CN106353499A - Kit for detecting lung cancer serum marker aptamer and gene at same time and application thereof - Google Patents
Kit for detecting lung cancer serum marker aptamer and gene at same time and application thereof Download PDFInfo
- Publication number
- CN106353499A CN106353499A CN201610825646.2A CN201610825646A CN106353499A CN 106353499 A CN106353499 A CN 106353499A CN 201610825646 A CN201610825646 A CN 201610825646A CN 106353499 A CN106353499 A CN 106353499A
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- China
- Prior art keywords
- lung cancer
- sera
- gene
- aptamers
- magnetic bead
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
Abstract
The invention discloses a kit for detecting lung cancer serum marker aptamer and gene at same time and application thereof and belongs to the field of protein and nucleic acid detection. By combining the aptamer with target molecules and converting target molecular signals into nucleic acid signals, the unity of lung cancer serum marker signals and nucleic acid signals is achieved, and detection with aRT-PCR is carried out; a detection method enables non-nucleic acid signals to be converted into nucleic acid signals through the aptamer, the advantages such as high speed, good simplicity, high sensitivity and high affinity are achieved, and multi-aptamer simultaneous detection is achieved.
Description
Technical field
The invention belongs to albumen and field of nucleic acid detection, particularly Sera of Lung Cancer mark aptamers and gene are examined simultaneously
Test agent box and application.
Background technology
Traditional protein quality detection is to be combined with protein to be detected with specific antibodies, by forming specific antigen-anti-
The detection technique that nanocrystal composition is realized., it is coated antibody on solid-phase media taking antibody capture albumen to be detected as a example, Ran Houyu
Protein sample incubation to be detected, washes away unconjugated albumen, finally goes detection to combine with the traget antibody of specific recognition antibody
Antibody, many detection methods are all indirect detection.Labelling includes radiosiotope, organic dyestuff etc..In detection method
Elisa detection (MBP enzyme linked immuno-adsorbent assay) is clinically wide variety of.
It is very universal phenomenon that nucleic acid and albumen interact in vivo.Single-stranded dna energy folding onto itself forms two grades
Structure, tertiary structure etc., can form the structures such as stem ring, pocket, hair fastener, and this has very to nucleic acid and protein interaction
Important effect.In-vitro separation blood serum designated object is carried out by selex technology (the aglucon phyletic evolution technology of index concentration)
Aptamers, the blood serum designated object of aptamers identification specificity, and be not combined with other albumen, (glimmering in real time by qrt-pcr
Light quantitation pcr) can dynamic detection blood serum sample, can quickly analyze the amount of sample target, and quantitation is carried out to it, enter
And be that lung cancer detection provides a kind of new molecular biology for detection.
Content of the invention
It is an object of the invention to provide a kind of Sera of Lung Cancer mark aptamers and gene detection kit simultaneously, pass through
Qrt-pcr detects aptamers and gene order simultaneously, realizes the technical method that blood serum designated object aptamers and gene detect simultaneously,
Significant to research proteomics, genomics.It is a further object of the present invention to provide a kind of Sera of Lung Cancer mark
The application process of thing aptamers and gene detection kit simultaneously.
The technical scheme is that Sera of Lung Cancer mark aptamers and gene be simultaneously for achieving the above object for the present invention
Detection kit, is characterized in that: described test kit mainly includes blood serum sample to be detected, agar magnetic bead, eluent, detection examination
Agent, qrt-pcr detection system；
Described blood serum sample to be detected contains detected mark aptamers and gene；
Described agar magnetic bead is used for forming agar magnetic bead-Sera of Lung Cancer complex；
Described eluent is 1 × binding buffer (combination buffer)+1%tween-20；
Described detectable is Sera of Lung Cancer mark aptamers+1 × binding buffer mixed liquor；
Described qrt-pcr detection system is the conventional sense reagent containing target molecule aptamers and gene probe and primer.
The application of Sera of Lung Cancer mark aptamers and gene detection kit simultaneously, is characterized in that: comprise the following steps:
(1) agar magnetic bead is added in blood serum sample to be detected, 37 DEG C of incubation 30min, formation agar magnetic bead-to be detected
Blood serum sample complex, using Magneto separate, isolates agar magnetic bead, is washed 4 times with eluent, each 1min, after retaining eluting
Agar magnetic bead；
(2) the agar magnetic bead after eluting in above-mentioned steps (1) is added in detectable, Sera of Lung Cancer in detectable
Mark aptamers form composite structure with agar magnetic bead-target protein, by the Sera of Lung Cancer mark adaptation of uncombined formation
Body and agar magnetic bead-target protein form composite structure eluent and wash off, wash 4 times, each 1min, discard eluent and retain fine jade
Fat magnetic bead；
(3) add 100 μ l pure water in the agar magnetic bead retaining in step (2), react 5min at 95 DEG C, in absorption
Clearly；
(4) supernatant in step (3) is carried out amplification gene routine monitoring.
Described blood serum sample to be detected is to remove hemocyte, the serum of blood fat.
Described gene is pathogen, antibacterial, the gene order in cell biological body.
Connect Sera of Lung Cancer mark and specificity aptamers on agar magnetic bead in described step (2), be combined with target protein
Form agar magnetic bead-Sera of Lung Cancer complex.
Described Sera of Lung Cancer mark aptamers are by the high specific blood serum designated object being obtained by selex technology screening
Aptamers form, and aptamers sequence can optimize further, thus improving the stability of aptamers.
Described amplification gene routine monitoring is one of qrt-pcr detection, gene amplification detection, biochip test.
Described Sera of Lung Cancer mark aptamers and gene detect to refer to screen simultaneously and obtain high specific, strong affinity
Sera of Lung Cancer mark aptamers, target protein signal is converted into nucleic acid signal, with gene unified detection on a molecular scale.
The application of Sera of Lung Cancer mark aptamers of the present invention and gene detection kit simultaneously, beneficial effect is:
The test kit of the present invention is to be combined with target molecule by aptamers target molecule signal is converted into nucleic acid signal to complete albumen and base
Method because detecting simultaneously, enters Mobile state and detection by quantitative using qrt-pcr.The present invention is easy and simple to handle, practical, can group
Dress up test kit or be built into biochip and can be widely used in basic research and clinical diagnosises, there is higher economic effect
Benefit and public and social interest.
Brief description
Fig. 1 is Sera of Lung Cancer nucleic acid-protein qrt-pcr detection method flow chart.
Fig. 2 is that Sera of Lung Cancer aptamers specificity identification real time fluorescent quantitative-pcr schemes.
In figure: 1- is positive；2- is negative；3- is blank；4,5- Sera of Lung Cancers；6,7- normal human serums
Specific embodiment
The present invention is described in detail with reference to the accompanying drawings and examples, but the invention is not limited in and be embodied as
Example.
Embodiment
Sera of Lung Cancer mark aptamers and gene detection kit simultaneously, mainly include blood serum sample to be detected, agar
Magnetic bead, eluent, detectable, qrt-pcr detection system；
Described blood serum sample to be detected contains detected mark aptamers and gene；
Described agar magnetic bead is used for forming agar magnetic bead-Sera of Lung Cancer complex；
Described eluent is 1 × binding buffer+1%tween-20；
Described detectable is Sera of Lung Cancer mark aptamers+1 × binding buffer mixed liquor；
Described qrt-pcr detection system is the conventional sense reagent containing target molecule aptamers and gene probe and primer.
Sera of Lung Cancer mark aptamers as shown in Figure 1 and gene detection kit application process simultaneously, including following
Step:
(1) prepare blood serum sample: blood sampling, 3000rpm is centrifuged 5min, separates serum, 4 DEG C of preservations；
(2) formation of agar magnetic bead-Sera of Lung Cancer marker complexes: be coated Sera of Lung Cancer agar magnetic bead 50 μ l, add
The blood serum sample that 200 μ l prepare, forms agar magnetic bead-serum complexes, 37 DEG C, is incubated 30min, washes out unconjugated supernatant
Liquid, is washed 4 times with 1 × binding buffer+1%tween-20 200 μ l, each 1min, adds serum tumor markers in lung cancer
Aptamers are incubated 30min, form agar magnetic bead-serum-adaptor complex structure, with 1 × binding buffer+1%
Tween-20 200 μ l washes 4 times, and each 1min, with Magneto separate, isolates agar magnetic bead, then adds 100 μ in agar magnetic bead
L pure water, 95 DEG C, 5min, draws supernatant；
(3) qrt-pcr detection: the supernatant collected last in (2) is added in qrt-pcr detection detection system, carries out
Dynamic detection and quantitative analyses；
(4) pass through computer data acquisition, analysis, disposal data, obtain the molecule copy number of blood serum designated object and gene.
Above content is to be further described it is impossible to assert invention with reference to what optimal technical scheme did to the present invention
It is embodied as being only limitted to these explanations.For general technical staff of the technical field of the invention, without departing from the present invention
Design on the premise of, can also make simple deduce and replace, all should be considered as protection scope of the present invention.
Claims (8)
1. Sera of Lung Cancer mark aptamers and gene detection kit simultaneously, is characterized in that: described test kit mainly includes treating
Detection blood serum sample, agar magnetic bead, eluent, detectable, qrt-pcr detection system；
Described blood serum sample to be detected contains detected mark aptamers and gene；
Described agar magnetic bead is used for forming agar magnetic bead-Sera of Lung Cancer complex；
Described eluent is 1 × binding buffer+1%tween-20；
Described detectable is Sera of Lung Cancer mark aptamers+1 × binding buffer mixed liquor；
Described qrt-pcr detection system is the conventional sense reagent containing target molecule aptamers and gene probe and primer.
2. the application of Sera of Lung Cancer mark aptamers according to claim 1 and gene detection kit simultaneously, it is special
Levying is: comprises the following steps:
(1) agar magnetic bead is added in blood serum sample to be detected, 37 DEG C of incubation 30min, forms agar magnetic bead-serum to be detected
Sample composites, using Magneto separate, isolate agar magnetic bead, are washed 4 times with eluent, each 1min, retain the agar after eluting
Magnetic bead；
(2) the agar magnetic bead after eluting in above-mentioned steps (1) is added in detectable, Sera of Lung Cancer mark in detectable
Thing aptamers and agar magnetic bead-target protein forms composite structure, by the Sera of Lung Cancer mark aptamers of uncombined formation with
Agar magnetic bead-target protein forms composite structure eluent and washes off, washes 4 times, each 1min, discards eluent and retains agar magnetic
Pearl；
(3) add 100 μ l pure water in the agar magnetic bead retaining in step (2), at 95 DEG C, react 5min, draw supernatant；
(4) supernatant in step (3) is carried out amplification gene routine monitoring.
3. the application of Sera of Lung Cancer mark aptamers according to claim 2 and gene detection kit simultaneously, it is special
Levying is: described blood serum sample to be detected is to remove hemocyte, the serum of blood fat.
4. the application of Sera of Lung Cancer mark aptamers according to claim 2 and gene detection kit simultaneously, it is special
Levying is: described gene is pathogen, antibacterial, the gene order in cell biological body.
5. the application of Sera of Lung Cancer mark aptamers according to claim 2 and gene detection kit simultaneously, it is special
Levying is: connects Sera of Lung Cancer mark and specificity aptamers on agar magnetic bead in described step (2), combines to form with target protein
Agar magnetic bead-Sera of Lung Cancer complex.
6. the application of Sera of Lung Cancer mark aptamers according to claim 2 and gene detection kit simultaneously, it is special
Levying is: described Sera of Lung Cancer mark aptamers are to be fitted by the high specific blood serum designated object being obtained by selex technology screening
Part forms, and aptamers sequence can optimize further, thus improving the stability of aptamers.
7. the application of Sera of Lung Cancer mark aptamers according to claim 2 and gene detection kit simultaneously, it is special
Levying is: described amplification gene routine monitoring is one of qrt-pcr detection, gene amplification detection, biochip test.
8. the application of Sera of Lung Cancer mark aptamers according to claim 2 and gene detection kit simultaneously, it is special
Levying is: described Sera of Lung Cancer mark aptamers and gene detect simultaneously and refer to screen the lung obtaining high specific, strong affinity
Cancer-serum mark aptamers, target protein signal is converted into nucleic acid signal, with gene unified detection on a molecular scale.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610825646.2A CN106353499A (en) | 2016-09-14 | 2016-09-14 | Kit for detecting lung cancer serum marker aptamer and gene at same time and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610825646.2A CN106353499A (en) | 2016-09-14 | 2016-09-14 | Kit for detecting lung cancer serum marker aptamer and gene at same time and application thereof |
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| CN106353499A true CN106353499A (en) | 2017-01-25 |
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| CN201610825646.2A Pending CN106353499A (en) | 2016-09-14 | 2016-09-14 | Kit for detecting lung cancer serum marker aptamer and gene at same time and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109804081A (en) * | 2018-11-08 | 2019-05-24 | 廖世奇 | A kind of compound target-tumour serum aptamer detection method and kit |
| CN110168376A (en) * | 2018-06-08 | 2019-08-23 | 廖世奇 | The method and kit that the more target molecules of magnetic bead-aptamer-detect simultaneously |
| CN112680452A (en) * | 2021-01-18 | 2021-04-20 | 燕山大学 | Oligonucleotide aptamer specifically binding to lung cancer serum and application thereof |
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| CN101368209A (en) * | 2008-09-12 | 2009-02-18 | 青岛科技大学 | Method for detecting target numerator based on nucleic acid aptamer and PCR amplification |
| RU2012129259A (en) * | 2012-07-10 | 2014-01-20 | Государственное бюджетное образовательное учреждение высшего профессионального образования "Красноярский государственный медицинский университет имени профессора В.Ф. Войно-Ясенецкого" Министерства здравоохранения и социального развития Российской Федерации (ГБОУ ВПО КрасГМУ им. проф. В.Ф. Войно-Ясе | DIAGNOSTIC METHOD FOR LUNG CANCER |
| CN104845975A (en) * | 2015-05-29 | 2015-08-19 | 郑州大学 | DNA aptamer of small cell lung cancer marker gastrin releasing peptide precursor polypeptide fragment |
| CN105018590A (en) * | 2015-01-30 | 2015-11-04 | 廖世奇 | Detection kit capable of simultaneous detection of protein ligand and genes and application thereof |
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2016
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Patent Citations (4)
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| CN101368209A (en) * | 2008-09-12 | 2009-02-18 | 青岛科技大学 | Method for detecting target numerator based on nucleic acid aptamer and PCR amplification |
| RU2012129259A (en) * | 2012-07-10 | 2014-01-20 | Государственное бюджетное образовательное учреждение высшего профессионального образования "Красноярский государственный медицинский университет имени профессора В.Ф. Войно-Ясенецкого" Министерства здравоохранения и социального развития Российской Федерации (ГБОУ ВПО КрасГМУ им. проф. В.Ф. Войно-Ясе | DIAGNOSTIC METHOD FOR LUNG CANCER |
| CN105018590A (en) * | 2015-01-30 | 2015-11-04 | 廖世奇 | Detection kit capable of simultaneous detection of protein ligand and genes and application thereof |
| CN104845975A (en) * | 2015-05-29 | 2015-08-19 | 郑州大学 | DNA aptamer of small cell lung cancer marker gastrin releasing peptide precursor polypeptide fragment |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110168376A (en) * | 2018-06-08 | 2019-08-23 | 廖世奇 | The method and kit that the more target molecules of magnetic bead-aptamer-detect simultaneously |
| WO2019232797A1 (en) * | 2018-06-08 | 2019-12-12 | Liao Shiqi | Method and kit for simultaneous detection of magnetic bead-nucleic acid aptamer-multi-target molecules |
| US11293918B2 (en) | 2018-06-08 | 2022-04-05 | Liao Shiqi | Method and kit for simultaneous detection of multi target molecules using magnetic bead-aptamer conjugate |
| CN109804081A (en) * | 2018-11-08 | 2019-05-24 | 廖世奇 | A kind of compound target-tumour serum aptamer detection method and kit |
| CN112680452A (en) * | 2021-01-18 | 2021-04-20 | 燕山大学 | Oligonucleotide aptamer specifically binding to lung cancer serum and application thereof |
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Application publication date: 20170125 |