CN109402233A - The application of gene parting detecting reagent, its method and the detection of vitamin A Utilization ability - Google Patents
The application of gene parting detecting reagent, its method and the detection of vitamin A Utilization ability Download PDFInfo
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- CN109402233A CN109402233A CN201811373174.7A CN201811373174A CN109402233A CN 109402233 A CN109402233 A CN 109402233A CN 201811373174 A CN201811373174 A CN 201811373174A CN 109402233 A CN109402233 A CN 109402233A
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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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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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Abstract
The embodiment of the invention discloses the applications on a kind of gene parting detecting reagent, its method and vitamin A Utilization ability.The kit includes: first beacon probe as shown in SEQ ID 3, as shown in SEQ ID 1 and SEQ ID 2, with the matched the first primer pair of the first beacon probe；Second beacon probe as shown in SEQ ID 6 is matched with second beacon probe, second primer pair as shown in SEQ ID 4 and SEQ ID 6；The third beacon probe as shown in SEQ ID 9 is matched with the third beacon probe, the third primer pair as shown in SEQ ID 7 and SEQ ID 8；The 4th beacon probe as shown in SEQ ID 12 is matched with the 4th beacon probe, the 4th primer pair as shown in SEQ ID 10 and SEQ ID 11 and detection matched reagent.It is with simple and convenient, the advantages of high sensitivity, cost is relatively low.
Description
Technical field
The present invention relates to technical field of gene detection more particularly to a kind of gene parting detecting reagents, its method and dimension
Application in raw element A Utilization ability detection.
Background technique
Vitamin A is also known as retinol or antiophthalmic factor, is a kind of liposoluble vitamin, it, which has, maintains vision, promotees
Into the functions such as growth and development, complete and sound, enhancing immunocompetence, the removing free radical that maintain epithelial structure.
Food rich in vitamin A has two classes: first is that provitamin A, i.e., various carrotene, are present in plant food
In, such as greenery vegetables, yellow vegetables and fruits.The another kind of vitamin A for coming from animal food.This kind is energy
Enough vitamin As being directly immediately used by the body, are primarily present in animal's liver, fish, marine product, milk and dairy produce (non-defatted milk)
And in the animal foods such as birds, beasts and eggs.
The vitamin A of human body excess intake will cause poisoning, and only when required, human body can just turn beta carotene
Change vitamin A into.This feature makes beta carotene become a safe sources of vitamin A, does not have and ingests because excessive
And cause vitamin A progressive poisoning phenomenon.In addition, β-carrotene also has preferably in the fertility and growth for promoting animal
Effect.Have now been found that there are about 50 kinds of natural carotenoids can be converted into vitamin A.It is wherein important to have β-carrot
Element, alpha-carotene, gamma carotene etc., with the active highest of beta carotene, it often with chlorophyll and deposit.By β-Hu Luo
The vitamin A that Bu Su is converted to accounts for about the 2/3 of body vitamin A requirement.
There is the BCMO1 gene for encoding beta carotene monoxidase in human body.It is responsible for plant type Hu trailing plants
Bu Su (especially beta carotene) is converted into the retinol that can be used by cell.It is existing the study found that on the gene
The gene pleiomorphism of rs12934922 (R267S) and rs7501331 (A379V) are related to the expression activity for the enzyme that it is encoded.Example
Such as, while the crowd of 267S and 379V is carried, beta carotene monoxidase activity can be reduced to 60%, that is to say, that this
The ability of the bata-carotene conversion active vitamin A taken in class crowd body is weaker, and internal beta carotene level is higher.
In addition, being located at the site rs6564851 of the upstream BCMO1, gene pleiomorphism can also influence the gene table of BCMO1
It reaches, to affect in human body to the ability of beta carotene conversion active vitamin A.
And the vitamin A taken in from animal food and the active vitamin A being converted by carotenoid, it is most of
All it is previously stored in liver.RBP4 gene is for encoding retinol-binding proteins.It is expressed in liver, is that Huang is regarded in blood
The idiosyncratic carrier of alcohol, retinol can be transported to come in peripheral tissues from liver transfer and be added on utilization, so that activity dimension life
Plain A can play its biological function.
Existing research shows that the polymorphism in the site rs10882272 on RBP4 gene has with the expression of its albumen
It closes, the other crowd of CC and CT genotype, the crowd of the retinol level ratio TT type in blood is lower.That is, this kind of people
Group is lower to the transhipment Utilization ability of active vitamin A, is easy the risk of vitamine A deficiency.
Although providing some using genetic test, to determine other different vitamin contents methods in the prior art.But
It is that there are no a kind of kits that the detection of people's vitamin A Utilization ability can be carried out with efficient quick.How correlation is effectively passed through
Kit carry out Genotyping detection, different crowd is provided and corresponding suggests being problem in the urgent need to address.
Summary of the invention
In view of the above technical problems, the embodiment of the invention provides a kind of gene parting detecting reagent, its method and dimensions
Application on raw element A Utilization ability, with solution there are currently no detection kit or correlation technique to vitamin A Utilization ability into
The problem of row detection.
The first aspect of the embodiment of the present invention provides a kind of beacon probe of Genotyping detection.Wherein, the beacon is visited
The nucleic acid sequence of needle is as shown in SEQ ID 3, SEQ ID 6, SEQ ID 9 and/or SEQ ID 12；The end 5` of the beacon probe
It is connect with fluorescent reporter molecule, the end 3` is connected with quencher.
Optionally, the fluorescent reporter molecule includes FAM, HEX, Texas Red and Cy5, and the quencher includes BHQ-1
And BHQ-2.
Optionally, the beacon probe as shown in nucleic acid sequence SEQ ID 3 is used to detect the rs12934922 of BCMO1 gene
Site.
Optionally, the beacon probe as shown in nucleic acid sequence SEQ ID 6 is used to detect the position rs7501331 of BCMO1 gene
Point.
Optionally, the beacon probe as shown in nucleic acid sequence SEQ ID 9 is used to detect the position rs6564851 of BCMO1 gene
Point.
The second aspect of the embodiment of the present invention additionally provides a kind of gene parting detecting reagent.Wherein, the kit
Include:
First beacon probe, the nucleic acid sequence of first beacon probe is as shown in SEQ ID 3, the end 5` and fluorescence report
Molecule FAM connection is accused, the end 3` is connect with quencher BHQ-1；
With the matched the first primer pair of the first beacon probe；The nucleic acid sequence of the first primer pair such as SEQ ID 1
With shown in SEQ ID 2；
Second beacon probe, the nucleic acid sequence of second beacon probe is as shown in SEQ ID 6, the end 5` and fluorescence report
Molecule HEX connection is accused, the end 3` is connect with quencher BHQ-1；
With matched second primer pair of second beacon probe；The nucleic acid sequence of second primer pair such as SEQ ID 4
With shown in SEQ ID 6；
Third beacon probe, the nucleic acid sequence of the third beacon probe is as shown in SEQ ID 9, the end 5` and fluorescence report
Molecule Texas Red connection is accused, the end 3` is connect with quencher BHQ-2；
With the matched third primer pair of the third beacon probe；The nucleic acid sequence of the third primer pair such as SEQ ID 7
With shown in SEQ ID 8；
4th beacon probe, the nucleic acid sequence of the 4th beacon probe is as shown in SEQ ID 12, the end 5` and fluorescence report
Molecule Cy5 connection is accused, the end 3` is connect with quencher BHQ-2；
With matched 4th primer pair of the 4th beacon probe；The nucleic acid sequence such as SEQ ID of 4th primer pair
10 and SEQ ID 11 is shown and detects matched reagent.
Optionally, the detection matched reagent includes: HS Taq polymerase, UNG enzyme, MgCl2, PCR buffer, dU
Plus dNTP MIX, VA-Gene standard control product and VA-Gene negative controls；The PCR buffer includes 20mM
(NH4) 2SO4,10mM Tri-HCl and 2mM MgCl2。
The third aspect of the embodiment of the present invention additionally provides a kind of Genotyping detection method.The detection method includes:
Obtain sample DNA；Using gene parting detecting reagent as described above, multiple fluorescence quantitative PCR is carried out to the sample DNA
Amplification, obtains the Tm value of corresponding solubility curve；According to the Tm value, rs12934922 in the sample DNA is judged,
The genotype of rs7501331, rs6564851, rs10882272.
The fourth aspect of the embodiment of the present invention additionally provides gene parting detecting reagent as described above and gives birth in people to dimension
Application in plain A Utilization ability detection.
In technical solution provided in an embodiment of the present invention, the Taqman probe of molecular beacon and optimization is selected, in conjunction with melting
Curve method carries out Genotyping detection, and time-consuming easy to operate is short compared with traditional gene chips, high sensitivity and repetition
Property is good.And compared with Taqman probe hydrolysis method, 1 SNP site only needs to design 1 molecular beacon probe or optimization
Taqman probe has saved one times of cost, so as to quickly, easily detect the genotype and human body dimension of four kinds of corresponding genes
The Utilization ability situation of raw element A.The Genotyping detection method is simple and convenient, and high sensitivity, cost is relatively low.
Detailed description of the invention
Fig. 1 is one embodiment schematic diagram of gene parting detecting reagent provided in an embodiment of the present invention；
Fig. 2 is one embodiment flow chart of Genotyping detection method provided in an embodiment of the present invention；
Fig. 3 is one embodiment schematic diagram of the melting curve analysis result provided in an embodiment of the present invention in the channel FAM；
Fig. 4 is one embodiment schematic diagram of the melting curve analysis result provided in an embodiment of the present invention in the channel HEX；
Fig. 5 is one embodiment of the melting curve analysis result provided in an embodiment of the present invention in the channel Texas Red
Schematic diagram；
Fig. 6 is one embodiment schematic diagram of the melting curve analysis result provided in an embodiment of the present invention in the channel Cy5.
In sequence table:
SEQ ID 1 is the forward primer of the first primer pair；
SEQ ID 2 is the reverse primer of the first primer pair；
SEQ ID 3 is the first molecular beacon probe (not including fluorescent reporter molecule and quencher)；
SEQ ID 4 is the forward primer of the second primer pair；
SEQ ID 5 is the reverse primer of the second primer pair；
SEQ ID 6 is the second molecular beacon probe (not including fluorescent reporter molecule and quencher)；
SEQ ID 7 is the forward primer of third primer pair；
SEQ ID 8 is the reverse primer of third primer pair；
SEQ ID 9 is third molecular beacon probe (not including fluorescent reporter molecule and quencher)；
SEQ ID 10 is the forward primer of the 4th primer pair；
SEQ ID 11 is the reverse primer of the 4th primer pair；
SEQ ID 12 is the 4th molecular beacon probe (not including fluorescent reporter molecule and quencher).
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, those skilled in the art's every other implementation obtained without creative efforts
Example, shall fall within the protection scope of the present invention.
It should be noted that be expressed " being fixed on " another element when element, it can directly on the other element,
Or there may be one or more elements placed in the middle therebetween.When an element is expressed " connection " another element, it can be with
It is directly to another element or there may be one or more elements placed in the middle therebetween.Used in this specification
The orientation or position of the instructions such as term "vertical", "horizontal", "left" and "right", "upper", "lower", "inner", "outside", " bottom "
Relationship is to be based on the orientation or positional relationship shown in the drawings, and is merely for convenience of description of the present invention and simplification of the description, without referring to
Show or imply that signified device or element must have a particular orientation, be constructed and operated in a specific orientation, therefore cannot manage
Solution is the limitation to invention.In addition, term " first ", " second " etc. are used for description purposes only, and it should not be understood as instruction or dark
Show relative importance.
Unless otherwise defined, technical and scientific term all used in this specification is led with technology of the invention is belonged to
The normally understood meaning of the technical staff in domain is identical.Used term is only in the description of the invention in this specification
The purpose of description specific embodiment is not intended to the limitation present invention.Term "and/or" used in this specification includes
Any and all combinations of one or more related listed items.In addition, invention described below difference is implemented
Technical characteristic involved in mode can be combined with each other as long as they do not conflict with each other.
Fig. 1 is the schematic diagram of gene parting detecting reagent provided in an embodiment of the present invention.As shown in Figure 1, the reagent
Box includes box body 11, sponge partition, the PCR MIX mixing liquid pipe 13 for installing sample, VA-Gene standard control quality control 14 and
VA-Gene negative control quality control 15.
Wherein, box cover to be opened/closed is provided on the box body 11, the sponge partition 12 is embedded into box body 11, sponge
The pore of certain size size is provided on partition 12, for mixing liquid pipe 13, VA-Gene standard items pair for the PCR MIX
According to quality control 14 and the insertion of VA-Gene negative control quality control 15 to fix the pipe that these contain reagent.
The PCR MIX mixing liquid pipe 13 is equipped with detection matched reagent relevant to PCR reaction.In some embodiments,
The reagent that PCR MIX mixing liquid pipe 13 is equipped with includes: the HS Taq polymerase of 1U, the UNG enzyme of 0.5U, 3.25mM's
MgCl2, 1 × PCR Buffer (20mM (NH4)2SO4, 10mM Tri-HCl, 2mM MgCl2), the dU plus dNTP of 200uM
MIX, the upstream and downstream primer of 4 gene locis (rs12934922, rs7501331, rs6564851, rs10882272) to be checked and
Corresponding probe.
VA-Gene standard control quality control 14 and VA-Gene negative control quality control 15 are then respectively provided with VA-Gene standard
Product reference substance and VA-Gene negative controls.
In the present embodiment, 4 gene locis to be detected are respectively by four pairs of upstream and downstream primers and corresponding molecular beacon
The probe Tapman probe of improvement (or) is detected.The end 5` of each molecular beacon probe is connect with fluorescent reporter molecule
(such as FAM, HEX, Texas Red perhaps Cy5) end 3` is connected with quencher (such as BHQ-1 or BHQ-2).
Wherein, the first primer to and the first molecular beacon probe for detecting the site people BCMO1 gene rs12934922.The
The DNA sequence dna of one primer pair is as shown in SEQ ID1 and SEQ ID2.The DNA sequence dna of first molecular beacon probe such as SEQ ID3 institute
Show, the end 5` of the first molecular beacon probe is connect with fluorescent reporter molecule FAM, and the end 3` is connect with quencher BHQ-1.
Second primer pair and the second molecular beacon probe are for detecting the site people BCMO1 gene rs7501331.Second primer
Pair DNA sequence dna as shown in SEQ ID4 and SEQ ID5.The DNA sequence dna of second molecular beacon probe is as shown in SEQ ID6, and second
The end 5` of molecular beacon probe is connect with fluorescent reporter molecule HEX, and the end 3` is connect with quencher BHQ-1.
Third primer pair and third molecular beacon probe are for detecting the site people BCMO1 upstream region of gene rs6564851.Third
The DNA sequence dna of primer pair is as shown in SEQ ID7 and SEQ ID8.The DNA sequence dna of third molecular beacon probe as shown in SEQ ID9,
Third molecular beacon probe is the Tapman probe of improvement, and the end 5` connect with fluorescent reporter molecule Texas Red, the end 3` with it is sudden
It goes out agent BHQ-2 connection.
4th primer pair and the 4th molecular beacon probe are for detecting the site people RBP4 upstream region of gene rs10882272.4th
The DNA sequence dna of primer pair is as shown in SEQ ID10 and SEQ ID11.The DNA sequence dna of 4th molecular beacon probe such as SEQ ID12 institute
Show, the 4th molecular beacon probe is the Tapman probe of improvement, and the end 5` is connect with fluorescent reporter molecule Cy5, the end 3` and quenching
Agent BHQ-2 connection.
Specifically, in the kit each component specific source are as follows:
PCR MIX mixed liquor each component source: HS Taq polymerase is to be purchased from Bao Yi Bioisystech Co., Ltd, HS
Taq enzyme has hi-fi, can guarantee the base mispairing rate of amplified production in the critical field that sequencing allows.
UNG enzyme is to be purchased from Bao Yi Bioisystech Co., Ltd, due to use this kit to expand PCR product in contain
Uracil reacts 10min at 25 DEG C, and UNG enzyme can cut off uracil in the amplified production being accidentally introduced into early period, makes it not
Can become amplification template, UNG enzyme 95 DEG C inactivate, not influence amplification of the following people DNA as template, thus effectively prevent by
False positive results caused by the pollution of PCR product.
1 × PCR Buffer is by being purchased from 10 × PCR buffer of Shanghai Sheng Gong bioengineering Co., Ltd (containing (NH4)
2SO4、20mM MgCl2) dilution 10 times obtains.
DU plus dNTP MIX is to be purchased from Bao Yi Bioisystech Co., Ltd, contains dATP (2.5mM), dCTP
(2.5mM), dGTP (2.5mM), dUTP (7.5mM).3.25mM MgCl2It is voluntarily to be prepared according to the effect of multiplex amplification.
VA-Gene standard reference material is respectively by the AT type plasmid of the BCMO1-rs12934922 synthesized, BCMO1-
The CT type plasmid of rs7501331, the GT type plasmid of BCMO1 upstream region of gene rs6564851, the CT type matter of RBP4-rs10882272
Grain is mixed according to 1:1:1:1 ratio, and final concentration is each 1uM.
VA-Gene negative controls are free from target fragment aqua sterilisa.
Below in conjunction with specific example, the use process of said gene parting detecting reagent is described in detail.In the present embodiment
In, it uses by sequencing, it is known that the sample of Genotyping is detected, to investigate the specificity of mentioned reagent box detection sample
And accuracy.
Table 1 is 5 samples used in the present embodiment in the corresponding gene type of 4 gene locis.Such as table
Shown in lattice 1,5 samples include 12 kinds of gene types in different gene locis.
Table 1
| Sample number | rs12934922 | rs7501331 | rs6564851 | rs10882272 |
| No. 1 | AT | TT | GT | TT |
| No. 2 | AA | CC | GG | CC |
| No. 3 | AA | CT | TT | TT |
| No. 4 | TT | CC | GT | TT |
| No. 5 | AT | CT | GG | CT |
Fig. 2 be it is provided in this embodiment, to the VA-Gene standard control product and VA- in above-mentioned sample and kit
The method flow diagram that Gene negative controls are detected.
As shown in Figure 2, which comprises
S100: sample DNA is extracted.
In " the E.Z.N.A.TM Forensic DNA Kit " that the present embodiment is produced using OMEGA company in human mouth
Chrotoplast carries out DNA extraction, obtains the DNA of sample.
S200: sample DNA quality inspection.
The detection of concentration and purity is carried out to the DNA of extraction using Nano-1000 ultramicrospectrophotometer.Quality inspection requirement
DNA concentration needs to be greater than 0.2ng/ul, and the range of purity A260/A280 is 1.7-2.0.
S300: PCR reaction solution is prepared.
Then plus the sample to be examined DNA or VA-Gene of 2ul the PCR MIX mixed liquor of 23ul is dispensed into fluorescent PCR pipe,
Standard reference material or VA-Gene negative controls.
Add 2 reference substances by, needing to detect in this present embodiment 5 samples.Therefore, it needs in total in 7 fluorescent PCR pipes
In plus 23ul PCR MIX mixed liquor, then respectively plus 1-5 sample and each 2ul of negative controls and standard reference material.
S400: the above-mentioned PCR reaction tube equipped with PCR reaction solution is placed on fluorescent PCR instrument (Bio-Rad CFX 96)
Carry out PCR amplification and melting curve analysis.
The specific response procedures are as follows:
(1) 25 DEG C of 10min, 95 DEG C of 7min；
(2) 95 DEG C of 30s → 65~56 DEG C 15s (- 1 DEG C/cycle) → 72 DEG C of 15s, 10 circulations, wherein 65~56 DEG C of 15s
Each circulation declines 1 DEG C；
(3) 95 DEG C of 15s → 55 DEG C 20s → 76 DEG C 15s, 40 circulations；
(4) 95 DEG C of 30s → 30 DEG C 30s → 35~79.5 DEG C, wherein 35~79.5 DEG C with the heating rate of 0.3 DEG C/5s
Carry out melting curve analysis, and the fluorescence letter of the channel corresponding to this phase acquisition probe (FAM, HEX, Texas Red, Cy5)
Number.
S500: interpretation of result.
Fig. 3-Fig. 6 be the embodiment of the present invention 5 sample to be examined and two reference substances each channel melting curve analysis
As a result.Wherein, Fig. 3 is the channel FAM, and Fig. 4 is the channel HEX, and Fig. 5 is the channel Texas Red, and Fig. 6 is the channel Cy5, and NO1-NO5 divides
Not Biao Shi No. 1 to No. 5 sample, Std indicate standard reference material, NC indicate negative controls.
The melting curve analysis software carried by fluorescent PCR instrument reads standard control and 5 samples in the Tm value in each channel,
Each Tm value of sample is compareed with Tm value interpretation table (table 2), reads the corresponding gene type of sample.
Table 2
Gene type shown in testing result and table 1 by 1 to No. 5 sample shown in table 2 is compared can be with
Find out, the genotype of 5 parts of samples of the present embodiment detection is consistent with the genotype of its sequencing result, and accuracy and specificity are
It is 100%.
Genetic test based on 5 samples is as a result, can to carry out simple analysis to it as follows:
No. 1, No. 3, No. 4 this 3 samples sites relevant to BCMO1 activity of gene expression carry risk genotype,
RBP4 gene loci does not carry risk genotype, it is possible to think that this 3 are converted into work to the bata-carotene of intake in vivo
The efficiency of property vitamin A is relatively low.
If there is vitamine A deficiency occurs, it is proposed that replace vegetal dimension with the vitamin A in Some Animals source
The supplement of raw element A original.
And the RBP4 gene loci of No. 2 samples carries risk genes, it is weaker to the turn-over capacity of retinol relatively slow in vivo,
BCMO1 gene expression is normal, it is possible to think No. 2 provitamin As that can normally take in vegetalitas source.Therefore, there is dimension
It when raw element A deficiency disease, can suitably increase the intake of the provitamin A in vegetalitas source, and suggest not taking in excessive move
The vitamin A in physical property source, be easy because transhipment it is slow due to accumulate excessive retinol and have the phenomenon that vitamin A poisoning.
Two genes of No. 5 samples carry risk genotype, it should be noted that the intake of vitamin A, appropriate can supplement
The intake of vitamin A increases the utilization rate of internal vitamin A, to reduce or avoid vitamine A deficiency.
In conclusion kit provided in an embodiment of the present invention is directed to conversion of the human body to vitamin A is obtained from food
4 strong gene locis with Utilization ability correlation, develop primed probe and PCR reagent, utilize multiple asymmetric PCR and probe
The combination of melting curve method carries out the detection of Genotyping, has the advantages of simple and convenient, high sensitivity, cost is relatively low.
It, can according to the technique and scheme of the present invention and this hair it is understood that for those of ordinary skills
Bright design is subject to equivalent substitution or change, and all these changes or replacement all should belong to the guarantor of appended claims of the invention
Protect range.
Sequence table
<110>Shenzhen, which is made innovations, merges Science and Technology Ltd.
<120>application of gene parting detecting reagent, its method and the detection of vitamin A Utilization ability
<141> 2018-11-19
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 339
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
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sccacagcat ccggagaatg agctgggctg tggmsansgg ataacaacag gaaacgagms 120
anscctgtga aaatctgccc cttmsanscc gcaaagtggc atctacaacg cggmsansac 180
gttcctgtgt attggtgtcm sansacatct gcctcattga ctggmsansc tgtgtatgtc 240
ccagtgctta amsansccct accttggact cgcaamsans aaaccactcc tgaactcact 300
cmsanscatt tataaaaatg ccatggaccc ttttaagag 339
Claims (10)
1. a kind of beacon probe of Genotyping detection, which is characterized in that the nucleic acid sequence of the beacon probe such as SEQ ID 3,
Shown in SEQ ID 6, SEQ ID 9 and/or SEQ ID 12；The end 5` of the beacon probe is connect with fluorescent reporter molecule, the end 3`
It is connected with quencher.
2. beacon probe according to claim 1, which is characterized in that the fluorescent reporter molecule include FAM, HEX,
Texas Red and Cy5, the quencher include BHQ-1 and BHQ-2.
3. beacon probe according to claim 1, which is characterized in that the beacon probe as shown in nucleic acid sequence SEQ ID 3
For detecting the site rs12934922 of BCMO1 gene.
4. beacon probe according to claim 1, which is characterized in that the beacon probe as shown in nucleic acid sequence SEQ ID 6
For detecting the site rs7501331 of BCMO1 gene.
5. beacon probe according to claim 1, which is characterized in that the beacon probe as shown in nucleic acid sequence SEQ ID 9
For detecting the site rs6564851 of BCMO1 gene.
6. beacon probe according to claim 1, which is characterized in that the beacon as shown in nucleic acid sequence SEQ ID 12 is visited
Needle is used to detect the site rs10882272 of RBP4 gene.
7. a kind of gene parting detecting reagent, which is characterized in that the kit includes:
First beacon probe, the nucleic acid sequence of first beacon probe is as shown in SEQ ID 3, the end 5` and fluorescence report point
Sub- FAM connection, the end 3` is connect with quencher BHQ-1；
With the matched the first primer pair of the first beacon probe；The nucleic acid sequence of the first primer pair such as 1 He of SEQ ID
Shown in SEQ ID 2；
Second beacon probe, the nucleic acid sequence of second beacon probe is as shown in SEQ ID 6, the end 5` and fluorescence report point
Sub- HEX connection, the end 3` is connect with quencher BHQ-1；
With matched second primer pair of second beacon probe；The nucleic acid sequence of second primer pair such as 4 He of SEQ ID
Shown in SEQ ID 6；
Third beacon probe, the nucleic acid sequence of the third beacon probe is as shown in SEQ ID 9, the end 5` and fluorescence report point
Sub- Texas Red connection, the end 3` is connect with quencher BHQ-2；
With the matched third primer pair of the third beacon probe；The nucleic acid sequence of the third primer pair such as 7 He of SEQ ID
Shown in SEQ ID 8；
4th beacon probe, the nucleic acid sequence of the 4th beacon probe is as shown in SEQ ID 12, the end 5` and fluorescence report point
Sub- Cy5 connection, the end 3` is connect with quencher BHQ-2；
With matched 4th primer pair of the 4th beacon probe；The nucleic acid sequence of 4th primer pair such as 10 He of SEQ ID
SEQ ID 11 is shown and detects matched reagent.
8. gene parting detecting reagent according to claim 7, which is characterized in that the detection matched reagent includes:
HS Taq polymerase, UNG enzyme, MgCl2, PCR buffer, dU plus dNTP MIX, VA-Gene standard control product and
VA-Gene negative controls；
The PCR buffer includes 20mM (NH4) 2SO4,10mM Tri-HCl and 2mM MgCl2。
9. a kind of Genotyping detection method characterized by comprising
Obtain sample DNA；
Using gene parting detecting reagent as claimed in claim 7 or 8, multiple fluorescence quantitative is carried out to the sample DNA
PCR amplification obtains the Tm value of corresponding solubility curve；
According to the Tm value, rs12934922 in the sample DNA, rs7501331, rs6564851 are judged, rs10882272's
Genotype.
10. the gene parting detecting reagent of such as claim 8 or 9 is in people to the application in the detection of vitamin A Utilization ability.
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