CN109283329A - A kind of method of the quick detection target of based on PCR and magnetic bead antibody beneficiation technologies - Google Patents

A kind of method of the quick detection target of based on PCR and magnetic bead antibody beneficiation technologies Download PDF

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Publication number
CN109283329A
CN109283329A CN201711130951.0A CN201711130951A CN109283329A CN 109283329 A CN109283329 A CN 109283329A CN 201711130951 A CN201711130951 A CN 201711130951A CN 109283329 A CN109283329 A CN 109283329A
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target
antibody
pcr
quick detection
magnetic bead
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孔惠
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GUANGZHOU FOODSAFE BIO-TECHNOLOGY Co Ltd
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GUANGZHOU FOODSAFE BIO-TECHNOLOGY Co Ltd
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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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00—Magnetic particle immunoreagent carriers
    • G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
    • G01N2446/90—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids characterised by small molecule linker used to couple immunoreagents to magnetic particles

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Abstract

The invention belongs to field of biotechnology, a kind of method of the quick detection target of based on PCR and magnetic bead antibody beneficiation technologies is disclosed, includes the following steps: step 1: can identify the antibody or aptamer of target in magnetic microsphere surface markers；Step 2: target being enriched with by antibody or aptamer in the magnetic microsphere surface that step 1 obtains；Step 3: will specific recognition target DNA marker in conjunction with the target for the magnetic microsphere that step 2 obtains；Step 4: by separating with antibody or aptamer-target-DNA marker sandwich structure for magnetic microsphere surface, and passing through PCR or isothermal duplication method DNA amplification marker；Step 5: the DNA marker after detection amplification obtains the concentration results of target.This method detection specificity is high, detection accuracy is high.

Description

A kind of method of the quick detection target of based on PCR and magnetic bead antibody beneficiation technologies
Technical field
The present invention relates to field of biotechnology, the quick detection of especially a kind of based on PCR and magnetic bead antibody beneficiation technologies The method of target.
Background technique
Malignant tumour is a kind of using cell paraplasm as the human diseases of essential characteristic.It is sent out along with tumour
Exhibition, macromolecular substances (including nucleic acid, albumen and polysaccharide etc.) can discharge into blood circulation by cancer cell, this Free (Circulating) macromolecular of class is as biomarker (Biomarkers) in diagnosing tumor, prognosis evaluation and the state of an illness Extensive clinical value is all had in follow-up.
But the existing problem low based on the generally existing detection accuracy of detection method of magnetic bead antibody beneficiation technologies, it can not Realize trace detection.
Summary of the invention
In order to solve the above shortcomings and deficiencies, the present invention provides a kind of based on PCR that detection accuracy is high and magnetic bead are anti- The method of the quick detection target of body beneficiation technologies.
Itself the specific scheme is that the quick detection target of a kind of based on PCR and magnetic bead antibody beneficiation technologies method, including Following steps:
Step 1: can identify the antibody or aptamer of target in magnetic microsphere surface markers；
Step 2: target being enriched with by antibody or aptamer in the magnetic microsphere surface that step 1 obtains；
Step 3: will specific recognition target DNA marker and the obtained target of magnetic microsphere of step 2 In conjunction with；
Step 4: magnetic microsphere surface is had into antibody or aptamer-target-DNA marker sandwich knot Structure separation, and pass through PCR or isothermal duplication method DNA amplification marker；
Step 5: the DNA marker after detection amplification obtains the concentration results of target.
In the method for the quick detection target of above-mentioned based on PCR and magnetic bead antibody beneficiation technologies, target is thin Bacterium, virus, pathogen, cell or protein.
In the method for the quick detection target of above-mentioned based on PCR and magnetic bead antibody beneficiation technologies, the magnetism of step 1 Microballoon is the magnetic microsphere by carboxylated.
In the method for the quick detection target of above-mentioned based on PCR and magnetic bead antibody beneficiation technologies, the DNA mark Remember that object is single-chain nucleic acid of the end with amino or carboxyl.
In the method for the quick detection target of above-mentioned based on PCR and magnetic bead antibody beneficiation technologies, the step 2 It is sub- to pass through carbonization two for the coupling of target and DNA marker in the coupling of middle antibody or aptamer and target, step 3 Amine is coupled.
In the method for the quick detection target of above-mentioned based on PCR and magnetic bead antibody beneficiation technologies, the step 5 DNA marker after middle amplification is detected by fluorescent quantitation method or nucleic acid colloidal-gold detecting-card.
In the method for the quick detection target of above-mentioned based on PCR and magnetic bead antibody beneficiation technologies, the magnetism The partial size of microballoon is 25nm.
The beneficial effects of the present invention are:
The present invention is combined by PCR and magnetic bead antibody beneficiation technologies, and trace detection, detection can be realized to target Precision is high.
Detailed description of the invention
Fig. 1 is the sensitivity experiment result figure of the embodiment of the present invention 1；
Fig. 2 is the specificity experiments result figure of the embodiment of the present invention 1.
Specific embodiment
With reference to embodiment, technical solution of the present invention is described in further detail, but do not constituted pair Any restrictions of the invention.
In order to which more clearly the present invention will be described, embodiment is listed below to illustrate superiority of the invention.
Embodiment 1
Be illustrated for detecting detection of Salmonella: the method detection sensitivity can reach 10cfu.
1, Biotin and anti-salmonella antibody marks
1) 10mM Sulfo-NHS-LC-Biotin is configured；
2) antibody anti-salmonella antibody is diluted to 0.5mg/mL with PBS；
3) be added in the anti-salmonella antibody of 300 μ L suitable 10mM Sulfo-NHS-LC-Biotin (according to EZ-Link NHS-Biotin Reagents specification calculates), it places and reacts 2 hours on ice；
4) liquid after reaction is transferred in 2.5mL ultra-filtration centrifuge tube (using the preceding ultrapure water rinse that pre-cooling is first added), Pipette tips cannot touch ultrafiltration membrane, 12000rpm, and 4 DEG C of centrifugation 20min discard liquid in collecting pipe；
5) 500 μ L PBS are added in super filter tube, is centrifuged again with 1000rpm, repeats 4 steps 2 to 3 times；
6) it is carefully mixed in super filter tube with pipette tips and is sucked out after protein liquid, be stored in -20 DEG C；
Bio-anti-salmonella antibody and Magpearl Streptavidin are coupled
It will according to the concentration ratio of 5:1 (bio-anti-salmonella antibody and Magpearl Streptavidin) Bio-anti-salmonella antibody and Magpearl Streptavidin are mixed 20 minutes, and midfeather a few minutes run up and down Centrifuge tube is shaken to be allowed to sufficiently combine；After having reacted the time, by centrifuge tube close to magnet stand, careful sucks supernatant, is added The PBS solution of original volume is resuspended；This step 3 time is repeated, to wash away free bio-anti-salmonella antibody molecule；Most It is resuspended eventually with isometric PBS, puts 4 DEG C of preservations.
2, anti-salmonella antibody and amplification aptamer are coupled
It is coupled with traditional EDC, NHS method.
1) 1OD nucleic acid is centrifuged 30s, and 100 μ L MES buffer solutions are added；
2) 0.5mg EDC, 0.5mg NHS are weighed respectively to be added in MES solution, is mixed, and room temperature reaction 3 hours is protected from light；
3) 10 μ L are added, the NaAc solution that concentration is 3M mixes；
4) 100 μ L isopropanols are added, mix, -20 DEG C of reaction overnights；
5) at 4 DEG C, 12000rpm is centrifuged 15min, discards supernatant；
6) 80% ethyl alcohol of 500 μ L pre-cooling is added, at 4 DEG C, 12000rpm is centrifuged 8min, goes after supernatant to be protected from light to dry；
7) the 0.5mg/mL anti-salmonella that volume is 30 μ L is added, is protected from light 2 hours；
8) it is dialysed with the PBS of pre-cooling at 4 DEG C, changes a not good liquor every 3 hours, change liquid 3-5 times；
3, experimentation
1) it is collected with LB culture medium in 37 DEG C of culture detection of Salmonella, then gradient dilution is added in sterile water for centrifugation, is formed 10cfu/ml,50cfu/ml,100cfu/ml,500cfu/ml,1000cfu/ml.It is each that EpCAM- magnetic bead is added, it is mixed by inversion 10min。
2) 10 μ L of anti-salmonella-amplification aptamer is added, mixes reaction 20min.
3) Magnetic Isolation is resuspended with PBS containing 0.05%tween, repeats this step 6 time, and 10 μ L of final volume is resuspended.
4) Q-PCR reacts.
Table 1 is sensitivity technique result corresponding with Fig. 1.
The sensitivity technique result corresponding with Fig. 1 of table 1
Cell quantity 0 10 100 500 1000
Ct value average value 24.79 19.54 17.20 18.19 21.20
Attached drawing 1 is the sensitivity experiment result figure of the present embodiment, i.e., different detection of Salmonella Concentration Testing results.
Attached drawing 2 is the specificity experiments result figure of the present embodiment, i.e. large intestine angstrom is let out in salmonella, E.sakazakii, cause Uncommon Salmonella, Listeria Monocytogenes, staphylococcus aureus, Shigella 100cfu comparison；Wherein, 1. sramana Bacterium；2. E.sakazakii；3. escherichia coli is let out in cause；4. Listeria Monocytogenes；5.Marke；6. golden yellow Staphylococcus；7. shigella dysenteriae.
Above-described is only presently preferred embodiments of the present invention, all made within the scope of the spirit and principles in the present invention What modifications, equivalent substitutions and improvements etc., should all be included in the protection scope of the present invention.

Claims (7)

1. a kind of method of the quick detection target of based on PCR and magnetic bead antibody beneficiation technologies, which is characterized in that including as follows Step:
Step 1: can identify the antibody or aptamer of target in magnetic microsphere surface markers；
Step 2: target being enriched with by antibody or aptamer in the magnetic microsphere surface that step 1 obtains；
Step 3: will specific recognition target DNA marker in conjunction with the target for the magnetic microsphere that step 2 obtains；
Step 4: by magnetic microsphere surface with antibody or aptamer-target-DNA marker sandwich structure point From, and pass through PCR or isothermal duplication method DNA amplification marker；
Step 5: the DNA marker after detection amplification obtains the concentration results of target.
2. the method for the quick detection target of based on PCR according to claim 1 and magnetic bead antibody beneficiation technologies, special Sign is that target is bacterium, virus, pathogen, cell or protein.
3. the method for the quick detection target of based on PCR according to claim 1 and magnetic bead antibody beneficiation technologies, special Sign is that the magnetic microsphere of step 1 is the magnetic microsphere by carboxylated.
4. the method for the quick detection target of based on PCR according to claim 3 and magnetic bead antibody beneficiation technologies, special Sign is that the DNA marker is single-chain nucleic acid of the end with amino or carboxyl.
5. the method for the quick detection target of based on PCR according to claim 4 and magnetic bead antibody beneficiation technologies, special Sign is, the coupling of antibody or aptamer and target in the step 2, target and DNA marker in step 3 Coupling is coupled by carbodiimides.
6. the method for the quick detection target of based on PCR according to claim 1 and magnetic bead antibody beneficiation technologies, special Sign is that the DNA marker after expanding in the step 5 is examined by fluorescent quantitation method or nucleic acid colloidal-gold detecting-card It surveys.
7. the method for the quick detection target of based on PCR according to claim 1 and magnetic bead antibody beneficiation technologies, special Sign is that the partial size of the magnetic microsphere is 25nm.
CN201711130951.0A 2017-11-15 2017-11-15 A kind of method of the quick detection target of based on PCR and magnetic bead antibody beneficiation technologies Pending CN109283329A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113567685A (en) * 2021-09-26 2021-10-29 瑞博奥(广州)生物科技股份有限公司 HGFR (human liver factor receptor) identification method based on nucleic acid aptamer probe and kit for detecting HGFR

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
*** 等: "琼脂磁珠介导的核酸信标配基免疫PCR检测HBsAg", 《SCIENCE DISCOVERY》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113567685A (en) * 2021-09-26 2021-10-29 瑞博奥(广州)生物科技股份有限公司 HGFR (human liver factor receptor) identification method based on nucleic acid aptamer probe and kit for detecting HGFR
CN113567685B (en) * 2021-09-26 2022-05-17 瑞博奥(广州)生物科技股份有限公司 HGFR (human liver factor receptor) identification method based on nucleic acid aptamer probe and kit for detecting HGFR

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Application publication date: 20190129