CN105296631A - Flow type liquid phase chip detection reagent kit for loca of acute mountain sickness prolyl hydroxylase gene rs480902 - Google Patents
Flow type liquid phase chip detection reagent kit for loca of acute mountain sickness prolyl hydroxylase gene rs480902 Download PDFInfo
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- CN105296631A CN105296631A CN201510743104.6A CN201510743104A CN105296631A CN 105296631 A CN105296631 A CN 105296631A CN 201510743104 A CN201510743104 A CN 201510743104A CN 105296631 A CN105296631 A CN 105296631A
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- 238000001514 detection method Methods 0.000 title claims abstract description 23
- 239000007791 liquid phase Substances 0.000 title claims abstract description 14
- 108010043005 Prolyl Hydroxylases Proteins 0.000 title claims abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 title abstract description 5
- 208000034388 Mountain sickness acute Diseases 0.000 title abstract 3
- 208000018315 acute mountain sickness Diseases 0.000 title abstract 3
- 239000000523 sample Substances 0.000 claims abstract description 27
- 238000009396 hybridization Methods 0.000 claims abstract description 16
- 239000004005 microsphere Substances 0.000 claims abstract description 14
- 239000000243 solution Substances 0.000 claims abstract description 11
- 239000011259 mixed solution Substances 0.000 claims abstract description 7
- 108010004729 Phycoerythrin Proteins 0.000 claims abstract description 4
- 108010090804 Streptavidin Proteins 0.000 claims abstract description 4
- 230000001154 acute effect Effects 0.000 claims description 20
- 208000008445 altitude sickness Diseases 0.000 claims description 19
- 238000012360 testing method Methods 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 9
- 239000012530 fluid Substances 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 3
- 238000013016 damping Methods 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 abstract description 18
- 238000003745 diagnosis Methods 0.000 abstract description 6
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000004132 cross linking Methods 0.000 abstract 1
- 239000007850 fluorescent dye Substances 0.000 abstract 1
- 238000001215 fluorescent labelling Methods 0.000 abstract 1
- 206010028980 Neoplasm Diseases 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 8
- 102100037249 Egl nine homolog 1 Human genes 0.000 description 6
- 101000881648 Homo sapiens Egl nine homolog 1 Proteins 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 238000011161 development Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 108010006785 Taq Polymerase Proteins 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 206010002660 Anoxia Diseases 0.000 description 1
- 241000976983 Anoxia Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 102000004079 Prolyl Hydroxylases Human genes 0.000 description 1
- 241001113283 Respirovirus Species 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000007953 anoxia Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 101150024923 da gene Proteins 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
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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
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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Abstract
The invention discloses a flow type liquid phase chip detection reagent kit for loca of an acute mountain sickness prolyl hydroxylase gene rs480902. The reagent kit comprises a specific fluorescent microsphere crosslinking probe microspere hybridization solution, a secific PCR primer mixed solution, a premixed solution and phycoerythrin fluorescence labeling streptavidin. The reagent kit has high specificity and sensitivity and capability of detecting multiple samples and multiple indexes at the same time, can be clinically used for detection and diagnosis of key susceptibility genes of acute mountain sickness and further can be applied to detection and parting of people who need to rush into a plateau in batches in a short time.
Description
Technical field
The present invention relates to human gene diagnosis's test kit, adopt the SNP site in streaming fluorescent hybridization legal detection acute high altitude sickness tumor susceptibility gene.Be applicable to the predictive diagnosis of Clinical Acute altitude sickness tumor susceptibility gene, also can be used for altitude acclimatization crowd and AMS patient gene organizes detection.
Background technology
Qinghai-Tibet Platean is vast in territory, produce enrich, and contains huge the economic development potentialities; Qinghai-Tibet Platean is located in limit, motherland southwest and sleeps, and be the important natural cover for defense, military geography position is important.But plateau Natural Survival bad environments, on height above sea level 3000 meters of plateaus, normal atmosphere is down to 537mmHg, be only 70% of sea level, atmosphericoxygen dividing potential drop is 103mmHg, and alveolar air oxygen partial pressure is 62mmHg, and high altitude anoxia causes body work capacity obviously to reduce, in areas of 4500 metres above sea level, the muscle power of people only has 60% of Plain.The special environmental factors in plateau can cause the generation of altitude sickness, and acute high altitude sickness (AcutemountainsieknessAMS) is the most common, endangers also maximum.Epidemiology survey shows, the multiple plateau being born in height above sea level more than 2500 meters of AMS, and the plateau of China's height above sea level more than 3000 meters just accounts for 1/6 of area, and resident population about more than 6,000 ten thousand.Current AMS has become the great public health problem of China highlands, seriously hampers the development of local economy, society and all our undertakings.Because AMS development is rapid, consequence is serious, lacks again very effective remedy measures at present, therefore, finds effective preventive measures and seem particularly urgent.AMS is a kind of multigenic disease, is environmental factors and the coefficient result of many minor effect Disease-causing genes of Altitude.Large quantity research shows, the expression of tumor susceptibility gene product between AMS patient and altitude acclimatization crowd that induction AMS is formed exists significant difference.If before entering plateau, by the detection of genetic marker, by carrying the individual screening of this susceptible genotype out, avoiding it to be exposed to environment of low oxygen plateau, just effectively can prevent the generation of AMS.But existing test in laboratory method all can not carry out susceptible genotype detection.In a word, the predictive diagnosis test kit researching and developing acute high altitude sickness tumor susceptibility gene remains one of challenge faced by people.
Streaming liquid-phase chip technology is also known as streaming fluorescence technique or suspension array streaming fluorescence technique, its principle be by fluorescent mark after unicellular (or particle) suspension enter flow chamber with sheath fluid, the drop that pressure forces sheath fluid to wrap up comprises single cell or particle perpendicular through detection zone.Fluorescently-labeled cell or particle send fluorescence under laser excitation, and the fluorescence marked according to itself and the fluorescence sent after exciting comprehensively are analyzed and detected.It is at small sample, high-throughput, easy and simple to handle, quick and precisely, susceptibility, the aspects such as specific degree is high are better than traditional analytical procedure, and it responds and carries out all in the liquid phase, keep native conformation, be conducive to probe and determinand fully reacts, all kinds of nucleic acid and Protein Detection are widely used at present, as gene type, antigen/antibody detects, infectious diseases diagnosis etc., wherein HPV detects and parting kit, Respirovirus detection kit, cytokine detection kits, tumor markers detection kit etc. are used for clinical detection in the U.S. and China through FDA and SFDA approval.
Summary of the invention
Object of the present invention mainly provides a kind of high specific degree, sensitivity, and has the test kit simultaneously detecting multiple sample, multiple index ability.
The present invention is achieved through the following technical solutions:
Streaming liquid-phase chip acute high altitude sickness prolyl hydroxylase gene rs480902 site detection kit, this test kit comprises specificity fluorescent micro-sphere crosslinked probe microballoon hybridization solution, Specific PCR primers mixed solution, premixed liquid, phycoerythrin fluorescent mark Streptavidin (SA-PE);
Wherein, the micro-sphere crosslinked probe of specificity fluorescent and Specific PCR primers and probe sequence as follows:
E-F5-2：5'-CAAGTGATCTCCCAGTGACTCTT-3'
E-F6-2：5'-CAAGTGATCTCCCAGTGACTCTC-3'；
E-R3：5'-GCAAAGTCAGAAAGCGGTG-3'；
E-P0707：5'-GGACCTTAGACCAGCACTTCT-3'。
Further, described premixed liquid is made up of 4XBuffer, dNTPs, TaqPolymerase.
Particularly, the consumption of premixed liquid is: 10 μ l, and the consumption of Specific PCR primers mixed solution is: 2 μ l.
In addition, described test kit also comprises ddH
2o.Particularly, ddH
2the consumption of O is: 6 μ l.When detecting, the consumption of DNA to be checked is: 2 μ l.
In addition, described specificity fluorescent micro-sphere crosslinked probe microballoon hybridization solution be coupled by probe E-P0707 and No. 22 fluorescent microsphere, prepared by TE damping fluid and tetramethyl-ammonia chloride hybridization buffer.
Mentioned reagent box PCR reaction conditions is in actual use: first warm start 95 DEG C of 10min, then enter 35 circulations; 94 DEG C of sex change 30s, 64 DEG C of annealing 30s, 72 DEG C extend 30s; After loop ends, 72 DEG C extend 10min.
Get PCR primer 3 μ l to mix with 22 μ l microballoon hybridization solutions, 95 DEG C of sex change 5min, after 48 DEG C of hybridization 30min, add SA-PE75 μ l, 48 DEG C of incubation 15min.Luminex200 streaming fluorescence analyser is used to detect hybrid product.
The present invention has the following advantages and beneficial effect:
Test kit of the present invention has high specific degree, sensitivity, and there is the ability simultaneously detecting multiple sample, multiple index, may be used for the checkout and diagnosis of Clinical Acute altitude sickness (AMS) emphasis tumor susceptibility gene, also can be applicable to tackle the crowd needing Acute Exposed Altitude in batches, carry out detection in the short period of time and somatotype.
Accompanying drawing explanation
Fig. 1 is No. 1 sample EGLN1 site sequencing result.
Fig. 2 is No. 2 sample EGLN1 site sequencing results.
Fig. 3 is No. 3 sample EGLN1 site sequencing results.
Fig. 4 is No. 4 sample EGLN1 site sequencing results.
Embodiment
Below in conjunction with embodiment, the present invention is described further, but embodiments of the present invention are not limited to this.
Embodiment
The selection of acute high altitude sickness (AMS) tumor susceptibility gene SNP site:
Use NCBI nucleic acid database public information and literature survey, the screening acute uncomfortable susceptible target gene in plateau and site, by information biology Comparison Study, screening acute high altitude sickness (AMS) tumor susceptibility gene, to sequence number inquiry and the genome sequence download of the AMS tumor susceptibility gene SNP site chosen.The selection principle of SNP site: this site is positioned at the protein-active such as gene coding region, control region region in close relations as far as possible, each SNP site Gene frequency distribution data is all from Chinese han population.According to SNP site selection principle, 1 SNP site of selected 1 tumor susceptibility gene.Selected SNP site is: prolyl hydroxylase (EGLN1) gene: rs480902.
By the selection to SNP site, the application investigated following test kit:
This test kit comprises specificity fluorescent micro-sphere crosslinked probe microballoon hybridization solution, Specific PCR primers mixed solution, premixed liquid, phycoerythrin fluorescent mark Streptavidin.
Described premixed liquid is made up of 4XBuffer, dNTPs, TaqPolymerase.This premixed liquid can from directly buying on the market.
Described specificity fluorescent micro-sphere crosslinked probe microballoon hybridization solution be coupled by probe (No. E-P0707) and No. 22 fluorescent microspheres, prepared by TE damping fluid and 170 μ l tetramethyl-ammonia chloride (TMAC) hybridization buffers.Other raw materials of its concrete preparation method and employing are all prior aries, and the present invention have employed different probes and fluorescent microsphere, and then preparation method is existing.
The micro-sphere crosslinked probe of above-mentioned specificity fluorescent and Specific PCR primers and probe sequence as follows:
E-F5-2：5'-CAAGTGATCTCCCAGTGACTCTT-3'
E-F6-2：5'-CAAGTGATCTCCCAGTGACTCTC-3'；
E-R3：5'-GCAAAGTCAGAAAGCGGTG-3'；
E-P0707：5'-GGACCTTAGACCAGCACTTCT-3'。
Particularly, the premixed liquid consumption in test kit is: 10 μ l, and Specific PCR primers mixed solution consumption is: 2 μ l.
In addition, described test kit also comprises ddH
2o.Particularly, ddH
2the consumption of O is: 6 μ l.When detecting, the consumption of DNA to be checked is: 2 μ l.That is the cumulative volume of the PCR reaction system of the application's test kit is 20 μ l.
But PCR reaction conditions is: first warm start 95 DEG C of 10min, then enter 35 circulations; 94 DEG C of sex change 30s, 64 DEG C of annealing 30s, 72 DEG C extend 30s; After loop ends, 72 DEG C extend 10min.Hybridization condition mixes with 22 μ l microballoon hybridization solutions for getting PCR primer 3 μ l, 95 DEG C of sex change 5min, after 48 DEG C of hybridization 30min, adds SA-PE75 μ l, 48 DEG C of incubation 15min.
When test kit of the present invention uses, primer pair is two right: 1. E-F5-2 E-R3,2. E-F6 E-R3.Luminex200 streaming fluorescence detector is adopted to detect.
Clinical verification of the present invention:
Gathering the 3 routine clinical diagnosises that in January, 2015, Tibet hospital accepted for medical treatment is the patient whole blood of acute high altitude sickness, extracts DNA and adopts AMS tumor susceptibility gene streaming liquid-phase chip to detect and somatotype.By amplification, hybridize and use Luminex200 streaming fluorescence detector to detect, detected result is as table 1.
The patient of table 1 acute high altitude sickness is run off liquid-phase chip detected result
Result shows, if only have primer pair 1. or primer pair products thereof result be 2. positive (MFI > 500), be then judged to be homozygous mutation, SNP type be primer pair 1. or primer pair 2. with base type.If 1. primer pair is all positive with primer pair products thereof result 2., be then judged to be heterozygous mutant.
Genomic dna is carried out sequence verification result (Hua Da gene) after detection, sequencing result is shown in Fig. 1 ~ 4.Case sample is numbered: 1,2,3,4, and sequencing result and detected result of the present invention fit like a glove.
In patient's sample of 4 routine acute high altitude sicknesses, EGLN1(rs480902) 4 examples of suddenling change; Wherein the case of numbering 1 is heterozygous mutant, and numbering 2 is homozygous mutation C, and the case of numbering 3 is heterozygous mutant, and the case of numbering 4 is C sudden change of isozygotying.Illustrate that the method and clinical diagnosis consistence are very good.
SEQUENCELISTING
<110> Wu Ai continuous heavy rain
<120> streaming liquid-phase chip acute high altitude sickness prolyl hydroxylase gene rs480902 site detection kit
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<170>PatentInversion3.3
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caagtgatctcccagtgactctt23
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<213>Artificial
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caagtgatctcccagtgactctc23
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<212>DNA
<213>Artificial
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gcaaagtcagaaagcggtg19
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ggaccttagaccagcacttct21
Claims (5)
1. streaming liquid-phase chip acute high altitude sickness prolyl hydroxylase gene rs480902 site detection kit, it is characterized in that, this test kit comprises specificity fluorescent micro-sphere crosslinked probe microballoon hybridization solution, Specific PCR primers mixed solution, premixed liquid, phycoerythrin fluorescent mark Streptavidin;
Wherein, the micro-sphere crosslinked probe of specificity fluorescent and Specific PCR primers and probe sequence as follows:
E-F5-2：5'-CAAGTGATCTCCCAGTGACTCTT-3'
E-F6-2：5'-CAAGTGATCTCCCAGTGACTCTC-3'；
E-R3：5'-GCAAAGTCAGAAAGCGGTG-3'；
E-P0707：5'-GGACCTTAGACCAGCACTTCT-3'。
2. streaming liquid-phase chip acute high altitude sickness prolyl hydroxylase gene rs480902 site according to claim 1 detection kit, it is characterized in that, the consumption of premixed liquid is: 10 μ l, and the consumption of Specific PCR primers mixed solution is: 2 μ l.
3. streaming liquid-phase chip acute high altitude sickness prolyl hydroxylase gene rs480902 site according to claim 1 and 2 detection kit, it is characterized in that, described test kit also comprises ddH
2o.
4. streaming liquid-phase chip acute high altitude sickness prolyl hydroxylase gene rs480902 site according to claim 3 detection kit, is characterized in that, ddH
2the consumption of O is: 6 μ l.
5. streaming liquid-phase chip acute high altitude sickness prolyl hydroxylase gene rs480902 site according to claim 1 detection kit, it is characterized in that, described specificity fluorescent micro-sphere crosslinked probe microballoon hybridization solution be coupled by probe E-P0707 and No. 22 fluorescent microsphere, prepared by TE damping fluid and tetramethyl-ammonia chloride hybridization buffer.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114740199A (en) * | 2022-03-18 | 2022-07-12 | 北京安奇生物医药科技有限公司 | SARS-CoV-2 neutralizing antibody reagent kit and its use |
| CN118762749A (en) * | 2024-09-06 | 2024-10-11 | 中国人民解放军总医院 | Computer device and computer readable storage medium for screening susceptible persons to acute mountain sickness |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2467689A1 (en) * | 2001-12-06 | 2003-06-19 | Fibrogen, Inc. | Stabilization of hypoxia inducible factor (hif) alpha using inhibitors of hif prolyl hydroxylase |
| CN104313166A (en) * | 2014-10-30 | 2015-01-28 | 中国人民解放军济南军区第四〇一医院 | Liquid-chip detection kit for multiple drug-resistant genes of pseudomonas aeruginosa |
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2015
- 2015-11-04 CN CN201510743104.6A patent/CN105296631A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2467689A1 (en) * | 2001-12-06 | 2003-06-19 | Fibrogen, Inc. | Stabilization of hypoxia inducible factor (hif) alpha using inhibitors of hif prolyl hydroxylase |
| CN104313166A (en) * | 2014-10-30 | 2015-01-28 | 中国人民解放军济南军区第四〇一医院 | Liquid-chip detection kit for multiple drug-resistant genes of pseudomonas aeruginosa |
Non-Patent Citations (3)
| Title |
|---|
| BUROKER NE ET AL.: "EPAS1 and EGLN1 associations with high altitude sickness in Han and Tibetan Chinese at the Qinghai-Tibetan Plateau", 《BLOOD CELLS MOL DIS》 * |
| 陈国柱等: "急性高原病的研究进展", 《军事医学》 * |
| 陈芃等: "EGLN1基因多态性位点rs479200、rs2790870与高原肺水肿的关系", 《新疆医科大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114740199A (en) * | 2022-03-18 | 2022-07-12 | 北京安奇生物医药科技有限公司 | SARS-CoV-2 neutralizing antibody reagent kit and its use |
| CN118762749A (en) * | 2024-09-06 | 2024-10-11 | 中国人民解放军总医院 | Computer device and computer readable storage medium for screening susceptible persons to acute mountain sickness |
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