CN102994638B - Sandwich immuno-PCR (polymerase chain reaction) detection method and kit for to-be-detected object in biological samples - Google Patents
Sandwich immuno-PCR (polymerase chain reaction) detection method and kit for to-be-detected object in biological samples Download PDFInfo
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Abstract
The invention discloses a sandwich immuno-PCR (polymerase chain reaction) detection method and a kit for the to-be-detected object in biological samples. The method comprises the following steps of: 1) immobilizing a first antibody or receptor; 2) specifically capturing the to-be-detected object in the samples; 3) connecting a second antibody or receptor, wherein the second antibody or receptor is labelled with first nucleotide sequences; 4) hybridizing: hybridizing second nucleotide sequences which are complementary with the first nucleotides sequence with the first nucleotide sequences; 5) transferring: separating the second nucleotide sequences and transferring the separated second nucleotide sequences in a PCR tube by means of enzyme digestion or nucleic acid displacement; and 6) quantitatively detecting the content of the second nucleotide sequences in the PCR tube via PCR, wherein the content reflects the content of the to-be-detected object. The kit comprises the main ingredients related by the detection method. The sandwich immuno-PCR detection method and the kit disclosed by the invention have the beneficial effects of being simple to operate and high in detection accuracy.
Description
Technical field
The present invention relates to the detection technique of thing to be checked in biological sample, relate in particular to sandwich immunoassay PCR detection method and the test kit of thing to be checked in a kind of biological sample.
Background technology
IFN-γ is a kind of immunoreactive protein with several functions, has the effects such as antiviral, antitumor, immunomodulatory and control apoptosis.The effect of Interferon, rabbit and relevant cell factor is the focus of biomedical research always.Yet the interior biological action of Interferon, rabbit body is very large but content is very low, and in normal human blood, every milliliter is only had several piks, even lower.Therefore, a kind of monitoring means efficient, convenient, accurate, that cost is low of research invention seems extremely important.At present, the method for conventional detection IFN-γ has instrumental method and immunodetection.
Instrumental method mainly comprises high performance liquid chromatography (HPLC) and mass spectrum, and sensitivity is very high, but it needs expensive plant and instrument, and consumptive material requires high and sample preprocessing, under professional's operation, just can carry out.This quasi-instrument analytical method can not reach modern measure to requirement quick, that convenient, cost is low.
Immune analysis method typically refer to specificity by antibody and antigen for feature antibody (or antigen) carry out the detection method of detectable antigens (or antibody).This species specificity is mutually identified and is not limited to antigen and antibody, acceptor and part, and enzyme-to-substrate, lectin and polysaccharide are also applicable.The classical way of immunodetection is enzyme-linked immunosorbent assay (ELISA).Traditional E LISA by antigen non-specific be attached on solid support, realized tested antigen and shifted from solution.Washing is removed after unnecessary (unconjugated) antigen, sealing plate hole.Yet on solid support, fixing antigen and antibody are connected to form an immunocomplex, unconjugated antibody is removed in washing, and enzyme can be directly connected on antibody, then surveys primary antibodie by enzyme joint inspection.Or add two resisting of being connected with plate subsequently.The enzymic activity that is connected to solid support be fixed on antigen in solid phase support and be certain proportionlity, for example, can be by the effect of a kind of chromogenic substrate and enzyme, the content of antigen on calculating upholder.The method is simple, economy, and specificity is good, and extensively adopts automated operation.But its detection sensitivity is low, the routine of Interferon, rabbit and cytokine is detected to poor effect.
And (Polymerase Chain Reaction initialism is: PCR) technology has very high detection sensitivity, and in order to strengthen the sensitivity of ELISA, immuno-PCR uses and gives birth in polymerase chain reaction.Immuno-PCR (Immuno-PCR, IPCR) is to be proposed by people such as Sano the earliest, similar with indirect ELISA, and different is that immuno-PCR is used section of DNA chain to substitute the enzyme in ELISA reaction as detection molecules.Document shows: the people such as Sano react the effect that has confirmed immunohistochemical methods with anti-BSA antibody by BSA, its by albumin A-avidin connected system by biotin labeled plasmid DNA mark on primary antibodie, the DNA molecular that pcr amplification is connected with antigen subsequently, 30 circulations of increasing, EB dyeing, then gel electrophoresis analysis.This method and ELISA ratio, immuno-PCR has significantly improved detection sensitivity.This sensitivity can be verified by purification system, but because it exists process loaded down with trivial details, the feature that step is many, is difficult to use in actual medical research.
The method of ' sandwich ' formula of employing is carried out immuno-PCR, be coated antibody on solid support as microwell plate, magnetic bead particles, catch the antigen in sample, and then add and detect antibody, detect on antibody and be marked with reporter molecules as radio isotope, fluorescein, DNA and enzyme, this has produced a special immunocomplex (antibody-antigen-antibody complex) and has been fixed on solid support surface, and repetitive scrubbing solid support surface, removes unconjugated Ag-Ab.In washing step, keep the integrity of immunocomplex very important, to cause strength of signal to maximize, reduce to greatest extent thus background.After washing, by examining report molecule, carry out quantitatively this fixing ' detection ' antibody.The amount of reporter molecules can demonstrate the amount of antigen.When this detection system is while being enough sensitive, the sensitivity minimization of system is conventionally by the degree decision of free ' detections ' antibody background that non-specific binding produces.
At diagnosis and bio-science field, need a strong detection method to remove to analyze the material that some low-level (lower concentrations) exist, and along with completing that human genome checks order and analyzes, this demand become more and more urgent.These albumen should be analyzed and qualitative, but use current detection technique can not quantize these albumen.Immuno-PCR is as a kind of highly sensitive detection method, for the highly sensitive detection of material provides an assurance, but so far, traditional E LISA exists sensitivity low, tradition immuno-PCR complicated operation and background are higher, cause detecting a part of material, therefore, the new technology of necessary exploitation solves these problems.
Summary of the invention
Technical problem to be solved by this invention is to provide sandwich immunoassay PCR detection method and the test kit of thing to be checked in a kind of biological sample, raising detection sensitivity, reduction operational requirement.
Technical problem of the present invention is solved by following technique means:
In biological sample, a sandwich immunoassay PCR detection method for thing to be checked, is characterized in that, comprises the following steps:
1) first antibody or acceptor is fixing: the first antibody or the acceptor that on upholder, connect thing to be checked in biological sample;
2) specificity catches biological sample: make the thing to be checked in biological sample pass through a connection site and described first antibody or receptors bind;
3) connect second antibody or acceptor: the second antibody of thing to be checked or acceptor are connected in another connection site of described thing to be checked, and wherein, described second antibody or receptor marker have the first nucleotide sequence;
4) hybridization: will there is complementary the second nucleotide sequence and described the first nucleotide sequence hybridization with described the first nucleotide sequence;
5) shift: adopt method that displacement or enzyme cut nucleic acid the second nucleotide sequence transferring in PCR pipe described in wash-out from described upholder;
6) detect: the content of the second nucleotide sequence in PCR pipe described in quantitative PCR detection, this content back has been answered the content of thing to be checked in described biological sample.
Preferably:
Described the first nucleotide sequence has the sequence shown in sequence table SEQ ID NO.1: 5 ＇-aagctcgatatcagaggaaggaggagggac-3 ＇;
Described the second nucleotide sequence has the sequence shown in sequence table SEQ ID NO.2: 5 ＇-tccttcctctgatatcgagcttggcgtaatcagggtcatagctgtttcctgtgtga aattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaa agcctgaagtgcctaatgagtg-3 ＇.
In described step 5), adopt method of replacing from described upholder described in wash-out the second nucleotide sequence comprise: adopt with displacement nucleic acid molecule and described second nucleotide sequence of described the first nucleotide sequence complementation and replace, described displacement nucleic acid molecule is that DNA sequence dna or the described displacement nucleic acid molecule with sequence shown in sequence table SEQ ID NO.5 are the peptide nucleic acid(PNA) with sequence shown in sequence table SEQ ID NO.6, shown in sequence table SEQ ID NO.5, sequence is: 5 ＇-gtccctcctccttcctctgatatcgagctt-3 ＇, shown in sequence table SEQ ID NO.6, sequence is: 5 ＇-tccttcctctgatatcgagctt-3 ＇, or in described the first nucleotide sequence and described the second nucleotide sequence, all comprise restriction enzyme site, in described step 5), adopt enzyme blanking method from described upholder described in wash-out the second nucleotide sequence adopt restriction enzyme to carry out wash-out.
Described thing to be checked is human gamma-interferon.
A sandwich immunoassay PCR detection kit for thing to be checked in biological sample, is characterized in that comprising:
Upholder;
The first antibody of thing to be checked or acceptor;
The second antibody of thing to be checked or acceptor, this second antibody or receptor marker have the first nucleotide sequence;
Single stranded DNA fragment, this single stranded DNA fragment has with described the first Nucleotide and has the second complementary nucleotide sequence;
And enzyme cut reagent or with the displacement nucleic acid molecule of described the first nucleotide sequence complementation;
Described enzyme is cut reagent and is comprised enzyme digestion reaction liquid and enzyme digestion reaction stop buffer
Preferably: described the first nucleotide sequence has the sequence shown in sequence table SEQ ID NO.1;
Described the second nucleotide sequence has the sequence shown in sequence table SEQ ID NO.2.
Described displacement nucleic acid molecule is that DNA sequence dna or the described displacement nucleic acid molecule with sequence shown in sequence table SEQ ID NO.5 are the peptide nucleic acid(PNA) with sequence shown in sequence table SEQ ID NO.6.
Compared with prior art, in the immuno-PCR testing process of the present invention's thing to be checked in biological sample, adopt DNA hybridization, mixture transfer techniques, by the single stranded DNA fragment of hybridization is discharged, and the single stranded DNA fragment of non-specific binding is still kept on original upholder, significantly reduced Experimental Background, significantly improved the sensitivity that human gamma-interferon detects, traditional E LISA method lowest detection is limited the quantity of generally in 5pg/mL left and right, linearity range 10 ~ 1000pg/mL, and the detection limit of the inventive method and test kit can reach 0.01pg/mL, linearity range is wider, between 0.05 ~ 1000pg/mL.
Preferred the first nucleotide sequence and the second nucleotide sequence show good interference free performance in practice, can further improve the accuracy of detection.
Preferred version adopts specific displacement nucleic acid molecule, can be conducive to the second nucleotide sequence and the displacement of replacing nucleic acid molecule, reduces or remove simultaneously the wash-out of non-specific binding thing.
Accompanying drawing explanation
Fig. 1 is that the method for the specific embodiment of the invention is at the structure principle chart of the formed mixture of hybridization step;
Fig. 2 is the typical case that adopts the human gamma-interferon Real-time PCR Analysis result of the specific embodiment of the invention;
Fig. 3 is the immuno-PCR typical curve of the specific embodiment of the invention;
Fig. 4 is the traditional E LISA typical curve of prior art.
Embodiment
Preferred embodiment the invention will be further described to contrast accompanying drawing combination below.
A sandwich immunoassay PCR detection method for thing to be checked in biological sample, mainly comprises the following steps:
1. the antibody or the acceptor that on upholder, connect a thing to be checked, be called the fixing of first antibody or acceptor, and upholder preferably adopts solid support;
2. first antibody specificity catches thing to be checked: thing to be checked is attached on first antibody or acceptor by a connection site;
3. second antibody or acceptor are connected on another site of thing to be checked, herein second antibody or the acceptor specific a bit of nucleotide sequence of mark (the first nucleotide sequence); Wherein, the second antibody of the specific a bit of nucleotide sequence of mark of the present embodiment or acceptor are second antibody coupling oligonucleotide, and this oligonucleotide sequence is the sequence shown in sequence table SEQ ID NO.1: 5 ＇-aagctcgatatcagaggaaggaggagggac-3 ＇.
4. long section Nucleotide (the second nucleotide sequence, the i.e. template DNA molecule) hybridization having with aforementioned a bit of nucleotide sequence complementary sequence is attached on the second receptor complex (product of step 3); Referring to the sequence shown in sequence table SEQ ID NO.2, the template DNA molecule of the present embodiment is single stranded DNA fragment: 5 ＇-tccttcctctgatatcgagcttggcgtaatcagggtcatagctgtttcctgtgtga aattgttatccgctcacaatt
ccacacaacatacgagccggaagcataaagtgtaaagcctgaagtgcctaatgagtg-3＇。
5) adopt method that displacement or enzyme cut nucleic acid the second nucleotide sequence transferring in PCR pipe described in wash-out from described upholder; If the method that adopts enzyme to cut nucleic acid, in described the first nucleotide sequence and described the second nucleotide sequence, all comprise restriction enzyme site, for example be illustrated as EcoRV restriction enzyme site, then utilize restriction enzyme as EcoR V, Alu I, EcoR I, Not I, Hind III etc., digest double-stranded bridge chain but be not limited to these enzymes, thereby carry out endonuclease reaction wash-out the second nucleotide sequence; If adopt the mode of displacement nucleic acid, need to adopt displacement nucleic acid molecule and described the second nucleotide sequence with described the first nucleotide sequence complementation to replace, described displacement nucleic acid molecule is that DNA sequence dna or the described displacement nucleic acid molecule with sequence shown in sequence table SEQ ID NO.5 are the peptide nucleic acid(PNA) (PNA) with sequence shown in sequence table SEQ ID NO.6, and shown in sequence table SEQ ID NO.5, sequence is: 5 ＇-gtccctcctccttcctctgatatcgagctt
-3 ＇, shown in sequence table SEQ ID NO.6, sequence is: 5 ＇-tccttcctctgatatcgagctt-3 ＇.
6) by quantitative PCR detection nucleotide sequence molecule, the existence of material to be detected in show sample.
The present embodiment also provides a kind of PCR detection kit based on aforesaid method, and it comprises:
Upholder, preferred solid support, " solid support " refer to and can be used for fixing, for example any solid phase carrier of analyte, antibody or a mixture.The well-known suitable solid phase carrier in this field, it comprises the hole wall of reactive tank, as microwell plate, test tube, polystyrene bead, paramagnetism or non-magnetic pearl, nitrocellulose membrane, nylon membrane; Particulate is as red blood cell of emulsion particle, sheep (or other animals) etc.The typical material of solid phase carrier comprises polyvinyl chloride (PVC), polystyrene, Mierocrystalline cellulose, nylon, latex and derivative thereof, but is not limited only to these.
The first antibody of thing to be checked or acceptor;
The second antibody of thing to be checked or acceptor, this second antibody or acceptor be the specific a bit of nucleotide sequence of mark; The present embodiment adopts the form of second antibody coupling oligonucleotide, and this oligonucleotide sequence is the sequence shown in sequence table SEQ ID NO.1: 5 ＇-aagctcgatatcagaggaaggaggagggac-3 ＇.
Single stranded DNA fragment (template DNA molecule), this single stranded DNA fragment has with described the first Nucleotide and has complementary nucleotide sequence.As shown in sequence table SEQ ID NO.2, described single stranded DNA fragment is: 5 ＇-tccttcctctgatatcgagcttggcgtaatcagggtcatagctgtttcctgtgtga aattgttatccgctcacaattccac
acaacatacgagccggaagcataaagtgtaaagcctgaagtgcctaatgagtg
-3＇
Enzyme cut reagent or with the displacement nucleic acid molecule of described the first nucleotide sequence complementation, wherein:
Described enzyme is cut reagent and is comprised enzyme digestion reaction liquid and enzyme digestion reaction stop buffer, and enzyme digestion reaction liquid of the present invention can adopt existing general enzyme digestion reaction liquid, its component for example: 0.1mol/L pH7.4 Tirs-HCl, 1mol/L NaCl, 0.07mol/L MgCl
2; Enzyme digestion reaction stop buffer of the present invention also can adopt existing general enzyme digestion reaction stop buffer, for example the enzyme digestion reaction stop buffer of following two kinds of components: (1) 0.1mol/L EDTA Na2,20%Ficoll 400, appropriate orange G.(2) 0.25% tetrabromophenol sulfonphthaleins, the blue or green FF of 0.25% dimethylbenzene (or claiming xylene blue AS), 40% aqueous sucrose solution (W/V) (or using 30% aqueous glycerin solution).
Described biological sample is often referred to aqua or from the resuspended liquid of water of biomaterial herein.The sample that the present invention analyzes, is that the form with material exists, and comprising: cell, tissue, homogenate, lysate, extract, purifying or partially purified albumen and other some biomolecules mixtures.The non-limitative example of the biological sample in method of the present invention, comprising: the body fluid of humans and animals is as whole blood, serum, blood plasma, urine, cerebrospinal fluid etc.Described thing to be checked may be comprised of albumen, peptide chain, carbohydrate, lipid, cell or antigenic substance herein.The present embodiment be take human gamma-interferon and is described as example, but the present invention is not limited to this concrete material.
Wherein, the preparation process of above-mentioned second antibody coupling oligonucleotide is as follows:
I. the full gene of amino labeled nucleotide chain is synthetic: 5 ＇-aagctcgatatcagaggaaggaggagggac-3 ＇ (NH
2c7)
Ii. use excessive thiosuccimide, 4 formyl radicals-N-isopropylbenzamide (Sulfo-s-4FB) is modified 3 ' end amido labeled oligonucleotide
1. the oligonucleotide of 10 OD260 is resuspended in 100 μ L lysates, with 200 rifle head, repeatedly inhales 30 times, until all dissolve.Vortex concussion 60 seconds, 1,000 x g collects centrifugate for centrifugal 10 seconds.
2. detect the concentration of amido modified oligonucleotide.
3. with 25 microlitre DMF(Dimethyl Fumarate, be dimethyl fumarate) dissolving Sulfo-S-4FB(1 x 1.5 mg), and added in the centrifuge tube of collecting oligonucleotide, whirlpool mixed for 1000 x g centrifugal 10 seconds, collect the whole liquid below centrifuge tube, incubated at room two hours.
4. in centrifugal end before 5 minutes, the revolving filter of prewetting.Buffer exchange 4FB-Oligo.
5. measure the concentration of 4FB-Oligo.
Iii. excessive succinimide, buzane nicotinamide (S-HYNIC) modified antibodies.
1. add 100 μ L diluents and dissolve the anti-Human IFN-r of 100 μ g antibody, the residual antibody in centrifugal 10 seconds receiving flasks of 1,000 x g.
2. detect antibody concentration
3. with 35 μ LDMF, dissolve a pipe buzane nicotinamide (S-HYNIC), get the buzane nicotinamide of 2 μ L dissolvings in antibody-solutions, mix.
4. with buzane nicotinamide (S-HYNIC) modified antibodies.
5. after S-HYNIC modification reaction completes, HyNic/IgG reaction solution is added to above the resin bed of column spinner, unscrew column spinner lid, the mark that makes to rotate on pillar deviates from whizzer rotating shaft, centrifugal 2 minutes of 1,500 x.
6. IgG HyNic-being modified transfers in the centrifuge tube of 1.5mL, and detects its content.
The using method of the detection kit of the present embodiment is as follows:
(1) formation of thing to be checked (the present embodiment is human gamma-interferon) double-antibody sandwich
I. use phosphate buffered saline buffer (Phosphate Buffered Saline, initialism: PBS) dilute coated antibody (first antibody), 100 μ L/well add in enzyme plate hole (solid support), and 4 ℃ are spent the night.
Ii. the PBS damping fluid containing 0.05% polysorbas20 with PBS-T() wash plate 3-5 time.
Iii. add confining liquid (containing 1%BSA) 200 μ L/well, 37 ℃ 2 hours.
Iv. the PBS damping fluid containing 0.05% polysorbas20 with PBS-T() wash plate 3-5 time.
V. add human gamma-interferon positive sample 100 μ L/well, 37 ℃ 1 ~ 3 hour, the present embodiment selects 2 hours.
Vi. the PBS damping fluid containing 0.05% polysorbas20 with PBS-T() wash plate 3-5 time.
Vii. add second antibody coupling oligonucleotide, 37 ℃ 1 ~ 3 hour, the present embodiment selects 2 hours.
Viii. use the polysorbas20 of PBS-T(0.05%) wash plate 3-5 time, the completing of IFN-γ double-antibody sandwich.
(2) Nucleotide hybridization
Template DNA molecule (5 * 10
11copies/ μ L) be dissolved in hybridization solution, add in enzyme mark hole and second antibody coupling oligonucleotide hybridization, hatch 1 ~ 2 hour for 37 ℃, the present embodiment is hatched 1 hour, and hybridization post-flush is unconjugated.As shown in Figure 1, wherein 100 is solid support to product structure after hybridization; 200 is first antibody or acceptor; 300 is antigen (that is: human gamma-interferon); 400 is second antibody or acceptor; 500 is oligonucleotide; 600 is restriction enzyme site or displacement position; 700 is template DNA molecule.
(3) enzyme is cut and is shifted or replace and shift
Add enzyme digestion reaction liquid, in 37 degree water-baths, enzymolysis 1 ~ 3 hour, the present embodiment enzymolysis 3 hours.
Add 5 ~ 10
μ Lenzyme reaction stop buffer, the present embodiment addition is 10
μ L, mix to stop enzyme reaction.
Get 1～10 μ L and transfer in PCR pipe, PCR reaction can adopt existing general technology, and the component that the present embodiment PCR reaction adopts comprises: PCR Master Mix(comprises 10 * PCR damping fluid, MgCl
2dNTP, archaeal dna polymerase, SYBR Green I etc.) 12.5 μ L, 0.5 μ upstream primer 5 ＇-gcttggcgtaatcagggtca t-3 ＇ (referring to sequence table SEQ ID NO.3), 0.5 μ L downstream primer 5 ＇-cactcattag gcacttcagg cttt-3 ＇ (referring to sequence table SEQ ID NO.4), supply 25 μ L with sterilizing ultrapure water.
Enzyme blanking method can adopt the method for displacement nucleic acid to replace, that is: need to adopt with displacement nucleic acid molecule and described second nucleotide sequence of described the first nucleotide sequence complementation and replace, described displacement nucleic acid molecule is that DNA sequence dna or the described displacement nucleic acid molecule with sequence shown in sequence table SEQ ID NO.5 are the peptide nucleic acid(PNA) with sequence shown in sequence table SEQ ID NO.6.
(4) real-time fluorescence quantitative PCR detects
Getting enzyme, to cut liquid be that template is carried out quantitative PCR reaction, PCR reaction parameter: 94 ℃ of denaturations 5 minutes; 94 ℃ of sex change 30 seconds; Anneal 30 seconds for 58 ℃; 72 ℃ are extended 20 seconds; 35 circulations; 72 ℃ are extended 5 minutes.Quantitative PCR analysis, obtains the content of human gamma-interferon in sample to be measured, the typical consequence of quantitative PCR analysis as shown 2.
The oligonucleotide sequence of the present embodiment, single stranded DNA fragment, displacement nucleic acid molecule also available other nucleotide sequences replace, but selected oligonucleotide sequence, single stranded DNA fragment and the displacement nucleic acid molecule of the present embodiment has extraordinary interference free performance, can improve the accuracy of detection.
As shown in Figure 3,4, the immuno-PCR typical curve of the present embodiment: linear relationship △ Ct=5.5714 * log (conc.)+13.381, R
2=0.997; Sensing range 0.05 ~ 1000pg/mL, sensitivity reaches 0.01pg/mL.And traditional E LISA typical curve: y=0.0017x+0.0975, R
2=0.997; Sensing range 15.6 ~ 1000pg/mL, sensitivity reaches 5pg/mL.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For those skilled in the art, without departing from the inventive concept of the premise, can also make some being equal to substitute or obvious modification, and performance or purposes identical, all should be considered as belonging to protection scope of the present invention.
It is biological Engineering Co., Ltd that <110> Shenzhen reaches section
Sandwich immunoassay PCR detection method and the test kit of thing to be checked in <120> biological sample
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<160> 6
<170> PatentIn version 3.3
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Claims (6)
1. a sandwich immunoassay PCR detection method for thing to be checked in biological sample, is characterized in that, comprises the following steps:
1) first antibody or acceptor is fixing: the first antibody or the acceptor that on upholder, connect thing to be checked in biological sample;
2) specificity catches biological sample: make the thing to be checked in biological sample pass through a connection site and described first antibody or receptors bind;
3) connect second antibody or acceptor: the second antibody of thing to be checked or acceptor are connected in another connection site of described thing to be checked, and wherein, described second antibody or receptor marker have the first nucleotide sequence;
4) hybridization: will there is complementary the second nucleotide sequence and described the first nucleotide sequence hybridization with described the first nucleotide sequence;
5) shift: adopt method that displacement or enzyme cut nucleic acid the second nucleotide sequence transferring in PCR pipe described in wash-out from described upholder;
6) detect: the content of the second nucleotide sequence in PCR pipe described in quantitative PCR detection, this content back has been answered the content of thing to be checked in described biological sample;
Described the first nucleotide sequence has the sequence shown in sequence table SEQ ID NO.1;
Described the second nucleotide sequence has the sequence shown in sequence table SEQ ID NO.2.
2. the sandwich immunoassay PCR detection method of thing to be checked in biological sample according to claim 1, it is characterized in that, in described step 5), adopt displacement nucleic acid method from described upholder described in wash-out the second nucleotide sequence comprise: adopt with displacement nucleic acid molecule and described second nucleotide sequence of described the first nucleotide sequence complementation and replace, described displacement nucleic acid molecule is that DNA sequence dna or the described displacement nucleic acid molecule with sequence shown in sequence table SEQ ID NO.5 are the peptide nucleic acid(PNA) with sequence shown in sequence table SEQ ID NO.6.
3. the sandwich immunoassay PCR detection method of thing to be checked in biological sample according to claim 1, it is characterized in that, in described the first nucleotide sequence and described the second nucleotide sequence, all comprise restriction enzyme site, in described step 5), adopt enzyme cut nucleic acid method from described upholder described in wash-out the second nucleotide sequence adopt restriction enzyme to carry out wash-out.
4. the sandwich immunoassay PCR detection method of thing to be checked in biological sample according to claim 1, is characterized in that: described thing to be checked is human gamma-interferon.
5. a sandwich immunoassay PCR detection kit for thing to be checked in biological sample, is characterized in that comprising:
Upholder;
The first antibody of thing to be checked or acceptor;
The second antibody of thing to be checked or acceptor, this second antibody or receptor marker have the first nucleotide sequence;
Single stranded DNA fragment, this single stranded DNA fragment has with described the first Nucleotide and has the second complementary nucleotide sequence;
And enzyme cut reagent or with the displacement nucleic acid molecule of described the first nucleotide sequence complementation;
Described enzyme is cut reagent and is comprised enzyme digestion reaction liquid and enzyme digestion reaction stop buffer;
Described the first nucleotide sequence has the sequence shown in sequence table SEQ ID NO.1;
Described the second nucleotide sequence has the sequence shown in sequence table SEQ ID NO.2.
6. the sandwich immunoassay PCR detection kit of thing to be checked in biological sample according to claim 5, is characterized in that: described displacement nucleic acid molecule is that DNA sequence dna or the described displacement nucleic acid molecule with sequence shown in sequence table SEQ ID NO.5 are the peptide nucleic acid(PNA) with sequence shown in sequence table SEQ ID NO.6.
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| HK13110398.2A HK1183064A1 (en) | 2012-11-29 | 2013-09-06 | Sandwich immuno-pcr detection method and kit of analyte in biological samples pcr |
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| CN105510591B (en) * | 2014-09-22 | 2019-02-12 | 程永升 | A kind of detection kit and detection method reacted using antibody modification immuno-PCR |
| CN106932590A (en) * | 2015-12-31 | 2017-07-07 | 复旦大学 | A kind of quantitative determination low-abundance protein and the thereafter method of modified protein |
| WO2018111782A1 (en) * | 2016-12-12 | 2018-06-21 | Cepheid | Integrated immuno-pcr and nucleic acid analysis in an automated reaction cartridge |
| CN110498858B (en) * | 2019-07-26 | 2024-01-23 | 深圳市达科为生物工程有限公司 | Method for dynamically detecting secretion condition of single-cell exoprotein |
| CN111733212A (en) * | 2020-06-22 | 2020-10-02 | 广州水石基因科技有限公司 | Hypersensitive molecule lock-key immune polymerase chain reaction detection method |
| CN112778426B (en) * | 2021-01-06 | 2023-12-12 | 深圳伯生生物传感技术有限公司 | Accurate antibody nucleic acid directional connection method |
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| CN102269759A (en) * | 2010-06-07 | 2011-12-07 | 常州楚天生物科技有限公司 | Multiplex detection method based on immuno-PCR and DNA melting curve analysis |
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| CN102269759A (en) * | 2010-06-07 | 2011-12-07 | 常州楚天生物科技有限公司 | Multiplex detection method based on immuno-PCR and DNA melting curve analysis |
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| CN102994638A (en) | 2013-03-27 |
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