CN102519925B - Kit for screening and checking glucose-6-phosphate dehydrogenase (G6PD) deficiency of neonates and preparation method for kit - Google Patents
Kit for screening and checking glucose-6-phosphate dehydrogenase (G6PD) deficiency of neonates and preparation method for kit Download PDFInfo
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Abstract
The invention discloses a kit for screening and checking glucose-6-phosphate dehydrogenase (G6PD) deficiency of neonates and a preparation method for the kit. The kit for screening and checking the glucose-6-phosphate dehydrogenase (G6PD) deficiency of the neonates consists of substrate reagent dry powder, a substrate redissolved reagent, a copper reagent, a reaction board and filter paper dried blood spot quality control material. The detection result of the kit is high in accuracy and sensitivity, and the kit has the advantages of high stability, economy, simplicity, high efficiency and the like; when the kit is applied to screening and checking for the neonates, the relevance ratio of heterozygote is improved; and the judgment accuracy is improved.
Description
Technical field
The present invention relates to the detection kit field, be specifically related to a kind of kit for the six phosphate dehydrogenase enzyme deficiency disease examinations of neonate's glucose and preparation method thereof.
Background technology
Glucose six phosphate dehydrogenase enzyme deficiency diseases are that red blood cell glucose six phosphate dehydrogenases (G6PD) significantly lack the one group of different substantiality disease that causes, and are commonly called as " favism ", are a kind of modal heredity enzyme defect diseases.Glucose six phosphate dehydrogenase enzyme deficiency diseases (Glucose-6-phosphate dehydrogenase G6PD) are nearly all high and low one of biological important special enzymes that waits, molecular weight is 59kDa, its activity form is made up of 2 or 4 subunits, and each subunit contains 515 amino acid.The rate-limiting step of this enzymatic cellular energy metabolic process hexose phosphate shunt pathway, and in this process the NADP(codehydrogenase) be reduced to NADPH, the latter is synthetic essential by reduced glutathione, is the important factor during erythrocyte antioxidant is reacted.
The shortage of G6PD enzymatic activity directly has influence on erythrocytic oxidation resistance, can cause sufferers such as hemolytic anemia, favism, the high courage protoheme of neonate mass formed by blood stasis, is human modal enzyme defect disease, involves 400,000,000 people in the global range approximately.Found more than 400 kind of G6PD variant at present, caused that by the sudden change of more than 150 kinds of G6PD genes the type of sudden change and frequency present tangible region or colony's specificity.This disease is mainly seen in malaria such as Africa, Mediterranean bank, the Middle East, Asia, Austronesia, South America once voguish torrid zone and subtropical zone, generally believes to select relevantly with the resistance of malaria, but lacks enough and necessary positive evidence always.China is one of this sick district occurred frequently, is characteristic distributions high in the south and low in the north, and morbidity rate is 0.2-44.8%.Mainly being distributed in each province on the south the Changjiang river, is height with provinces such as Hainan, Guangdong, Guangxi, Yunnan, Guizhou, Sichuan.G6PD deficiency disease pathogenic factor is that red blood cell can not be resisted oxidative damage and wreck, and causes hemolytic anemia because the G6PD gene mutation causes this enzymatic activity to reduce.
The clinical manifestation of G6PD deficiency disease is roughly the same with general hemolytic anemia.Divide Clinical types such as icterus neonatorum, favism, medicine haemolysis, infectious haemolysis, nonspherocytic hemolytic anemia.The light and heavy degree difference of this sick clinical manifestation, most of patients, particularly women's heterozygote is not fallen ill at ordinary times, no conscious sympton, part patient can show as the chronic hemolytic anemia symptom.Normal because eating broad bean, taking or contact some drugs, infection etc. and bring out acute hemolytic reactions such as hemoglobinuria, jaundice, anaemia.Too much because of hematoclasis because of the serious acute hemolytic anemia that the G6PD shortage is brought out, as untimely processing, can cause liver, kidney or heart failure, in addition dead.Once broke out the popular of G6PD deficiency disease in area, Xingning, Guangdong in the broad bean harvest season sixties, causes many patients' death.The G6PD deficiency disease is again the main cause of pathological jaundice of newborn.A statistics according to middle mountain medical university shows, suffers among the neonate of G6PD deficiency disease, and icterus neonatorum can appear in about 50% infant, wherein about 12% can develop into nuclear icterus, causes the brain infringement, causes feeblemindedness.
When the G6PD deficiency disease is not fallen ill in no inducement, the same with the normal person, need not special processing.The key of control is prevention, the healthy prescription of adhere rigidly to, prevention outbreak.Next be to third trimester of pregnancy pregnant woman or neonate take low dose of phenobarbital, can effectively lower the generation of kernicterus of newborn.Secondly the most effective therapy when blood transfusion is this sick acute attack is to correct acidosis, handle renal failure.Light moderate haemolysis patient generally uses fluid-supplement therapy.
At present, the direct ratioing technigue (UVST) of the 6-phosphogluconate dehydrogenase (G6PD/6PGD) of WHO recommendation is the best approach that the G6PD deficiency disease is made a definite diagnosis by clinical labororatory in the world.And generally using of Chinese neonates G6PD deficiency disease examination is fluorescence spot and fluorometry, wherein the most frequently used method is fluorometry, this method principle is that the G-6-P substrate changes 6PG under G6PD catalysis, follow generation NADPH(to fluoresce simultaneously) increase, measure glucose 6 phosphate dehydrogenase enzymatic activitys by the amount that detects NADPH.Above-mentioned reaction can take place at mixed at room temperature 30min in examination blood cake sample and substrate reagent G6P and NADP, adds copper solution and makes reaction terminating, with luminoscope assaying reaction thing fluorescence under wavelength 355nm exciting light and wavelength 460nm emission light.Draw G6PD concentration (U/gHb) in unknown sample and the quality-control product by typical curve, the G6PD activity is lower than the normal value, and the person lacks for G6PD.But fluorometry is for anaemia, haemolysis, and blood coagulation and outmoded blood sample sample false positive rate height, accuracy rate is low.
Existing G6PD/6PGD ratioing technigue technology is detecting glucose 6 phosphate dehydrogenase deficiency, is mainly used in biochemical instruments or the manual G6PD of packed red cells in the whole blood sample and the quantitative ratio of 6PGD activity measured.Its principle of improvement glucose-6-phosphate dehydrogenase (G6PD) mensuration kit (quantitative ratio method) of producing as Guangzhou Mi Ji company is: glucose six phosphoric acid (6PG) in the G6PD catalytic reagent generate six phosphogluconates (6PG), with reduced diphosphopyridine nucleotide I(NADPH) generation, the H of NADPH passs under the hydrogen effect PMS's, with the NBT(yellow of oxidized form) change reduced form NBT(blueness into).The blue depth and the growing amount of NADPH are proportional, with spectrophotometer 650nm colorimetric (S1).Isoenzymes 6PGD as G6PD also has NAD I(NADP+) change into the effect of NADPH, record the amount (S2) of NADPH with method, draw the relative content of NADPH by the ratio of S1/S2.
Also the measuring principle of some technology is: in the test sample in the G6PD catalytic reagent 6PG generate six phosphogluconates (6PG), simultaneously the NADP+ in the reagent is converted into NADPH.Along with increasing of NADPH, detecting instrument can calculate the activity of G6PD in the sample by the speed that monitoring 340nm place absorbance rises.Owing to contain 6PGD in the sample, it can generate five ribulose monophosphates by catalysis 6PG, simultaneously also NADP+ is reduced into NADPH, absorbance is risen, by detecting the coefficient absorbance rate of change of G6PD+6PGD and the G6PD rate of change of effect separately respectively, both subtract each other, and obtain the activity of real glucose six phosphate dehydrogenases.
Summary of the invention
The objective of the invention is to according to exist in the existing G6PD deficiency disease detection method for anaemia, haemolysis, blood coagulation and outmoded blood sample sample false positive rate height, defectives such as accuracy rate is low, provide a kind of to heterozygote recall rate height, the kit that is used for the six phosphate dehydrogenase enzyme deficiency disease examinations of neonate's glucose of the low and good reproducibility of false positive rate.
Another purpose of the present invention is to provide the preparation method of mentioned reagent box.
A further object of the invention is to provide the using method of mentioned reagent box.
Above-mentioned purpose of the present invention is achieved by the following technical programs:
Kit provided by the invention, principle is: the G6P(6-glucose 1-phosphate1-) substrate changes 6PG(6-glucose 1-phosphate1-acid esters under G6PD catalysis), follow generation NADPH(to fluoresce simultaneously) increase, NADPH can measure its fluorescence intensity by luminoscope under wavelength 355nm exciting light and wavelength 460nm emission light.6PGD as the G6PD isoenzymes also has the effect that NADP is converted into NADPH, and human 6PGD shortage is very rare.By detecting the fluorescence intensity of these two kinds of enzymes respectively, ratio calculated G6PD/6PGD as interior Quality Control, namely reacts G6PD active reduction if ratio reduces with 6PGD, can correctly judge glucose six phosphate dehydrogenase enzyme deficiency disease or minimizings.
A kind of kit for the six phosphate dehydrogenase enzyme deficiency disease examinations of neonate's glucose comprises following component:
(1) the G6P substrate reagent dry powder of 16ml/ bottle;
(2) the 6PG substrate reagent dry powder of 16ml/ bottle;
(3) cupferron of 35ml/ bottle: make with copper sulphate, sodium potassium tartrate tetrahydrate and sodium carbonate;
(4) G6PD filter paper dried blood spot quality-control product, the C1 positive, C2 feminine gender;
(5) substrate of 35ml/ bottle redissolution reagent: use MgCl
2, double-glycine and Tris-Hcl make;
(6) micropore white reaction plate.
The preparation method of above-mentioned kit for the six phosphate dehydrogenase enzyme deficiency disease examinations of neonate's glucose comprises the steps:
(1) preparation G6P and two kinds of substrate reagent dry powder of 6PG, substrate redissolution reagent, cupferron;
(2) preparation filter paper dried blood spot quality-control product;
(3) the above-mentioned substrate reagent dry powder of packing, substrate redissolution reagent, cupferron;
(4) label;
(5) be assembled into the finished product kit;
In the step (1), the preparation of G6P substrate reagent is that the G6P with 1.2g adds purified water, and fully stirring and dissolving after treating to dissolve fully, adds the NADPNa of 1.6 g
2, supply purified water to 100ml, mixing; The preparation of 6PG substrate reagent is that the 6PG with 1.5 g adds purified water, and fully stirring and dissolving after treating to dissolve fully, adds the NADPNa of 1.6 g
2, supply purified water to 100ml, mixing; The preparation of substrate redissolution reagent is that 0.2 g Tris and 5.4 g NaCl are added purified water, and fully stirring and dissolving after treating to dissolve fully, adds 1.8 g MgCl
2With 2.5 g double-glycines, supply purified water to 100ml, mixing; The preparation of cupferron solution is respectively with 0.03gCuSO
45H
2O, 0.05gNa
2CO
3Join in the container that purified water is housed with the 0.03g sodium potassium tartrate tetrahydrate, after treating to dissolve fully, supply purified water to 100ml, mixing;
In the step (2), the preparation method of described filter paper dried blood spot quality-control product is as follows: with citric acid anti-freezing sheep whole blood low speed centrifuge 3000rmp, centrifugal 10 ~ 15min isolates blood plasma and red blood cell.Press red blood cell: blood plasma=1.1: 1.0 adjustment packed cell volumes; Add the pure product raw material of G6PD and 6PGD respectively, make G6PD/6PGD in the C1 whole blood＜1.0, G6PD/6PGD in the C2 whole blood＞1.0; C1, C2 are placed on constantly stirring, fully mixing on the magnetic stirring apparatus respectively; Hang on the top of the shelf cutting out good 903# filter paper, with pipettor quality-control product is added drop-wise on the filter paper.Getting liquid measure is the 50ul/ blood cake, and centering adjustment drips, and room temperature was dried in the shade 3 ~ 4 hours, can not overlappingly put, and the storage bag of packing into after drying in the shade is put drying agent in the bag, puts into 2 ~ 8 ℃ of refrigerators and preserves standby;
In the step (3) the G6P substrate reagent, 6GP substrate reagent, substrate redissolution reagent, the cupferron that prepare in the step (2) are carried out packing, G6P substrate reagent, 6GP substrate reagent that branch is installed utilize freeze dryer to make freeze-dried powder.
The using method of above-mentioned kit for the six phosphate dehydrogenase enzyme deficiency disease examinations of neonate's glucose comprises the steps:
(1) reagent is prepared: G6P substrate reagent dry powder, 6PG substrate reagent dry powder add 16ml substrate redissolution reagent respectively, and mixing left standstill 20 minutes;
(2) checked operation:
(a) application of sample: lay down two parts in the sample that diameter is 3mm or 1/8 inch with perforating plier from sample filter paper dried blood spot, laying the scraps of paper must be soaked into by blood, and adds respectively successively in the blank micropore of G6PD and 6PGD test, and every hole a slice is corresponding one by one;
(b) add substrate: in the micropore that detects the G6PD fluorescent value, add the good G6P substrate reagent 150 μ l of redissolution; In the micropore that detects the 6PGD fluorescent value, add the good 6PG substrate reagent 150 μ l of redissolution and stick on mounting;
(c) hatch: at room temperature, slowly vibrated 30 minutes;
(d) add cupferron: hatch finish after, add the cold cupferron working fluid in 150 μ l/ holes immediately, mixing;
(e) detect: use corresponding program to detect, testing was finished in 15 minutes, excitation wavelength 355nm, emission wavelength 460nm;
(f) interpretation of result: the fluorescent value (S1) of the G6PD that detection is obtained and the fluorescent value (S2) of 6PGD, calculate the value of S1/S2, when S1/S2≤1.0, the result is judged as the positive, and when S1/S2＞1.0, the result is judged as feminine gender.
The determination methods as a result that the present invention is used for the kit of neonate's glucose six phosphate dehydrogenase enzyme deficiency disease examinations comprises the steps:
(1) detect: the micro reaction plate that will add cupferron uses corresponding program to detect, and testing was finished in 15 minutes, excitation wavelength 355nm, emission wavelength 460nm;
(2) interpretation of result: the fluorescent value (S1) of the G6PD that detection is obtained and the fluorescent value (S2) of 6PGD, calculate the value of S1/S2, when S1/S2≤1.0, the result is judged as the positive, and when S1/S2＞1.0, the result is judged as feminine gender.
Compared with prior art, the present invention has following beneficial effect:
Neonate's glucose six phosphate dehydrogenase examinations mensuration kit of the present invention is mainly used in the mensuration to G6PD/6PGD fluorescence ratio in neonate's filter paper dried blood spot sample, recall rate height to heterozygote, reduced the false positive rate of sample greatly, for world initiative, filled the domestic gaps.This method adopts particular device, is determined at the 460nm emitting fluorescence signal that produces under the 355nm excitation, measures the variation of absorbance than classic method, has that background is low, accuracy is high, the advantage of good reproducibility has higher susceptibility and specificity simultaneously; Adopt micro reaction plate as detecting carrier, easy and simple to handlely save time, spend low, be particularly suitable for large sample is carried out the examination of newborn infant diseases, removed the loaded down with trivial details of the independent examination of classic method from; Kit also contains yin and yang attribute contrast quality-control product in design, for the quality control in the product application process is given security; With the filter paper dried blood spot as sample, need not sample pre-treatments such as centrifugal and washing, alleviate staff's burden greatly, and one person-portion blood sheet can be applicable to four kinds of congenital hereditary metabolic diseases of examination (congenital thyroid gland machine hypofunction, congenital adrenal cortical hyper plasia, phenylketonuria and G6PD deficiency disease) simultaneously, be worth very much application clinically (table 1).
Table 1
| Britain's Landau | Guangzhou rice base | Patent | This reagent | |
| Method | Speed A method | The ratio end-point method | The blank method of sample speed | The fluorescence ratio method |
| Purposes | The diagnosis favism | The diagnosis favism | The diagnosis favism | The diagnosis favism |
| Advantage | Holding time is long | Need not be centrifugal, need not detect hemoglobin concentration. | 1 year term of validity, biliquid need not be prepared direct use.Need not measure hemoglobin concentration. | Use the filter paper dried blood spot to detect, the preservation transportation of convenience sample; Can detect great amount of samples simultaneously, can be used for neonatal screening; Need not measure hemoglobin concentration; Can be with to doing. |
| Shortcoming | Need to detect hemoglobin concentration. | Full manual operations, the running time is long, and the reagent preparation back holding time is short. | Require to use packed red cells. | Need 30 minutes incubation time. |
| The sample pre-treatment | With physiological saline Washed Red Blood Cells three times. | Need not | Need not | Need not |
| The heterozygote recall rate | Low | High | High | High |
Embodiment
Further explain the present invention below in conjunction with embodiment, but embodiment does not do any type of restriction to the present invention.
G6P-Na among the embodiment
2Purchase the company in Sigma; 96 hole micropore blank plates (8 * 12 hole) are the Therom product; The 6PG(6 glucose 1-phosphate1-) is ORIENTAL company product; β-NADP-Na
2Available from Regal Biotechnology Company company; Double-glycine (GLY-GLY) is purchased the company in Sigma; G6PD is available from WORTHINGTON company; 6PGD is available from PROSPEC company.
Other reagent are that homemade analysis is pure; Time resolved fluoro-immunoassay instrument (PE company 1420 types), constant temperature shaker are that Feng Hua biological company limited in Guangzhou produces.
Embodiment 1
Preparing G6PD mensuration kit (fluorescence ratio method) component that detects simultaneously of the present invention comprises:
(1) G6P substrate reagent dry powder: make packing and freeze-drying with G-6-P and NADP;
(2) 6PG substrate reagent dry powder: make packing and freeze-drying with 6-PG and NADP;
(3) cupferron: make with copper sulphate, sodium potassium tartrate tetrahydrate and sodium carbonate;
(4) G6PD filter paper dried blood spot quality-control product, the C1 positive, C2 feminine gender: be prepared into the filter paper dried blood spot with containing anti-freezing sheep whole blood red blood cell;
(5) substrate redissolves reagent: with MgCl2, Gly-Gly(double-glycine) and Tris-Hcl make.
(6) scraps of paper quality-control product: with citric acid anti-freezing sheep whole blood low speed centrifuge 3000rmp, centrifugal 10～15min isolates blood plasma and red blood cell.(red blood cell: blood plasma=1.2: 1.0) adjust packed cell volume in proportion; Add the pure product raw material of G6PD and 6PGD respectively, make G6PD/6PGD in the C1 whole blood＜1.0, G6PD/6PGD in the C2 whole blood＞1.0.C1, C2 are placed on constantly stirring (avoiding producing bubble), fully mixing on the magnetic stirring apparatus respectively.Hang on the top of the shelf cutting out good 903# filter paper, with pipettor quality-control product is added drop-wise on the filter paper.Getting liquid measure is the 50ul/ blood cake, and centering adjustment drips.Room temperature is dried in the shade (3～4 hours), can not overlappingly put.The storage bag (bag in put drying agent) of packing into after drying in the shade is put into 2～8 ℃ of refrigerators and can be preserved standby.
(7) micropore white reaction plate.
The main technique that the present invention detects G6PD deficiency disease kit is prepared as follows:
(1) preparation of G6P substrate reagent dry powder: the G6P of 1.2 g in special barrel, is added an amount of purified water, fully stirring and dissolving.After treating to dissolve fully, add the NADPNa of 1.6g
2, supply purified water to 100ml, mixing.The packing freeze-drying gets final product.
(2) preparation of 6PG substrate reagent dry powder: the 6PG of 1.5 g in special barrel, is added an amount of purified water, fully stirring and dissolving.After treating to dissolve fully, add the NADPNa of 1.6 g
2, supply purified water to 100ml, mixing.The packing freeze-drying gets final product.
(3) preparation of cupferron: respectively with 0.03gCuSO
45H
2O, 0.05gNa
2CO
3Join in the special container that purified water is housed with the 0.03g sodium potassium tartrate tetrahydrate, after treating to dissolve fully, supply purified water to 100ml, mixing.
(4) preparation of substrate redissolution reagent: 0.2 g Tris and 5.4 g NaCl are added purified water, and fully stirring and dissolving after treating to dissolve fully, adds 1.8 g MgCl
2With the 2.5g double-glycine, supply purified water to 100ml, mixing.
(5) G6PD quality-control product, the C1 positive, the preparation of C2 feminine gender: with citric acid anticoagulated whole blood low speed centrifuge 3000rmp, centrifugal 10～15min isolates blood plasma and red blood cell is stand-by.With citric acid anti-freezing sheep whole blood low speed centrifuge 3000rmp, centrifugal 10～15min isolates blood plasma and red blood cell.(red blood cell: blood plasma=1.1: 1.0) adjust packed cell volume in proportion; Add the pure product raw material of G6PD and 6PGD respectively, make G6PD/6PGD in the C1 whole blood＜1.0, G6PD/6PGD in the C2 whole blood＞1.0.C1, C2 are placed on constantly stirring (avoiding producing bubble), fully mixing on the magnetic stirring apparatus respectively.Hang on the top of the shelf cutting out good 903# filter paper, with pipettor quality-control product is added drop-wise on the filter paper.Getting liquid measure is the 50ul/ blood cake, and centering adjustment drips.Room temperature is dried in the shade (3～4 hours), can not overlappingly put.The storage bag (bag in put drying agent) of packing into after drying in the shade is put into 2～8 ℃ of refrigerators and can be preserved standby.
(6) micropore white reaction plate;
(7) label;
(8) finished product assembling.
The packing of above-mentioned steps products obtained therefrom is semi-manufacture.Extract 3 parts of process specificitys, accuracy, sensitivity and stable assay approvals out and just can be assembled into neonatal screening G6PD kit (fluorescence ratio method).Assembling also need be inspected by random samples after finishing and just can dispatch from the factory after qualified.
The using method of embodiment 2 kits of the present invention
The concrete operations of the neonate G6PD kit for screening (fluorescence ratio method) of above embodiment 1 preparation are as follows:
Reagent is prepared:
The redissolution of substrate: G6P substrate reagent dry powder, 6PG substrate reagent dry powder add 16ml substrate redissolution reagent respectively, and mixing left standstill 20 minutes;
The cooling of cupferron: it is standby to put into 4 ℃ of refrigerator and cooled before reagent uses.
Checked operation:
Application of sample: from sample filter paper dried blood spot, lay down diameter with perforating plier and be about 3mm(1/8 inch) two parts in sample (laying the scraps of paper must be soaked into by blood), and add respectively successively in the blank micropore of G6PD and 6PGD test, every hole a slice, corresponding one by one.The consistance of control punching makes the scraps of paper consistent as far as possible.
Add substrate: in the micropore that detects the G6PD fluorescent value, add the good G6PD substrate reagent 150 μ l of redissolution; In the micropore that detects the 6PGD fluorescent value, add the good 6PGD substrate reagent 150 μ l of redissolution and stick on mounting.
Hatch: at room temperature, slowly vibrated 30 minutes.
Add cupferron: hatch finish after, add the cold cupferron working fluid in 150 μ l/ holes (just taking out from refrigerator, not rewarming) immediately, mixing.
Detect: use corresponding program to detect, (excitation wavelength 355nm, emission wavelength 460nm) finished in testing in 15 minutes.
Interpretation of result: the fluorescent value (S1) of the G6PD that detection is obtained and the fluorescent value (S2) of 6PGD, calculate the value of S1/S2, when S1/S2≤1.0, the result is judged as the positive, and when S1/S2＞1.0, the result is judged as feminine gender.
The analytical performance evaluation index of embodiment 3 kits of the present invention
The performance evaluation index that the neonatal screening of above embodiment 1 preparation is measured G6PD kit (fluorescence ratio method) is as follows:
Sensitivity: measure positive reference material 1 cover of being set up by the company laboratory.Positive reference material is made up of 100 parts of samples for be defined as G6PD defective disease patient's sample through clinical detection.
Positive reference material coincidence rate (the %)=positive reference material quantity/10*100% of actual measurement.
The sensitivity test of table 2 kit of the present invention
| ? | Positive (ratio≤1.0) | Negative (ratio＞1.0) |
| Testing result | 100 | 0 |
Continuing to get positive coincidence rate by last table is 100%.
Specificity:
Negative reference material coincidence rate is measured: overlapped by the negative reference material 1 that the company laboratory is set up.Negative reference material is made up of 100 parts of samples for to determine the normal sample of G6PD through clinical detection.
Negative reference material coincidence rate (the %)=negative reference material quantity/10*100% of actual measurement.
The specificity test of table 3 kit of the present invention
| ? | Positive (ratio≤1.0) | Negative (ratio＞1.0) |
| Testing result | 0 | 100 |
Can get negative match-rate by last table calculating is 100%.
Accuracy:
Withinrun precision:
Use a batch of reagent, 10 hole replicate determination feminine gender and positive quality control product, the yin and yang attribute degree of conformity of calculating measurement result.
The withinrun precision test of table 4 kit of the present invention
Calculate by last table and can get positive coincidence rate and negative match-rate is 100%.
Betweenrun precision:
Use three different batches reagent, same batch of positive and negative quality-control product measured in 10 holes respectively, calculates the yin and yang attribute degree of conformity of measurement result.
The betweenrun precision test of table 5 kit of the present invention
Calculate by last table and can get positive coincidence rate and negative match-rate is 100%.
Thermal stability (test accelerates the failure):
Kit detects positive reference material and negative reference material after 37 ℃ of bakings in 3 days, calculate the yin and yang attribute coincidence rate.
The heat stability testing of table 6 kit of the present invention
Calculate by last table and can get positive coincidence rate and negative match-rate is 100%.
Above performance index show that " neonatal screening G6PD kit (fluorescence ratio method) " is highly sensitive, high specificity, accuracy is good and Heat stability is good.
Claims (1)
1. kit that is used for the examination of neonate's glucose six phosphate dehydrogenases (G6PD) deficiency disease is characterized in that comprising following component:
(1) 16ml/ bottle is by glucose six phosphoric acid (G6P) and NADP+ disodium (NADPNa
2) the G6P substrate reagent dry powder formed;
(2) 16ml/ bottle is by six phosphogluconates (6PG) and NADPNa
2The 6PG substrate reagent dry powder of forming;
(3) cupferron of 35ml/ bottle: make with copper sulphate, sodium potassium tartrate tetrahydrate and sodium carbonate;
(4) G6PD filter paper dried blood spot quality-control product comprises positive quality control product and negative quality-control product;
(5) substrate of 35ml/ bottle redissolution reagent: use MgC1
2, double-glycine and Tris make, and uses the HCl adjust pH;
(6) micropore white reaction plate;
The preparation method of described kit for the six phosphate dehydrogenase enzyme deficiency disease examinations of neonate's glucose comprises the steps:
(1) preparation G6P and two kinds of substrate reagent dry powder of 6PG, substrate redissolution reagent, cupferron;
(2) preparation filter paper dried blood spot quality-control product;
(3) the above-mentioned substrate reagent dry powder of packing, substrate redissolution reagent, cupferron;
(4) label;
(5) be assembled into the finished product kit;
In the step (1), the preparation of G6P substrate reagent is that the G6P with 1.2g adds purified water, and fully stirring and dissolving after treating to dissolve fully, adds the NADPNa of 1.6 g
2, supply purified water to l00ml, mixing, the G6P substrate reagent that branch is installed utilizes freeze dryer to make freeze-dried powder; The preparation of 6PG substrate reagent is that the 6PG with 1.5g adds purified water, and fully stirring and dissolving after treating to dissolve fully, adds the NADPNa of 1.6 g
2, supply purified water to l00ml, mixing, the 6GP substrate reagent that branch is installed utilizes freeze dryer to make freeze-dried powder; The preparation of substrate redissolution reagent is that 0.2 g Tris and 5.4 g NaCl are added purified water, and fully stirring and dissolving after treating to dissolve fully, adds 1.8 g MgC1
2With 2.5 g double-glycines, supply purified water to l00ml, mixing; The preparation of cupferron solution is respectively with 0. 03gCuSO
45H
2O, 0.05gNa
2CO
3Join in the container that purified water is housed with the 0.03g sodium potassium tartrate tetrahydrate, after treating to dissolve fully, supply purified water to l00ml, mixing;
In the step (2), the preparation method of described filter paper dried blood spot quality-control product is as follows: with citric acid anti-freezing sheep whole blood low speed centrifuge 3000rmp, centrifugal 10 ~ 15min isolates blood plasma and red blood cell; Press red blood cell: blood plasma=1.1:1.0 adjusts packed cell volume; Add G6PD and the pure product raw material of six glucose phosphate dehydrogenases (6PGD) respectively, make fluorescent value S2＜1.0 of the fluorescent value S1/6PGD of G6PD in the positive whole blood, the fluorescent value S2 of the fluorescent value S1/6PGD of G6PD in the negative whole blood 1.0; Positive whole blood, negative whole blood are placed on constantly stirring, fully mixing on the magnetic stirring apparatus respectively; Hang on the top of the shelf cutting out good 903# filter paper, be added drop-wise to positive whole blood, negative whole blood on the filter paper respectively with pipettor; Getting liquid measure is 50 μ l/ blood cakes, and centering adjustment drips, and room temperature was dried in the shade 3 ~ 4 hours, can not overlappingly put, and the storage bag of packing into after drying in the shade is put drying agent in the bag, puts into 2 ~ 8 ℃ of refrigerators and preserves standby;
The using method of described kit for the six phosphate dehydrogenase enzyme deficiency disease examinations of neonate's glucose comprises the steps:
(1) reagent is prepared: G6P substrate reagent dry powder, 6PG substrate reagent dry powder add 16ml substrate redissolution reagent respectively, and mixing left standstill 20 minutes;
(2) checked operation:
(a) application of sample: from sample filter paper dried blood spot, lay down two parts in the sample that diameter is 3mm or 1/8 inch with perforating plier,
Laying the scraps of paper must be soaked into by blood, and adds respectively successively in the micropore that detects the G6PD fluorescent value and the micropore that detects the 6PGD fluorescent value, and every hole a slice is corresponding one by one;
(b) add substrate: in the micropore that detects the G6PD fluorescent value, add the good G6P substrate reagent 150 μ l of redissolution; In the micropore that detects the 6PGD fluorescent value, add the good 6PG substrate reagent 150 μ l of redissolution and stick on mounting;
(c) hatch: at room temperature, slowly vibrated 30 minutes;
(d) add cupferron: hatch finish after, add the cupferron working fluid of 1501 μ l/Kong Leng, mixing immediately;
(e) detect: the micro reaction plate that will add cupferron uses corresponding program to detect, and testing was finished in 15 minutes, excitation wavelength 355nm, emission wavelength 460nm;
(f) interpretation of result: the fluorescent value S1 of the G6PD that detection is obtained and the fluorescent value S2 of 6PGD, calculate the value of S1/S2, when S1/S2≤1.0, the result is judged as the positive, as S1/S2〉1.0 the time, the result is judged as feminine gender.
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| BR112017002247A2 (en) * | 2014-08-05 | 2018-01-16 | Becton Dickinson Co | methods, compositions and kit for analysis of glucose-6-phosphate dehydrogenase activity in blood samples |
| CN105758700B (en) * | 2016-03-28 | 2019-01-15 | 广西壮族自治区人民医院 | Whole blood control and preparation method thereof is lyophilized in glucose-6-phosphate dehydrogenase (G6PD) |
| CN108008131A (en) * | 2017-12-29 | 2018-05-08 | 广州市丰华生物工程有限公司 | It is a kind of to detect kit of creatine kinase and preparation method thereof in Filter Paper Dry Blood piece |
| CN109115859A (en) * | 2018-10-23 | 2019-01-01 | 广东祥岳生物技术有限公司 | A kind of newborn's Screening for Thalassemia outcome quality control method |
| CN111060503B (en) * | 2020-01-02 | 2022-02-01 | 青岛汉唐生物科技有限公司 | G-6-PD detection kit free from anemia and detection method thereof |
| CN115078341B (en) * | 2022-08-22 | 2022-11-29 | 上海执诚生物科技有限公司 | Reagent for detecting glucose-6-phosphate dehydrogenase and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1243163A (en) * | 1999-06-02 | 2000-02-02 | 暨南大学 | Diagnosis box for checking glucose-6-phosphodehydrogenase deficiency disease, and preparation method thereof |
| CN101059421A (en) * | 2007-05-24 | 2007-10-24 | 杜传书 | Red cell G 6PD deficiency disease double enzyme ratio detection method and its reagent kit and usage method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5492815A (en) * | 1990-02-20 | 1996-02-20 | Iatron Laboratories, Inc. | Method of determining glucose-6-phosphate and composition therefor |
-
2011
- 2011-12-02 CN CN 201110394189 patent/CN102519925B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1243163A (en) * | 1999-06-02 | 2000-02-02 | 暨南大学 | Diagnosis box for checking glucose-6-phosphodehydrogenase deficiency disease, and preparation method thereof |
| CN101059421A (en) * | 2007-05-24 | 2007-10-24 | 杜传书 | Red cell G 6PD deficiency disease double enzyme ratio detection method and its reagent kit and usage method |
Non-Patent Citations (8)
| Title |
|---|
| 一种灵敏的干血斑葡萄糖-6-磷酸脱氢酶荧光分析法的建立;汤永平等;《热带医学杂志》;20091130;第9卷(第11期);1238-1240 * |
| 应用荧光法筛查葡萄糖6磷酸脱氢酶缺乏症;朱文斌等;《检验医学》;20051231;第20卷(第6期);508-510 * |
| 新生儿干血片葡萄糖-6-磷酸脱氢酶测定的影响因素分析;朱文斌等;《检验医学》;20090430;第24卷(第4期);245-247 * |
| 曹伟锋等.用定量荧光法测定干血斑葡萄糖-6-磷酸脱氢酶活性.《广东医学》.2008,第29卷(第3期),362-363. |
| 朱文斌等.应用荧光法筛查葡萄糖6磷酸脱氢酶缺乏症.《检验医学》.2005,第20卷(第6期),508-510. |
| 朱文斌等.新生儿干血片葡萄糖-6-磷酸脱氢酶测定的影响因素分析.《检验医学》.2009,第24卷(第4期),245-247. |
| 汤永平等.一种灵敏的干血斑葡萄糖-6-磷酸脱氢酶荧光分析法的建立.《热带医学杂志》.2009,第9卷(第11期),1238-1240. |
| 用定量荧光法测定干血斑葡萄糖-6-磷酸脱氢酶活性;曹伟锋等;《广东医学》;20080331;第29卷(第3期);362-363 * |
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