CN106501201B - A kind of enzyme round-robin method for being used to detect homocysteine - Google Patents
A kind of enzyme round-robin method for being used to detect homocysteine Download PDFInfo
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Abstract
It is a kind of to include the circular response of two reactants for detecting the enzyme round-robin method of homocysteine, it is tetrahydrofolic acid circular response and 5 respectively, 10 methyl tetrahydrofolate circular responses；After the tetrahydrofolic acid circular response reacts generation tetrahydrofolic acid and L methionines with L homocysteine for 5 methyl tetrahydrofolates, then using tetrahydrofolic acid as reaction substrate and glycine and NAD+Reaction 5,10 methyl tetrahydrofolates of generation and NADH；Described 5,10 methyl tetrahydrofolate circular responses are that 5,10 methyl tetrahydrofolates that will be generated in tetrahydrofolic acid circular response are reaction substrate, and tetrahydrofolic acid and serine are generated with glycine reactant；By detecting the absorbance rising degree of NADH at 340nm wavelength, the concentration measurement result of L homocysteine is calculated.This method is that circular response is generated based on tetrahydrofolic acid, generates NADH amplification detection signals by accumulation, is measured in wavelength 340nm, NADH is directly proportional to the homotype semicystinol concentration investigating for participating in reaction.
Description
Technical field
The present invention relates to the detection method technical fields of biological micromolecule homocysteine, are used for more particularly, to one kind
Detect the enzyme round-robin method of homocysteine.
Background technology
Homocysteine (Homocysteine, Hcy) or for homocysteine, is the different of amino acid cysteine
Kind, sulfydryl (- SH) is contained in side chain.Hcy is mainly derived from the methionine of diet regimen, is methionine and cysteine metabolism
An important intermediate product in the process, itself does not participate in the synthesis of protein.Mccully in 1969 is from heredity homotype
It is found in cysteine urine disease death children's postmortem, there are extensive Arterial thrombosis and atherosclerosis in body circulation
(AS) thus pathological manifestations propose that hyperhomocysteinemiainjury (hyperhomocysteinemia, HHCY) can cause to move
The hypothesis of pulse atherosclerosis vascular conditions.Hcy can directly or indirectly lead to vascular endothelial cell damage, and blood vessel is promoted to put down
Sliding muscle cell multiplication, influences the oxidation of low-density lipoprotein, enhances platelet function, promotes thrombosis.
The assay method of homocysteine is in addition to traditional high performance liquid chromatography, enzyme immunoassay (EIA), fluorescence polarization
Method etc. is outer, also occurs the Enzymatic cycling based on methionine and adenosyl homocysteine in recent years (referring to Chinese special
Sharp CN200480026009.4), but since the production technology of critical materials is difficult to grasp and cost is very high, it is difficult to forming market should
With.
The fundamental analysis principle of traditional enzyme immunoassay (EIA) is that the antigen that enzymic-labelled antibody or enzymic-labelled antibody carry out resists
Then precursor reactant generates color reaction by enzyme-to-substrate, for quantitative determining, step is：Enzyme is first used by institute in blood sample
The Hcy for having form is transformed into s- adenosine-L-Hcy, then adds in the monoclonal or Anti-TNF-α of the anti-s- adenosines-L-Hcy of enzyme target
Body, using the principle of competitive binding, by the standard items of different level and enzyme labelled antibody competitive binding, then with colour reagent and end
Only reagent is developed the color and is terminated respectively, produces the standard curve of hcy concentration and coloring intensity.Sample is also by same steps
Reason can find the concentration of its Hcy on standard curve.
Invention content
In order to overcome the shortcomings of in background technology, the invention discloses be different from traditional assay method and existing Enzymatic cycling
Body fluid in homocysteine assay method, this method is that circular response is generated based on tetrahydrofolic acid, passes through accumulation
Reduced Coenzyme I (NADH) amplification detection signal is generated, is measured in wavelength 340nm, NADH and the homotype half for participating in reacting
Cystine concentration is directly proportional.Key enzyme involved in the reaction includes：Methionine synthetase (EC 2.1.1.13), amino methyl
Transferase (EC 2.1.2.10), glycine hydroxymethyl transferase (EC 2.1.2.1) are examined for the cycle of homocysteine
It surveys.The present invention also discloses the reagents of the homocysteine detection based on above-mentioned detection method, which can be extensive
Detection applied to homocysteine in clinic.
In order to realize the goal of the invention, the present invention adopts the following technical scheme that：One kind is used to detect homocysteine
Enzyme round-robin method, include the circular response of two reactants, be tetrahydrofolic acid circular response and 5,10- methyl tetrahydrochysene leaf respectively
Sour circular response；
The tetrahydrofolic acid circular response for 5-methyltetrahydrofolate reacted with L- homocysteine generate tetrahydrofolic acid and
After l-methionine, then using tetrahydrofolic acid as reaction substrate and glycine and NAD+Reaction generation 5,10- methyl tetrahydrofolates and
NADH；
5, the 10- methyl tetrahydrofolates circular response is 5, the 10- methyl four that will be generated in tetrahydrofolic acid circular response
Hydrogen folic acid is reaction substrate, and tetrahydrofolic acid and serine are generated with glycine reactant；
By detecting the absorbance rising degree of NADH at 340nm wavelength, the concentration of L- homocysteine is calculated
Measurement result.
In order to be further improved technical solution, in tetrahydrofolic acid circular response of the present invention, 5-methyltetrahydrofolate with
In the reaction step of L- homocysteine reaction generation tetrahydrofolic acid and l-methionine, the enzyme used is synthesized for methionine
Enzyme, the reaction equation of the reaction are：5-methyltetrahydrofolate+L- homocysteine → tetrahydrofolic acid+l-methionine.
In order to be further improved technical solution, in tetrahydrofolic acid circular response of the present invention, using tetrahydrofolic acid as reaction
Substrate and glycine and NAD+In the reaction step of reaction generation 5,10- methyl tetrahydrofolates and NADH, the enzyme used is amino
Transmethylase；The reaction equation of the reaction is：Glycine+tetrahydrofolic acid+NAD+→ 5,10- methyl tetrahydrofolate+NH3+CO2+
NADH+H+。
In order to be further improved technical solution, in 5,10- methyl tetrahydrofolates circular response of the present invention, 5,10- first
Base tetrahydrofolic acid is generated with glycine reactant in the reaction step of tetrahydrofolic acid and serine, and the enzyme used is glycine methylol
Transferase；The reaction equation of the reaction is：5,10- methyl tetrahydrofolates+glycine+H2O → tetrahydrofolic acid+serine.
A kind of enzyme round-robin method for being used to detect homocysteine, it is characterized in that：Its testing procedure is：
S1:Reagent preparation box：The kit reagent includes following each component：Buffer solution：Tris, reaction substrate 5- methyl
Calcium leucovorin, reaction substrate glycine, reaction substrate NAD+, enzyme activition ION Mg Cl2, methionine synthetase, amino methyl
Transferase, glycine hydroxymethyltransferase；
S2：The buffer solution Tris in step S1 is taken, is dissolved in purified water, and pH value is adjusted with concentrated hydrochloric acid；
S3：5-methyltetrahydrofolate, glycine, the NAD in step S1 are weighed successively+、MgCl2, and add in the molten of step S2
In liquid, stirring is to being completely dissolved；
S4：Take prepared reagent methionine synthetase, Aminomethyltransferase and glycine methylol in step S1
After transferase and respectively independent dissolving, add in the solution described in step S3, stir and evenly mix；
S5：The pH value of the mixing solution of determination step S4, and pH value is adjusted, purified water constant volume is added in, then filters postposition
It is spare in 2-8 DEG C；
S6：Solution after being filtered in step S5 is placed in after water-bath balance and obtains reaction solution, and compound concentration is 0.05mM's
L-HCY solution is as detectable substance；
S7：By reaction solution：Detectable substance in proportion, sets response parameter on Biochemical Analyzer, and records each read point
Absorbance, for calculating absorbance change rate.
In order to be further improved technical solution, each component is a concentration of in the kit of step S1 of the present invention：Buffering
Liquid Tris：20mM-50mM；Reaction substrate 5-methyltetrahydrofolate calcium：5mM-10mM；Reaction substrate glycine：10mM-20mM；
NAD+1mM-5mM；Enzyme activition ION Mg Cl2：2mM-10mM；Methionine synthetase：5-15kU/L；Aminomethyltransferase：1-
4kU/L；Glycine hydroxymethyltransferase：2-6kU/L.
In order to be further improved technical solution, the pH value after being adjusted in step S2 and step S5 of the present invention is to 8.00
±0.05；Purified water is added in the step S5 and is settled to 500ml, solution after constant volume is passed through into 0.22 μm of membrane filtration.
In order to be further improved technical solution, bath temperature is 37 DEG C in step S6 of the present invention, equilibration time 15-
20min。
In order to be further improved technical solution, reaction solution in step S7 of the present invention：The ratio of detectable substance is 50:1.
In order to be further improved technical solution, reagent Tris of the present invention, 5-methyltetrahydrofolate calcium, glycine, NAD+And MgCl2, it is the reagent that Sigma companies produce.
By adopting the above-described technical solution, the present invention has the advantages that：Detection half Guang of homotype of the present invention
The enzyme round-robin method of propylhomoserin includes the circular response of two reactants, mainly passes through tetrahydrofolic acid and 5,10- methyl tetrahydrochysene leaves
Two-way Cycle reaction between acid, makes NADH (reduced Coenzyme I) constantly accumulate under the startup of L- homocysteine, that is, exists
Absorbance is continuously increased at 340nm wavelength, and detection signal is made constantly to enhance, and letter obtains to participate in the L- homocysteine of reaction
Concentration.Each reaction is as follows：
1) 5-methyltetrahydrofolate+L- homocysteine → tetrahydrofolic acid+l-methionine in the reaction process, uses
Methionine synthetase is as reaction enzymes；
2) glycine+tetrahydrofolic acid+NAD+→ 5,10- methyl tetrahydrofolate+NH3+CO2+NADH+H+, the reaction process
In, using Aminomethyltransferase as reaction enzymes；
3) 5,10- methyl tetrahydrofolates+glycine+H2O → tetrahydrofolic acid+serine in the reaction process, uses sweet ammonia
Sour hydroxymethyl transferases are as reaction enzymes.
Two-way Cycle of the present invention, first circular response：Above-mentioned reaction 1) and reaction 2) in tetrahydrofolic acid follow
Ring reacts, and tetrahydrofolic acid is product in reaction 1), is substrate in reaction 2)；Second circular response：It is above-mentioned reaction 2)
With 5, the 10- methyl tetrahydrofolates cycle in reaction 3), 5,10- methyl tetrahydrofolates are product in reaction 2, in reaction 3
For substrate；When reacting startup, the concentration of tetrahydrofolic acid and 5,10- methyl tetrahydrofolate can maintain certain level, constantly disappear
Consumption and generation, and NADH (final indicant) concentration reacted in 2 then will continue to accumulate, and increase the suction at wavelength 340nm
Luminosity, and it is related to the L- homocysteine concentration in reaction 1, increase the sensitivity of measure L- homocysteine.
When reaction is acyclic reaction, sensitivity is mainly related to initial reactant concentration, can only be in certain model
Enclose interior increase；When reaction is single cycle reaction, sensitivity is mainly related to initial reactant and circular response object concentration,
Relatively acyclic reaction can greatly improve sensitivity；When reaction is Two-way Cycle reaction, reaction signal is obviously improved, is enhanced
Reaction sensitivity, sensitivity is mainly related to initial reactant and circular response object concentration, can with respect to single cycle reaction
Greatly improve sensitivity.
Description of the drawings
Fig. 1 is response curve figure of the embodiment one to embodiment three.
Fig. 2 be embodiment one to embodiment three 22-23 range read points response curve figure.
Specific embodiment
Explanation that can be detailed by the following examples is of the invention, and the open purpose of the present invention is intended to protect model of the present invention
Enclose all interior technological improvements.
Embodiment one
S1：Reagent preparation box, the kit reagent include the substance of following each component：The Tris bufferings of a concentration of 20mM
Liquid；The 5-methyltetrahydrofolate calcium of a concentration of 5mM；The glycine of a concentration of 10mM；The NAD of a concentration of 1mM+；A concentration of 2mM's
MgCl2；The methionine synthetase of a concentration of 5kU/L；The Aminomethyltransferase of a concentration of 1kU/L；A concentration of 2kU/L's is sweet
Propylhomoserin hydroxymethyl transferases.
S2：The Tris buffer solutions in 1.211g steps S1 are weighed, are dissolved in 300ml purified waters, and adjusted with concentrated hydrochloric acid
PH to 8.00 ± 0.05；
S3：Weigh the 5-methyltetrahydrofolate calcium 1.244g in step S1, glycine 0.375g, NAD+0.331g and
MgCl20.203g is added in the buffer solution of step S2, and stirring is to being completely dissolved；
S4：Weigh methionine synthetase 2.5kU, Aminomethyltransferase 0.5kU, the glycine methylol in step S1
Transferase 1kU, and individually after dissolving, add in the solution of step S3, stir to abundant mixing；
S5：Above-mentioned solution ph is measured, and adjusts pH to 8.00 ± 0.05, purified water constant volume is added in using 500ml graduated cylinders
To 500ml.By solution after constant volume by 0.22 μm of membrane filtration, be placed in 2~8 DEG C it is spare.
S6:Solution after being filtered in step S5 is placed in 37 DEG C of water-baths after balancing 15min and obtains reaction solution, and prepare
The L- homocysteine solution of a concentration of 0.05mM is as detectable substance；
S7：By reaction solution：Detectable substance presses 50：1 ratio sets response parameter on Biochemical Analyzer, and records each
The absorbance of read point, for calculating absorbance change rate.
Embodiment two
S1：Reagent preparation box, the kit reagent include the substance of following each component：The Tris bufferings of a concentration of 35mM
Liquid；The 5-methyltetrahydrofolate calcium of a concentration of 7.5mM；The glycine of a concentration of 15mM；The NAD of a concentration of 3mM+；It is a concentration of
The MgCl of 3.5mM2；The methionine synthetase of a concentration of 7.5kU/L；The Aminomethyltransferase of a concentration of 3kU/L；It is a concentration of
The glycine hydroxymethyltransferase of 4kU/L.
S2：The Tris buffer solutions in 2.120g steps S1 are weighed, are dissolved in 300ml purified waters, and adjusted with concentrated hydrochloric acid
PH to 8.00 ± 0.05；
S3：Weigh the 5-methyltetrahydrofolate calcium 1.866g in step S1, glycine 0.563g, NAD+0.994g and
MgCl20.356g is added in the buffer solution of step S2, and stirring is to being completely dissolved；
S4：Weigh methionine synthetase 3.75kU in step S1, Aminomethyltransferase 1.5kU, glycine hydroxyl first
Based transferase 2kU, and individually after dissolving, add in the solution of step S3, stir to abundant mixing；
S5：Above-mentioned solution ph is measured, and adjusts pH to 8.00 ± 0.05, purified water constant volume is added in using 500ml graduated cylinders
To 500ml.By solution after constant volume by 0.22 μm of membrane filtration, be placed in 2-8 DEG C it is spare.
S6:Solution after being filtered in step S5 is placed in 37 DEG C of water-baths after balancing 15min and obtains reaction solution, and prepare
The L- homocysteine solution of a concentration of 0.05mM is as detectable substance；
S7：By reaction solution：Detectable substance presses 50：1 ratio sets response parameter on Biochemical Analyzer, and records each
The absorbance of read point, for calculating absorbance change rate.
Embodiment three
S1：Reagent preparation box, the kit reagent include the substance of following each component：The Tris bufferings of a concentration of 50mM
Liquid；The 5-methyltetrahydrofolate calcium of a concentration of 10mM；The glycine of a concentration of 20mM；The NAD of a concentration of 5mM+；A concentration of 5mM
MgCl2；The methionine synthetase of a concentration of 10kU/L；The Aminomethyltransferase of a concentration of 5kU/L；A concentration of 6kU/L
Glycine hydroxymethyltransferase.
S2：The Tris buffer solutions in 3.029g steps S1 are weighed, are dissolved in 300ml purified waters, and adjusted with concentrated hydrochloric acid
PH to 8.00 ± 0.05；
S3：Weigh the 5-methyltetrahydrofolate calcium 2.488g in step S1, glycine 0.751g, NAD+1.656g and
MgCl20.508g is added in the buffer solution of step S2, and stirring is to being completely dissolved；
S4：The methionine synthetase 5kU in step S1, Aminomethyltransferase 2.5kU, glycine methylol is weighed to turn
Enzyme 3kU is moved, and individually after dissolving, add in the solution of step S3, stir to abundant mixing；
S5：Above-mentioned solution ph is measured, and adjusts pH to 8.00 ± 0.05, purified water constant volume is added in using 500ml graduated cylinders
To 500ml.By solution after constant volume by 0.22 μm of membrane filtration, be placed in 2-8 DEG C it is spare.
S6:Solution after being filtered in step S5 is placed in 37 DEG C of water-baths after balancing 15min and obtains reaction solution, and prepare
The L- homocysteine solution of a concentration of 0.05mM is as detectable substance；
S7：By reaction solution：Detectable substance presses 50：1 ratio sets response parameter on Biochemical Analyzer, and records each
The absorbance of read point, for calculating absorbance change rate.
The measurement result of embodiment one to three is as shown in the table：
It is shown by reaction result, three embodiments show preferable linear relationship, are shown in embodiment one
In the concentration range of each reactant, which can implement, and when reactant concentration increases, reactions change rate (Δ A/
Min) can also increase, but when concentration increases to the formula upper limit, Δ A/min is excessively high, can make extinction when measuring high concentration sample
Degree is more than the readable upper limit of instrument.Embodiment three the results show that the reaction later stage change greatly, may when testing high concentration sample
It is that absorbance reaches biochemical instruments detection threshold value, each reactant concentration is more than that the value that embodiment 3 provides may cause absorbance to exceed
Instrument detection threshold value.So as to which test result is not allowed.Therefore in 1 given concentration range of table, linear and reaction sensitivity is equal
It can meet the requirements.
Part not in the detailed description of the invention is the prior art.
Claims (5)
1. it is a kind of for detecting the enzyme round-robin method of homocysteine, it is characterized in that：Include the circular response of two reactants,
It is tetrahydrofolic acid circular response and 5,10- methyl tetrahydrofolate circular response respectively；
The tetrahydrofolic acid circular response reacts generation tetrahydrofolic acid and L- first with L- homocysteine for 5-methyltetrahydrofolate
After methyllanthionine, then using tetrahydrofolic acid as reaction substrate and glycine and NAD+Reaction generation 5,10- methyl tetrahydrofolates and NADH；
In the tetrahydrofolic acid circular response, 5-methyltetrahydrofolate reacts generation tetrahydrofolic acid and L- first with L- homocysteine
In the reaction step of methyllanthionine, the enzyme used is methionine synthetase, and the reaction equation of the reaction is：5-methyltetrahydrofolate
+ L- homocysteine=tetrahydrofolic acid+l-methionine；
In the tetrahydrofolic acid circular response, using tetrahydrofolic acid as reaction substrate and glycine and NAD+Reaction generation 5,10- methyl
In the reaction step of tetrahydrofolic acid and NADH, the enzyme used is Aminomethyltransferase；The reaction equation of the reaction is：Glycine
+ tetrahydrofolic acid+NAD+=5,10- methyl tetrahydrofolate+NH3+CO2+NADH+H+；
5, the 10- methyl tetrahydrofolates circular response is 5, the 10- methyl tetrahydrochysene leaves that will be generated in tetrahydrofolic acid circular response
Acid is reaction substrate, and tetrahydrofolic acid and serine are generated with glycine reactant；
In 5, the 10- methyl tetrahydrofolates circular response, 5,10- methyl tetrahydrofolates generate tetrahydrofolic acid with glycine reactant
In the reaction step of serine, the enzyme used is glycine hydroxymethyltransferase；The reaction equation of the reaction is：5,10- first
Base tetrahydrofolic acid+glycine+H2O=tetrahydrofolic acids+serine；
By detecting the absorbance rising degree of NADH at 340nm wavelength, calculate L- homocysteine concentration and then
Obtain measurement result；
The enzyme round-robin method for being used to detect homocysteine, testing procedure are：
S1:Reagent preparation box：The kit reagent includes following each component：Buffer solution Tris, reaction substrate 5- methyl tetrahydrochysenes
Calcium Folinate-SF, reaction substrate glycine, reaction substrate NAD+, enzyme activition ION Mg Cl2, methionine synthetase, amino methyl transfer
Enzyme, glycine hydroxymethyltransferase；
Each component is a concentration of in the kit of the step S1：Buffer solution Tris：20mM-50mM；Reaction substrate 5- methyl four
Hydrogen Calcium Folinate-SF：5mM-10mM；Reaction substrate glycine：10mM-20mM；NAD+：1mM-5mM；Enzyme activition ION Mg Cl2：2mM-
10mM；Methionine synthetase：5-15kU/L；Aminomethyltransferase：1-4kU/L；Glycine hydroxymethyltransferase：2-6kU/
L；
S2：The buffer solution Tris in step S1 is taken, is dissolved in purified water, and pH value is adjusted with concentrated hydrochloric acid；
S3：5-methyltetrahydrofolate calcium, glycine, the NAD in step S1 are weighed successively+、MgCl2, and add in the solution of step S2
In, stirring is to being completely dissolved；
S4：Take prepared reagent methionine synthetase in step S1, Aminomethyltransferase and the transfer of glycine methylol
After enzyme and respectively independent dissolving, add in the solution described in step S3, stir and evenly mix；
S5：The pH value of the mixing solution of determination step S4, and pH value is adjusted, purified water constant volume is added in, then filtering is placed on 2-8
It is DEG C spare；
S6:Solution after being filtered in step S5 is placed in after water-bath balance and obtains reaction solution, and the L- high that compound concentration is 0.05mM
Cysteine solution is as detectable substance；
S7：By reaction solution：Detectable substance in proportion, sets response parameter on Biochemical Analyzer, and records the extinction of each read point
Degree, for calculating absorbance change rate.
2. it is as described in claim 1 for detecting the enzyme round-robin method of homocysteine, it is characterized in that：The step S2 and
PH value after being adjusted in step S5 is to 8.00 ± 0.05；Purified water is added in the step S5 and is settled to 500ml, after constant volume
Solution passes through 0.22 μm of membrane filtration.
3. it is as described in claim 1 for detecting the enzyme round-robin method of homocysteine, it is characterized in that：In the step S6
Bath temperature is 37 DEG C, equilibration time 15-20min.
4. it is as described in claim 1 for detecting the enzyme round-robin method of homocysteine, it is characterized in that：In the step S7
Reaction solution：The ratio of detectable substance is 50:1.
5. it is as described in claim 1 for detecting the enzyme round-robin method of homocysteine, it is characterized in that：The reagent
Tris, 5-methyltetrahydrofolate calcium, glycine, NAD+And MgCl2, it is the reagent that Sigma companies produce.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1849513A (en) * | 2003-07-10 | 2006-10-18 | 通用原子公司 | Methods and compositions for assaying homocysteine |
| CN102095696A (en) * | 2011-02-14 | 2011-06-15 | 王学忠 | Homocysteine measuring method and reagent |
| CN103184269A (en) * | 2011-12-28 | 2013-07-03 | 协和干细胞基因工程有限公司 | Kit for detecting SNP sites related to homocysteine metabolism and amplification method and detection method thereof |
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| CN101089599A (en) * | 2006-06-12 | 2007-12-19 | 苏州艾杰生物科技有限公司 | Homotype semicystinol diagnostic kit and homotype semicystinol concentration investigating method |
| CN101089603A (en) * | 2006-06-12 | 2007-12-19 | 苏州艾杰生物科技有限公司 | Homotype semicystinol diagnostic kit and homotype semicystinol concentration investigating method |
| CN101762522A (en) * | 2008-12-10 | 2010-06-30 | 苏州艾杰生物科技有限公司 | Homocysteine diagnosis/determination reagent (kit) and homocysteine concentration determination method |
| CN101750356A (en) * | 2008-12-10 | 2010-06-23 | 苏州艾杰生物科技有限公司 | Homocysteine diagnosing/measuring reagent (kit) and homocysteine concentration measuring method |
| AR083468A1 (en) * | 2010-10-25 | 2013-02-27 | Metabolic Explorer Sa | INCREASING NADPH'S AVAILABILITY FOR METIONIN PRODUCTION |
-
2016
- 2016-12-31 CN CN201611266287.8A patent/CN106501201B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1849513A (en) * | 2003-07-10 | 2006-10-18 | 通用原子公司 | Methods and compositions for assaying homocysteine |
| CN102095696A (en) * | 2011-02-14 | 2011-06-15 | 王学忠 | Homocysteine measuring method and reagent |
| CN103184269A (en) * | 2011-12-28 | 2013-07-03 | 协和干细胞基因工程有限公司 | Kit for detecting SNP sites related to homocysteine metabolism and amplification method and detection method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 叶酸代谢通路相关基因SNPS与孕妇同型半胱氨酸代谢能力的相关性分析;梁爽;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20130315(第3期);34-35 * |
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