EPO4 erythropoietin stimulant kit based on high-affinity stimulant aptamer and detection method thereof

Abstract

The invention relates to an erythropoietin EPO kit based on an aptamer fluorescent probe; meanwhile, the invention also relates to a method for measuring the concentration of the erythropoietin EPO as well as constitution and components of a measuring reagent, and belongs to the technical field of medical examination measurement. The main components of the kit provided by the invention comprise erythrocyte lysate, a phosphatic buffer solution PBS, an erythropoietin EPO standard product and an erythropoietin EPO aptamer fluorescent probe; the concentration of the erythropoietin EPO is measured by combining blood sample pyrolysis and mixed egg cultivation with fluorospectro photometer detection. The kit has the advantages that a sample is treated simply, the operation is simple and convenient, the detection time is short, the detection specificity is strong, the sensitivity is high, the detection result repeatability is high, and the like.

Description

technical field

[0001] The invention belongs to the technical field of medical examination and measurement, in particular to an erythropoietin kit and a detection method based on a nucleic acid aptamer fluorescent probe EPO4. Background technique

[0002] EPO is the English abbreviation of ErythropoietEP. Erythropoietin in humans is a hormone-like substance secreted by the kidneys and liver that stimulates red blood cell production. Taking erythropoietin can increase blood flow specific solubility (that is, increase the percentage of red blood cells in the blood) in patients with nephrotic anemia. This drug has entered the commercial market in recent years. When the human body is hypoxic, the production of this hormone increases and causes red blood cell hyperplasia. EPO stimulants are artificially synthesized according to the principle of erythropoietin, which can promote oxygen production in muscles, thereby making muscles stronger and working longer.

[0003] Erythrop...

Examples

Embodiment 1

[0033] After dissolving and preparing the required reagent components according to Example 1 in Table 1, divide into bottles and freeze-dry to make a dry powder reagent; before use, add ultrapure water and use after reconstitution. Three parallels were set for each sample, and the detection steps were as follows:

[0034] (1) Blood sample lysing: Mix heparin anticoagulated whole blood and erythrocyte lysate at a volume ratio of 1:0.5, let stand for 30 minutes, then centrifuge at a medium speed for 7 minutes, and collect the supernatant;

[0035] (2) Mixed egg incubation: take 20 μl of the supernatant obtained in step 1) and mix with 45ul of the erythropoietin nucleic acid aptamer fluorescent probe reagent obtained by dissolving the erythropoietin nucleic acid aptamer fluorescent probe in 0.2M phosphate buffer, Incubate the eggs at room temperature for 5 minutes to obtain the test solution;

[0036] (3) Fluorescence detection: 50ul of the test solution obtained in the detectio...

Embodiment 2

[0040] After dissolving and preparing the required reagent components according to Example 2 in Table 1, divide into bottles and freeze-dry to make a dry powder reagent; before use, add ultrapure water and use after reconstitution. Three parallels were set for each sample, and the detection steps were as follows:

[0041] (1) Blood sample lysis: Mix peripheral blood and red blood cell lysate at a volume ratio of 1:2.5, let stand for 5 minutes, then centrifuge at a medium speed for 6 minutes, and collect the supernatant;

[0042] (2) Mixed egg incubation: Take 50μl of the supernatant obtained in step 1) and 30ul of

[0043] The erythropoietin nucleic acid aptamer fluorescent probe reagent obtained by dissolving the erythropoietin nucleic acid aptamer fluorescent probe in 0.2M phosphate buffer was mixed, and the eggs were incubated at room temperature for 6 minutes to obtain the test solution;

[0044] (3) Fluorescence detection: 50ul of the test solution obtained in the detect...

Embodiment 3

[0047] After dissolving and preparing the required reagent components according to Example 3 in Table 1, divide into bottles and freeze-dry to make a dry powder reagent; before use, add ultrapure water and use after reconstitution. Three parallels were set for each sample, and the detection steps were as follows:

[0048] (1) Blood sample lysis: Mix peripheral blood and red blood cell lysate at a volume ratio of 1:3.5, let stand for 10 minutes, then centrifuge at a medium speed for 5 minutes, and collect the supernatant;

[0049] (2) Mixed egg incubation: Take 75μl of the supernatant obtained in step 1) and 45ul of

[0050] The erythropoietin nucleic acid aptamer fluorescent probe reagent obtained by dissolving the erythropoietin nucleic acid aptamer fluorescent probe in 0.2M phosphate buffer was mixed, and the eggs were incubated at room temperature for 7 minutes to obtain the test solution;

[0051] (3) Fluorescence detection: 50ul of the test solution obtained in the detec...

technical field

[0001] The invention belongs to the technical field of medical examination and measurement, in particular to an erythropoietin kit and a detection method based on a nucleic acid aptamer fluorescent probe EPO4. Background technique

[0002] EPO is the English abbreviation of ErythropoietEP. Erythropoietin in humans is a hormone-like substance secreted by the kidneys and liver that stimulates red blood cell production. Taking erythropoietin can increase blood flow specific solubility (that is, increase the percentage of red blood cells in the blood) in patients with nephrotic anemia. This drug has entered the commercial market in recent years. When the human body is hypoxic, the production of this hormone increases and causes red blood cell hyperplasia. EPO stimulants are artificially synthesized according to the principle of erythropoietin, which can promote oxygen production in muscles, thereby making muscles stronger and working longer.

[0003] Erythrop...

Examples

Embodiment 1

[0033] After dissolving and preparing the required reagent components according to Example 1 in Table 1, divide into bottles and freeze-dry to make a dry powder reagent; before use, add ultrapure water and use after reconstitution. Three parallels were set for each sample, and the detection steps were as follows:

[0034] (1) Blood sample lysing: Mix heparin anticoagulated whole blood and erythrocyte lysate at a volume ratio of 1:0.5, let stand for 30 minutes, then centrifuge at a medium speed for 7 minutes, and collect the supernatant;

[0035] (2) Mixed egg incubation: take 20 μl of the supernatant obtained in step 1) and mix with 45ul of the erythropoietin nucleic acid aptamer fluorescent probe reagent obtained by dissolving the erythropoietin nucleic acid aptamer fluorescent probe in 0.2M phosphate buffer, Incubate the eggs at room temperature for 5 minutes to obtain the test solution;

[0036] (3) Fluorescence detection: 50ul of the test solution obtained in the detectio...

Embodiment 2

[0040] After dissolving and preparing the required reagent components according to Example 2 in Table 1, divide into bottles and freeze-dry to make a dry powder reagent; before use, add ultrapure water and use after reconstitution. Three parallels were set for each sample, and the detection steps were as follows:

[0041] (1) Blood sample lysis: Mix peripheral blood and red blood cell lysate at a volume ratio of 1:2.5, let stand for 5 minutes, then centrifuge at a medium speed for 6 minutes, and collect the supernatant;

[0042] (2) Mixed egg incubation: Take 50μl of the supernatant obtained in step 1) and 30ul of

[0043] The erythropoietin nucleic acid aptamer fluorescent probe reagent obtained by dissolving the erythropoietin nucleic acid aptamer fluorescent probe in 0.2M phosphate buffer was mixed, and the eggs were incubated at room temperature for 6 minutes to obtain the test solution;

[0044] (3) Fluorescence detection: 50ul of the test solution obtained in the detect...

Embodiment 3

[0047] After dissolving and preparing the required reagent components according to Example 3 in Table 1, divide into bottles and freeze-dry to make a dry powder reagent; before use, add ultrapure water and use after reconstitution. Three parallels were set for each sample, and the detection steps were as follows:

[0048] (1) Blood sample lysis: Mix peripheral blood and red blood cell lysate at a volume ratio of 1:3.5, let stand for 10 minutes, then centrifuge at a medium speed for 5 minutes, and collect the supernatant;

[0049] (2) Mixed egg incubation: Take 75μl of the supernatant obtained in step 1) and 45ul of

[0050] The erythropoietin nucleic acid aptamer fluorescent probe reagent obtained by dissolving the erythropoietin nucleic acid aptamer fluorescent probe in 0.2M phosphate buffer was mixed, and the eggs were incubated at room temperature for 7 minutes to obtain the test solution;

[0051] (3) Fluorescence detection: 50ul of the test solution obtained in the detec...

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