CN111060503A - G-6-PD detection kit free from anemia and detection method thereof - Google Patents
G-6-PD detection kit free from anemia and detection method thereof Download PDFInfo
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- CN111060503A CN111060503A CN202010001809.1A CN202010001809A CN111060503A CN 111060503 A CN111060503 A CN 111060503A CN 202010001809 A CN202010001809 A CN 202010001809A CN 111060503 A CN111060503 A CN 111060503A
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Abstract
The invention belongs to the technical field of biochemical detection, and particularly relates to a G-6-PD detection kit which is not affected by anemia and a detection method thereof, wherein the kit comprises a sample detection device, hemolytic agent dry powder, hemoglobin test paper, a sample adding colorimetric card, G-6-PD test paper and a detection colorimetric card; the sample detection device comprises a sample adding pipe, a first clamping groove and a second clamping groove. The kit and the detection method thereof provided by the invention can conveniently determine the anemia degree of a subject, quickly confirm the sample adding amount, are used for screening detection of erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD) deficiency, and can compensate the sample adding amount according to a hemoglobin test result.
Description
Technical Field
The invention belongs to the technical field of in-vitro diagnostic reagent detection, and particularly relates to a G-6-PD detection kit which is not affected by anemia and a detection method thereof.
Background
Anemia is a clinical syndrome in which peripheral red blood cell volume is lower than normal for various reasons, and refers to a condition in which the amount of hemoglobin counted by red blood cells and the hematocrit are lower than normal standards in a certain volume of circulating blood, and among them, hemoglobin (Hb) is the most important. China haematologists consider that in China sea level areas, Hb of adult males is less than 120g/L (12.0g/dl), Hb of adult females is less than 110g/L (11.0g/dl), and Hb of pregnant women is less than 120g/L, and anemia exists. Erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD) is derived from erythrocytes, catalyzes glucose-6-phosphate, and the generated NAD-PH is a coenzyme of glutathione reductase, and reduced Glutathione (GSH) is a necessary condition for keeping the stability of hemoglobin and the integrity of erythrocyte membranes. In the case of G-6-PD deficiency of erythrocytes, the function of the erythrocyte membrane is impaired due to loss of thiol protection, resulting in hemolysis. The existing G-6-PD detection method comprises G-6-PD activity detection, a methemoglobin cyanide detection method, an immune dot hybridization method and the like on a biochemical analyzer. At present, most primary hospitals in China adopt a methemoglobin reduction test as a screening test for erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD) deficiency, and the method has the advantages of complex operation, long time, large blood consumption, unstable result and high false positive rate. The accuracy of the tetrazolium blue quantitative method and the fluorescent spot method is high, but the tetrazolium blue quantitative method and the fluorescent spot method are difficult to popularize in most hospitals due to complex operation and high technical requirements.
In recent years, a G-6-PD test paper method is adopted to carry out a sieving test of erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD) deficiency, the sieving test is initially popularized, fingertip blood can be used for direct test, the operation is convenient, the result is more accurate, but the detection method still has defects. Because the result of the detection method shows the enzyme activity in the unit hemoglobin, the detection result of anemic patients has a certain deviation, and in order to compensate the deviation result, the sample adding amount of the test needs to be changed according to different anemia degrees of anemic patients. However, the existing test paper method G-6-PD test kit in the market cannot detect anemia, and other means, such as a blood cell analyzer, a hemoglobin meter, etc., are needed to assist in determining whether anemia and the degree of anemia, and some unconditional mechanisms may directly test without changing the sample loading amount, resulting in differences in the detection results of anemia patients. Therefore, a method for detecting G-6-PD with accurate results without being affected by anemia is needed.
Disclosure of Invention
The invention aims to solve the problem of inaccurate detection result of anemic patients in the prior art, and provides a G-6-PD detection kit and a detection method thereof, which can conveniently determine the anemia degree of a subject and quickly confirm the sample addition amount, are used for screening and detecting the deficiency of erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD), can compensate the sample addition amount according to the hemoglobin test result, and have the advantages of less blood consumption, flexible and convenient test and improved accuracy of the detection result.
The technical scheme of the invention is as follows:
a G-6-PD detection kit which is not affected by anemia comprises a sample detection device, hemolytic agent dry powder, hemoglobin test paper, a sample adding colorimetric card, G-6-PD test paper and a detection colorimetric card; the sample detection device comprises a sample adding pipe, a first clamping groove and a second clamping groove.
The sample adding pipe can be used for containing hemolytic agent dry powder and is used as a reaction container for dissolving the hemolytic agent dry powder and then fully and uniformly mixing the hemolytic agent dry powder with a sample; the sample adding tube can be a transparent plastic test tube with a buckle cover and is fixed on the sample detection device through a buckle; the reaction vessel may be made of other materials and fixed to the sample detection device.
Further, the preparation of the sample adding colorimetric card comprises the following steps: and (3) testing a sample assigned by the hemoglobin tester by using the hemoglobin test paper, determining the color development conditions of the testees with different anemia degrees according to the color development of the experimental result, converting corresponding sample adding amount according to a proportion, and marking the sample adding amount below the corresponding color of the color card to obtain the sample adding colorimetric card.
Further, the hemoglobin test paper is slidably connected with a first card slot, and the sample adding colorimetric card is fixedly arranged on the sample detection device at a position close to the first card slot; the G-6-PD test paper is connected with the second clamping groove in a sliding mode, and the detection colorimetric card is fixedly arranged at a position, close to the second clamping groove, on the sample detection device.
Further, the G-6-PD test paper is prepared by adopting a dry chemical method, and is specifically prepared by fixedly sticking a G-6-PD detection block to one end of a carrier, wherein the G-6-PD detection block is obtained by soaking a reaction membrane in a G-6-PD detection solution to saturation, drying and cutting.
Further, the G-6-PD detection solution comprises a Tris-HCl buffer solution, glucose-6-phosphate or glucose-6-phosphate, oxidized coenzyme II, nitro blue tetrazolium and phenazine methyl sulfate; the pH value of the buffer solution is 7-8; the buffer solution is composed of tris (hydroxymethyl) aminomethane or potassium hydrogen phthalate.
Further, the hemoglobin test paper is prepared by adopting a dry chemical method, specifically, a hemoglobin detection block is fixedly attached to one end of a carrier, and the hemoglobin detection block is obtained by soaking and saturating a reaction membrane in hemoglobin detection liquid, drying and cutting. The hemoglobin test paper is used for judging whether the test subject is anaemic, and determining the sample adding amount required by the anaemia test subject sample according to the color comparison with the sample adding colorimetric card.
Further, the hemoglobin detection solution comprises PBS buffer solution, sodium lauryl sulfate and surfactant.
A method for detecting G-6-PD not affected by anemia, comprising the steps of:
(1) dissolving hemolytic agent dry powder in distilled water to obtain sample treatment solution, adding appropriate amount of whole blood or peripheral blood sample of subject into sample adding tube, adding sample treatment solution into sample adding tube, and mixing completely;
(2) fixing a hemoglobin test paper on a first clamping groove, taking a proper amount of the sample solution uniformly mixed in the step (1), dropwise adding the sample solution on the hemoglobin test paper, judging whether the addition amount needs to be adjusted according to the color comparison between the color development of the test paper and the color of the sample adding colorimetric card, directly supplementing the corresponding sample amount in a channel if the addition amount needs to be adjusted, and fully and uniformly mixing;
(3) and (3) dropwise adding a proper amount of the uniformly mixed sample solution obtained in the step (2) onto G-6-PD test paper, incubating at 37 ℃ for 10-30min, taking out, washing with distilled water, fixing the G-6-PD test paper in a second card slot, and comparing with a detection color comparison card to obtain a detection result.
The invention has the beneficial effects that:
(1) the G-6-PD detection kit provided by the invention has less blood consumption in the detection process, and can meet the detection requirement by adopting fingertip blood or neonatal heel blood; when the kit is used for detection, the operation is flexible and convenient, sporadic samples can be detected, sample batch detection can also be performed, results can be obtained through visual inspection, instruments are not needed, manpower and material resources are saved, and the cost is low.
(2) The detection method provided by the invention can conveniently determine the anemia degree of a subject through the combined detection of the hemoglobin and the G-6-PD, quickly confirm the compensation sample adding amount, correct the measurement error caused by the anemia and improve the test accuracy.
Drawings
FIG. 1 is a schematic diagram of a G-6-PD detection kit provided by an embodiment of the present invention;
fig. 2 is a schematic view of a sample-adding colorimetric card according to an embodiment of the present invention;
in the above figures: 1. a sample adding pipe; 2. a first card slot; 3. and a second card slot.
Detailed Description
The technical solutions of the present invention will be described in detail and fully with reference to the following specific embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
For a further understanding of the present invention, reference will now be made in detail to the following examples.
Examples
Preparation of G-6-PD test paper: soaking the reaction membrane in a G-6-PD detection solution until the reaction membrane is saturated, drying the reaction membrane, cutting the reaction membrane into a shape to obtain a G-6-PD detection block, and fixedly adhering the G-6-PD detection block to one end of a carrier to obtain test paper;
preparing a G-6-PD detection solution: 0.20mol/L Tris-HCl buffer (pH7.4); 0.15% phenazine methosulfate (M-PMS); 0.75% Nitrotetrazolium Blue (NBT); 12.5mmol/L glucose-6-phosphate disodium salt (G6P-Na)2) (ii) a 12mmol/L coenzyme II (NADP); 0.6mol/L MgCl2And mixing the components in proportion to obtain the G-6-PD detection solution.
Preparing the hemoglobin test paper: soaking the reaction membrane in hemoglobin detection solution to saturation, drying, cutting and forming to obtain a hemoglobin detection block, and fixedly adhering the hemoglobin detection block to one end of a carrier to obtain the test paper;
preparing a hemoglobin detection solution: 0.1mol/L PBS buffer solution (pH6.5), 0.15% lauryl sodium sulfate, surfactant Triton X-100, etc. are mixed according to a certain proportion to obtain the hemoglobin detection solution.
Sample adding colorimetric card: assigning a sample to be measured by using a hemoglobin measuring instrument, selecting samples with measurement values of 140g/L, 120g/L, 100g/L, 80g/L, 60g/L and 40g/L, testing by using prepared hemoglobin test strips, reading the color development of 30s to prepare a colorimetric card, converting corresponding sample addition amount according to a proportion, and then labeling the sample addition amount below the corresponding color of the colorimetric card to prepare the sample addition colorimetric card, wherein the sample addition amount is shown in FIG. 2. According to the different sample adding amount, the color shades of the corresponding color blocks on the sample adding colorimetric card are different, and can be obviously distinguished.
The use method of the kit comprises the following steps:
(1) unsealing the kit, taking a bag of hemolytic agent dry powder, unsealing, pouring into a sample adding tube, adding 1mL of distilled water, fully dissolving to obtain sample treatment solution, adding 10 μ L of whole blood or peripheral blood of a subject into the sample adding tube, fully mixing, and standing for 1 min;
(2) fixing a hemoglobin test paper on a first clamping groove, taking 20 mu L of the sample solution which is uniformly mixed and stood in the step (1), dropwise adding the sample solution on the hemoglobin test paper, observing the color development condition of the hemoglobin test paper after 30s, searching a color block of the sample-adding colorimetric card closest to the color displayed by the test paper, judging whether the addition amount needs to be adjusted or not by comparing the color block with the color of the sample-adding colorimetric card, calculating the addition amount according to the graph 2 if the addition amount needs to be adjusted, directly supplementing the corresponding sample amount in the sample-adding tube, fully mixing the sample amount and the sample amount, and standing the sample solution for 1;
(3) and (3) taking 20 mu L of the sample solution uniformly mixed and stood in the step (2), dropwise adding the sample solution on G-6-PD test paper, removing redundant samples, incubating at 37 ℃ for 10-30min, taking out, washing with distilled water, fixing the test paper on a second clamping groove, and comparing the test paper with a detection color comparison card in the kit to obtain a detection result.
The above description is only for the preferred embodiment of the present invention and should not be taken as limiting the invention, and any modifications, equivalents, improvements and the like made within the scope of the present invention should be included in the patent protection scope of the present invention.
Claims (8)
1. A G-6-PD detection kit which is not affected by anemia is characterized by comprising a sample detection device, hemolytic agent dry powder, hemoglobin test paper, a sample adding colorimetric card, G-6-PD test paper and a detection colorimetric card; the sample detection device comprises a sample adding pipe, a first clamping groove and a second clamping groove.
2. The kit of claim 1, wherein the sample application color comparison card is prepared by: and (3) testing a sample assigned by the hemoglobin tester by using the hemoglobin test paper, determining the color development conditions of the testees with different anemia degrees according to the color development of the experimental result, converting corresponding sample adding amount according to a proportion, and marking the sample adding amount below the corresponding color of the color card to obtain the sample adding colorimetric card.
3. The kit of claim 1, wherein the hemoglobin test strip is slidably coupled to a first card slot and the G-6-PD test strip is slidably coupled to a second card slot.
4. The kit according to claim 1, wherein the G-6-PD test paper is prepared by fixedly adhering a G-6-PD detection block to a carrier, and the G-6-PD detection block is prepared by drying a reaction membrane after the reaction membrane is soaked and saturated in a G-6-PD detection solution.
5. The kit of claim 4, wherein the G-6-PD test solution comprises Tris-HCl buffer, glucose-6-phosphate or glucose-6-phosphate, oxidized coenzyme II, nitrotetrazolium blue, phenazine methosulfate; the pH value of the buffer solution is 7-8.
6. The kit according to claim 1, wherein the hemoglobin test strip is prepared by adhering a hemoglobin test block onto a carrier, and the hemoglobin test block is prepared by drying a reaction membrane after the reaction membrane is saturated in a hemoglobin test solution.
7. The kit of claim 6, wherein the hemoglobin detection solution comprises PBS buffer, sodium lauryl sulfate, and a surfactant.
8. A method for detecting G-6-PD not affected by anemia, comprising the steps of:
(1) dissolving hemolytic agent dry powder in distilled water to obtain sample treatment solution, adding appropriate amount of whole blood or peripheral blood sample of subject into sample adding tube, adding sample treatment solution into sample adding tube, and mixing completely;
(2) fixing a hemoglobin test paper on a first clamping groove, taking a proper amount of the sample solution uniformly mixed in the step (1), dropwise adding the sample solution on the hemoglobin test paper, judging whether the addition amount needs to be adjusted according to the color comparison between the color development of the test paper and the color of the sample adding colorimetric card, directly supplementing the corresponding sample amount in a channel if the addition amount needs to be adjusted, and fully and uniformly mixing;
(3) and (3) taking a proper amount of the uniformly mixed sample solution in the step (2), dropwise adding the sample solution on G-6-PD test paper, incubating at 37 ℃ for 10-30min, taking out, washing with distilled water, fixing the test paper in a second clamping groove, and comparing with a detection color comparison card to obtain a detection result.
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Cited By (2)
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| CN111638212A (en) * | 2020-06-15 | 2020-09-08 | 江南大学 | Method for detecting content of glucose-6-phosphate based on nano enzyme |
| CN115078341A (en) * | 2022-08-22 | 2022-09-20 | 上海执诚生物科技有限公司 | Reagent for detecting glucose-6-phosphate dehydrogenase and application thereof |
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