CN108715887A - The detection method of DNA rapid extracting methods and Methylene tetrahydrofolate reductase gene polymorphism for fluorescent PCR detection - Google Patents
The detection method of DNA rapid extracting methods and Methylene tetrahydrofolate reductase gene polymorphism for fluorescent PCR detection Download PDFInfo
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- 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
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- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention discloses the detection methods of the DNA rapid extracting methods and Methylene tetrahydrofolate reductase gene polymorphism that are detected for fluorescent PCR.The rapid extracting method of DNA for fluorescent PCR detection is to take detection sample whole blood that lysate is added；It is incubated for the first time at 52-57 DEG C；It is incubated for second at 92-98 DEG C；It is then centrifuged for obtaining DNA to be checked；Then fluorescence quantitative PCR detection is carried out.The method of the present invention is easy to operate, time-saving and efficiency, and Idiotype is strong, high sensitivity, stability are good, is suitable for large-scale clinical application.
Description
Technical field
The present invention relates to polymorphic detection fields, more particularly, to the DNA rapid extracting methods of fluorescent PCR detection and Asia
The detection method of methylenetetrahydrofreductase reductase gene polymorphism.
Background technology
Methylenetetrahydrofolate reductase (5,10-methylenetetrahydrofolate reductase, MTHFR),
One of the important enzyme during folic acid-methionine metabolism, it can make the homocysteine (Hcy) in blood maintain compared with
Low level.There are three types of Genotypings in 677 sites by MTHFR：Wild type CC, heterozygous mutant CT and homozygous mutant TT.It should
Site mutates, and enzymatic activity may be made to reduce, influence folic acid metabolism, causes Hcy levels in blood to increase, to cause the heart
Vascular diseases, cranial vascular disease etc..Therefore for people at highest risk, such as primary hypertension patient, with homocysteine liter
High hypertensive patient, with the risk factors such as hyperlipidemia and hyperglycemia hypertensive patient, have occurred and that cardiovascular and cerebrovascular disease
Patient, crowd, the pregnant woman etc. that have cardiovascular and cerebrovascular disease family history carry out the detections of MTHFR C677T gene pleiomorphisms, can
The generation of prevention of cardiovascular disease, cranial vascular disease, for judging that it is important that hypertension type and subsequent administrations guidance all have
Meaning.Also pregnant woman's reasonable supplement folic acid, newborns' birth such as prediction harelip, Down's syndrome, neural tube defect can be instructed to lack
It falls into, is suitable for preventive test before normal Mr. and Mrs fertility.However, traditional MTHFR detection methods be required for DNA carry out it is pure
Change, and DNA purification process is very complicated, and the human and material resources of consuming, resource are huge, and has cross contamination between sample
Problem.
Invention content
The purpose of the present invention is to provide a kind of time-saving and efficiency, Idiotype is strong, high sensitivity, stability are good, is suitable for big
The detection method of methylenetetrahydrofolate reductase (MTHFR) gene pleiomorphism of the clinical application of scale.
To achieve the above object, the present invention provides a kind of rapid extracting method of the DNA for fluorescent PCR detection, special
Sign is, takes detection sample whole blood that lysate is added；It is incubated for the first time at 52-57 DEG C；It is incubated for second at 92-98 DEG C；Then
Centrifugation.
Further, the Tris-HCl, the EDTA of 1~50mM, 0.01~0.5%w/ that the cracking formula of liquid is 5~100mM
The surfactant of v, the Proteinase K of 50~250 μ g, the ammonium chloride of 20~200mM, the sodium chloride of 10~200mM, pH value are
6.5~8.5；Preferably, surfactant is SDS or NP-40 or Brj-35；
Optional, the volume ratio for detecting sample whole blood and lysate is 1：(8~15)；Preferably, detection sample whole blood with
The volume ratio of lysate is 1：(8~12)；It is furthermore preferred that the volume ratio of detection sample whole blood and lysate is 1：11.
Further, the temperature that the first time is incubated is 53~56 DEG C；Preferably, the temperature being incubated for the first time is 55 DEG C；
Optional, the time that the first time is incubated is 8~12 minutes；Preferably, the time being incubated for the first time is 10 points
Clock；
The temperature that the first time is incubated is 93~97 DEG C；Preferably, the temperature being incubated for the first time is 95 DEG C；
Optional, second of the time being incubated is 3~8 minutes；Preferably, second of the time being incubated is 5 minutes；
Optional, the time of centrifugation is 0.5~2 minute；Preferably, the time of centrifugation is 1 minute.
The present invention also provides a kind of detection methods of Methylene tetrahydrofolate reductase gene polymorphism, which is characterized in that
Fluorescence quantitative PCR detection is carried out after extracting DNA using the above method.
Further, the primer and probe of the quantitative fluorescent PCR is SEQ ID NO:Sequence shown in 1-4；
Optional, 20 μ L reaction systems of the quantitative fluorescent PCR are：2 × TaqMan Fast Advanced of 10 μ L
Master Mix, the primer of a concentration of 300nM, the probe of a concentration of 250nM, 2 μ L samples to be tested, surplus is sterile water；
Optional, the response procedures of the quantitative fluorescent PCR are：50℃2min；95℃20s；95 DEG C of 3s, 60 DEG C of 30s, are followed
Ring 40 times；50℃1min.
The present invention also provides a kind of kit for Methylene tetrahydrofolate reductase gene polymorphic detection, features
It is, including lysate, SEQ ID NO:The primer and probe of sequence shown in 1-4.
Further, the Tris-HCl, the EDTA of 1~50mM, 0.01~0.5%w/ that the cracking formula of liquid is 5~100mM
The surfactant of v, the Proteinase K of 50~250 μ g, the ammonium chloride of 20~200mM, the sodium chloride of 10~200mM, pH value are
6.5~8.5；Preferably, surfactant is SDS or NP-40 or Brj-35.
The present invention also provides a kind of kits for Methylene tetrahydrofolate reductase gene polymorphic detection
Application method, which is characterized in that take detection sample whole blood that lysate is added；It is incubated for the first time at 52-57 DEG C；At 92-98 DEG C
Secondary incubation；It is then centrifuged for obtaining DNA；Fluorescence quantitative PCR detection again.
Further, the Tris-HCl, the EDTA of 1~50mM, 0.01~0.5%w/ that the cracking formula of liquid is 5~100mM
The surfactant of v, the Proteinase K of 50-250 μ g, the ammonium chloride of 20-200mM, the sodium chloride of 10-200mM, pH value be 6.5~
8.5；Preferably, surfactant is SDS or NP-40 or Brj-35；
Optional, the volume ratio for detecting sample whole blood and lysate is (8~15)；Preferably, it detects sample whole blood and splits
The volume ratio for solving liquid is 1：(8~12)；It is furthermore preferred that the volume ratio of detection sample whole blood and lysate is 1：11；
Optional, the temperature that the first time is incubated is 53-56 DEG C；Preferably, the temperature being incubated for the first time is 55 DEG C；
Optional, the time that the first time is incubated is 8~12 minutes；Preferably, the time being incubated for the first time is 10 points
Clock；
The temperature that the first time is incubated is 93~97 DEG C；Preferably, the temperature being incubated for the first time is 95 DEG C；
Optional, second of the time being incubated is 3~8 minutes；Preferably, second of the time being incubated is 5 minutes；
Optional, the time of centrifugation is 0.5~2 minute；Preferably, the time of centrifugation is 1 minute.
Further, 20 μ L reaction systems of the quantitative fluorescent PCR are：2 × TaqMan Fast Advanced of 10 μ L
Master Mix, the primer of a concentration of 300nM, the probe of a concentration of 250nM, 2 μ L samples to be tested, surplus is sterile water；
Optional, the response procedures of the quantitative fluorescent PCR are：50℃2min；95℃20s；95 DEG C of 3s, 60 DEG C of 30s, are followed
Ring 40 times；50℃1min.
Proteinase K in lysate of the present invention is using preceding addition.
In actual mechanical process, nucleic acid extraction is the rate-limiting step of detection of nucleic acids.Existing kit for detecting nucleic acid is logical
Common two methods extract nucleic acid：(1) paramagnetic particle method and (2) silica gel embrane method.Two methods are required for cracking whole blood using lysate
Nucleic acid is released, recycles magnetic bead or pellosil to adsorb nucleic acid, Nucleic Acid Elution is got off for subsequent again after removing impurity
In detection of nucleic acids.Cracking, absorption, removal of impurities, elution process need multiple pipetting, and it is more to there is (1) operating procedure, take
It is long；(2) the problems such as error is easy when being manually operated.When handling multiple samples (48 or 96 samples) at the same time, above problem meeting
More highlight.
The present invention proposes, with after enzymatic cleavage liquid lytic cell, directly pyrolysis product to be used in follow-up PCR, greatly letter
Change nucleic acid purification step, shortened the operating time, and essentially eliminates the possibility of maloperation.Compared to traditional lysate,
The lysate of this method is milder, contains only a small amount of surfactant, and pyrolysis product is allow to be directly added into PCR reactions
Without influencing subsequent gene parting.Cracking is divided into two stages：(1) the enzymic digestion stage.At this stage, surfactant and egg
White enzyme K destroys cell membrane, nuclear membrane structure release goes out nucleic acid, while Proteinase K also digests the histone and cell in nucleosome
DNA enzymatic in matter further makes nucleic acid separate out, and prevents DNA degradation.(2) the heat inactivation stage.At this stage, Proteinase K
By high temperature deactivation, the archaeal dna polymerase in its follow-up PCR reaction of degrading is prevented.Also, thermal change occurs for some albumen in lysate
Property formed precipitation, removal can be facilitated after centrifugation, reduce the interference to subsequent reactions.If without first step enzymic digestion rank
Section, nucleic acid release is insufficient, can reduce the sensitivity of follow-up PCR reaction, and if without second stage, remaining excess
Archaeal dna polymerase during active protease can react PCR impacts.
In design, the primer amplification segment of this method covers the SNP institutes that will be detected to the primer and probe of the present invention
Parting is carried out to amplified production in site, while using the saltant type probe of the wild-type probe of FAM labels and HEX labels.Expand
Increasing primer has good specificity, and to genome, other genes do not respond to.Pass through the design to two probes, two probes
It only reacts to special amplified production, can clearly distinguish homozygous wildtype, heterozygous and homozygous mutant.
The primer sequence of the present invention is as follows：
Advantageous effect of the present invention is：The method of the present invention is easy to operate, time-saving and efficiency, and Idiotype is strong, high sensitivity, steady
It is qualitative good, it is suitable for large-scale clinical application.Embodiment 4-7 is demonstrated to blood sample, low leucocyte blood sample, height urine
Sour blood sample, high triglyceride blood sample, can realize Genotyping.
Description of the drawings
Fig. 1 is the result figure of influence of the pH value of Tris buffer solutions in embodiment 1 to nucleic acid extraction.
Fig. 2 is influence result figure of the surfactant to nucleic acid extraction in embodiment 2.
Fig. 3 is the result figure of influence of 3 Proteinase K of embodiment to nucleic acid extraction.
Fig. 4 is the result figure that embodiment 4 carries out blood sample Genotyping.
Fig. 5 is the result figure that embodiment 5 carries out low leucocyte blood sample Genotyping
Fig. 6 is the result figure that embodiment 6 carries out high lithemia blood sample Genotyping.
Fig. 7 is the result figure that embodiment 7 carries out high triglyceride blood sample Genotyping.
Specific implementation mode
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end
Same or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attached
The embodiment of figure description is exemplary, it is intended to for explaining the present invention, and is not considered as limiting the invention.Embodiment
In particular technique or condition person is not specified, according to technology or condition described in document in the art or according to the description of product
Book carries out.Reagents or instruments used without specified manufacturer, being can be with conventional products that are commercially available.
The rapid extracting method of a kind of DNA for fluorescent PCR detection, which is characterized in that detection sample whole blood is taken to be added
Lysate；It is incubated for the first time at 52-57 DEG C；It is incubated for second at 92-98 DEG C；It is then centrifuged for.
Further, the Tris-HCl, the EDTA of 1~50mM, 0.01~0.5%w/ that the cracking formula of liquid is 5~100mM
The surfactant of v, the Proteinase K of 50-250 μ g, the ammonium chloride of 20-200mM, the sodium chloride of 10-200mM, pH value be 6.5~
8.5；Preferably, surfactant is SDS or NP-40 or Brj-35；
Optional, the volume ratio for detecting sample whole blood and lysate is 1：(8~15)；Preferably, detection sample whole blood with
The volume ratio of lysate is 1：(8~12)；It is furthermore preferred that the volume ratio of detection sample whole blood and lysate is 1：11.
Further, the temperature that the first time is incubated is 53~56 DEG C；Preferably, the temperature being incubated for the first time is 55 DEG C；
Optional, the time that the first time is incubated is 8~12 minutes；Preferably, the time being incubated for the first time is 10 points
Clock；
The temperature that the first time is incubated is 93~97 DEG C；Preferably, the temperature being incubated for the first time is 95 DEG C；
Optional, second of the time being incubated is 3~8 minutes；Preferably, second of the time being incubated is 5 minutes；
Optional, the time of centrifugation is 0.5~2 minute；Preferably, the time of centrifugation is 1 minute.
The present invention also provides a kind of detection methods of Methylene tetrahydrofolate reductase gene polymorphism, which is characterized in that
Fluorescence quantitative PCR detection is carried out after extracting DNA using the above method.
Further, the primer and probe of the quantitative fluorescent PCR is SEQ ID NO:Sequence shown in 1-4；
Optional, 20 μ L reaction systems of the quantitative fluorescent PCR are：2 × TaqMan Fast Advanced of 10 μ L
Master Mix, the primer of a concentration of 300nM, the probe of a concentration of 250nM, 2 μ L samples to be tested, surplus is sterile water；
Optional, the response procedures of the quantitative fluorescent PCR are：50℃2min；95℃20s；95 DEG C of 3s, 60 DEG C of 30s, are followed
Ring 40 times；50℃1min.
The present invention also provides a kind of kit for Methylene tetrahydrofolate reductase gene polymorphic detection, features
It is, including lysate, SEQ ID NO:The primer and probe of sequence shown in 1-4.
Further, the Tris-HCl, the EDTA of 1~50mM, 0.01~0.5%w/ that the cracking formula of liquid is 5~100mM
The surfactant of v, the Proteinase K of 50-250 μ g, the ammonium chloride of 20-200mM, the sodium chloride of 10-200mM, pH value be 6.5~
8.5；Preferably, surfactant is SDS or NP-40 or Brj-35.
The present invention also provides a kind of kits for Methylene tetrahydrofolate reductase gene polymorphic detection
Application method, which is characterized in that take detection sample whole blood that lysate is added；It is incubated for the first time at 52-57 DEG C；At 92-98 DEG C
Secondary incubation；It is then centrifuged for obtaining DNA；Fluorescence quantitative PCR detection again.
Further, the Tris-HCl, the EDTA of 1~50mM, 0.01~0.5%w/ that the cracking formula of liquid is 5~100mM
The surfactant of v, the Proteinase K of 50-250 μ g, the ammonium chloride of 20-200mM, the sodium chloride of 10-200mM, pH value be 6.5~
8.5；Preferably, surfactant is SDS or NP-40 or Brj-35；
Optional, the volume ratio for detecting sample whole blood and lysate is (8~15)；Preferably, it detects sample whole blood and splits
The volume ratio for solving liquid is 1：(8~12)；It is furthermore preferred that the volume ratio of detection sample whole blood and lysate is 1：11；
Optional, the temperature that the first time is incubated is 53-56 DEG C；Preferably, the temperature being incubated for the first time is 55 DEG C；
Optional, the time that the first time is incubated is 8-12 minutes；Preferably, the time being incubated for the first time is 10 points
Clock；
The temperature that the first time is incubated is 93-97 DEG C；Preferably, the temperature being incubated for the first time is 95 DEG C；
Optional, second of the time being incubated is 3-8 minutes；Preferably, second of the time being incubated is 5 minutes；
Optional, the time of centrifugation is 0.5-2 minutes；Preferably, the time of centrifugation is 1 minute.
Further, 20 μ L reaction systems of the quantitative fluorescent PCR are：2 × TaqMan Fast Advanced of 10 μ L
Master Mix, the primer of a concentration of 300nM, the probe of a concentration of 250nM, 2 μ L samples to be tested, surplus is sterile water；
Optional, the response procedures of the quantitative fluorescent PCR are：50℃2min；95℃20s；95 DEG C of 3s, 60 DEG C of 30s, are followed
Ring 40 times；50℃1min.
Proteinase K in lysate of the present invention is using preceding addition.
Embodiment 1：Influence of the pH value of Tris buffer solutions to nucleic acid extraction
The Tris-HCl that the cracking formula of liquid is 1M, pH is respectively 6.0,6.5,7.0,7.5,8.0,8.5,9.0.50mM
EDTA, the surfactant of 0.5%w/v, the Proteinase K of 50 μ g, 200mM NH4Cl, 10mM NaCl；Preferably, surface is lived
Property agent be Brj-35 or NP-40 or SDS.
It is CC, CT, the human serum of TT types, concrete operations to choose genotype respectively：10 μ L of EDTA anticoagulations are extracted, addition is split
110 μ L of liquid are solved, mixing is shaken, 55 DEG C incubate 10 minutes, and 95 DEG C incubate 5 minutes, and 12000rpm is centrifuged 1 minute.Take 2 μ L of supernatant into
Row qPCR Genotypings.The 20 μ L reaction systems of qPCR are：2 × TaqMan Fast Advanced Master Mix of 10 μ L,
Concentration is respectively primer (the i.e. SEQ ID NO of 300nM:Sequence shown in 1-2), concentration be respectively 250nM probe (i.e. SEQ ID
NO:Sequence shown in 3-4), 2 μ L samples to be tested, surplus is sterile water；The response procedures of qPCR are：50℃2min；95℃20s；95
DEG C 3s, 60 DEG C of 30s are recycled 40 times；50℃1min.
Experimental result is shown in Fig. 1, from Fig. 1 it can be found that pH<6.5 or>When 8.5, qPCR ct values compared with other lysate highers,
When proving between pH6.5-8.0, lysate nucleic acid extraction efficiency highest.
Embodiment 2：Influence of the surfactant to nucleic acid extraction
The Tris-HCl that the cracking formula of liquid is 50mM, pH is respectively the EDTA of 8.0,50mM, 0.05~0.5%w/v
Surfactant, the Proteinase K of 50 μ g, 200mM NH4Cl, 10mM NaCl,；Preferably, surfactant be Brj-35 or
NP-40 or SDS.
It is CC, CT, the human serum of TT types, concrete operations to choose genotype respectively：10 μ L of EDTA anticoagulations are extracted, addition is split
110 μ L of liquid are solved, mixing is shaken, 55 DEG C incubate 10 minutes, and 95 DEG C incubate 5 minutes, and 12000rpm is centrifuged 1 minute.Take 2 μ L of supernatant into
Row qPCR Genotypings.The 20 μ L reaction systems of qPCR are：2 × TaqMan Fast Advanced Master Mix of 10 μ L,
Concentration is respectively primer (the i.e. SEQ ID NO of 300nM:Sequence shown in 1-2), concentration be respectively 250nM probe (i.e. SEQ ID
NO:Sequence shown in 3-4), 2 μ L samples to be tested, surplus is sterile water；The response procedures of qPCR are：50℃2min；95℃20s；95
DEG C 3s, 60 DEG C of 30s are recycled 40 times；50℃1min.
Experimental result is shown in Fig. 2, and from Fig. 2 it can be found that when surfactant concentration is more than 0.2%, qPCR reactions cannot
Occur, when surfactant is less than 0.1%, ct values are compared with other lysate highers, it was demonstrated that at high concentrations can in surfactant
Inhibit DNA polymerase activity, and when concentration is too low, cell cannot crack completely, reduce nucleic acid extraction efficiency.So choosing 0.1
When the surfactant of~0.2%w/v, effect is preferable.
Embodiment 3：Influence of the Proteinase K to nucleic acid extraction
The Tris-HCl that the cracking formula of liquid is 50mM, pH is respectively the EDTA of 8.0,50mM, the surface of 0.5%w/v
Activating agent, the Proteinase K of 50 μ g or is added without Proteinase K, 200mM NH4Cl, 10mM NaCl,；Preferably, surfactant
For Brj-35 or NP-40 or SDS.
It is CC, CT, the human serum of TT types, concrete operations to choose genotype respectively：10 μ L of EDTA anticoagulations are extracted, addition is split
110 μ L of liquid are solved, mixing is shaken, 55 DEG C incubate 10 minutes, and 95 DEG C incubate 5 minutes, and 12000rpm is centrifuged 1 minute.Take 2 μ L of supernatant into
Row qPCR Genotypings.The 20 μ L reaction systems of qPCR are：2 × TaqMan Fast Advanced Master Mix of 10 μ L,
Concentration is respectively primer (the i.e. SEQ ID NO of 300nM:Sequence shown in 1-2), concentration be respectively 250nM probe (i.e. SEQ ID
NO:Sequence shown in 3-4), 2 μ L samples to be tested, surplus is sterile water；The response procedures of qPCR are：50℃2min；95℃20s；95
DEG C 3s, 60 DEG C of 30s are recycled 40 times；50℃1min.
Experimental result is shown in Fig. 3, are found from Fig. 3, and when there are Proteinase K, qPCR ct values are significantly lower than and are not added with Proteinase K
Lysate, it was demonstrated that Proteinase K can with fully degraded blood in foreign protein, discharge nucleic acid, improve the amplification efficiency of qPCR.
Embodiment 4：Genotyping is carried out to blood sample
The Tris-HCl that the cracking formula of liquid is 50mM, pH is respectively the EDTA of 8.0,50mM, 0.05~0.5%w/v
Surfactant, the Proteinase K of 50 μ g, 200mM NH4Cl, 10mM NaCl,；Preferably, surfactant be Brj-35 or
NP-40 or SDS.
It is CC, CT, the human serum of TT types, concrete operations to choose genotype respectively：10 μ L of EDTA anticoagulations are extracted, addition is split
110 μ L of liquid are solved, mixing is shaken, 55 DEG C incubate 10 minutes, and 95 DEG C incubate 5 minutes, and 12000rpm is centrifuged 1 minute.Take 2 μ L of supernatant into
Row qPCR Genotypings.The 20 μ L reaction systems of qPCR are：2 × TaqMan Fast Advanced Master Mix of 10 μ L,
Concentration is respectively primer (the i.e. SEQ ID NO of 300nM:Sequence shown in 1-2), concentration be respectively 250nM probe (i.e. SEQ ID
NO:Sequence shown in 3-4), 2 μ L samples to be tested, surplus is sterile water；The response procedures of qPCR are：50℃2min；95℃20s；95
DEG C 3s, 60 DEG C of 30s are recycled 40 times；50℃1min.Experimental result is shown in Fig. 1.Wherein A is that the wild homozygote CC of MTHFR are different
DNA extraction method fluorescence quantitive PCR typing result and sequencing result；B is that MTHFR heterozygotes CT is glimmering with different DNA extraction methods
Fluorescent Quantitative PCR genotyping result；C is the different DNA extraction method fluorescence quantitive PCR typing knots of the wild no mutant homozygote TT of MTHFR
Fruit and sequencing result；
The column formulation step of comparative example：Genomic DNA is extracted using business whole blood genome extracts kit.Step：
(1) 10 μ L of EDTA anticoagulations are extracted, 20 μ L Proteinase Ks are added, 500 μ L BB3 are in whole blood, vortex 15s mixings,
It is incubated at room temperature 10min.
(2) centrifugal column is added in whole pyrolysis products by of short duration centrifugation, and 12000g centrifuges 1min, abandons efflux.
(3) 500 μ L CB3,12000g centrifugation 30s are added, abandon efflux.
(4) 500 μ L WB3,12000g centrifugation 30s are added, abandon efflux.
(5) step 4 is repeated.
(6) 12000g centrifuges 2min, thoroughly removes remaining WB3.
(7) centrifugal column is placed in clean centrifuge tube, 50 μ L deionized waters is added in column center, are stored at room temperature 1min,
12000g centrifuges 1min.Eluted dna.
The DNA of 2 μ L elutions is taken to carry out qPCR Genotypings.QPCR reaction systems are same as above with condition.From fig. 4, it can be seen that
The DNA of the method (rapid fractionation method i.e. in figure) of the present invention and the extraction of column formulation, qPCR react ct values, fluorescence intensity, parting
As a result all same.And it is consistent with sequencing result, three figures of right column are corresponding sequencing result.Prove this method and commercial silica gel film
Centrifugal column method is compared, and the nucleic acid of extraction is equally applicable to qPCR Genotypings.And pipetting step needed for this method, behaviour
Make the time far less than commercial DNA extraction method.Time-saving and efficiency is further embodied, Idiotype is strong, high sensitivity, stability are good
Advantage.
Embodiment 5：Genotyping is carried out to low leucocyte blood sample
The Tris-HCl that the cracking formula of liquid is 50mM, pH is respectively the EDTA of 8.0,50mM, 0.05~0.5%w/v
Surfactant, the Proteinase K of 50 μ g, 200mM NH4Cl, 10mM NaCl,；Preferably, surfactant be Brj-35 or
NP-40 or SDS.
It chooses low leucocyte blood sample, extracts 10 μ L of EDTA anticoagulations, 110 μ L of lysate are added, shake mixing, 55 DEG C
It incubates 10 minutes, 95 DEG C incubate 5 minutes, and 12000rpm is centrifuged 1 minute.2 μ L of supernatant are taken to carry out qPCR Genotypings.The 20 of qPCR
μ L reaction systems are：2 × TaqMan Fast Advanced Master Mix of 10 μ L, concentration are respectively the primer of 300nM
(i.e. SEQ ID NO:Sequence shown in 1-2), concentration be respectively 250nM probe (i.e. SEQ ID NO:Sequence shown in 3-4), 2 μ L
Sample to be tested, surplus are sterile water；The response procedures of qPCR are：50℃2min；95℃20s；95 DEG C of 3s, 60 DEG C of 30s, cycle 40
It is secondary；50℃1min.Experimental result is shown in Fig. 5.Wherein A is column formulation (column formulation of the method and step with embodiment 4) and the present invention
Rapid fractionation method extracts DNA quantitative fluorescent PCR genotypic results respectively；B is different white blood cell concentrations to quantitative fluorescent PCR
The influence of CT values.
From fig. 5, it can be seen that for same sample, method of the invention (rapid fractionation method i.e. in figure) is carried with column formulation
The DNA taken, qPCR react ct values, fluorescence intensity, genotyping result all same (A).As quantity of leucocyte reduces, fluorescent quantitation
The ct values of PCR are increasing, and for same sample, are centrifuged than commercial silica gel film with the nucleic acid (grey dot) that the method for the present invention is extracted
The ct values that column carries nucleic acid (black side's point) want small, it was demonstrated that the method for the present invention is more efficient when extracting low leukocyte samples nucleic acid.
Meanwhile linearly also superior to commercial silica gel film centrifugal column method, it was demonstrated that this method has good stability.
Embodiment 6：Genotyping is carried out to high lithemia blood sample
The Tris-HCl that the cracking formula of liquid is 50mM, pH is respectively the EDTA of 8.0,50mM, 0.05~0.5%w/v
Surfactant, the Proteinase K of 50 μ g, 200mM NH4Cl, 10mM NaCl,；Preferably, surfactant be Brj-35 or
NP-40 or SDS.
High lithemia blood sample is chosen, 10 μ L of EDTA anticoagulations is extracted, 110 μ L of lysate is added, shake mixing, 55 DEG C of temperature
It educates 10 minutes, 95 DEG C incubate 5 minutes, and 12000rpm is centrifuged 1 minute.2 μ L of supernatant are taken to carry out qPCR Genotypings.The 20 μ L of qPCR
Reaction system is：2 × TaqMan Fast Advanced Master Mix of 10 μ L, concentration be respectively the primer of 300nM (i.e.
SEQ ID NO:Sequence shown in 1-2), concentration be respectively 250nM probe (i.e. SEQ ID NO:Sequence shown in 3-4), 2 μ L it is to be measured
Sample, surplus are sterile water；The response procedures of qPCR are：50℃2min；95℃20s；95 DEG C of 3s, 60 DEG C of 30s are recycled 40 times；
50℃1min.Experimental result is shown in Fig. 6.Wherein A is column formulation (column formulation of the method and step with embodiment 4) and of the invention quick
Extraction method extracts DNA quantitative fluorescent PCR genotypic results respectively；B is different uric acid levels to quantitative fluorescent PCR CT values
It influences.
From fig. 6, it can be seen that for same sample, method of the invention (rapid fractionation method i.e. in figure) is carried with column formulation
The DNA taken, qPCR react ct values, fluorescence intensity, genotyping result all same (A).With the raising of uric acid level, fluorescent quantitation
The ct values of PCR are basically unchanged, and illustrate that uric acid will not impact extraction and qPCR reactions, it was demonstrated that this method has good stabilization
Property.
Embodiment 7：Genotyping is carried out to high triglyceride blood sample
The Tris-HCl that the cracking formula of liquid is 50mM, pH is respectively the EDTA of 8.0,50mM, 0.05~0.5%w/v
Surfactant, the Proteinase K of 50 μ g, 200mM NH4Cl, 10mM NaCl,；Preferably, surfactant be Brj-35 or
NP-40 or SDS.
High triglyceride blood sample is chosen, 10 μ L of EDTA anticoagulations is extracted, 110 μ L of lysate is added, shakes mixing, 55
DEG C incubate 10 minutes, 95 DEG C incubate 5 minutes, 12000rpm centrifuge 1 minute.2 μ L of supernatant are taken to carry out qPCR Genotypings.QPCR's
20 μ L reaction systems are：2 × TaqMan Fast Advanced Master Mix of 10 μ L, concentration are respectively the primer of 300nM
(i.e. SEQ ID NO:Sequence shown in 1-2), concentration be respectively 250nM probe (i.e. SEQ ID NO:Sequence shown in 3-4), 2 μ L
Sample to be tested, surplus are sterile water；The response procedures of qPCR are：50℃2min；95℃20s；95 DEG C of 3s, 60 DEG C of 30s, cycle 40
It is secondary；50℃1min.Experimental result is shown in Fig. 7.Wherein A is column formulation (column formulation of the method and step with embodiment 4) and the present invention
Rapid fractionation method extracts DNA quantitative fluorescent PCR genotypic results respectively；B is different triglyceride levels to quantitative fluorescent PCR
The influence of CT values.
From figure 7 it can be seen that for same sample, method of the invention (rapid fractionation method i.e. in figure) is carried with column formulation
The DNA taken, qPCR react ct values, fluorescence intensity, genotyping result all same (A).With the raising of triglyceride level, fluorescence is fixed
The ct values of amount PCR are basically unchanged, and illustrate that triglycerides will not impact extraction and qPCR reactions, it was demonstrated that this method has very well
Stability.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art are not departing from the principle of the present invention and objective
In the case of can make changes, modifications, alterations, and variations to the above described embodiments within the scope of the invention.
SEQUENCE LISTING
<110>Shenzhen University
<120>DNA rapid extracting methods and Methylene tetrahydrofolate reductase gene polymorphism for fluorescent PCR detection
Detection method
<130> SZDX-18001-CNI
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 24
<212> DNA
<213>It is artificial synthesized
<400> 1
tgacctgaag cacttgaagg agaa 24
<210> 2
<211> 24
<212> DNA
<213>It is artificial synthesized
<400> 2
ggaagaatgt gtcagcctca aaga 24
<210> 3
<211> 15
<212> DNA
<213>It is artificial synthesized
<400> 3
aatcggctcc cgcag 15
<210> 4
<211> 19
<212> DNA
<213>It is artificial synthesized
<400> 4
tgatgaaatc gactcccgc 19
Claims (10)
1. the rapid extracting method of DNA for fluorescent PCR detection a kind of, which is characterized in that detection sample whole blood addition is taken to split
Solve liquid；It is incubated for the first time at 52-57 DEG C；It is incubated for second at 92-98 DEG C；It is then centrifuged for.
2. the rapid extracting method described in claim 1 for the DNA of fluorescent PCR detection, which is characterized in that the lysate
Formula be 5~100mM Tris-HCl, the surfactant of the EDTA of 1~50mM, 0.01~0.5%w/v, 50~250 μ g's
Proteinase K, the ammonium chloride of 20~200mM, the sodium chloride of 10~200mM, pH value are 6.5~8.5；Preferably, surfactant
For SDS or NP-40 or Brj-35；
Optional, the volume ratio for detecting sample whole blood and lysate is 1：(8~15)；Preferably, detection sample whole blood and cracking
The volume ratio of liquid is 1：(8~12)；It is furthermore preferred that the volume ratio of detection sample whole blood and lysate is 1：11.
3. the rapid extracting method described in claim 1 for the DNA of fluorescent PCR detection, which is characterized in that the first time incubates
The temperature educated is 53~56 DEG C；Preferably, the temperature being incubated for the first time is 55 DEG C；
Optional, the time that the first time is incubated is 8~12 minutes；Preferably, the time being incubated for the first time is 10 minutes；
The temperature that the first time is incubated is 93~97 DEG C；Preferably, the temperature being incubated for the first time is 95 DEG C；
Optional, second of the time being incubated is 3~8 minutes；Preferably, second of the time being incubated is 5 minutes；
Optional, the time of centrifugation is 0.5~2 minute；Preferably, the time of centrifugation is 1 minute.
4. a kind of detection method of Methylene tetrahydrofolate reductase gene polymorphism, which is characterized in that use claim 1-3
Fluorescence quantitative PCR detection is carried out after any one the method extraction DNA.
5. the detection method of Methylene tetrahydrofolate reductase gene polymorphism described in claim 4, which is characterized in that described glimmering
The primer and probe of Fluorescent Quantitative PCR is SEQ ID NO:Sequence shown in 1~4；
Optional, 20 μ L reaction systems of the quantitative fluorescent PCR are：2 × TaqMan Fast Advanced of 10 μ L
Master Mix, the primer of a concentration of 300nM, the probe of a concentration of 250nM, 2 μ L samples to be tested, surplus is sterile water；
Optional, the response procedures of the quantitative fluorescent PCR are：50℃2min；95℃20s；95 DEG C of 3s, 60 DEG C of 30s, cycle 40
It is secondary；50℃1min.
6. a kind of kit for Methylene tetrahydrofolate reductase gene polymorphic detection, which is characterized in that including cracking
Liquid, SEQ ID NO:The primer and probe of sequence shown in 1-4.
7. being used for the kit of Methylene tetrahydrofolate reductase gene polymorphic detection described in claim 6, which is characterized in that
The formula of the lysate is the Tris-HCl of 5~100mM, the surface-active of the EDTA of 1~50mM, 0.01~0.5%w/v
Agent, the Proteinase K of 50~250 μ g, the ammonium chloride of 20~200mM, the sodium chloride of 10~200mM, pH value are 6.5~8.5；It is preferred that
, surfactant is SDS or NP-40 or Brj-35.
8. the user described in a kind of claim 6 for the kit of Methylene tetrahydrofolate reductase gene polymorphic detection
Method, which is characterized in that take detection sample whole blood that lysate is added；It is incubated for the first time at 52-57 DEG C；It is incubated for the second time at 92-98 DEG C
It educates；It is then centrifuged for obtaining DNA；Fluorescence quantitative PCR detection again.
9. the application method described in claim 8 for the kit of Methylene tetrahydrofolate reductase gene polymorphic detection,
It is characterized in that, Tris-HCl, the EDTA of 1~50mM that the cracking formula of liquid is 5~100mM, 0.01~0.5%w/v's
Surfactant, the Proteinase K of 50~250 μ g, the ammonium chloride of 20~200mM, the sodium chloride of 10-200mM, pH value be 6.5~
8.5；Preferably, surfactant is SDS or NP-40 or Brj-35；
Optional, the volume ratio for detecting sample whole blood and lysate is (8~15)；Preferably, sample whole blood and lysate are detected
Volume ratio be 1：(8~12)；It is furthermore preferred that the volume ratio of detection sample whole blood and lysate is 1：11；
Optional, the temperature that the first time is incubated is 53~56 DEG C；Preferably, the temperature being incubated for the first time is 55 DEG C；
Optional, the time that the first time is incubated is 8~12 minutes；Preferably, the time being incubated for the first time is 10 minutes；
The temperature that the first time is incubated is 93~97 DEG C；Preferably, the temperature being incubated for the first time is 95 DEG C；
Optional, second of the time being incubated is 3~8 minutes；Preferably, second of the time being incubated is 5 minutes；
Optional, the time of centrifugation is 0.5~2 minute；Preferably, the time of centrifugation is 1 minute.
10. the application method described in claim 8 for the kit of Methylene tetrahydrofolate reductase gene polymorphic detection,
It is characterized in that, 20 μ L reaction systems of the quantitative fluorescent PCR are：2 × TaqMan Fast Advanced of 10 μ L
Master Mix, the primer of a concentration of 300nM, the probe of a concentration of 250nM, 2 μ L samples to be tested, surplus is sterile water；
Optional, the response procedures of the quantitative fluorescent PCR are：50℃2min；95℃20s；95 DEG C of 3s, 60 DEG C of 30s, cycle 40
It is secondary；50℃1min.
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| CN114990110A (en) * | 2022-07-19 | 2022-09-02 | 江西农业大学 | Non-destructive sampling method for field butterfly monitoring |
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