CN117965679A - G6PD enzyme activity detection kit and preparation method thereof - Google Patents
G6PD enzyme activity detection kit and preparation method thereof Download PDFInfo
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The application discloses a G6PD enzyme activity detection kit and a preparation method thereof, wherein the detection kit comprises a reaction tube and a detection card, the reaction tube contains a detection reagent, the detection reagent comprises D-glucose-6-disodium phosphate, nicotinamide adenine dinucleotide phosphate, phenazine methosulfate and tetrazolium blue, the detection card comprises a panel, a bonding pad, an adsorption pad and a bottom plate which are sequentially stacked, the panel is also provided with a sample adding hole, the sample adding hole is positioned above the bonding pad, the bonding pad is configured to intercept a color-developing substance after reaction and nucleic acid in a blood sample to be detected, and the adsorption pad is configured to adsorb liquid and hemoglobin in the blood sample to be detected. According to the application, the interference of hemoglobin interference on result interpretation can be eliminated, and the result interpretation is carried out independently of instruments and equipment; in addition, if further G6PD nucleic acid detection is required, the detected conjugate pad can be used for nucleic acid extraction, thereby reducing secondary sampling or preservation of blood samples.
Description
Technical Field
The application relates to the technical field of biochemical detection, in particular to a G6PD enzyme activity detection kit and a preparation method thereof.
Background
Glucose-6-phosphate dehydrogenase (G6 PD) deficiency is a genetic disease, and is caused by G6PD deficiency of erythrocyte membranes, so that the production of reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH), which is a coenzyme of glutathione reductase in the pentose phosphate pathway of erythrocytes, is reduced, so that the reduced glutathione production which maintains the stability of erythrocyte membranes is reduced and cannot resist oxidative damage, acute hemolytic anemia and hyperbilirubinemia can occur under the conditions of infection, oxidative stress, food or drug induction and the like of patients, and severe patients can cause nuclear jaundice and even endanger life.
Currently, a fluorescence quantitative analysis method is generally adopted for the G6PD enzyme activity detection, which is also a recommended method for the G6PD screening of the newborn at present, but the result reading of the method depends on instruments and equipment, and for the area with poor infrastructure, the problem of hardware and technology is faced when the G6PD screening is to be carried out, for example, the method disclosed in CN201110394189.3 depends on fluorescence excitation, and the fluorescence is required to be read by matched equipment. Although the BinaxNOW G6PD test kit of yaban can be read directly, the color of blood and the color of the reaction of the substrate are mixed together, which is unfavorable for the interpretation of the result. In the product CARESTART ™ of Access Bio company and the patent US9575056, the enzyme of G6PD reacts with the substrate in the chromatographic process, blood and reaction products are gradually separated by a chromatographic method, and the reaction products are diluted into long strips and are unevenly distributed, so that the interpretation is not facilitated; in addition, the blood sample and the chromatographic liquid need to be respectively added into different holes, and compared with the reaction in the liquid, the reaction in the chromatographic process has no liquid reaction and has high reliability. In addition, in clinical practice, genotyping is often required for samples suspected of defects by nucleic acid detection, however, the reaction products after detection of the prior art are treated as waste and the reagents cannot be used for secondary detection.
In view of the foregoing, it is desirable to provide a detection product that is independent of instrumentation, facilitates reading of results, and is capable of G6PD enzymatic activity in combination with G6PD nucleic acid detection.
Disclosure of Invention
The application aims to provide a G6PD enzyme activity detection kit and a preparation method thereof.
The application adopts the following technical scheme:
One aspect of the application discloses a detection kit for G6PD enzyme activity, comprising: the reaction tube contains detection reagent in, detection reagent contains D-glucose-6-disodium phosphate, nicotinamide adenine dinucleotide phosphate, phenazine methyl sulfate and tetrazolium blue, detection card is including panel, binding pad, adsorption pad and the bottom plate of laminating the setting in proper order, the panel still is equipped with the application of sample hole just the application of sample hole is located binding pad top, wherein, the binding pad is configured to the nucleic acid in chromogenic substance and the sample that awaits measuring after the interception reaction, the adsorption pad is configured to adsorb liquid with the hemoglobin in the sample that awaits measuring.
In the detection kit of the present application, when a blood sample to be measured is added to a reaction tube and brought into contact with a detection reagent, if the activity of G6PD in the blood sample to be measured is normal, D-glucose-6-phosphate disodium salt and nicotinamide adenine dinucleotide phosphate (NAPD +) generate NADPH by the action of the enzyme, NADPH is used as an electron donor, phenazine methosulfate can be used as an electron shuttle carrier, and electrons transferred from NADPH are received and transferred to tetrazolium blue, so that tetrazolium blue generates a bluish-violet crystal substance (i.e., a chromogenic substance). And (3) dripping the reacted sample into a sample adding hole of the detection card, wherein soluble hemoglobin can penetrate through the binding pad and be adsorbed by the adsorption pad, and a chromogenic substance cannot pass through the binding pad and remain on the binding pad, so that visual interference of the hemoglobin on a chromogenic result can be reduced or eliminated, and the result can be directly read by naked eyes without ultraviolet irradiation or depending on instrument equipment. In addition, nucleic acid in the blood sample to be tested is intercepted by the binding pad, thereby enabling sampling from the binding pad for nucleic acid detection, avoiding secondary sampling or preservation of the blood sample.
In one implementation of the present application, the bonding pad is a nylon membrane having a pore size of 0.4 μm to 1.2 μm. The nylon membrane is an ideal nucleic acid solid phase support, and the nucleic acid is a long-chain polymer and is easy to combine with the membrane; the pore size nylon membrane is capable of intercepting the chromogenic substance and passing hemoglobin.
In one implementation mode of the application, the water absorption parameter of the adsorption pad is more than or equal to 3000g/m 2. In this case, the liquid can be adsorbed well, and the color development of the color-developing substance is facilitated.
In one implementation mode of the application, the bottom plate is provided with a clamping groove, and the clamping groove is used for fixing the adsorption pad. In this case, the stability of the test card can be improved.
In one embodiment of the present application, the detection reagent is prepared by adding a solution containing the detection reagent to a reagent pad and drying the solution, or is prepared by freeze-drying a solution containing the detection reagent. In this case, the stability of the detection reagent can be improved, and the stability of the detection can be maintained for a long time even when the detection kit is stored at normal temperature, that is, the detection kit can be stored at normal temperature, and the transportation and storage costs can be reduced.
In one embodiment of the present application, the detection reagent is prepared by applying a solution containing the detection reagent to a glass fiber paper and drying the same, or by adding polyethylene glycol to a solution containing the detection reagent to a final concentration of 0.01g/mL and lyophilizing the same.
In one implementation mode of the application, the detection reagent comprises a first dry reagent obtained by drying or freeze-drying a first solution and a second dry reagent obtained by drying or freeze-drying a second solution, wherein the first solution contains phenazine methosulfate, magnesium chloride, D-glucose-6-phosphate disodium salt, nicotinamide adenine dinucleotide phosphate and trehalose, and the second solution contains tetrazolium blue and saponin. It should be noted that, the detection reagent is divided into the first dry reagent and the second dry reagent, so that the stability of the reagent can be further improved, specifically, the two reagents are mixed together, and react under the condition of being wet or having an electron donor, so that the validity period of the reagent is shortened and invalid, the reagent is split into the two dry reagents, the reaction between the reagents can be avoided, and the stability of the reagent at room temperature is improved. Wherein, magnesium ions of magnesium chloride can activate and promote G6PD enzyme to play a role, and promote reaction; the trehalose can improve the stability of the dried reagent, thereby improving the accuracy of enzyme activity detection; the saponin can be favorable for cracking red blood cells, and increases the water solubility and dispersibility of the dry reagent, thereby promoting the reaction and improving the detection accuracy.
In one implementation of the application, the first solution contains 0.1 to 1mg/mL of phenazine methosulfate, 1.5 to 20mM of magnesium chloride, 1 to 15mg/mL of D-glucose-6-phosphate disodium salt, 1 to 20mg/mL of nicotinamide adenine dinucleotide phosphate and 1 to 6% of trehalose by mass fraction, and the second solution contains 0.4 to 10mg/mL of tetrazolium blue and 0.1 to 5% of saponin by mass fraction. In this case, it is advantageous to provide a suitable amount of the detection reagent, and thus, the reaction is facilitated and the accuracy of the test is improved. In the present application, the mass fraction means a mass-to-volume ratio (w/v), for example, a mass fraction of 1% means a concentration of 1g/100mL.
In one implementation mode of the application, the first solution contains 0.5mg/mL phenazine methosulfate, 5mM magnesium chloride, 5mg/mL D-glucose-6-phosphate disodium salt, 7mg/mL nicotinamide adenine dinucleotide phosphate and 4% trehalose by mass, and the second solution contains 4mg/mL tetrazolium blue and 1% saponin by mass.
The application further discloses a preparation method of a G6PD enzyme activity detection kit, which comprises the steps of placing a detection reagent in a reaction tube, and sequentially laminating a panel, a bonding pad, an adsorption pad and a bottom plate to obtain a detection card, wherein the detection reagent comprises D-glucose-6-disodium phosphate, nicotinamide adenine dinucleotide phosphate, phenazine methosulfate and tetrazolium, the panel is provided with a sample adding hole, the sample adding hole is positioned above the bonding pad, the bonding pad is configured to intercept a chromogenic substance after reaction and nucleic acid in a sample to be detected, and the adsorption pad is configured to adsorb liquid and hemoglobin in the sample to be detected.
In the detection kit prepared by the preparation method, when a blood sample to be detected is added into a reaction tube to be contacted with a detection reagent, if the activity of G6PD in the blood sample to be detected is normal, D-glucose-6-disodium phosphate and nicotinamide adenine dinucleotide phosphate (NAPD +) generate NADPH through the action of enzyme, the NADPH is used as an electron donor, phenazine methosulfate can be used as an electron shuttle carrier to receive electrons transferred from the NADPH, and the electrons are further transferred to tetrazolium blue, so that the tetrazolium blue generates a blue-violet crystalline substance (namely a chromogenic substance). And (3) dripping the reacted sample into a sample adding hole of the detection card, wherein soluble hemoglobin can penetrate through the binding pad and be adsorbed by the adsorption pad, and a chromogenic substance cannot pass through the binding pad and remain on the binding pad, so that visual interference of the hemoglobin on a chromogenic result can be reduced or eliminated, and the result can be directly read by naked eyes without ultraviolet irradiation or depending on instrument equipment. In addition, nucleic acid in the blood sample to be tested is intercepted by the binding pad, thereby enabling sampling from the binding pad for nucleic acid detection, avoiding secondary sampling or preservation of the blood sample.
The application has the beneficial effects that:
The application can provide a detection kit for G6PD enzyme activity and a preparation method thereof, and the detection kit can be beneficial to eliminating interference of hemoglobin interference on result interpretation, does not depend on instrument and equipment, improves the usability of products, and reduces the threshold of G6PD enzyme activity test; in addition, the detected detection card can be stored at normal temperature, and if G6PD nucleic acid detection is required to be further carried out, the detected binding pad is used for nucleic acid extraction, so that secondary sampling or preservation of a blood sample can be reduced.
Drawings
Fig. 1 is a schematic structural view of a test card according to the present application.
FIG. 2 is a schematic structural view of a reaction tube according to the present application.
FIG. 3 illustrates a schematic of the results of a partial test card development.
Detailed Description
The application will be described in further detail below with reference to the drawings by means of specific embodiments. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted in various situations, or replaced by other materials, methods. In some instances, related operations of the present application have not been shown or described in the specification in order to avoid obscuring the core portions of the present application, and may be unnecessary to describe in detail those related operations by those skilled in the art, given the description herein and the general knowledge of one skilled in the art.
Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.
For the detection of G6PD enzyme activity, the current domestic fluorescent quantitative analysis method is adopted by most neonatal screening laboratories and is also the recommended method for the current neonatal G6PD screening. However, this method relies on instrumentation and is technically problematic for areas of poor infrastructure where G6PD screening is desired. CN201110394189.3 discloses that the method relies on fluorescence excitation, requiring a companion device to read the fluorescence. Although the BinaxNOW G6PD test kit of yaban can be read directly, the color of blood and the color of the reaction of the substrate are mixed together, which is unfavorable for the interpretation of the result. In the product CARESTART ™ of Access Bio and the patent US9575056, the enzyme of G6PD reacts with the substrate in the chromatographic process, blood and reaction products are gradually separated by the chromatographic method, and the reaction products are diluted into long strips and are unevenly distributed, so that the interpretation is not facilitated. Also, the blood sample and the chromatographic liquid need to be added separately to different wells. The reaction in the chromatographic process is less reliable than the reaction in the liquid. In addition, in clinical practice, genotyping is often required for samples suspected of defects by nucleic acid detection, and the detection reaction products of the prior art are treated as waste and the reagents are not available for secondary detection.
In view of the above, the application creatively adopts the G6PD enzyme activity detection kit based on the percolation technology, can reduce or even eliminate the interference of hemoglobin on color development, can read the result without depending on instruments and equipment, can be used together with a G6PD nucleic acid detection method, improves the usability of the product, and reduces the threshold of G6PD test. The detection kit has the following advantages:
1) Based on the percolation method, a large amount of water-soluble hemoglobin is absorbed by the adsorption pad, which is beneficial to eliminating the visual interference of the hemoglobin on the color development result.
2) The result of the reaction is limited to a specified regular shape through the cooperation of the cartridge and the filter membrane, and is easy to observe and interpret.
3) The result can be read directly by naked eyes, ultraviolet irradiation is not needed, and the method is independent of instruments and equipment.
4) The same sample can be used for enzyme activity detection and nucleic acid detection, and the nucleic acid combined on the combining pad in reaction can be further used for detecting the nucleic acid, so that the sample is saved, and secondary sampling and blood sample preservation are avoided.
5) Can be preserved at room temperature and detected at room temperature; the reagent does not need refrigeration, so that the usability of the reagent is improved, and the transportation cost is reduced.
6) The kit has higher sensitivity, specificity and accuracy, and in one embodiment, the sensitivity of the kit is 98.68%, the specificity is 95.16%, and the accuracy is 97.66%.
The following describes the detection kit and the preparation method thereof in detail with reference to the drawings and the specific embodiments.
Fig. 1 is a schematic structural view of a detection card according to the present application, and fig. 2 is a schematic structural view of a reaction tube according to the present application.
The present application relates to a kit for detecting G6PD enzymatic activity (hereinafter, sometimes simply referred to as "detection kit" or "kit").
In a specific embodiment, the detection object of the detection kit may be a whole blood sample. For example, it may be a fresh whole blood sample, EDTA-or heparin-anticoagulated whole blood sample.
In a specific embodiment, the test kit may include a reaction tube 50 and a test card 10.
As shown in fig. 1 and 2, in one embodiment, the reaction tube 50 may include a tube body 501 and a tube cover 502. The tube 501 has a space for accommodating a detection reagent, and the tube cover 502 is used to form a closed space for the tube 501.
In one embodiment, the reaction tube 50 may contain a detection reagent. The detection reagent may comprise D-glucose-6-phosphate disodium salt, nicotinamide adenine dinucleotide phosphate, phenazine methosulfate, and tetrazolium blue. In this case, by adding the blood sample to be measured to the reaction tube to be brought into contact with the detection reagent, if the activity of the G6PD enzyme in the blood sample to be measured is normal, D-glucose-6-phosphate disodium salt and nicotinamide adenine dinucleotide phosphate (NAPD +) generate NADPH by the action of the enzyme, NADPH is used as an electron donor, phenazine methosulfate can be used as an electron shuttle carrier, electrons transferred from NADPH are received, and the electrons are further transferred to tetrazolium blue, so that the tetrazolium blue generates a blue-violet crystalline substance (i.e., a chromogenic substance).
In one embodiment, the detection reagent may be prepared by drying a solution containing the detection reagent.
In one embodiment, the reagent pad may be prepared by adding a solution containing a detection reagent to the reagent pad and drying the solution, or may be prepared by freeze-drying a solution containing a detection reagent.
In one embodiment, the solution containing the detection reagent may be obtained by dissolving the detection reagent in a buffer. The buffer may be Tris-HCl buffer.
In one embodiment, the reagent pad may be fiberglass paper. The solution containing the detection reagent may be uniformly applied to the glass fiber paper and then dried in a drying room (humidity < 25%) at 25 ℃ to 30 ℃ for 8 hours or more.
In one embodiment, after drying, the glass fiber paper may be cut into small pieces of glass fiber paper having a length and a width of 3mm to 8mm and placed in a reaction tube for storage.
In one embodiment, polyethylene glycol may be added to the solution containing the detection reagent to a final concentration of 1% (w/v) and lyophilized.
In one embodiment, the polyethylene glycol-added solution may be lyophilized into a liquid with a diameter of 3+ -1 mm using a granulator, pre-frozen into a microsphere solid state by liquid nitrogen, and then lyophilized at a low temperature using a lyophilizer. Wherein the lyophilization procedure can comprise: pre-freezing: -50 ℃ for 150min; primary drying: -40 ℃ for 480min; -30 ℃ for 240min; and (3) analysis and drying: 25 ℃ for 240min.
In one embodiment, the detection reagent may further comprise magnesium chloride.
In one embodiment, the detection reagent may further comprise trehalose.
In one embodiment, the detection reagent may further comprise saponin.
In one embodiment, the detection reagent may be prepared from a solution containing D-glucose-6-phosphate disodium salt, nicotinamide adenine dinucleotide phosphate, phenazine methosulfate, tetrazolium blue, magnesium chloride, trehalose, and saponin by drying or lyophilizing.
In one embodiment, D-glucose-6-phosphate disodium salt, nicotinamide adenine dinucleotide phosphate, phenazine methosulfate, tetrazolium blue, magnesium chloride, trehalose, and saponin may be dissolved using a buffer to obtain a solution, and then dried or lyophilized. The buffer may be Tris-HCl buffer.
In a particular embodiment, the detection reagent may include a first dry reagent 503 and a second dry reagent 504. The first dry reagent 503 may be prepared from a first solution by drying or lyophilizing, and the second dry reagent 504 may be prepared from a second solution by drying or lyophilizing.
In one embodiment, the first solution may be obtained by dissolving phenazine methosulfate, magnesium chloride, D-glucose-6-phosphate disodium salt, nicotinamide adenine dinucleotide phosphate, and trehalose in Tris-HCl buffer. The second solution can be obtained by dissolving tetrazolium blue and saponin in Tris-HCL buffer.
In a specific embodiment, the first solution contains 0.1 to 1mg/mL of phenazine methosulfate. Preferably, the first solution contains 0.5 mg/mL phenazine methosulfate.
In a specific embodiment, the first solution contains 1.5 to 20mM magnesium chloride. Preferably, the first solution contains 5mM magnesium chloride.
In a specific embodiment, the first solution contains 1 to 15mg/mL of disodium D-glucose-6-phosphate. Preferably, the first solution contains 5mg/mL of disodium D-glucose-6-phosphate.
In a specific embodiment, the first solution contains 1 to 20mg/mL nicotinamide adenine dinucleotide phosphate. Preferably, the first solution contains 7mg/mL nicotinamide adenine dinucleotide phosphate.
In one embodiment, the first solution contains 1% to 6% trehalose by mass. Preferably, the first solution contains trehalose in an amount of 4% by mass.
In one embodiment, the second solution contains 0.4 to 10mg/mL tetrazolium blue. Preferably, the second solution contains 4mg/mL tetrazolium blue.
In one embodiment, the second solution contains 0.1% to 5% by mass of saponin. Preferably, the second solution contains 1% by mass of saponin.
In one embodiment, the test card 10 may include a panel 100, a bonding pad 200, an adsorption pad 300, and a bottom plate 400.
In one embodiment, the panel 100, the bonding pad 200, the adsorption pad 300, and the base plate 400 may be sequentially stacked. In other words, the panel 100, the bonding pad 200, the adsorption pad 300, and the bottom plate 400 may be sequentially stacked from top to bottom.
In one embodiment, the faceplate 100 is provided with a loading well 101. The loading well 101 is for receiving a sample in a liquid state.
In one embodiment, the adsorption pad 300 is configured to intercept the reacted chromogenic material. In this case, the color-developing substance remains on the adsorption pad 300, and the color-developing result can be observed through the sample addition well.
In one embodiment, the absorbent pad 300 is configured to detect nucleic acids in a blood sample. In this case, it is possible to sample from the conjugate pad 200 for nucleic acid detection, avoiding secondary use or preservation of the blood sample.
In one embodiment, the absorbent pad 300 may be a nylon membrane.
In one embodiment, the nylon membrane may have a pore size of 0.4 μm to 1.2 μm.
In one embodiment, the test card 10 after detection can be stored at room temperature (25 ℃) for 2 to 3 weeks, and the conjugate pad 200 can be removed for nucleic acid extraction when further nucleic acid detection is desired.
In one embodiment, the sample is applied dropwise to the conjugate pad 200 through the application well 101.
In one embodiment, the absorbent pad 300 is configured to absorb a liquid.
In one embodiment, the adsorption pad 300 is configured to adsorb hemoglobin in a sample.
In one embodiment, the absorbent pad 300 may be absorbent paper.
In one embodiment, the absorbent pad 300 has a water absorption parameter of 3000g/m 2.
In one embodiment, the adsorption pad 300 completely covers the bonding pad 200 along the bottom-up projection.
In one embodiment, the base plate 400 may be provided with a card slot 401. The card slot 401 may accommodate the adsorption pad 300 to improve the firmness of the adsorption pad 300.
In one embodiment, the well 101 is in intimate contact with the conjugate pad 200. In this case, the result of the reaction can be restricted to a specified regular shape by the cooperation of the loading well 101 and the conjugate pad 200, so that the result can be easily observed and interpreted.
In one embodiment, the panel 100 and the bottom plate 400 may be detachably assembled and combined. In this case, the panel 100 can be easily detached to take out the bonding pad 200, thereby facilitating nucleic acid detection.
In one embodiment, the loading well 101 may be closely contacted with the bonding pad 200 by assembling the panel 100 and the bottom plate 400. Thus, the firmness of the bonding pad 200 and the adsorption pad 300 can be improved, and the result of the reaction can be limited to a specified regular shape, thereby facilitating the observation and interpretation of the result.
In a specific embodiment, the test kit may further comprise a diluent. The diluent is used for being added into the reaction tube 50, so that the reaction reagent (dry reagent) in the reaction tube 50 forms a solution, thereby facilitating the reaction.
In one embodiment, the diluent may be pure water.
In a specific embodiment, the test kit may further comprise at least one of a sealed bag, a desiccant, a disposable sterile alcohol swab, a lancet, a quantitative micropipette, and instructions for use. The sealed bag may be an aluminum foil bag and is used to contain a desiccant and reaction tube 50.
In one embodiment, the test kit is a qualitative test.
The kit has higher sensitivity, specificity and accuracy, and in one embodiment, the sensitivity of the kit is 98.68%, the specificity is 95.16%, and the accuracy is 97.66%.
The application also relates to a preparation method of the G6PD enzyme activity detection kit (hereinafter, sometimes simply referred to as a preparation method). It should be noted that, the preparation method of the present application is the preparation method of the detection kit described above, and the content related to the detection kit may refer to the foregoing content, which is not described herein.
In a specific embodiment, the method of making may include making the test card 10 and making the reaction tube 50.
In one embodiment, preparing the test card 10 may include sequentially stacking the panel 100 provided with the loading hole 101, the bonding pad 200, the adsorption pad 300, and the bottom plate 400 provided with the card slot 401. And the bonding pad 200 and the adsorption pad 300 are firmly fixed to the test card 10 by the assembly and combination of the panel 100 and the bottom plate 400.
In one embodiment, preparing the reaction tube 50 may include placing the reactant in the tube body 501 and then covering the tube cover 502 to seal the reactant in the reaction tube 50.
In one embodiment, the preparation method of the reaction reagent may include preparing phenazine methosulfate, magnesium chloride, D-glucose-6-phosphate disodium salt, nicotinamide adenine dinucleotide phosphate and trehalose into a first solution, and drying or lyophilizing to obtain a first dry reagent 503; tetrazolium blue and saponin are formulated into a second solution, which is then dried or lyophilized to produce a second dry reagent 504. The contents of each component in the first solution and the second solution may refer to the description of the detection kit part, and the step of drying or freeze-drying may refer to the description of the detection kit part, which is not repeated herein.
The present application also relates to a method for detecting the activity of G6PD (hereinafter, sometimes simply referred to as "detection method").
In one embodiment, the detection method may include adding a blood sample to be detected to the reaction tube 50 to mix the blood sample to be detected with a detection reagent to obtain a sample to be detected, and then adding the sample to be detected to the sample adding hole 101 of the detection card 10 to observe the color development result, and interpreting the result.
In one embodiment, the diluent may be added to the detection reagent in the reaction tube 50 to form a solution containing the detection reagent, and then the blood sample to be tested is added to the reaction tube 50 to mix the blood sample to be tested with the detection reagent, so as to facilitate the reaction.
In one embodiment, the diluent may be added in an amount of 200 to 400uL.
In one embodiment, a blood sample to be tested may be collected by a lancing straw and immediately used for testing; or can be collected and then placed in an EDTA tube or heparin anticoagulation tube for later detection.
In one embodiment, the blood sample to be tested may be used in an amount of 10 to 20uL.
In one embodiment, the blood sample to be tested is added to the reaction tube 50 and then mixed and then waits for 8 to 12 minutes to obtain the sample to be tested. The sample to be measured is then added to the well 101 for observation and color development.
In one embodiment, the sample to be measured is added to the well 101 for about 1 minute to observe and develop color and interpret the detection result.
In one embodiment, the detection method is a qualitative detection. If the test card 10 is shown to be pink, yellow or white, the result is insufficient G6PD enzyme activity; if the test card 10 is blue, blue-violet or color-giving, the G6PD enzyme activity is normal.
The application is further illustrated by the following examples. The following examples are merely illustrative of the present application and should not be construed as limiting the application. In this example, unless otherwise specified, all reagents and equipment used were commercially available and the experimental procedures were carried out according to the product specifications and conventional experimental specifications.
Example 1: preparation of detection kit
① Preparation of test card
According to FIG. 1, a piece of absorbent paper (Shanghai Jiening Co., ltd., size: 20 cm. Times.30 cm) was cut according to the size of the card slot on the bottom plate of the test card, and 20 mm. Times.14 mm absorbent paper was cut, and the absorbent paper was 3 layers thick and placed in the card slot as an absorbent pad; cutting the bonding pad (1.2 μm nylon membrane of PALL company) to a size larger than that of the sample-adding hole, but smaller than or equal to that of the adsorption pad, placing into the clamping groove, cutting nylon membrane to a size of 14×14mm, and placing the cut nylon membrane on absorbent paper; finally, the panel of the detection card is covered.
② Preparation of the reaction tube
Phenazine methosulfate, magnesium chloride, D-glucose-6-phosphate disodium salt, nicotinamide adenine dinucleotide phosphate, tetrazolium blue, trehalose, saponin, and Tris-HCl buffer were prepared. Tris-HCl buffer solution is a solution of Tris (hydroxymethyl) aminomethane and hydrochloric acid, and the concentration is 20mM, and the pH is 8.0.
The first and second solutions were then prepared according to the following tables 1 and 2 using Tris-HCl buffer, respectively.
TABLE 1 first solution Components
| Reagent name | Concentration of |
| Phenazine methosulfate | 0.5 mg/mL |
| Magnesium chloride | 5 mM |
| D-glucose-6-phosphate disodium salt | 5 mg/mL |
| Nicotinamide adenine dinucleotide phosphate | 7 mg/mL |
| Trehalose | 4%（w/v） |
TABLE 2 second solution Components
| Reagent name | Concentration of |
| Tetrazolium blue | 4 mg/mL |
| Saponin | 1%（w/v） |
Cutting glass fiber paper (Shanghai Jiening organism) into strips of 7mm×300mm, mixing the first solution and the second solution, uniformly coating on two glass fiber strips according to 1 mL/strip, and drying the glass fiber strips in a drying room (humidity < 25%) at 25-30deg.C for more than 8 hr. After the drying was completed, the dried glass fiber strands were cut into 7mm×4mm small pieces of glass fiber, thereby preparing a first dry reagent and a second dry reagent.
The first dry reagent and the second dry reagent are filled into a reaction tube and stored with a cover.
Example 2: use of detection kit
According to the test kit prepared in example 1, the reaction tube and the diluent (pure water) were taken out, the tube cap of the reaction tube was opened, 300 uL of water was added to the treatment tube, and shaking was performed so that the reaction reagent on the glass fiber paper was dissolved in the liquid.
Taking a 10uL blood sample, adding the blood sample into the treatment tube, uniformly mixing, starting timing, and waiting for 10 minutes to obtain a sample to be detected.
And taking out the detection card, adding a sample to be detected to the sample-adding Kong Di, and observing the color development after 1 minute.
Example 3: detection performance of detection kit
The detection was performed using 214 heparinized or EDTA-anticoagulated whole blood samples using a commercial G6PD enzyme activity quantitative detection kit (guangdong family biology), and the detection showed 152G 6PD normal samples and 62G 6PD defective samples. The 214 samples were simultaneously tested using the test kit of example 1 and the test method of example 2, and FIG. 3 illustrates a schematic representation of the results of the development of a portion of the test card. And the 214 samples were tested using the qualitative detection kit BinaxNOW ® G6PD test (Abbott corporation) on the market. The detection performance tables of the detection kit and the control kit (BinaxNOW ® G6PD test, abbott) of the present invention were obtained, respectively.
TABLE 3 detection Performance of the detection kit of the examples of the present invention
| / | Normal (example) | Defects (examples) |
| Normal (example) | 150 | 3 |
| Defects (examples) | 2 | 59 |
According to table 3, the sensitivity of the detection kit of example 1 of the present invention: 98.68% (95.33% to 99.84%,95% CI); specificity: 95.16% (86.50% to 98.99%,95% CI); accuracy: 97.66% (94.63% to 99.24%,95% CI).
Table 4 detection performance of control kit
| / | Normal (example) | Defects (examples) |
| Normal (example) | 147 | 9 |
| Defects (examples) | 6 | 52 |
According to table 4, sensitivity of the control kit: 96.08% (91.66% to 98.55%,95% CI); specificity: 85.25% (73.83% to 93.02%, 95% CI); accuracy: 92.99% (88.70% to 96.02%,95% CI).
Therefore, the quantitative kit is used as a judgment standard of the normal and the defect, and compared with other qualitative kits, the detection kit of the embodiment 1 of the invention has obvious performance improvement in the aspects of sensitivity, specificity and accuracy, and reduces false positive and false negative.
Example 4: extraction of nucleic acid samples
Taking the detected detection card, removing the bonding pad, cutting out small discs at round spots by using a 3mm diameter puncher, taking 6 small discs, placing the small discs into a 1.5mL centrifuge tube, extracting a sample by using a nucleic acid extraction kit (Yue Shen mechanical equipment 20230509), adding 300 mu L of treatment liquid in the kit into the centrifuge tube, adding 35 mu L of proteinase K, carrying out vortex oscillation for 30 seconds, placing the mixture into a constant-temperature oscillation metal bath at 65 ℃ and carrying out pyrolysis for 30 minutes at 900 rpm. Subsequently, 300. Mu.L of the sample was removed and the nucleic acid was extracted manually using the magnetic bead method:
a. In a 1.5mL centrifuge tube, 20 [ mu ] L of magnetic bead liquid, 600 [ mu ] L of lysis liquid and 200 [ mu ] L of the sample treated by the proteinase K are uniformly mixed by vortex for 10 seconds. And standing for 5-10 minutes at room temperature, and reversing and uniformly mixing for a plurality of times.
B. transferring to a magnetic rack, standing for 5 minutes to adsorb magnetic beads, and carefully sucking all the solutions.
C. 500 μl of washing liquid was added and vortexed for 10 seconds. Transferring to a magnetic rack, standing for 1 min, and adsorbing magnetic beads. The solution was pipetted off.
D. 500 μl of washing liquid was added and vortexed for 10 seconds. Transferring to a magnetic rack, standing for 1 min, and adsorbing magnetic beads. The solution was pipetted off.
E. and (5) centrifuging briefly, sucking all the solution, and air-drying for 10 minutes.
F. Adding 30-100 mu L of eluent AVE, and vortex scattering the magnetic beads. Standing for 5-10 minutes, and vortexing for several times during which the nucleic acid is dissolved.
G. Transferred to a magnetic rack and left to stand for 3 minutes. Transfer the DNA/RNA solution to a new 1.5mL centrifuge tube to obtain the final nucleic acid extraction product.
H. Quantification of the extracted nucleic acid using Qubit Fluorometer (Thermo FISHER SCIENTIFIC) showed an extracted nucleic acid concentration of 4.2ng/uL and a260/a280 of 1.7, i.e., successful extraction of the nucleic acid.
The foregoing is a further detailed description of the application in connection with specific embodiments, and it is not intended that the application be limited to such description. It will be apparent to those skilled in the art that several simple deductions or substitutions can be made without departing from the spirit of the application.
Claims (10)
1. A detection kit for detecting G6PD enzymatic activity in a blood sample to be tested, comprising: the reaction tube contains detection reagent in, detection reagent contains D-glucose-6-disodium phosphate, nicotinamide adenine dinucleotide phosphate, phenazine methyl sulfate and tetrazolium blue, detection card is including panel, binding pad, adsorption pad and the bottom plate of range upon range of setting in proper order, the panel still is equipped with the application of sample hole just the application of sample hole is located binding pad top, wherein, the binding pad is configured to intercept the chromogenic substance after the reaction with nucleic acid in the blood sample that awaits measuring, the adsorption pad is configured to adsorb liquid with the hemoglobin in the blood sample that awaits measuring.
2. The test kit according to claim 1, wherein the conjugate pad is a nylon membrane having a pore size of 0.4 μm to 1.2 μm.
3. The detection kit according to claim 1 or 2, wherein the absorption coefficient of the absorption pad is not less than 3000g/m 2.
4. The test kit of claim 1, wherein the bottom plate is provided with a clamping groove for fixing the adsorption pad.
5. The detection kit according to claim 1, wherein the detection reagent is prepared by adding a solution containing the detection reagent to a reagent pad and drying it, or is prepared by lyophilizing a solution containing the detection reagent.
6. The test kit according to claim 5, wherein the test reagent is prepared by applying a solution containing the test reagent to glass fiber paper and drying the same, or by adding polyethylene glycol to a solution containing the test reagent to a final concentration of 0.01g/mL and lyophilizing the same.
7. The detection kit according to claim 5 or 6, wherein the detection reagent comprises a first dry reagent obtained by drying or lyophilizing a first solution containing phenazine methosulfate, magnesium chloride, disodium D-glucose-6-phosphate, nicotinamide adenine dinucleotide phosphate, and trehalose, and a second dry reagent obtained by drying or lyophilizing a second solution containing tetrazolium blue and saponin.
8. The test kit according to claim 7, wherein the first solution contains 0.1 to 1mg/mL of phenazine methosulfate, 1.5 to 20mM of magnesium chloride, 1 to 15mg/mL of D-glucose-6-phosphate disodium salt, 1 to 20mg/mL of nicotinamide adenine dinucleotide phosphate, and 1% to 6% by mass of trehalose, and the second solution contains 0.4 to 10mg/mL of tetrazolium blue and 0.1% to 5% by mass of saponin.
9. The test kit of claim 7, wherein the first solution comprises 0.5mg/mL phenazine methosulfate, 5mM magnesium chloride, 5mg/mL disodium D-glucose-6-phosphate, 7mg/mL nicotinamide adenine dinucleotide phosphate, and 4% trehalose, and the second solution comprises 4mg/mL tetrazolium blue and 1% saponin.
10. The preparation method of the G6PD enzyme activity detection kit is characterized by comprising the steps of placing a detection reagent in a reaction tube, enabling a panel, a bonding pad, an adsorption pad and a bottom plate to be sequentially stacked to obtain a detection card, wherein the detection reagent comprises D-glucose-6-disodium phosphate, nicotinamide adenine dinucleotide phosphate, phenazine methosulfate and tetrazolium, a sample adding hole is further formed in the panel and is positioned above the bonding pad, the bonding pad is configured to intercept a chromogenic substance after reaction and nucleic acid in a blood sample to be detected, and the adsorption pad is configured to adsorb liquid and hemoglobin in the blood sample to be detected.
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