CN108103181A - The detection method and detection kit in hypertriglyceridemia mutational site - Google Patents
The detection method and detection kit in hypertriglyceridemia mutational site Download PDFInfo
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- CN108103181A CN108103181A CN201711349756.7A CN201711349756A CN108103181A CN 108103181 A CN108103181 A CN 108103181A CN 201711349756 A CN201711349756 A CN 201711349756A CN 108103181 A CN108103181 A CN 108103181A
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- 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
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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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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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
- C12Q1/6869—Methods for sequencing
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a kind of detection methods in hypertriglyceridemia mutational site, target fragment is amplified using Single-tube multiplex-PCR by designing specific primer, and designs sequencing primer and obtained target fragment is sequenced using the Sanger PCR sequencing PCRs of non-rubber tapping purifying.The invention also discloses a kind of detection kits in hypertriglyceridemia mutational site.The present invention realizes the sequencing of the genes such as multiplex amplification LPL and non-rubber tapping purifying；The present invention only expands target gene, has the advantages that at low cost, target is clear and definite；The present invention expands tens segments simultaneously by single tube, and detection flux is high, and overcome substance PCR takes time and effort problem；The present invention is easy to operate quick, and routine experimentation personnel can be operated to specifications；During present invention sequencing, need not tap rubber purifying, it is meant that easy to operate and PCR tuples can be enough and identical or close without worry stripe size.The present invention is easy to promote, wide market.
Description
Technical field
The present invention relates to biomedical sector, more particularly to a kind of detection method in hypertriglyceridemia mutational site and
Detection kit.
Background technology
Lipoproteinesterase (lipoprotein lipase, LPL) is the key enzyme of hydrolyzing triglyceride, which obstacle occurs
It can make blood plasma chylomicron and the lipoprotein aggregation rich in triglycerides, clinical manifestation is chylomicronemia, blood triglyceride
Rise.Research shows that lpl gene defect or mutation can cause the generation of severe hypertriglyceridemia (HTG).In addition to LPL,
The gene mutations such as LMF1, GPIHBP1, APOA5, GPD1 or defect can also cause severe primary hypertriglyceridemia.Above-mentioned base
Variation/defect of cause can influence LPL activity, hinder triglyceride hydrolysis, ultimately result in severe hypertriglyceridemia.For base
Because of mutation/defect patient, become hypertriglyceridemia from now on using Molecular screening, efficient diagnosis and accurate treatment in time and examine
Treat the recent tendency of development.But still lack targetedly Molecular screening product both at home and abroad.LPL、GPIHBP1、APOA5、
Catastrophe of the gene mutations/defect such as LMF1, GPD1 in HTG patient is still not clear.It is sieved due to a lack of corresponding gene mutation
Technology is looked into, and then hinders the research and development and clinical research of accurate medicine.It is contemplated that primary hypertriglyceridemiapatients patients
The gene mutation for screening such as LPL, GPIHBP1, APOA5, LMF1, GPD1 are unfolded, carries out personalized precisely diagnosis and treatment for such patient and establishes
Fixed clinical and scientific research basis.
The sequencing approach that Frederick Sanger and his colleague had devised dideoxy chain-termination in 1977, it is former
Reason is that the ddNTP (dideoxy nucleotide) of fluorescent marker is mixed into synthesis material dNTP (deoxynucleotide) so that reaction is eventually
Only at the ddNTP of insertion, generate with a series of nucleotide of A, T, C, G four groups of different lengths terminated, then in high score
Electrophoresis is detected in the PAGE glue distinguished, so as to obtain visible DNA base sequence.Sanger sequencing technologies have passed through 30 years
Continue to develop with it is perfect, the DNA fragmentation for being up to 1,000bp can be sequenced now, and to the reading of each base
Accuracy rate is taken to be up to 99.999%, this causes its great clinical value in gene mutation or SNP detections, but its flux
It is not high so that when multiple target areas are sequenced, time-consuming, work is heavy, efficiency-cost ratio is low, therefore limit single amplification
Sanger PCR sequencing PCRs the widely using clinically of son.Also there is the report that Sanger is sequenced after multiplex PCR, but it reacts tuple
It is low, and need to tap rubber and purify and then be sequenced again, still belong to the Sanger PCR sequencing PCRs of single amplicon.Rubber tapping purifying is so that sequencing
It is complicated for operation and limit PCR tuples.In order to ensure to tap rubber successfully, it is necessary to which the stripe size difference of amplification is apparent, this is limited
The design of primer.
With the rise of high-flux sequence or next-generation sequencing (NGS), genome sequencing is allowed to be possibly realized, but it is next-generation
(NGS) is sequenced, and there is the shortcomings of read tablet is short, price is high.Although sequencing price had in past more than 10 years to be significantly reduced,
Still without reaching the acceptable degree of clinical practice.In order to reduce cost, full sequencing of extron group and target sequence PCR sequencing PCR quilt
Developed, and mixed by multiple samples and be sequenced further to reduce sequencing cost, such as designed for cancer and
The application of the gene panel of other complex diseases, but cumbersome sample prepares and collects, builds storehouse, sequencing, assembling, analysis waited
Time-consuming for journey, is unfavorable for the quick diagnosis and tracking and monitoring of disease, which has limited its application clinically, and more by with
In clinical or biological study.In addition to sequencing technologies, there are qPCR technologies, genetic chip skill for the method for genetic test
Art etc., they cannot provide complete sequence information.QPCR can detect rare mutation, and still its flux is relatively low and can only examine
Survey known mutations.The technical costs of genetic chip is high, sensitivity is low.
A kind of inexpensive, high-throughput, instant convenience, the sequencing approach that target is clear and definite, error rate is low are needed in clinical practice,
And it is sequenced using the Sanger that non-glue purification is carried out after multiplex PCR and meets this demand well.For tens target regions,
Sanger sequencings have many advantages, such as that at low cost, detection flux is higher, convenient to use, interpretation of result is simple after multiplex PCR.It can
To say, it has filled up the vacancy between the Sanger sequencings of single amplicon and high-flux sequence, inherits Sanger sequencings
Precisely, the advantage of read tablet length overcomes the shortcomings that its flux is low and the next-generation sequencing deficiency that (NGS) is of high cost, process is cumbersome,
Become a kind of possible ways for clinically detecting multiple mutation site
The content of the invention
In view of the above-mentioned deficiencies in the prior art, the technical problem to be solved by the present invention is that provide a kind of high glycerine three
The detection method and detection kit in ester mass formed by blood stasis mutational site.
This method need not tap rubber purifying in sequencing, but the enzymatic process of operation easy to use, it is then direct on
Machine be sequenced, this not only it is extremely convenient also imply that PCR tuples can it is enough and without worry stripe size it is identical or close, from
And detection flux, flexibility, simplicity are substantially increased, while this also requires that the non-specific amplification of multiplex PCR will be extremely
Without set peak phenomenon when few and sequencing primer specificity very high will ensure to be sequenced with this.
LPL, GPIHBP1, APOA5-1, LMF1, GPD1 gene are target by the present invention, are designed and are developed for this 5
The target sequence sequencing approach of 15 exon regions of gene.Multiple PCR technique can realize this 15 segments of Single tube amplification, instead
Product directly upper machine sequencing after enzymatic is answered, obtains the sequence of high quality, analysis can find position and the species of mutation.
This will provide the guidance of accurate medicine for the prevention of hypertriglyceridemia, diagnose and treat.
The technical solution adopted by the present invention is：A kind of detection method in hypertriglyceridemia mutational site, passes through design
Going out specific primer recycles Single-tube multiplex-PCR amplified reaction to obtain target fragment, and designs sequencing primer and cut again using non-
Obtained target fragment is sequenced in the Sanger PCR sequencing PCRs of glue purification.
Preferably, the specific primer includes：
For expanding the specific primer of LPL-1 segments, forward primer is：SEQ ID Nos：1 or 2, reverse primer is
SEQ ID Nos：3 or 4；
For expanding the specific primer of LPL-2 segments, forward primer is：SEQ ID Nos：5 or 6, reverse primer is
SEQ ID Nos：7 or 8；
For expanding the specific primer of LPL-3 segments, forward primer is：SEQ ID Nos：9 or 10, reverse primer is
SEQ ID Nos：11 or 12；
For expanding the specific primer of LPL-4 segments, forward primer is：SEQ ID Nos：13 or 14, reverse primer is
SEQ ID Nos：15 or 16；
For expanding the specific primer of LPL-5 segments, forward primer is：SEQ ID Nos：17 or 18, reverse primer is
SEQ ID Nos：19 or 20；
For expanding the specific primer of LPL-6 segments, forward primer is：SEQ ID Nos：21 or 22, reverse primer is
SEQ ID Nos：23 or 24；
For expanding the specific primer of LPL-7 segments, forward primer is：SEQ ID Nos：25 or 26, reverse primer is
SEQ ID Nos：27 or 28；
For expanding the specific primer of LPL-8 segments, forward primer is：SEQ ID Nos：29 or 30, reverse primer is
SEQ ID Nos：31 or 32；
For expanding the specific primer of LPL-9 segments, forward primer is：SEQ ID Nos：33 or 34, reverse primer is
SEQ ID Nos：35 or 36；
For expanding the specific primer of LPL-10 segments, forward primer is：SEQ ID Nos：37 or 38, reverse primer
For SEQ ID Nos：39 or 40.
For expanding the specific primer of APOA5-1 segments, forward primer is：SEQ ID Nos：41 or 42, reverse primer
For SEQ ID Nos：43 or 44；
For expanding the specific primer of GPIHBP1-1 segments, forward primer is：SEQ ID Nos：45 or 46, reversely draw
Object is SEQ ID Nos：47 or 48；
For expanding the specific primer of GPIHBP1-2 segments, forward primer is：SEQ ID Nos：49 or 50, reversely draw
Object is SEQ ID Nos：51 or 52；
For expanding the specific primer of LMF1 segments, forward primer is：SEQ ID Nos：53 or 54, reverse primer is
SEQ ID Nos：55 or 56；
For expanding the specific primer of GPD1 segments, forward primer is：SEQ ID Nos：57 or 58, reverse primer is
SEQ ID Nos：59 or 60.
Preferably, the sequencing primer includes：
For the sequencing primer of LPL-1 segments, sequence is SEQ ID Nos：61；
For the sequencing primer of LPL-2 segments, sequence is SEQ ID Nos：62；
For the sequencing primer of LPL-3 segments, sequence is SEQ ID Nos：63；
For the sequencing primer of LPL-4 segments, sequence is SEQ ID Nos：64；
For the sequencing primer of LPL-5 segments, sequence is SEQ ID Nos：65；
For the sequencing primer of LPL-6 segments, sequence is SEQ ID Nos：66；
For the sequencing primer of LPL-7 segments, sequence is SEQ ID Nos：67；
For the sequencing primer of LPL-8 segments, sequence is SEQ ID Nos：68；
For the sequencing primer of LPL-9 segments, sequence is SEQ ID Nos：69；
For the sequencing primer of LPL-10 segments, sequence is SEQ ID Nos：70；
For the sequencing primer of APOA5-1 segments, sequence is SEQ ID Nos：71；
For the sequencing primer of GPIHBP1-1 segments, sequence is SEQ ID Nos：72；
For the sequencing primer of GPIHBP1-2 segments, sequence is SEQ ID Nos：73；
For the sequencing primer of LMF1 segments, sequence is SEQ ID Nos：74；
For the sequencing primer of GPD1 segments, sequence is SEQ ID Nos：75.
Preferably, the response procedures of the Single-tube multiplex-PCR amplification are：95 DEG C of Taq enzymes activate 15 minutes；95℃DNA
Denaturation 15 seconds, 60 DEG C of primer annealings 20 seconds, 70 DEG C of primer extends 90 seconds repeat 45 cycles；70 DEG C extend 10 minutes：4 DEG C of drops
Temperature 10 minutes.
Preferably, the Sanger PCR sequencing PCRs of the non-rubber tapping purifying comprise the following steps：After crossing column purification PCR product,
A certain amount of target fragment is extracted, is sequenced using directly upper machine after enzymatic.
A kind of detection kit in hypertriglyceridemia mutational site, uses above-mentioned hypertriglyceridemia to be mutated
The detection method in site carries out hypertriglyceridemia mutational site detection.
Preferably, the detection kit includes reaction mixture, Taq enzyme and negative quality-control product.
Preferably, the reaction mixture includes：The MgCl2 solution of 1-6mM, the PCR of the dNTPs of 0.1-0.5mM, 1X
Buffer, the specific primer that 30 concentration are 0.05-1.0 μM；The Tris-HCl that the feminine gender quality-control product is 10-100mM is molten
Liquid or deionized water.
The beneficial effects of the invention are as follows：The present invention realizes the sequencing side of the genes such as multiplex amplification LPL and non-rubber tapping purifying；
The present invention is different from genome sequencing, and the method only expands target gene, therefore has the advantages that at low cost, target is clear and definite；
The present invention expands tens segments simultaneously by single tube, and detection flux is high, and overcome substance PCR takes time and effort problem；This hair
Bright easy to operate quick, routine experimentation personnel can be operated to specifications；During present invention sequencing, it need not tap rubber pure
Change, it is meant that easy to operate and PCR tuples can be enough and identical or close without worry stripe size；The present invention uses
Sanger PCR sequencing PCRs be genetic test goldstandard, read tablet is long, error rate is low, can detect known and unknown mutation, as a result
Analysis letter is convenient.The present invention is easy to promote, wide market.
Description of the drawings
Fig. 1 is the running gel figure of the multi-PRC reaction in a kind of embodiment of the present invention；
Fig. 2 is the Sequencing chromatogram of the LPL-6 in a kind of embodiment of the present invention；
Fig. 3 is the Sequencing chromatogram of the LPL-9 in a kind of embodiment of the present invention；
Fig. 4 is the Sequencing chromatogram of the GPD1 in a kind of embodiment of the present invention；
Fig. 5 is that the LPL-4 segments in a kind of embodiment of the present invention compare the mutational site found；
Fig. 6 is that the LPL-6 segments in a kind of embodiment of the present invention compare the mutational site found.
Specific embodiment
With reference to embodiment, the present invention is described in further detail, to make those skilled in the art with reference to specification
Word can be implemented according to this.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein are not precluded from one or more
The presence or addition of a other elements or its combination.
A kind of detection method in hypertriglyceridemia mutational site of the present invention, comprises the following steps：
1) sample DNA is extracted；
2) specific primer is designed, then Single-tube multiplex-PCR amplified reaction is carried out to the sample DNA of extraction and obtains target patch
Section；
3) sequencing primer is designed, obtained target fragment is surveyed using the Sanger PCR sequencing PCRs of non-rubber tapping purifying
Sequence；
4) sequencing result is analyzed, is then again compared gene order on NCBI therewith with sequence alignment program
It is right, the site of heterozygosis or homozygous mutation is obtained, completes the detection in hypertriglyceridemia mutational site.
In the present invention, the gene informations such as LPL are obtained on NCBI, are included for the isogenic exon 1s of LPL and part
Sub-district designs the primer of multipair specific amplification, and the primer mixing selected carries out multiple reaction, wherein continuing to optimize suitably
The reaction conditions such as primer concentration, magnesium ion concentration, enzyme concentration, PCR annealing temperatures, to obtain satisfied amplification.Amplification effect
Fruit is confirmed by gel electrophoresis and sequencing result.Figure one gives a kind of electrophoresis of the multi-PRC reaction obtained by embodiment
Glue figure is multi-PRC reaction running gel figure (the 4% gel M of the DNA profiling containing 5ng in 30 μ L reaction systems：
marker；N：Negative control)
The present invention for 10 extrons of LPL-1,2 extrons of GPIHBP1,1 of APOA5-1, LMF1, GPD1
Extron designs multipair specific primer, and Tm values are at 60 DEG C or so, and length 18-24bp, G/C content is in 30-70%, primer
Evaluation display does not have serious hairpin structure and dimer generates.The specific primer sequence of 15 amplified fragments designs altogether
It is classified as：
Specific primer sequence table
Then target fragment is amplified using Single-tube multiplex-PCR by the specific primer designed.In amplification procedure not
It is disconnected to optimize the reaction conditions such as suitable primer concentration, magnesium ion concentration, enzyme concentration, PCR annealing temperatures, to obtain satisfied amplification
As a result.Wherein, a kind of preferred pcr amplification reaction program is：95 DEG C of Taq enzymes activate 15 minutes；95 DEG C of DNA are denatured 15 seconds, and 60
DEG C primer annealing 20 seconds, 70 DEG C of primer extends 90 seconds, repeats 45 cycles；70 DEG C extend 10 minutes：4 DEG C cool down 10 minutes.
Sequencing primer is finally designed to survey obtained target fragment using the Sanger PCR sequencing PCRs of non-rubber tapping purifying
Sequence.The method that the sequencing of the present invention is purified without rubber tapping, but remaining primer, dNTP directly in enzymatic removal PCR product
Deng.The Sanger PCR sequencing PCRs for the non-rubber tapping purifying that the present invention uses comprise the following steps：Cross column purification PCR product, extraction 2-10 μ
The target fragment of L obtains sequencing mixed liquor using 30-50 μ L are diluted to after enzymatic, then takes 1-2 in each sequencing reaction
The sequencing mixed liquor of μ L and the sequencing primer of 1.5-3 μ L are sequenced.Sequencing primer sequence is：
Sequencing primer sequence table
The present invention also provides a kind of detection kits in hypertriglyceridemia mutational site, use above-mentioned high glycerine
The detection method in three ester mass formed by blood stasis mutational sites carries out hypertriglyceridemia mutational site detection.The detection kit includes reaction
Mixed liquor, Taq enzyme and negative quality-control product.Wherein, the reaction mixture includes：The MgCl2 solution of 3-5mM, 0.2-0.4mM's
The PCR buffer of dNTPs, 1X, the specific primer that 30 concentration are 0.05-1.0 μM；The feminine gender quality-control product is 10-
The Tris-HCl solution or deionized water of 100mM.
The specific embodiment of the present invention presented below, a kind of detection method in hypertriglyceridemia mutational site,
Comprise the following steps：
1. prepared by sample DNA and quality-control product：
Commodity in use DNA extraction kit extracts the DNA of Jurkat cell, measures the concentration of DNA of extraction again by it
It is diluted to 0.5-10ng/ μ L.The negative quality-control product of Tris-HCl conducts for producing 10mM is to be melted using preceding room temperature, vortex shakes
It swings and of short duration centrifugation.
2. multiplexed PCR amplification：
2.1st, according to the reaction condition reagent preparation having determined：Reaction mixture and Taq enzyme taking-up are placed in room in advance
Temperature, vortex vibration and of short duration centrifugation after thawing.Determine stoichiometric number N, N=sample number (n) to be checked × 3+ quality-control products number+1.It calculates anti-
The addition of mixed liquor and Taq enzyme is answered, is calculated as follows：
| Component | Reaction mixture | Taq enzyme |
| Volume (μ L) | 24.52×N | 0.48×N |
A sterile centrifugation tube is taken to prepare above-mentioned reaction system, reagent is all added thereto rear vortex and vibrates 10 seconds, of short duration
Centrifugation.Then above-mentioned mixed liquor is dispensed by 25 μ L/ pipes into PCR reaction tubes
2.2nd, it is loaded：The sample DNA and negative quality-control product for taking 5 μ L are added in PCR reaction tubes, concussion mixing, of short duration centrifugation
Prepare reaction afterwards.
2.3rd, PCR reactions are carried out on ABIPCR instrument：Response procedures are arranged to：
3. carry out Sanger sequencing reactions：Using a certain amount of enzymatic PCR product and 30 μ L are diluted to, each sequencing is anti-
2 μ L of loading, sequencing primer additive amount are (10 μM) 2 μ L in answering.Sequencing machine is ABI 3730.
4. carry out sequence analysis：Sequencing result is analyzed using Ugene, obtains DNA sequence dna information；Again with Vector NTI with
The upper gene orders of NCBI compare the site for obtaining heterozygosis or homozygous mutation；Complete the detection in hypertriglyceridemia mutational site.
The mutation position that LPL-4 segments compare the mutational site found and LPL-6 segments compare discovery is respectively illustrated with reference to Fig. 5 and Fig. 6
Point.
Although the embodiments of the present invention have been disclosed as above, but its be not restricted in specification and embodiment it is listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, it is of the invention and unlimited
In specific details.
Denomination of invention：A kind of detection method and detection kit in hypertriglyceridemia mutational site
SEQ ID Nos 1： 5'-CTCTTCTCGTTGGCAGGGTTG-3'
SEQ ID Nos 2： 5'-GGCAGGGTTGATCCTCATTA-3'
SEQ ID Nos 3： 5'-TCTCATCCTCAGTTCGGGTGG-3'
SEQ ID Nos 4： 5'-AACCCGGCAGAGAGAAAAAG-3'
SEQ ID Nos 5： 5'-CGTTGGAGCATCTGTTGTTCT-3'
SEQ ID Nos 6： 5'-TGTCTCTCTGTCAACTTTGCTCA-3'
SEQ ID Nos 7： 5'-CATGAACCTGTGGCTTCAG-3'
SEQ ID Nos 8： 5'-CATGAACCTGTGGCTTCAG-3'
SEQ ID Nos 9： 5'-GCCGTTCCTCAACTCAACTC-3'
SEQ ID Nos 10： 5'-CCTTGGAAGGGACAGACCT-3'
SEQ ID Nos 11： 5'-AATATCCACGCTGATTCTGAAG-3'
SEQ ID Nos 12： 5'-TTGAATGCCCCCAGAAAATA-3'
SEQ ID Nos 13： 5'-CGGAAAAGTGAAACAAAAGAAAA-3'
SEQ ID Nos 14： 5'-CTCTCTCTTACCTGTAACAC-3'
SEQ ID Nos 15： 5'-GTCATGTCTCCGGGAAGAAC-3'
SEQ ID Nos 16： 5'-CAGTGAGAGCGTCTGTGTTTTT-3'
SEQ ID Nos 17： 5'-ACCATGACTGTAGAATAGGAGCTAA-3'
SEQ ID Nos 18： 5'-GGAGCCAAGCCTCCTTTTA-3'
SEQ ID Nos 19： 5'-GGCTCTAAGGTGGTCATGCT-3'
SEQ ID Nos 20： 5'-GCCATTATTGAGGTCAGTGGA-3'
SEQ ID Nos 21： 5'-GCCGCTACCACCAAGAATATC-3'
SEQ ID Nos 22： 5'-CACAGGACTATATCCTTGGGTGA-3'
SEQ ID Nos 23： 5'-CCAGGGCTCAGGGTTTAGTA-3'
SEQ ID Nos 24： 5'-GGAACAGAGATGATGACTG-3'
SEQ ID Nos 25： 5'-TCCGGTTTGAGTGCTAGTGA-3'
SEQ ID Nos 26： 5'-GGTTCCATGTGTGTGCACTT-3'
SEQ ID Nos 27： 5'-CTAGGCATCGCTCTCTGCTT-3'
SEQ ID Nos 28： 5'-CAATATCAAATAGAGGAAAGACCTCA-3'
SEQ ID Nos 29： 5'-ATGCCATCGACCTTCATTTT-3'
SEQ ID Nos 30： 5'-GGATAAATGCTGGAATGTGGAT-3'
SEQ ID Nos 31： 5'-GAAGACTCCTAAAGAAAATCTACATCA-3'
SEQ ID Nos 32： 5'-TGAAATACAAAAATACTGTCACTTCC-3'
SEQ ID Nos 33： 5'-TGATTCTGATGTGGCCTGAG-3'
SEQ ID Nos 34： 5'-TCTCTTGGTGAAAGCCCAGT-3'
SEQ ID Nos 35： 5'-TACATATGCAGAAGGACTAAG-3'
SEQ ID Nos 36： 5'-TGATCAAAGCATTCAATCAG-3'
SEQ ID Nos 37： 5'-CCCCTGGGTTTATTCTCACA-3'
SEQ ID Nos 38： 5'-CCTGCCATTCTCTGATCCAT-3'
SEQ ID Nos 39： 5'-CAAGGGTAGGGCTGGGATTA-3'
SEQ ID Nos 40： 5'-GGATGTGCTATTTGGCCACT-3'
SEQ ID Nos 41： 5'-TGGGTCTCCGACCCTGACTT-3'
SEQ ID Nos 42： 5'-GACTTCAACGTGGGGGTGTG-3'
SEQ ID Nos 43： 5'-GCGAGCCATCTTCTGCTGAT-3'
SEQ ID Nos 44： 5'-ATCTTCTGCTGATGGATCTGCT-3'
SEQ ID Nos 45： 5'-CTCACGCACACAGCACAGCTTA-3'
SEQ ID Nos 46： 5'-GCTAGGCTTTGGGAGCACA-3'
SEQ ID Nos 47： 5'-TCCTGGACTCGGGAGCTTT-3'
SEQ ID Nos 48： 5'-GACATTGCACAGGCTGGACT-3'
SEQ ID Nos 49： 5'-AGAGCAGGTGTCCTCCATAC-3'
SEQ ID Nos 50： 5'-ATGCTTGCCCAGAGCAGGTGTC-3'
SEQ ID Nos 51： 5'-GCCTGCTGGCTTCCATCACAC-3'
SEQ ID Nos 52： 5'-AGCAGGACTGTGGCCTGCT-3'
SEQ ID Nos 53： 5'-TCATGCAGCCCCTCTGTC-3'
SEQ ID Nos 54： 5'-CTGCTGGGGGTCTACCTGT-3'
SEQ ID Nos 55： 5'-CTCTCCACGTCTCTCTTGG-3'
SEQ ID Nos 56： 5'-TTGGCGATGCCCAGCTTG-3'
SEQ ID Nos 57： 5'-GCTCCACATGGGGCCTATA-3'
SEQ ID Nos 58： 5'-AGGAGGGGGTCTTTTCTCAC-3'
SEQ ID Nos 59： 5'-ATCAGGTCAGCAACACCACA-3'
SEQ ID Nos 60： 5'-CATAGCAGGTAGTGATCAGGTCA-3'
SEQ ID Nos 61： 5'-GGCGTGAAAACAGTGTCAGA-3'
SEQ ID Nos 62： 5'-CGTTGGAGCATCTGTTGTTCT-3'
SEQ ID Nos 63： 5'-AATATCCACGCTGATTCTGAAG-3'
SEQ ID Nos 64： 5'-GTCATGTCTCCGGGAAGAAC-3'
SEQ ID Nos 65： 5'-GGCTCTAAGGTGGTCATGCT-3'
SEQ ID Nos 66： 5'-CCAGGGCTCAGGGTTTAGTA-3'
SEQ ID Nos 67： 5'-TCCGGTTTGAGTGCTAGTGA-3'
SEQ ID Nos 68： 5'-ATGCCATCGACCTTCATTTT-3'
SEQ ID Nos 69： 5'-TGATTCTGATGTGGCCTGAG-3'
SEQ ID Nos 70： 5'-CCCCTGGGTTTATTCTCACA-3'
SEQ ID Nos 71： 5'-GACTTCAACGTGGGGGTGTG-3'
SEQ ID Nos 72： 5'-GCTAGGCTTTGGGAGCACA-3'
SEQ ID Nos 73： 5'-ATGCTTGCCCAGAGCAGGTGTC-3'
SEQ ID Nos 74： 5'-CTGCTGGGGGTCTACCTGT-3'
SEQ ID Nos 75： 5'-AGGAGGGGGTCTTTTCTCAC-3'
Claims (7)
1. a kind of detection method in hypertriglyceridemia mutational site, which is characterized in that by designing specific primer again
Target fragment is obtained using Single-tube multiplex-PCR amplified reaction, and designs sequencing primer again using the Sanger of non-rubber tapping purifying
Obtained target fragment is sequenced in PCR sequencing PCR.
2. the detection method in hypertriglyceridemia mutational site according to claim 1, which is characterized in that described special
Property primer includes：
For expanding the specific primer of LPL-1 segments, forward primer is：SEQ ID Nos：1 or 2, reverse primer is SEQ ID
Nos：3 or 4；
For expanding the specific primer of LPL-2 segments, forward primer is：SEQ ID Nos：5 or 6, reverse primer is SEQ ID
Nos：7 or 8；
For expanding the specific primer of LPL-3 segments, forward primer is：SEQ ID Nos：9 or 10, reverse primer SEQ
ID Nos：11 or 12；
For expanding the specific primer of LPL-4 segments, forward primer is：SEQ ID Nos：13 or 14, reverse primer SEQ
ID Nos：15 or 16；
For expanding the specific primer of LPL-5 segments, forward primer is：SEQ ID Nos：17 or 18, reverse primer SEQ
ID Nos：19 or 20；
For expanding the specific primer of LPL-6 segments, forward primer is：SEQ ID Nos：21 or 22, reverse primer SEQ
ID Nos：23 or 24；
For expanding the specific primer of LPL-7 segments, forward primer is：SEQ ID Nos：25 or 26, reverse primer SEQ
ID Nos：27 or 28；
For expanding the specific primer of LPL-8 segments, forward primer is：SEQ ID Nos：29 or 30, reverse primer SEQ
ID Nos：31 or 32；
For expanding the specific primer of LPL-9 segments, forward primer is：SEQ ID Nos：33 or 34, reverse primer SEQ
ID Nos：35 or 36；
For expanding the specific primer of LPL-10 segments, forward primer is：SEQ ID Nos：37 or 38, reverse primer SEQ
ID Nos：39 or 40.
For expanding the specific primer of APOA5-1 segments, forward primer is：SEQ ID Nos：41 or 42, reverse primer is
SEQ ID Nos：43 or 44；
For expanding the specific primer of GPIHBP1-1 segments, forward primer is：SEQ ID Nos：45 or 46, reverse primer is
SEQ ID Nos：47 or 48；
For expanding the specific primer of GPIHBP1-2 segments, forward primer is：SEQ ID Nos：49 or 50, reverse primer is
SEQ ID Nos：51 or 52；
For expanding the specific primer of LMF1 segments, forward primer is：SEQ ID Nos：53 or 54, reverse primer SEQ
ID Nos：55 or 56；
For expanding the specific primer of GPD1 segments, forward primer is：SEQ ID Nos：57 or 58, reverse primer SEQ
ID Nos：59 or 60.
3. the detection method in hypertriglyceridemia mutational site according to claim 1, which is characterized in that the sequencing
Primer includes：
For the sequencing primer of LPL-1 segments, sequence is SEQ ID Nos：61；
For the sequencing primer of LPL-2 segments, sequence is SEQ ID Nos：62；
For the sequencing primer of LPL-3 segments, sequence is SEQ ID Nos：63；
For the sequencing primer of LPL-4 segments, sequence is SEQ ID Nos：64；
For the sequencing primer of LPL-5 segments, sequence is SEQ ID Nos：65；
For the sequencing primer of LPL-6 segments, sequence is SEQ ID Nos：66；
For the sequencing primer of LPL-7 segments, sequence is SEQ ID Nos：67；
For the sequencing primer of LPL-8 segments, sequence is SEQ ID Nos：68；
For the sequencing primer of LPL-9 segments, sequence is SEQ ID Nos：69；
For the sequencing primer of LPL-10 segments, sequence is SEQ ID Nos：70；
For the sequencing primer of APOA5-1 segments, sequence is SEQ ID Nos：71；
For the sequencing primer of GPIHBP1-1 segments, sequence is SEQ ID Nos：72；
For the sequencing primer of GPIHBP1-2 segments, sequence is SEQ ID Nos：73；
For the sequencing primer of LMF1 segments, sequence is SEQ ID Nos：74；
For the sequencing primer of GPD1 segments, sequence is SEQ ID Nos：75.
4. the detection method in hypertriglyceridemia mutational site according to claim 1, which is characterized in that the single tube
The response procedures of multiplexed PCR amplification are：95 DEG C of Taq enzymes activate 15 minutes；95 DEG C of DNA are denatured 15 seconds, 60 DEG C of primer annealings 20 seconds,
70 DEG C of primer extends 90 seconds repeat 45 cycles；70 DEG C extend 10 minutes：4 DEG C cool down 10 minutes.
5. a kind of detection kit in hypertriglyceridemia mutational site, which is characterized in that it is used as in claim 1-4
The detection method in the hypertriglyceridemia mutational site described in any one carries out hypertriglyceridemia mutational site detection.
6. the detection kit in hypertriglyceridemia mutational site according to claim 5, which is characterized in that it includes
Reaction mixture, Taq enzyme and negative quality-control product.
7. the detection kit in hypertriglyceridemia mutational site according to claim 6, which is characterized in that described anti-
Mixed liquor is answered to include：The MgCl2 solution of 1-6mM, the PCR buffer of the dNTPs of 0.1-0.5mM, 1X, 30 concentration are 0.05-
1.0 μM of specific primer；The Tris-HCl solution or deionized water that the feminine gender quality-control product is 10-100mM.
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| CN117384912A (en) * | 2023-12-13 | 2024-01-12 | 中国人民解放军东部战区总医院 | GPIHBP1 mutant gene and application thereof |
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