole elisa Plates to use
20 solution of PBS-Tween flushes three times, and activates chemical group thereon, designs and synthesizes the DNA of 5 ' terminal modified amino or sulfydryl
Sequence is captured, is connected on ELISA Plate by esterification or amide reaction by sequence is captured, then BSA protein solutions is used to close
The chemical group of reaction is had neither part nor lot on ELISA Plate, after PBS is cleaned three times, ELISA Plate is covered with plastic foil, is stored in 4 DEG C of environment
In, wait for next step；
4) nucleic acid hybridizes to form ELISA Plate-chimeric probe-miRNA compounds：It will hybridize with target miRNA in step 2)
Chimeric probe be added step 3) in have connected DNA capture sequence ELISA Plate hole in, be added contain the surfaces Triton X-100
The Tris-HCl buffer solutions of activating agent, according to the principle of base pair complementarity, the part the DNA of chimeric probe be connected to ELISA Plate
On capture sequence generate hybridization, formed ELISA Plate-chimeric probe-miRNA compounds, pass through the hybridization between nucleic acid chains
Chimeric probe and target miRNA are fixed on ELISA Plate；
5) RNase ONE ribalgilases are checked：RNase ONE ribalgilases in double-stranded RNA of degrading by sending out
The single stranded RNA of raw mispairing or partial complementarity, checks the miRNA hybridized in chimeric probe, even only differing 1 with target miRNA
The same miRNAs of a nucleotide can also be identified by it and degrade；
6) chemoluminescence method detection target miRNA amounts and theophylline amount：SA-HRP is added, the albumen and has passed through RNase
ONE ribalgilase inspections, with the chimeric probe Biotin of target miRNA complete complementaries labels it is rapid combined with, then add
Luminol, hydrogen peroxide and chemiluminescence intensifier utilize the HRP and Luminol-H connected on the albumen2O2It is anti-that enzymatic occurs
It answers, generates strong 425nm fluorescence, fluorescence microplate reader detects its fluorescence intensity level, it is extrapolated according to different fluorescence intensity levels
Corresponding target miRNA amounts；Theophylline RNA aptamer are blocked as two parts and are combined with nanogold simultaneously, are overcome
RNA holds degradation-labile defect, and the concentration of theophylline is had detected in serum.
According to above-mentioned technical proposal, Tris-HCl buffer solutions need to be pre-processed with DEPC in the step 1), also need
Add RNase inhibitor.
According to above-mentioned technical proposal, step 2) the middle probe quantity is determined by the target miRNA quantity to be detected.
According to above-mentioned technical proposal, the step 4) needs the let-7a to let- in external artificial synthesized let-7 families
7h tests RNase ONE ribalgilases with miRNAs for eight kinds totally, verifies ability of its resolution with miRNAs.
Compared with prior art, the beneficial effects of the invention are as follows：The present invention is according to the pre-treatment in Direct round pcrs
Scheme carries out chemistry and biology processing to biological cell or clinical lung cancer sample, decomposes protein and DNA ingredients, discharge
Go out total serum IgE therein, without carrying out the extraction of RNA, this way can not only simplify experiment flow, shorten detection time, and
And precious clinical tumor sample can be saved；Simultaneously the detection technique be not necessarily to special detecting instrument and analysis software, without into
The extraction of row miRNA is not necessarily to nucleic acid amplification；Required reagent and consumptive material can largely be provided by domestic production quotient, part consumptive material
It is repeatable to utilize, therefore, use cost is greatly reduced, which completes detection, inspection using the ELISA Plate that surface is modified
Biochemical reagents needed for surveying are identical, and according to different target miRNA, corresponding nucleic acid capture sequence and chimeric probe are connected per hole
Hybridized, multiple target miRNA in the same tumor sample can be detected only with a fluorescence microplate reader；Together
When the detection technique can realize that the parallel detection to a variety of miRNA tumor markers, a clinical tumor sample only carry out once
A variety of miRNA tumor markers can be detected after processing simultaneously, effectively promote detection efficiency.
Description of the drawings
Fig. 1 is the flow chart of the method for the theophylline in the detection serum of the present invention；
Specific implementation mode
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation describes, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Referring to Fig. 1, the present invention provides a kind of side using the theophylline in RNA aptamers and nanogold detection serum
Method includes the following steps：
1) pre-treatment of biology and clinical sample：Rectum cancer cell Tris-HCl buffer solutions are diluted, Proteinase K is added
It with EDTA, digests 2 hours at 37 DEG C, is mixed 30 minutes after adding SDS and 20 surfactants of Tween, removal package nucleic acid
Protein discharges nucleic acid；It is added followed by DNA enzymatic I, 37 DEG C are reacted 1 hour, after degrading the DNA in genome, are remained in solution
Remaining is the total serum IgE for including target miRNA；
2) chimeric probe hybridizes with target miRNA：The different DNA-RNA that equivalent is added in the sample solution handled well are embedding
Close probe, the corresponding target miRNA complete complementaries in the parts RNA of probe and label Biotin, parts DNA of probe and will
Be connected to corresponding DNA on ELISA Plate and capture sequence complete complementary, after being thoroughly mixed, solution is warming up to 90 DEG C, then by
It is gradually down to room temperature, is annealed, the parts RNA of probe is made to hybridize with its target miRNA；
3) connection captures sequence and is modified on ELISA Plate in surface：Acid anhydrides or amide surface are modified 96/384 hole elisa Plates to use
20 solution of PBS-Tween flushes three times, and activates chemical group thereon, designs and synthesizes the DNA of 5 ' terminal modified amino or sulfydryl
Sequence is captured, is connected on ELISA Plate by esterification or amide reaction by sequence is captured, then BSA protein solutions is used to close
The chemical group of reaction is had neither part nor lot on ELISA Plate, after PBS is cleaned three times, ELISA Plate is covered with plastic foil, is stored in 4 DEG C of environment
In, wait for next step；
4) nucleic acid hybridizes to form ELISA Plate-chimeric probe-miRNA compounds：It will hybridize with target miRNA in step 2)
Chimeric probe be added step 3) in have connected DNA capture sequence ELISA Plate hole in, be added contain the surfaces Triton X-100
The Tris-HCl buffer solutions of activating agent, according to the principle of base pair complementarity, the part the DNA of chimeric probe be connected to ELISA Plate
On capture sequence generate hybridization, formed ELISA Plate-chimeric probe-miRNA compounds, pass through the hybridization between nucleic acid chains
Chimeric probe and target miRNA are fixed on ELISA Plate；
5) RNase ONE ribalgilases are checked：RNase ONE ribalgilases in double-stranded RNA of degrading by sending out
The single stranded RNA of raw mispairing or partial complementarity, checks the miRNA hybridized in chimeric probe, even only differing 1 with target miRNA
The same miRNAs of a nucleotide can also be identified by it and degrade；
6) chemoluminescence method detection target miRNA amounts and theophylline amount：SA-HRP is added, the albumen and has passed through RNase
ONE ribalgilase inspections, with the chimeric probe Biotin of target miRNA complete complementaries labels it is rapid combined with, then add
Luminol, hydrogen peroxide and chemiluminescence intensifier utilize the HRP and Luminol-H connected on the albumen2O2It is anti-that enzymatic occurs
It answers, generates strong 425nm fluorescence, fluorescence microplate reader detects its fluorescence intensity level, it is extrapolated according to different fluorescence intensity levels
Corresponding target miRNA amounts；Theophylline RNA aptamer are blocked as two parts and are combined with nanogold simultaneously, are overcome
RNA holds degradation-labile defect, and the concentration of theophylline is had detected in serum.
According to above-mentioned technical proposal, Tris-HCl buffer solutions need to be pre-processed with DEPC in step 1), it is also necessary to add
Add RNase inhibitor.
According to above-mentioned technical proposal, step 2) middle probe quantity is determined by the target miRNA quantity to be detected.
According to above-mentioned technical proposal, step 4) needs the let-7a to let-7h in external artificial synthesized let-7 families total
Eight kinds are tested RNase ONE ribalgilases with miRNAs, verify ability of its resolution with miRNAs.
Based on above-mentioned, it is an advantage of the current invention that the present invention is according to the pre-treatment scheme in Direct round pcrs, to life
Object cell or clinical lung cancer sample carry out chemistry and biology processing, decompose protein and DNA ingredients, release therein total
RNA, without carrying out the extraction of RNA, this way can not only simplify experiment flow, shorten detection time, and can save treasure
Expensive clinical tumor sample；The detection technique is not necessarily to special detecting instrument and analysis software simultaneously, without carrying out carrying for miRNA
It takes, is not necessarily to nucleic acid amplification；Required reagent and consumptive material can largely be provided by domestic production quotient, and part consumptive material is repeatable to be utilized,
Therefore, use cost is greatly reduced, which completes detection using the ELISA Plate that surface is modified, and detects required biochemistry
Reagent is identical, according to different target miRNA, corresponding nucleic acid capture sequence is connected per hole and is hybridized with chimeric probe, only
Multiple target miRNA in the same tumor sample can be detected using a fluorescence microplate reader；The detection skill simultaneously
Art can be realized can after the parallel detection to a variety of miRNA tumor markers, a clinical tumor sample only carry out single treatment
A variety of miRNA tumor markers are detected simultaneously, effectively promote detection efficiency.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understanding without departing from the principles and spirit of the present invention can carry out these embodiments a variety of variations, modification, replace
And modification, the scope of the present invention is defined by the appended.
Claims (4)
1. a kind of method using the theophylline in RNA aptamers and nanogold detection serum, it is characterised in that：Including as follows
Step：
1) pre-treatment of biology and clinical sample：By rectum cancer cell with Tris-HCl buffer solutions dilute, be added Proteinase K with
EDTA digests 2 hours at 37 DEG C, is mixed 30 minutes after adding SDS and 20 surfactants of Tween, the egg of removal package nucleic acid
White matter discharges nucleic acid；It is added followed by DNA enzymatic I, 37 DEG C are reacted 1 hour, remaining in solution after degrading the DNA in genome
Be include target miRNA total serum IgE；
2) chimeric probe hybridizes with target miRNA：The chimeric spies of different DNA-RNA that equivalent will be added in the sample solution handled well
Needle, the corresponding target miRNA complete complementaries in the parts RNA of probe and label Biotin, the parts DNA of probe with will connect
The corresponding DNA being connected on ELISA Plate captures sequence complete complementary, and after being thoroughly mixed, solution is warming up to 90 DEG C, then gradually drops
It to room temperature, anneals, the parts RNA of probe is made to hybridize with its target miRNA；
3) connection captures sequence and is modified on ELISA Plate in surface：Acid anhydrides or amide surface are modified 96/384 hole elisa Plates PBS-
20 solution of Tween flushes three times, and activates chemical group thereon, and the DNA for designing and synthesizing 5 ' terminal modified amino or sulfydryl is captured
Sequence is connected to sequence is captured on ELISA Plate by esterification or amide reaction, then uses BSA protein solution sealase marks
The chemical group of reaction is had neither part nor lot on plate, after PBS is cleaned three times, ELISA Plate is covered with plastic foil, is stored in 4 DEG C of environment, etc.
Pending next step；
4) nucleic acid hybridizes to form ELISA Plate-chimeric probe-miRNA compounds：It is embedding by hybridizing with target miRNA in step 2)
It closes probe to be added in step 3) in the ELISA Plate hole for having connected DNA capture sequences, is added and contains Triton X-100 surface-actives
The Tris-HCl buffer solutions of agent, according to the principle of base pair complementarity, the part the DNA of chimeric probe be connected on ELISA Plate
It captures sequence and generates hybridization, form ELISA Plate-chimeric probe-miRNA compounds, it will be embedding by the hybridization between nucleic acid chains
Probe is closed to be fixed on ELISA Plate with target miRNA；
5) RNase ONE ribalgilases are checked：RNase ONE ribalgilases are wrong by occurring in double-stranded RNA of degrading
Match or the single stranded RNA of partial complementarity, the miRNA hybridized in chimeric probe is checked, even only differing 1 core with target miRNA
The same miRNAs of thuja acid can also be identified by it and degrade；
6) chemoluminescence method detection target miRNA amounts and theophylline amount：SA-HRP is added, the albumen and has passed through RNase ONE cores
Ribonuclease T. inspection is combined with the chimeric probe Biotin of target miRNA complete complementaries labels are rapid, then addition Luminol,
Hydrogen peroxide and chemiluminescence intensifier utilize the HRP and Luminol-H connected on the albumen2O2Enzymatic reaction occurs, generates strong
Strong 425nm fluorescence, fluorescence microplate reader detect its fluorescence intensity level, and the mesh corresponding to it is extrapolated according to different fluorescence intensity levels
Mark miRNA amounts；Theophylline RNA aptamer are blocked as two parts and are combined with nanogold simultaneously, RNA is overcome and is easy degradation
Defect, the concentration of theophylline is had detected in serum.
2. a kind of method using the theophylline in RNA aptamers and nanogold detection serum according to claim 1, special
Sign is：Tris-HCl buffer solutions need to be pre-processed with DEPC in the step 1), it is also necessary to add RNase inhibitor.
3. a kind of method using the theophylline in RNA aptamers and nanogold detection serum according to claim 1, special
Sign is：Step 2) the middle probe quantity is determined by the target miRNA quantity to be detected.
4. a kind of method using the theophylline in RNA aptamers and nanogold detection serum according to claim 1, special
Sign is：The step 4) need the let-7a to let-7h in external artificial synthesized let-7 families totally eight kinds with miRNAs pair
RNase ONE ribalgilases are tested, and ability of its resolution with miRNAs is verified.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810194965.7A CN108414760A (en) | 2018-03-09 | 2018-03-09 | A method of utilizing the theophylline in RNA aptamers and nanogold detection serum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810194965.7A CN108414760A (en) | 2018-03-09 | 2018-03-09 | A method of utilizing the theophylline in RNA aptamers and nanogold detection serum |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108414760A true CN108414760A (en) | 2018-08-17 |
Family
ID=63130898
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810194965.7A Pending CN108414760A (en) | 2018-03-09 | 2018-03-09 | A method of utilizing the theophylline in RNA aptamers and nanogold detection serum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108414760A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002059137A1 (en) * | 2000-11-03 | 2002-08-01 | Isis Pharmaceuticals, Inc. | Nuclease-based method for detecting and quantitating oligonucleotides |
| US20020137060A1 (en) * | 2000-08-09 | 2002-09-26 | Brown-Augsburger Patricia Lea | Combined hybridization-detection assays for determining nucleic acid concentrations in biological fluids |
| CN103397090A (en) * | 2013-07-30 | 2013-11-20 | 武汉中帜生物科技有限公司 | A kind of microRNA detection kit and the method for multi-biotin molecule detection microRNA |
| CN104569394A (en) * | 2014-12-24 | 2015-04-29 | 深圳市老年医学研究所 | Multiple miRNA tumor marker detecting method and application thereof |
| CN104877999A (en) * | 2015-05-19 | 2015-09-02 | 深圳市人民医院 | Liquid phase chip miRNA detection kit |
| CN107099612A (en) * | 2017-06-21 | 2017-08-29 | 深圳市展行生物有限公司 | A kind of method based on short nucleotide chain connecting detection cancer patient's serum miRNA |
-
2018
- 2018-03-09 CN CN201810194965.7A patent/CN108414760A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020137060A1 (en) * | 2000-08-09 | 2002-09-26 | Brown-Augsburger Patricia Lea | Combined hybridization-detection assays for determining nucleic acid concentrations in biological fluids |
| WO2002059137A1 (en) * | 2000-11-03 | 2002-08-01 | Isis Pharmaceuticals, Inc. | Nuclease-based method for detecting and quantitating oligonucleotides |
| CN103397090A (en) * | 2013-07-30 | 2013-11-20 | 武汉中帜生物科技有限公司 | A kind of microRNA detection kit and the method for multi-biotin molecule detection microRNA |
| CN104569394A (en) * | 2014-12-24 | 2015-04-29 | 深圳市老年医学研究所 | Multiple miRNA tumor marker detecting method and application thereof |
| CN104877999A (en) * | 2015-05-19 | 2015-09-02 | 深圳市人民医院 | Liquid phase chip miRNA detection kit |
| CN107099612A (en) * | 2017-06-21 | 2017-08-29 | 深圳市展行生物有限公司 | A kind of method based on short nucleotide chain connecting detection cancer patient's serum miRNA |
Non-Patent Citations (1)
| Title |
|---|
| XIAOYI MA，ET AL: "Colorimetric theophylline aggregation assay using an RNA aptamer and non-crosslinking gold nanoparticles", 《MICROCHIMICA ACTA》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106906211B (en) | Molecular joint and application thereof | |
| US20110015080A1 (en) | Solution-based methods for RNA expression profiling | |
| RU2270254C2 (en) | Identification of transgenic dna sequences in plant material and products made of the same, oligonucleotide kit and bioarray therefor | |
| Zhang et al. | An ultrasensitive label-free electrochemical biosensor for microRNA-21 detection based on a 2′-O-methyl modified DNAzyme and duplex-specific nuclease assisted target recycling | |
| AU2014406026A1 (en) | Isolated oligonucleotide and use thereof in nucleic acid sequencing | |
| CN112662771B (en) | Targeting capture probe of tumor fusion gene and application thereof | |
| Liu et al. | Research progress in molecular biology related quantitative methods of MicroRNA | |
| CN106191214A (en) | A kind of multicolor fluorescence melting curve PCR detection method | |
| CN103388024B (en) | Bridge-PCR-based method for detecting DNA hydroxymethylation | |
| CN104569394A (en) | Multiple miRNA tumor marker detecting method and application thereof | |
| CN107828914B (en) | RAA constant temperature fluorescence detection method and reagent for Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) | |
| CN104611471B (en) | For detecting gene chip and its detection method of foot and mouth disease viruses | |
| CN101613766A (en) | A RT-LAMP kit for Peste des petits ruminants virus | |
| CN110387401A (en) | A kind of detection method of miRNA-21 | |
| CN106282176A (en) | One is used for identifying AGA926-928the primer combination of the ARMS-qPCR of sudden change and application thereof | |
| WO2024006765A2 (en) | Universal method for parasite and eukaryotic endosymbiont identification | |
| CN107099612A (en) | A kind of method based on short nucleotide chain connecting detection cancer patient's serum miRNA | |
| CN110453010A (en) | A kind of LAMP primer set, reagent and kit for detecting African swine fever virus ASFV | |
| CN108414760A (en) | A method of utilizing the theophylline in RNA aptamers and nanogold detection serum | |
| CN1188531C (en) | Prawn white spot complex virogene diagnostic kit and detecting method thereof | |
| CN107988205A (en) | A kind of nucleic acid extraction and bisulfite conversion and the kit of nucleic acid purification | |
| CN105452488A (en) | Compositions and methods for detecting HEV nucleic acid | |
| CN105039564B (en) | MiRNA detection chips and preparation method thereof and application | |
| CN102191328B (en) | Nucleic acid sequence analysis method based on magnetic separation and high-fidelity polymerase correction function | |
| CN106906279A (en) | A kind of reaction system for detecting the bilharzial nucleic acid of oncomelania In vivo infection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180817 |
|
| RJ01 | Rejection of invention patent application after publication |