CN107475404A - A kind of primer pair for detecting hoptoglobin parting and its application - Google Patents
A kind of primer pair for detecting hoptoglobin parting and its application Download PDFInfo
- Publication number
- CN107475404A CN107475404A CN201710833598.6A CN201710833598A CN107475404A CN 107475404 A CN107475404 A CN 107475404A CN 201710833598 A CN201710833598 A CN 201710833598A CN 107475404 A CN107475404 A CN 107475404A
- Authority
- CN
- China
- Prior art keywords
- final concentration
- temperature
- seconds
- hoptoglobin
- primer pair
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Pathology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a kind of primer pair for detecting hoptoglobin parting, above-mentioned primer pair is：Forward primer (F):5’‑GAGGGGAGCTTGCCTTTCCATTG‑3’；Reverse primer (R):5’‑ACCAGCCAGGGCTCAAAAATCTC‑3’.The present patent application establishes a kind of method analyzed from gene angle Hp, provide a kind of primer pair for detecting hoptoglobin parting, this method carries out gene magnification by designing specific primer, by being that can determine that Hp genotype to amplified production electrophoresis detection, it is easy to operate quick, accurate using primer pair detection hoptoglobin parting, it can be applied to clinical practice.
Description
Technical field
The present invention relates to biomedicine field, more particularly, to a kind of primer pair for detecting hoptoglobin parting and
It is applied.
Background technology
People with diabetes increasingly increases at present, also gradually tends to rejuvenation, and these patients have very high concurrent painstaking effort
The risk of pipe disease, the diabetic that rough Statistics there are about 75% die from angiocardiopathy.In order to mitigate patient's hyperglycemia
Symptom, reduce the generation of capilary and macrovascular complications, enabled the patient to normally live, using suitable medicine
Prevention of cardiovascular complication is particularly important.Because diabetic's complicated with cardiovascular disease and vivo oxidation stress have certain phase
Guan Xing, therefore American Society of Cardiology and WHO (WHO) recommend the antioxidants such as vitamin E to prevent
Diabetic's cardiovascular complication.
Clinical discovery, different diabetics are very big for difference on effect caused by vitamin E supplement.Some trouble
Person's replenishing vitamins E can substantially reduce risk of cardiovascular diseases；And some patients after replenishing vitamins E on the contrary, on the contrary can
Increase the risk of complicated with cardiovascular disease, death incident is resulted even in when serious.
A large amount of scientific researches find, the concurrent close phase of cardiopathic risk of the genotype of hoptoglobin and diabetic
Close.Wherein, it is higher that diabetic's suffering from heart disease complication, the risk of apoplexy even death of Hp2-2 genotype are carried, about
To carry 5 times of the diabetic of Hp1-1 genotype；And the heart disease for carrying the diabetic of Hp2-1 genotype is concurrent
Risk falls between.And in terms of the therapeutic effect after replenishing vitamins E, for carrying hoptoglobin Hp2-2 genes
The diabetic of type, after 18 months vitamin E treatments, the probability ratio of heart disease and stroke breaking-out does not take vitamin E
Similar patient few 50%, and do not occur side effect.And to the diabetic of Hp2-1 or Hp1-1 genotype, replenishing vitamins
E can then improve the risk of its complicated with cardiovascular disease on the contrary.
Hoptoglobin (Haptoglobin, Hp) is also known as haptoglobin, is one kind sugar in Serum A 2- globulin fractions
Protide, it is a kind of anti-oxidant albumen of the stable erythrocyte hemoglobin of energy, vascular wall can be protected to avoid being inflamed.The mankind
The gene of hoptoglobin is located at Chromosome 16q 22.1, and common Hp has three kinds of genotype in crowd, is Hp1-1, Hp2- respectively
1 and Hp2-2, a pair of alleles Hp1 and Hp2 being controlled by Hp seats, in codominant inheritance.Different regions and ethnic group
Hp1 and HP2 gene frequencies are different, and the statistics of west crowd is shown, the probability of Hp1-1 genotype is 16%, Hp2-2
The probability of genotype is 36%, and the probability of Hp2-1 genotype is then 48%.Asian is in the majority with Hp2-2, is secondly Hp2-
1, and Hp1-1 is minimum, Han nationality of China ethnic group Hp1 genotype frequencies are relatively low, more based on Hp2.
In summary, the diabetic's complicated with cardiovascular disease for carrying hoptoglobin Hp2-2 genotype is even dead
It is higher to die risk, its risk can be reduced by taking vitamin E；Although carry the diabetic of Hp2-1 or Hp1-1 genotype simultaneously
It is relatively low to send out risk of cardiovascular diseases, but its coincidence risk can improve on the contrary after taking vitamin E.Therefore, in order to preferably analyze
Judge the coincidence risk of diabetic's angiocardiopathy, reasonably carry out early intervention, and clinically give more reliable standard
True medication guide, the Genotyping that globin is combined for people with diabetes are very necessary.
The method of detection hoptoglobin parting is mostly electrophoresis at present, first makes testing sample and hemoglobin shape before electrophoresis
Into Hp-Hb (hoptoglobin-hemoglobin) compound.The peroxidase activity of hemoglobin can be utilized after electrophoresis is complete
Benzidine is set to be oxidized to benzidine blue in the presence of hydrogen peroxide, dyeing plain edition can use polyacrylamide gel eletrophoresis
Parting.Hp hypotypes then need the Hp in first purification of samples, then are denatured the α peptide chains in Hp components, and common electricity can be used after reduction
Swimming or isoelectric focusing technique carry out parting, and operation is excessively complicated.Because Hp can be preserved for quite a long time in blood stain, putting into practice
In can by detect the Hp in blood stain carry out Classification Identification, but due to hemoglobin excessive in blood stain can cover Hp swimming spectrum, because
And the individual identification ability of blood stain is limited to a certain extent.
Therefore have one kind to be supplied easy to operate quick, accurate, the detection hoptoglobin point of clinical practice is can be applied to again
The primer pair of type.
The content of the invention
The invention provides a kind of primer pair for detecting hoptoglobin parting and its application, at least to solve prior art
The technical problem that middle detection hoptoglobin classifying method is complicated, accuracy rate is low.
The invention provides a kind of primer pair for detecting hoptoglobin parting, above-mentioned primer pair is：
Forward primer (F):5’-GAGGGGAGCTTGCCTTTCCATTG-3’；
Reverse primer (R):5’-ACCAGCCAGGGCTCAAAAATCTC-3’.
Application of the above-mentioned primer pair in hoptoglobin parting is detected.
Optionally, above-mentioned primer pair detection determines that the parting of hoptoglobin is Hp1 hypotypes or Hp2 hypotypes.
Optionally, when the parting that above-mentioned primer pair detects determination hoptoglobin is Hp2 hypotypes, above-mentioned primer pair enters one
Step detection determines that the parting of hoptoglobin is Hp2-1 hypotypes or Hp2-2 hypotypes.
Optionally, gene magnification is carried out to the sample to be tested comprising above-mentioned hoptoglobin gene using above-mentioned primer pair to obtain
Amplified production, it is that can determine that the above-mentioned hoptoglobin of above-mentioned testing gene by carrying out electrophoresis detection to above-mentioned amplified production
Genotype.
Optionally, said gene expands, i.e., polymerase chain reaction (PCR) comprises the following steps：In 90 DEG C to 100 DEG C of temperature
Spend lower pre-degeneration 3-5 minutes；It is denatured 30 seconds to 50 seconds at a temperature of 90 DEG C to 100 DEG C, is moved back at a temperature of 45 DEG C to 60 DEG C
Fire 20 seconds to 50 seconds, extends 90 seconds to 240 seconds at a temperature of 70 DEG C to 74 DEG C, wherein above-mentioned denaturing step, above-mentioned annealing step
Rapid and above-mentioned extension step is 25 to 40 circulations；Then extend eventually 3 to 10 minutes at a temperature of 70 DEG C to 74 DEG C；Finally
Keeping temperature is 3 DEG C to 10 DEG C.
Optionally, annealed 20 seconds to 50 seconds at a temperature of 47 DEG C to 52 DEG C.
Optionally, said gene expands, i.e., polymerase chain reaction (PCR) comprises the following steps：It is pre- at a temperature of 94 DEG C
Denaturation 5 minutes；It is denatured 50 seconds at a temperature of 94 DEG C, is annealed 30 seconds at a temperature of 50 DEG C, extend 150 at a temperature of 72 DEG C
Second, wherein above-mentioned denaturing step, above-mentioned annealing steps and above-mentioned extension step are 38 circulations；Then at a temperature of 72 DEG C
Extension 6 minutes eventually；Last keeping temperature is 4 DEG C.
Optionally, said gene expands, i.e., the mixed solution for the buffer solution that polymerase chain reaction (PCR) uses into subpackage
Include：
Three (methylol) methylglycines (Tricine), final concentration of 20mmol/L-35mmol/L,
PH is 8.4-8.8；
Potassium acetate, final concentration of 50mmol/L-80mmol/L；
Magnesium acetate, final concentration of 1mmol/L-1.7mmol/L；
Glycerine, final concentration of 6%-10%；
Dimethyl sulfoxide (DMSO) (DMSO), final concentration of 0.5%-1.8%；
Taq enzyme, final concentration of 0.8U-2.2U；
Deoxyribonucleoside triphosphate (dNTP), final concentration of 150umol/L-400umol/L.
Optionally, said gene expands, i.e., the mixed solution for the buffer solution that polymerase chain reaction (PCR) uses into subpackage
Include：
Three (methylol) methylglycines (Tricine), final concentration of 25mmol/L, pH 8.7；
Potassium acetate, final concentration of 80mmol/L；
Magnesium acetate, final concentration of 1.2mmol/L；
Glycerine, final concentration of 8%；
Dimethyl sulfoxide (DMSO) (DMSO), final concentration of 1%；
Taq enzyme, final concentration of 2U；
Deoxyribonucleoside triphosphate (dNTP), final concentration of 400umol/L.
The present patent application establishes a kind of method analyzed from gene angle Hp, there is provided one kind detection combines pearl
The primer pair of albumen parting, this method carries out gene magnification by designing specific primer, by being to amplified production electrophoresis detection
Hp genotype is can determine that, it is easy to operate quick, accurate using primer pair detection hoptoglobin parting, it can be applied to clinic
Practice.
Brief description of the drawings
Fig. 1 is Ago-Gel image after a kind of electrophoresis that the embodiment of the present invention 1 provides；
Fig. 2 is Ago-Gel image after a kind of electrophoresis that the embodiment of the present invention 2 provides；
Fig. 3 is Ago-Gel image after a kind of electrophoresis that the embodiment of the present invention 3 provides.
Embodiment is used to further illustrate but be not limited to the present invention below, and example below is only that the present invention is a kind of
It is preferably carried out mode.
Embodiment
Sample to be tested in following embodiments is same individual saliva and peripheral blood sample, and check sample is clinical bright
The really sample of identification Hp genotype.
Embodiment 1：
Step 1：Saliva sample gathers and processing
Gargle within 30 minutes before sampling to remove swill and residual microorganism, not fed in 30 minutes after gargling,
Drink water, brush teeth, smoking or chewing gum；Loosen cheek, and gently rub the 15-30 seconds to produce saliva, collect 2ml salivas to nothing
In the collector of bacterium.
Step 2：Saliva sample DNA (DNA) is extracted
(1) 500ul (microlitre) saliva sample is taken, adds 1ml (milliliter) buffer solution (100mmol/L (mM/l)
Tris-HCl (tris-HCI buffer), pH8.0,0.5%SDS (dodecyl sodium sulfate), 10mmol/L
EDTA (ethylenediamine tetra-acetic acid)) and 6ul 20mg/ul (milligram/microlitre) Proteinase K, shake in vortex instrument and mix；
(2) saliva after step (1) processing is subjected to 55 DEG C of water bath processing 20min；
(3) 600ul phenol is added：Chloroform：Isoamyl alcohol (25:24:1), overturn and mix, 10000rpm (rev/min) centrifugation
5min, take supernatant；
(4) 600ul chloroform is added into the supernatant of step (3)：Isoamyl alcohol (24:1) 10000rpm after mixing, is overturned
5min is centrifuged, takes supernatant；
(5) isometric isopropanol is added into the supernatant fluid of step (4), 14000rpm is centrifuged after gently mixing
10min, abandon supernatant；
(6) wash above-mentioned precipitation once with 500ul70% ethanol, 14000rpm centrifugation 10min, abandon supernatant；
(7) room temperature dries DNA, is dissolved with 20ul aqua sterilisas.
Step 3：Gene PCR (polymerase chain reaction) expands and product identification
(1) PCR is expanded：
Using PCR buffer solutions, its composition be comprising three (methylol) methylglycines (Tricine), it is final concentration of
25mmol/L, pH8.7；Potassium acetate, final concentration of 80mmol/L；Magnesium acetate, final concentration of 1.2mmol/L；Glycerine, it is final concentration of
8%；Dimethyl sulfoxide (DMSO) (DMSO), final concentration of 1%；Taq enzyme, final concentration of 2U；Deoxyribonucleoside triphosphate (dNTP), eventually
Concentration is 400umol/L, and the DNA that saliva sample extracts to obtain enters performing PCR amplification as template.
Amplimer is as follows：
F:5’-GAGGGGAGCTTGCCTTTCCATTG-3’；
R:5’-ACCAGCCAGGGCTCAAAAATCTC-3’。
PCR response procedures are as follows：
94℃5min；94 DEG C of 50s, 50 DEG C of 30s, 72 DEG C of 150s, 38 circulations；72℃6min；4℃∝.
Pre-degeneration 5 minutes i.e. at a temperature of 94 DEG C；It is denatured 50 seconds at a temperature of 94 DEG C, is annealed at a temperature of 50 DEG C
30 seconds, extend 150 seconds at a temperature of 72 DEG C, wherein the denaturing step, the annealing steps and the extension step are
38 circulations；Then extend eventually at a temperature of 72 DEG C 6 minutes；Last keeping temperature is 4 DEG C.
(2) agarose gel electrophoresis detects
As a result it is Ago-Gel image after a kind of electrophoresis that the embodiment of the present invention 1 provides to see Fig. 1, Fig. 1；
Wherein swimming lane 1 is DNA DNA marker (DNA marker), and swimming lane 2 is Hp2-2 genotype check samples, and swimming lane 3 is
Hp2-1 genotype check samples, swimming lane 4 are Hp1-1 genotype check samples, and swimming lane 5 is sample to be tested；(DNA marker bars
Band is followed successively by 100bp, 250bp, 500bp, 750kb, 1kb, 2kb, 3kb, 5kb from top to bottom).
Step 4：Determine Hp genotype
Hp genotype is determined according to electrophoresis result, Hp1-1 genotype band is 1.7kb in theory, and Hp2-1 genotype is
1.7kb and the bands of 3.8kb two, Hp2-2 genotype band are 3.8kb.Understand, the Hp of the present embodiment sample to be tested genotype is
Hp2-1 genotype.
The method for the detection hoptoglobin parting that the primer that the present embodiment provides is established can by Hp1-1, Hp2-1 and
Hp2-2 hypotypes are clearly distinguished, and simple to operate, for the gene of the hoptoglobin of clinical accurate judgement diabetic
Type has important value, so as to which for diabetic, whether replenishing vitamins E provides important evidence.
Embodiment 2：
Step 1：Peripheral blood sample present treatment extracts with DNA
(1) the peripheral blood 2ml of the anti-coagulants containing EDTA is taken, is placed in the centrifuge tube of 10ml sterilizings, adds 6mL 0.1mM
EDTA, processing 3000rpm centrifugation 10min, are abandoned supernatant, collect precipitation for 15 minutes with broken red blood cell；
(2) into precipitation add 1ml 0.1mM EDTA, be transferred to after mixing 2ml sterilizing centrifuge tube in, 5400rpm from
Heart 5min, abandons supernatant；
(3) 1ml buffer solutions (100mmol/LTris-HCl, pH8.0,0.5%SDS, 10mmol/L are added into precipitation
EDTA) and 6ul 20mg/ul Proteinase K, 55 DEG C of water bath processing 20min after concussion mixes in vortex instrument；
(4) 600ul phenol is added：Chloroform：Isoamyl alcohol (25:24:1), overturn and mix, 10000rpm centrifugation 5min, take
Clearly；
(5) 600ul chloroform is added into the supernatant of step (4)：Isoamyl alcohol (24:1) 10000rpm after mixing, is overturned
5min is centrifuged, takes supernatant；
(6) isometric isopropanol is added into the supernatant fluid of step (5), 14000rpm is centrifuged after gently mixing
10min, abandon supernatant；
(7) wash above-mentioned precipitation once with 500ul70% ethanol, 14000rpm centrifugation 10min, abandon supernatant；
(8) room temperature dries DNA, is dissolved with 30ul aqua sterilisas.
Step 2：Gene PCR (polymerase chain reaction) expands and product identification
(1) PCR is expanded：
It is as shown in the table using buffer components, wherein numbering is consistent with final track numbering,
Enter performing PCR amplification using the DNA that peripheral blood sample is extracted to obtain as template.
Amplimer is as follows：
F:5’-GAGGGGAGCTTGCCTTTCCATTG-3’；
R:5’-ACCAGCCAGGGCTCAAAAATCTC-3’。
PCR response procedures are as follows：
92℃5min；92 DEG C of 40s, 49 DEG C of 50s, 74 DEG C of 90s, 40 circulations；74℃4min；8℃∝.
Pre-degeneration 5 minutes i.e. at a temperature of 92 DEG C；It is denatured 40 seconds at a temperature of 92 DEG C, is annealed at a temperature of 49 DEG C
50 seconds, extend 90 seconds at a temperature of 74 DEG C, wherein the denaturing step, the annealing steps and the extension step are 40
Individual circulation；Then extend eventually at a temperature of 74 DEG C 4 minutes；Last keeping temperature is 8 DEG C.
(2) agarose gel electrophoresis detects
As a result Fig. 2 is seen.Fig. 2 is Ago-Gel image after a kind of electrophoresis that the embodiment of the present invention 2 provides；
Wherein swimming lane 1 be DNA Marker (its band be followed successively by from top to bottom 100bp, 250bp, 500bp, 750bp,
1kb、2kb、3kb、5kb)。
According to electrophoresis result it can be seen that condition number is preferable for 3-11 PCR reaction solution conditions, the gene of sample to be tested
Type is Hp2-1 types.
Embodiment 3：
Step 1：With the step 1 of embodiment 2.
Step 2：Gene PCR (polymerase chain reaction) expands and product identification
(1) PCR is expanded：
Using PCR buffer solutions, its composition be comprising three (methylol) methylglycines (Tricine), it is final concentration of
25mmol/L, pH8.7；Potassium acetate, final concentration of 80mmol/L；Magnesium acetate, final concentration of 1.2mmol/L；Glycerine, it is final concentration of
8%；Dimethyl sulfoxide (DMSO) (DMSO), final concentration of 1%；Taq enzyme, final concentration of 1.5U；Deoxyribonucleoside triphosphate (dNTP),
Final concentration of 300umol/L.
Amplimer is as follows：
F:5’-GAGGGGAGCTTGCCTTTCCATTG-3’；
R:5’-ACCAGCCAGGGCTCAAAAATCTC-3’。
PCR response procedures are as follows：
95℃4min；95 DEG C of 40s, annealing temperature set gradient, and anneal 30s, 70 DEG C of 200s, 38 circulations；70℃8min；
6℃∝。
Pre-degeneration 4 minutes i.e. at a temperature of 95 DEG C；It is denatured 40 seconds at a temperature of 95 DEG C, under the annealing temperature of setting
Annealing 30 seconds, extends 200 seconds at a temperature of 70 DEG C, wherein the denaturing step, the annealing steps and the extension step
It is rapid to be circulated for 38；Then extend eventually at a temperature of 70 DEG C 8 minutes；Last keeping temperature is 6 DEG C.
The annealing temperature of wherein PCR reaction conditions is respectively 45,47,49,51,52,54,57,60,63.
(2) agarose gel electrophoresis detects
As a result it is Ago-Gel image after a kind of electrophoresis that the embodiment of the present invention 2 provides to see Fig. 3, Fig. 2.Wherein electrophoresis
It is as shown in the table that swimming lane in figure corresponds to annealing temperature：
| Track | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Annealing temperature (DEG C) | 63 | 60 | 57 | 54 | 45 | 47 | 49 | 51 | 52 |
Wherein swimming lane 1 be DNA Marker (its band be followed successively by from top to bottom 500bp, 750bp, 1kb, 2kb, 3kb,
5kb)
According to electrophoresis result it can be seen that 45-60 DEG C of annealing temperature is all effective, wherein 47-52 DEG C of annealing temperature is most
Excellent, the genotype of sample to be tested is Hp2-1 types.
The primer that above-described embodiment provides can not only distinguish Hp1 hypotypes and Hp2 hypotypes, and can by Hp2-1 and
Hp2-2 hypotypes are clearly distinguished, therefore PCR method provided by the invention identification Hp hypotypes are reliably effective, are applicable to clinic
Identification.The primer that especially the present patent application embodiment 1 provides clearly can effectively distinguish Hp2-1 and Hp2-2 hypotypes, and operate letter
It is single, there is important value for the genotype of the hoptoglobin of clinical accurate judgement diabetic, so as to be diabetic
Whether replenishing vitamins E provides important evidence.
A kind of method analyzed from gene angle Hp that the embodiment of the present invention is established, the above method are special by designing
Determine primer and carry out gene magnification, by being that can determine that Hp genotype to amplified production electrophoresis detection, this method is easy to operate fast
It is fast, accurate, it can be applied to clinical practice.
Claims (10)
- A kind of 1. primer pair for detecting hoptoglobin parting, it is characterised in that：The primer pair is：Forward primer (F):5’-GAGGGGAGCTTGCCTTTCCATTG-3’；Reverse primer (R):5’-ACCAGCCAGGGCTCAAAAATCTC-3’.
- 2. application of the primer pair in hoptoglobin parting is detected described in claim 1.
- 3. application according to claim 2, it is characterised in that：The primer pair detection determines that the parting of hoptoglobin is Hp1 hypotypes or Hp2 hypotypes.
- 4. application according to claim 3, it is characterised in that：When the primer pair detects the parting of determination hoptoglobin For Hp2 hypotypes when, the primer pair further detect determine hoptoglobin parting be Hp2-1 hypotypes or Hp2-2 hypotypes.
- 5. according to any described application of claim 2 to 4, it is characterised in that：Using the primer pair to including the combination The sample to be tested of globin gene carries out gene magnification and obtains amplified production, by carrying out electrophoresis detection to the amplified production Determine the genotype of the hoptoglobin of the testing gene.
- 6. application according to claim 5, it is characterised in that：The gene magnification, i.e. polymerase chain reaction (PCR) include Following steps：The pre-degeneration 3-5 minutes at a temperature of 90 DEG C to 100 DEG C；It is denatured 30 seconds to 50 at a temperature of 90 DEG C to 100 DEG C Second, anneal 20 seconds to 50 seconds at a temperature of 45 DEG C to 60 DEG C, extend 90 seconds to 240 seconds at a temperature of 70 DEG C to 74 DEG C, its Described in denaturing step, the annealing steps and it is described extension step be 25 to 40 circulation；Then at 70 DEG C to 74 DEG C At a temperature of extend eventually 3 to 10 minutes；Last keeping temperature is 3 DEG C to 10 DEG C.
- 7. application according to claim 6, it is characterised in that：Annealed 20 seconds to 50 seconds at a temperature of 47 DEG C to 52 DEG C.
- 8. application according to claim 6, it is characterised in that：The gene magnification, i.e. polymerase chain reaction (PCR) include Following steps：Pre-degeneration 5 minutes at a temperature of 94 DEG C；It is denatured 50 seconds at a temperature of 94 DEG C, is annealed at a temperature of 50 DEG C 30 seconds, extend 150 seconds at a temperature of 72 DEG C, wherein the denaturing step, the annealing steps and the extension step are 38 circulations；Then extend eventually at a temperature of 72 DEG C 6 minutes；Last keeping temperature is 4 DEG C.
- 9. application according to claim 5, it is characterised in that：The gene magnification, i.e. polymerase chain reaction (PCR) use The composition of mixed solution of buffer solution include：Three (methylol) methylglycines (Tricine), final concentration of 20mmol/L-35mmol/L,PH is 8.4-8.8；Potassium acetate, final concentration of 50mmol/L-80mmol/L；Magnesium acetate, final concentration of 1mmol/L-1.7mmol/L；Glycerine, final concentration of 6%-10%；Dimethyl sulfoxide (DMSO) (DMSO), final concentration of 0.5%-1.8%；Taq enzyme, final concentration of 0.8U-2.2U；Deoxyribonucleoside triphosphate (dNTP), final concentration of 150umol/L-400umol/L.
- 10. application according to claim 8, it is characterised in that：The gene magnification, i.e. polymerase chain reaction (PCR) make The composition of the mixed solution of buffer solution includes：Three (methylol) methylglycines (Tricine), final concentration of 25mmol/L, pH 8.7；Potassium acetate, final concentration of 80mmol/L；Magnesium acetate, final concentration of 1.2mmol/L；Glycerine, final concentration of 8%；Dimethyl sulfoxide (DMSO) (DMSO), final concentration of 1%；Taq enzyme, final concentration of 2U；Deoxyribonucleoside triphosphate (dNTP), final concentration of 400umol/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710833598.6A CN107475404A (en) | 2017-09-15 | 2017-09-15 | A kind of primer pair for detecting hoptoglobin parting and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710833598.6A CN107475404A (en) | 2017-09-15 | 2017-09-15 | A kind of primer pair for detecting hoptoglobin parting and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107475404A true CN107475404A (en) | 2017-12-15 |
Family
ID=60584522
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710833598.6A Withdrawn CN107475404A (en) | 2017-09-15 | 2017-09-15 | A kind of primer pair for detecting hoptoglobin parting and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107475404A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101142320A (en) * | 2004-07-12 | 2008-03-12 | 拉帕波特家族医学研究院 | Method for detecting the phenotype of a polymorphic protein |
-
2017
- 2017-09-15 CN CN201710833598.6A patent/CN107475404A/en not_active Withdrawn
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101142320A (en) * | 2004-07-12 | 2008-03-12 | 拉帕波特家族医学研究院 | Method for detecting the phenotype of a polymorphic protein |
Non-Patent Citations (2)
| Title |
|---|
| SUKAINA AL-BALAGHEE ET AL: "Determination of Haptoglobin Genotype in an Iranian Population with Idiopathic Generalized Epilepsy", 《REPORTS OF BIOCHEMISTRY & MOLECULAR BIOLOGY》 * |
| WERNER KOCH，ET AL: "Genotyping of the Common Haptoglobin Hp1/2 Polymorphism Based on PCR", 《CLINICAL CHEMISTRY》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lorey et al. | Universal newborn screening for Hb H disease in California | |
| CN104232770B (en) | PKU (Phenylketnuria) gene detection kit | |
| CN107988376A (en) | A kind of application of relevant SNP marker of the colour of skin in black-bone chicken Genotyping | |
| US20200270690A1 (en) | Serum/plasma LncRNA marker composition associated with auxiliary diagnosis of intrahepatic cholestasis of pregnancy and application thereof | |
| CN105506118A (en) | Primer pair used for detecting CYP2C19 genotypes, fluorescent probe, reagent kit and method | |
| CN105441439A (en) | Primers, probe and kit for detecting ALDH2 (aldehyde dehydrogenase gene2) gene polymorphism | |
| CN106521023B (en) | A kind of kit and method detecting Chinese HPFH and δ β-thalassemia | |
| CN104673891B (en) | A kind of detection method and kit of spinal muscular atrophy associated gene mutation | |
| CN106520967A (en) | Method and primer for detecting hereditary blood coagulation factor XI(F11) genes | |
| CN106834434B (en) | Nucleic acid, kit and method for detecting COX-1, COX-2 and GPIIIa gene polymorphism | |
| CN115074443A (en) | Human gene CHD1 site mutation and kit for detecting multiple site mutations of CDH1 gene | |
| CN107475404A (en) | A kind of primer pair for detecting hoptoglobin parting and its application | |
| JPWO2019074004A1 (en) | SNP detection method | |
| CN107400726A (en) | A kind of method for detecting hoptoglobin parting | |
| CN107475405A (en) | A kind of kit for detecting hoptoglobin parting | |
| CN108004309A (en) | Detect primer, method and the kit of MTHFR677, MTHFR1298 and MTRR66 | |
| CN104928297B (en) | The LPL new mutations Disease-causing gene of the familial hypertriglyceridemia disease of separation and the kit for detecting the gene | |
| CN106086215A (en) | The method in detection No. 4 full exon sequence mutational site of dyskeratosis congenita disease NHP2 gene and primer | |
| CN106399503A (en) | Primers, kit and method for detection of SJS/TEN caused by AEDs | |
| CN107400725A (en) | A kind of method for detecting hoptoglobin parting | |
| CN107400724A (en) | A kind of primer pair and its application for being used to detect hoptoglobin parting | |
| CN107475406A (en) | A kind of kit for being used to detect hoptoglobin parting | |
| CN105861657A (en) | Cow mastitis resistance related CNV fragment and applications thereof | |
| CN106939345B (en) | PCR-RFLP method and application for detecting type II diabetes susceptibility gene CREB1 single nucleotide polymorphism | |
| Elsaid et al. | Association of NOS3 (rs 2070744) and SOD2Val16Ala (rs4880) gene polymorphisms with increased risk of ESRD among Egyptian patients |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20171215 |