CN106501534A - Using the anti-depressant test kit of nucleic acid aptamer fluorescence probe quick detection polypeptide type and its detection method - Google Patents
Using the anti-depressant test kit of nucleic acid aptamer fluorescence probe quick detection polypeptide type and its detection method Download PDFInfo
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Abstract
The present invention relates to a kind of erythropoietin EPO1 test kits based on nucleic acid aptamer fluorescence probe, while constituting and composition the invention further relates to determining the method for erythropoietin EPO concentration, measure reagent, belong to medical test determination techniques field.The test kit main component of the present invention includes：Erythrocyte cracked liquid, phosphate buffer PBS, erythropoietin EPO standard substance, erythropoietin EPO nucleic acid aptamer fluorescence probes；Cracked by blood sample, mixing ovum educates process, combined with fluorescent spectrophotometer is detected, so as to calculate the concentration of erythropoietin EPO.The present invention has the advantages of sample treatment is simple, and easy to operate, detection time is short, detects high specificity, and sensitivity is high, testing result repeatability is high.
Description
Technical field
The invention belongs to medical test determination techniques field, the more particularly to rush based on nucleic acid aptamer fluorescence probe EPO1
Erythropoietin test kit and its detection method.
Background technology
EPO is erythropoietin（ErythropoietEP）English abbreviation.Erythropoietin in human body
It is a kind of hormonelike material by kidney and hepatic secretion, erythropoiesis can be promoted.Taking erythropoietin can make
The patient for suffering from nephropathy anemia increases blood flow and compares solubility（Increase erythrocyte percentage ratio in blood）.This medicine enters business in recent years
Market.During human body anoxia, this kind of hormone is generated to be increased, and causes red blood cell proliferation.EPO analeptic is exactly given birth to according to promoting erythrocyte
The principle synthetic of Cheng Su, it can promote in muscle oxygen to generate so that muscle energeticallyer, the working time longer.
Erythropoietin is the body fluid sex factor that the generation to erythrocyte has potentiation.For molecular weight 6-7's ten thousand
Glycoprotein, the content of sugar are more, it has therefore proved that have its presence in blood and urine.Undifferentiated stem cell differentiation erythroblastic cell line is dry thin
Born of the same parents（erythropoietin responsive cell）, erythropoietin here plays a role, is allowed to be changed into front Cheng Hong
Cell.To further maturation is hemocytoblast, reticulocyte again, the synthesis of hemoglobin and inflow peripheral vessel etc. are
There is facilitation.Typically in anemia and hypoxia, according to needs of the bodily tissue to oxygen, the supply of erythropoietin
Amount will increase, but its content is then very low in kidney anemia.Though which generates organ and mechanism is not yet clear, but as certain kidney
The dirty factor（renal erythropoietic factor）The saying for producing glomerule with the substrate reaction of blood plasma is strong.
Additionally, playing in the factor equivalent to erythropoietin effect, promotion is leukopoietic colony
Stimulating factor, promote thrombopoietic for thrombopoietin, including including erythropoietin, system
Referred to as hemopoietic promotive factor.
Increase erythrocytic number, for anemia, histodialysis, premature infant, in terms of cancerology and hematology.For controlling
Treat the anemia of Chronic Renal Failure Patients.Treat and observe within initial 1～3 week that hematocrit increases, and and dose proportional, 4
Between in～6 weeks, hematocrit levels are in different researchs up to 30%～40%, the quality of life of patient improves, including
The raising of physical tolerance, emotion, daily life mode and sexual function.Research table to Anemic patients that chronic kidney hypofunction need not be dialysed
Bright, it is also advantageous using this product.
The detection method of erythropoietin has three major types at present：Chromatography, Immunological Method, Enzymatic cycling.Chromatography
Sensitivity is high, specificity is good, but sample treatment, separation condition, chromatographic column prepare all multi-Varis so as to be difficult to standardization；And
Hplc device is expensive, technical conditions have high demands, and needs special attendant so as to Difficulty.Immunological Method needs also
Former free EPO forms, antibody fluorescence analytic process and turbidimetry are unable to direct detection sequestered Homocysteine, can only detect blood
Total glycated protein is starched, being educated in 37 DEG C of half an hour ovum with reducing agent carries out reduction treatment to blood sample.Immunological Method is needed more than one hour
Result, complex operation step can just be gone out, since it is desired that carry out reduction treatment can be affected by some uncertain factors.Enzymatic cycling
Process is loaded down with trivial details, and test limit is low, it is larger to produce error, expensive, therefore cannot promote.
Aptamer is the new identification molecule of a class that developed recently gets up, is subject to the extensive pass of scientist in recent years
Note, the aptamer for important physiologically active molecule is screened out in a large number；The various analysis sides based on aptamer
Method and technology are reported；Aptamer medicine " Macugen " was also listed by FDA official approvals in 2005.SELEX skills
The oligonucleotide sequence that art screening is obtained is referred to as aptamer, and domestic its is translated as aptamer, nucleic acid aptamer, nucleic acid to be known
Body or aptamer etc..SELEX technology refers to that applied chemistry method synthesizes jumbo random oligonucleotide (consolidating by two ends
Sequencing row and middle random sequences composition) library, by applying selection pressure (in conjunction with target, elutriation and target height
The process of specific bond fragment), and Amplification Technologies are combined, the circulation selective enrichment through excessively taking turns is obtained and target substance
The oligonucleotide molecules that high special is combined, can be RNA can also be DNA, length is generally 25 ~ 60 nucleotide.
From the foregoing, it will be observed that aptamer is combined presented hypersensitivity and high specific with target substance so as to examine in disease
Have a good application prospect in disconnected, although clinical practice report ripe at present is less, apply fit detection globulin
Research be but on the increase, also continuously emerged based on fit new detecting technique.But it is currently based on being directed to for aptamer
The efficient specific recognition research of erythropoietin also lacks very much, and be directed to erythropoietin aptamer and
Which screens preparation method there is not yet report.
Content of the invention
The purpose of the present invention is to be also easy to produce that operating technology error, the not good enough, interference factor of repeatability be a lot, behaviour for existing
Make the deficiencies such as loaded down with trivial details, testing cost is high, there is provided a kind of erythropoietin test kit based on nucleic acid aptamer fluorescence probe and
Its detection method.
The solution of the present invention is by being achieved in that：
A kind of erythropoietin test kit based on nucleic acid aptamer fluorescence probe, it is characterised in that main component includes：Contain
NH4Cl、Tris、EDTA-Na2Erythrocyte cracked liquid；Contain MgCl20.2M phosphate buffers；Erythropoietin standard
Product；Erythropoietin nucleic acid aptamer fluorescence probe；The erythropoietin nucleic acid aptamer fluorescence probe is 5' and 3'
The nucleotide single-chain of mark fluorescent pyrene molecule monomer is distinguished at two ends, and the nucleotide single-chain sequence is：catctggtga
cgtttagagc gagagactgc gcatatacc.The sequence designations are EPO1.
A kind of above-described erythropoietin test kit based on nucleic acid aptamer fluorescence probe, described containing NH4Cl、
Tris、EDTA-Na2Erythrocyte cracked liquid be containing 1 ~ 280 mmol/L NH4Cl, 1~34 mmol/L Tris,1~2mmol/
LEDTA-Na2, pH 7.0~7.2；Described containing MgCl20.2M phosphate buffers be containing 1 ~ 10mmol/L MgCl2.
A kind of erythropoietin test kit based on nucleic acid aptamer fluorescence probe described in any of the above, its feature exist
In described containing NH4Cl、Tris、EDTA-Na2Erythrocyte cracked liquid, contain MgCl20.2M phosphate buffers, promoting erythrocyte life
It is to prepare the liquid reagent that directly uses or use front into plain standard substance, erythropoietin nucleic acid aptamer fluorescence probe
The dry powder that need to be dissolved in water.
A kind of above-described erythropoietin test kit based on nucleic acid aptamer fluorescence probe, it is characterised in that
The test kit is used for detecting anticoagulant heparin whole blood or the Erythropoietin in peripheral blood.
A kind of erythropoietin test kit with described in any of the above based on nucleic acid aptamer fluorescence probe is detecting rush
The method of Erythropoietin, it is characterised in that method and step includes：
（1）Blood sample is cracked：By blood sample and containing NH4Cl、Tris、EDTA-Na2Erythrocyte cracked liquid press 1:0.5 ~ 5 volume ratio is mixed
Even, 5 ~ 30min, then 5 ~ 10min of medium-speed centrifuge is stood, supernatant is collected；
（2）Mixing ovum is educated：Mixing ovum is educated：Take 20 ~ 100 μ l steps 1）The supernatant for obtaining and the use of 30 ~ 50ul contain MgCl2's
0.2M phosphate buffers, dissolve the erythropoietin aptamer that erythropoietin nucleic acid aptamer fluorescence probe is obtained
Fluorescent probe reagent mixes, and under room temperature, ovum educates 5 ~ 15min, makes erythropoietin nucleic acid aptamer fluorescence probe abundant with blood sample
In conjunction with obtaining test fluid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation；
（4）As a result calculate：Using biomedical mapping software, the standard made with reference to erythropoietin standard sample is bent
Line, in conjunction with step 3）In the fluorescent value that detects calculate the concentration of erythropoietin in blood sample.
Above-described biomedical software is Sigma plot softwares, in being commercially available on the market.
A kind of above-described erythropoietin test kit with based on nucleic acid aptamer fluorescence probe is red to detect rush
The method of erythropoietin concentration, it is characterised in that promote in the erythropoietin nucleic acid aptamer fluorescence probe reagent red
Erythropoietin nucleic acid aptamer fluorescence probe concentration is 200 ~ 400 nmol/L, and its characteristic also resides in, and the promoting erythrocyte is generated
Plain nucleic acid aptamer fluorescence probe distinguishes the nucleotide single-chain of mark fluorescent pyrene molecule monomer, the nucleotide single-chain for 5' and 3' two ends
Sequence is：catctggtga cgtttagagc gagagactgc gcatatacc.
When erythropoietin nucleic acid aptamer fluorescence probe is not combined with erythropoietin, aptamer be in than
Structure loosely, the pyrene molecule monomer at 5' and 3' two ends are mutually dissociated, and after fluorescence excitation, launch wavelength is in 370 ~ 400nm
Between；When erythropoietin nucleic acid aptamer fluorescence probe is combined with erythropoietin, erythropoietin is induced
Its recurring structure changes, and the pyrene molecule monomer at aptamer 5' and 3' two ends is close to each other, forms excited state dimer, and fluorescence swashs
Rear excited state dimer launch wavelength is sent out 480 between 500nm.
A kind of above-described erythropoietin test kit with based on nucleic acid aptamer fluorescence probe is red to detect rush
The method of erythropoietin concentration, it is characterised in that described blood sample is anticoagulant heparin whole blood or peripheral blood, described containing NH4Cl、
Tris、EDTA-Na2Erythrocyte cracked liquid be containing 1 ~ 280 mmol/L NH4Cl, 1~34 mmol/L Tris,1~2mmol/
LEDTA-Na2, pH 7.0~7.2；Described containing MgCl20.2M phosphate buffers be containing 1 ~ 10mmol/L MgCl2；Described
Fluorescence detector is the fluorescence detector with time-resolved fluorometry.
A kind of above-described erythropoietin test kit with based on nucleic acid aptamer fluorescence probe is red to detect rush
The method of erythropoietin concentration, it is characterised in that the detection method is used for detecting anticoagulant heparin whole blood or the rush in peripheral blood
Erythropoietin.
The present invention principle be：When not with erythropoietin, aptamer is in more open structure, 5'
Mutually dissociate with the pyrene molecule monomer at 3' two ends, fluorescence emission wavelengths are 370 between 400nm；Give birth to promoting erythrocyte
When Cheng Su is combined, erythropoietin induction aptamer structure changes, the pyrene point at the 5' and 3' two ends of aptamer
Sub- monomer is close to each other, forms dimer, and pyrene excited state emission wavelength dimer is 480 between 500nm.Pyrene excited state
Dimer fluorescence lifetime has up to 100 ns, than the biological sample autofluorescence life-span（About 5 ns）Long, red by detecting rush
Fluorescence intensity and fluorescence lifetime after erythropoietin aptamer probe and sample mix, calculates promoting erythrocyte in sample and generates
The concentration of element.
The present invention substantive distinguishing features and marked improvement be：
（1）Detection is simple to operate quick, processes without the need for complex sample and separates, aptamer probe is directly added into after cracking
Blood sample liquid, with the fluorescent value that can just detect in the spectrofluorophotometer short time at 480 ~ 500nm；
（2）This test kit and its detection method have sensitivity high, and testing result repeatability is high, sample detection error 0.01 ~
Between 0.1%, high specificity, when erythropoietin nucleic acid aptamer fluorescence probe is not combined with erythropoietin, nucleic acid
Fit in more open structure, the pyrene molecule monomer at 5' and 3' two ends mutually dissociates, and after fluorescence excitation, launch wavelength exists
Between 370 ~ 400nm；When which is combined with erythropoietin, erythropoietin induces which to change, and nucleic acid is fitted
The pyrene molecule monomer at body 5' and 3' two ends is close to each other, forms dimer, after fluorescence excitation dimer launch wavelength 480 ~
Between 500nm.
（3）Reagent used can make to prepare the liquid reagent that can be used directly or use using after being front dissolved in water
Dry powder, detectable can be with storage at normal temperature, convenient transportation.
Specific embodiment
Below in conjunction with the erythropoietin EPO of table 1 and the embodiment description present invention based on nucleic acid aptamer fluorescence probe
Test kit and its detection method.
Kit reagent in 1. embodiment of table is into being grouped into
Embodiment 1
After agent formulations dissolving is prepared needed for embodiment 1 in table 1, bottle is distributed into, lyophilization is carried out, make dry powder examination
Agent；Using front, ultra-pure water is added, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows：
（1）Blood sample is cracked：By anticoagulant heparin whole blood, erythrocyte cracked liquid by volume 1:0.5 mixes, and stands 30min, then middling speed
Centrifugation 7min, collects supernatant；
（2）Mixing ovum is educated：Take 20 μ l steps 1）The use 0.2M phosphate buffer dissolving promoting erythrocyte life of the supernatant 45ul for obtaining
The erythropoietin nucleic acid aptamer fluorescence probe reagent obtained into plain nucleic acid aptamer fluorescence probe mixes, and under room temperature, ovum is educated
5min, obtains test fluid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation；
（4）As a result calculate：Using biomedical mapping software, the standard made with reference to erythropoietin standard sample is bent
Line, in conjunction with step 3）In the fluorescent value that detects calculate the concentration of erythropoietin in blood sample.
3 parallel assay errors of sample are 0.03 ± 0.01%.
Embodiment 2
After agent formulations dissolving is prepared needed for embodiment 2 in table 1, bottle is distributed into, lyophilization is carried out, make dry powder examination
Agent；Using front, ultra-pure water is added, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows：
（1）Blood sample is cracked：By peripheral blood, erythrocyte cracked liquid by volume 1:2.5 mix, and stand 5min, then medium-speed centrifuge
6min, collects supernatant；
（2）Mixing ovum is educated：Take 50 μ l steps 1）The supernatant 30ul's for obtaining
The erythropoietin core that erythropoietin nucleic acid aptamer fluorescence probe is obtained is dissolved with 0.2M phosphate buffers
Fluorescent probe reagent mixing that acid is fit, under room temperature, ovum educates 6min, obtains test fluid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation；
（4）As a result calculate：Using biomedical mapping software, the standard made with reference to erythropoietin standard sample is bent
Line, in conjunction with step 3）In the fluorescent value that detects calculate the concentration of erythropoietin in blood sample.3 parallel surveys of sample
Error is determined for 0.05 ± 0.01%.
Embodiment 3
After agent formulations dissolving is prepared needed for embodiment 3 in table 1, bottle is distributed into, lyophilization is carried out, make dry powder examination
Agent；Using front, ultra-pure water is added, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows：
（1）Blood sample is cracked：By peripheral blood, erythrocyte cracked liquid by volume 1:3.5 mix, and stand 10min, then medium-speed centrifuge
5min, collects supernatant；
（2）Mixing ovum is educated：Take 75 μ l steps 1）The supernatant 45ul's for obtaining
The erythropoietin core that erythropoietin nucleic acid aptamer fluorescence probe is obtained is dissolved with 0.2M phosphate buffers
Fluorescent probe reagent mixing that acid is fit, under room temperature, ovum educates 7min, obtains test fluid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation；
（4）As a result calculate：Using biomedical mapping software, the standard made with reference to erythropoietin standard sample is bent
Line, in conjunction with step 3）In the fluorescent value that detects calculate the concentration of erythropoietin in blood sample.
3 parallel assay errors of sample are 0.03 ± 0.01%.
Embodiment 4
After agent formulations dissolving is prepared needed for embodiment 4 in table 1, bottle is distributed into, lyophilization is carried out, make dry powder examination
Agent；Using front, ultra-pure water is added, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows：
（1）Blood sample is cracked：By anticoagulant heparin whole blood, erythrocyte cracked liquid by volume 1:0.5 mixes, and stands 25min, then middling speed
Centrifugation 8min, collects supernatant；
（2）Mixing ovum is educated：Take 100 μ l steps 1）The use 0.2M phosphate buffer dissolving promoting erythrocyte life of the supernatant 50ul for obtaining
The erythropoietin nucleic acid aptamer fluorescence probe reagent obtained into plain nucleic acid aptamer fluorescence probe mixes, and under room temperature, ovum is educated
8min, obtains test fluid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation；
（4）As a result calculate：Using biomedical mapping software, the standard made with reference to erythropoietin standard sample is bent
Line, in conjunction with step 3）In the fluorescent value that detects calculate the concentration of erythropoietin in blood sample.
3 parallel assay errors of sample are 0.06 ± 0.01%.
Embodiment 5
After agent formulations dissolving is prepared needed for embodiment 5 in table 1, bottle is distributed into, lyophilization is carried out, make dry powder examination
Agent；Using front, ultra-pure water is added, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows：
（1）Blood sample is cracked：By anticoagulant heparin whole blood, erythrocyte cracked liquid by volume 1:1.0 mix, and stand 20min, then middling speed
Centrifugation 9min, collects supernatant；
（2）Mixing ovum is educated：Take 80 μ l steps 1）The use 0.2M phosphate buffer dissolving promoting erythrocyte life of the supernatant 30ul for obtaining
The erythropoietin nucleic acid aptamer fluorescence probe reagent obtained into plain nucleic acid aptamer fluorescence probe mixes, and under room temperature, ovum is educated
9min, obtains test fluid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation；
（4）As a result calculate：Using biomedical mapping software, the standard made with reference to erythropoietin standard sample is bent
Line, in conjunction with step 3）In the fluorescent value that detects calculate the concentration of erythropoietin in blood sample.
3 parallel assay errors of sample are 0.06 ± 0.01%.
Embodiment 6
After agent formulations dissolving is prepared needed for embodiment 6 in table 1, bottle is distributed into, lyophilization is carried out, make dry powder examination
Agent；Using front, ultra-pure water is added, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows：
（1）Blood sample is cracked：By anticoagulant heparin whole blood, erythrocyte cracked liquid by volume 1:4.5 mix, and stand 15min, then middling speed
Centrifugation 10min, collects supernatant；
（2）Mixing ovum is educated：Take 30 μ l steps 1）The use 0.2M phosphate buffer dissolving promoting erythrocyte life of the supernatant 40ul for obtaining
The erythropoietin nucleic acid aptamer fluorescence probe reagent obtained into plain nucleic acid aptamer fluorescence probe mixes, and under room temperature, ovum is educated
10min, obtains test fluid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation；
（4）As a result calculate：Using biomedical mapping software, the standard made with reference to erythropoietin standard sample is bent
Line, in conjunction with step 3）In the fluorescent value that detects calculate the concentration of erythropoietin in blood sample.
3 parallel assay errors of sample are 0.08 ± 0.01%.
Embodiment 7
After agent formulations dissolving is prepared needed for embodiment 7 in table 1, bottle is distributed into, lyophilization is carried out, make dry powder examination
Agent；Using front, ultra-pure water is added, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows：
（1）Blood sample is cracked：By peripheral blood, erythrocyte cracked liquid by volume 1:3.0 mix, and stand 18min, then medium-speed centrifuge
8min, collects supernatant；
（2）Mixing ovum is educated：Take 50 μ l steps 1）The use 0.2M phosphate buffer dissolving promoting erythrocyte life of the supernatant 20ul for obtaining
The erythropoietin nucleic acid aptamer fluorescence probe reagent obtained into plain nucleic acid aptamer fluorescence probe mixes, and under room temperature, ovum is educated
12min, obtains test fluid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation；
（4）As a result calculate：Using biomedical mapping software, the standard made with reference to erythropoietin standard sample is bent
Line, in conjunction with step 3）In the fluorescent value that detects calculate the concentration of erythropoietin in blood sample.
3 parallel assay errors of sample are 0.1 ± 0.01%.
Embodiment 8
After agent formulations dissolving is prepared needed for embodiment 8 in table 1, bottle is distributed into, lyophilization is carried out, make dry powder examination
Agent；Using front, ultra-pure water is added, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows：
（1）Blood sample is cracked：By peripheral blood, erythrocyte cracked liquid by volume 1:2.0 mix, and stand 26min, then medium-speed centrifuge
6min, collects supernatant；
（2）Mixing ovum is educated：Take 60 μ l steps 1）The use 0.2M phosphate buffer dissolving promoting erythrocyte life of the supernatant 25ul for obtaining
The erythropoietin nucleic acid aptamer fluorescence probe reagent obtained into plain nucleic acid aptamer fluorescence probe mixes, and under room temperature, ovum is educated
13min, obtains test fluid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation；
（4）As a result calculate：Using biomedical mapping software, the standard made with reference to erythropoietin standard sample is bent
Line, in conjunction with step 3）In the fluorescent value that detects calculate the concentration of erythropoietin in blood sample.
3 parallel assay errors of sample are 0.02 ± 0.01%.
Embodiment 9
After agent formulations dissolving is prepared needed for embodiment 9 in table 1, bottle is distributed into, lyophilization is carried out, make dry powder examination
Agent；Using front, ultra-pure water is added, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows：
（1）Blood sample is cracked：By peripheral blood, erythrocyte cracked liquid by volume 1:4.0 mix, and stand 8min, then medium-speed centrifuge
7min, collects supernatant；
（2）Mixing ovum is educated：Take 40 μ l steps 1）The use 0.2M phosphate buffer dissolving promoting erythrocyte life of the supernatant 35ul for obtaining
The erythropoietin nucleic acid aptamer fluorescence probe reagent obtained into plain nucleic acid aptamer fluorescence probe mixes, and under room temperature, ovum is educated
15min, obtains test fluid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation；
（4）As a result calculate：Using biomedical mapping software, the standard made with reference to erythropoietin standard sample is bent
Line, in conjunction with step 3）In the fluorescent value that detects calculate the concentration of erythropoietin in blood sample.
3 parallel assay errors of sample are 0.01 ± 0.01%.
Sequence table
<110>Liuzhou Li Jie Science and Technology Ltd.s
<120>Using the anti-depressant test kit of nucleic acid aptamer fluorescence probe quick detection polypeptide type and its detection method
<130> 2016
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 40
<212> DNA
<213>Artificial sequence
<400> 1
catctggtga cgtttagagc gagagactgc gcatatacc 40
Claims (9)
1. a kind of erythropoietin test kit based on nucleic acid aptamer fluorescence probe, it is characterised in that main component includes：
Contain NH4Cl、Tris、EDTA-Na2Erythrocyte cracked liquid；Contain MgCl20.2M phosphate buffers；Erythropoietin mark
Quasi- product；Erythropoietin nucleic acid aptamer fluorescence probe；The erythropoietin nucleic acid aptamer fluorescence probe be 5' and
The nucleotide single-chain of mark fluorescent pyrene molecule monomer is distinguished at 3' two ends, and the nucleotide single-chain sequence is：catctggtga
cgtttagagc gagagactgc gcatatacc.
2. a kind of erythropoietin test kit based on nucleic acid aptamer fluorescence probe according to claim 1, its are special
Levy and be, described containing NH4Cl、Tris、EDTA-Na2Erythrocyte cracked liquid be containing 1～280 mmol/L NH4Cl, 1~34
mmol/L Tris,1~2mmol/LEDTA-Na2, pH 7.0~7.2；Described containing MgCl20.2M phosphate buffers be containing 1 ~
10mmol/L MgCl2.
3. a kind of erythropoietin test kit based on nucleic acid aptamer fluorescence probe according to claim 1 and 2, its
It is characterised by, described containing NH4Cl、Tris、EDTA-Na2Erythrocyte cracked liquid, contain MgCl20.2M phosphate buffers, promote red
Erythropoietin standard substance, erythropoietin nucleic acid aptamer fluorescence probe be prepare the liquid reagent that directly uses or
Using the front dry powder that need to be dissolved in water.
4. a kind of erythropoietin test kit based on nucleic acid aptamer fluorescence probe according to claim 1 ~ 3, its
It is characterised by, the test kit is used for detecting anticoagulant heparin whole blood or the Erythropoietin in peripheral blood.
5. one kind detects rush with the arbitrary erythropoietin test kit based on nucleic acid aptamer fluorescence probe of right 1 ~ 3
The method of Erythropoietin, it is characterised in that method and step includes：
（1）Blood sample is cracked：By blood sample and containing NH4Cl、Tris、EDTA-Na2Erythrocyte cracked liquid press 1:0.5 ~ 5 volume ratio is mixed
Even, 5 ~ 30min, then 5 ~ 10min of medium-speed centrifuge is stood, supernatant is collected；
（2）Mixing ovum is educated：Take 20 ~ 100 μ l steps 1）The supernatant for obtaining and the use of 30 ~ 50ul contain MgCl20.2M phosphoric acid delay
Liquid is rushed, the erythropoietin nucleic acid aptamer fluorescence probe examination that erythropoietin nucleic acid aptamer fluorescence probe is obtained is dissolved
Agent mixes, and under room temperature, ovum educates 5 ~ 15min, erythropoietin nucleic acid aptamer fluorescence probe is fully combined with blood sample, obtains
Test fluid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation；
（4）As a result calculate：Using biomedical mapping software, the standard made with reference to erythropoietin standard sample is bent
Line, in conjunction with step 3）In the fluorescent value that detects calculate the concentration of erythropoietin in blood sample.
6. a kind of erythropoietin test kit with based on nucleic acid aptamer fluorescence probe according to claim 5 is examining
The method for surveying Erythropoietin, it is characterised in that the erythropoietin nucleic acid aptamer fluorescence probe reagent
Middle erythropoietin nucleic acid aptamer fluorescence probe concentration be 200 ~ 400 nmol/L, the erythropoietin nucleic acid fit
The nucleotide single-chain of mark fluorescent pyrene molecule monomer distinguished by body fluorescent probe for 5' and 3' two ends, and the nucleotide single-chain sequence is：
ggcatgggag gtgtaatggc cgagcgattc tactaggaca tcgatgacc agtctgcg.
7. a kind of erythropoietin test kit with based on nucleic acid aptamer fluorescence probe according to claim 6 is examining
The method for surveying Erythropoietin, it is characterised in that described erythropoietin nucleic acid aptamer fluorescence probe is not
When being combined with erythropoietin, aptamer is in more open structure, and the pyrene molecule monomer at 5' and 3' two ends is mutual
Free, after fluorescence excitation, launch wavelength is between 370 ~ 400nm；Described erythropoietin aptamer fluorescence is visited
When pin is combined with erythropoietin, erythropoietin induces its recurring structure to change, aptamer 5' and 3' two ends
Pyrene molecule monomer close to each other, formed excited state dimer, after fluorescence excitation, excited state dimer launch wavelength is arrived 480
Between 500nm.
8. a kind of erythropoietin test kit with based on nucleic acid aptamer fluorescence probe according to claim 5 is examining
The method for surveying Erythropoietin, it is characterised in that described blood sample is anticoagulant heparin whole blood or peripheral blood, described contains
NH4Cl、Tris、EDTA-Na2Erythrocyte cracked liquid be containing 1 ~ 280 mmol/L NH4Cl, 1~34 mmol/L Tris,1~
2mmol/LEDTA-Na2, pH 7.0~7.2；Described containing MgCl20.2M phosphate buffers be containing 1 ~ 10mmol/L
MgCl2；The fluorescence detector is the fluorescence detector with time-resolved fluorometry.
9. a kind of erythropoietin test kit with based on nucleic acid aptamer fluorescence probe according to claim 5 ~ 7 come
The method of detection Erythropoietin, it is characterised in that the detection method is used for detecting anticoagulant heparin whole blood or tip
Erythropoietin in blood.
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| CN114292843A (en) * | 2021-12-03 | 2022-04-08 | 中国科学院精密测量科学与技术创新研究院 | CRISPR/Cas12a detection system of gene stimulant and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191250A (en) * | 2010-03-19 | 2011-09-21 | 中国人民解放军军事医学科学院毒物药物研究所 | DNA (Desoxyribonucleic Acid) ligand for specific binding with EPO (erythropoietin), preparation method thereof and application thereof |
| CN105651755A (en) * | 2016-03-25 | 2016-06-08 | 徐大鹏 | Nucleic acid aptamer fluorescent probe based alpha-fetoprotein AFP1 kit and detection method |
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2016
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191250A (en) * | 2010-03-19 | 2011-09-21 | 中国人民解放军军事医学科学院毒物药物研究所 | DNA (Desoxyribonucleic Acid) ligand for specific binding with EPO (erythropoietin), preparation method thereof and application thereof |
| CN105651755A (en) * | 2016-03-25 | 2016-06-08 | 徐大鹏 | Nucleic acid aptamer fluorescent probe based alpha-fetoprotein AFP1 kit and detection method |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114292843A (en) * | 2021-12-03 | 2022-04-08 | 中国科学院精密测量科学与技术创新研究院 | CRISPR/Cas12a detection system of gene stimulant and application thereof |
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