CN105132566A - Preparation method and system for toxin accumulation type obese gene individualized intervention beverage - Google Patents
Preparation method and system for toxin accumulation type obese gene individualized intervention beverage Download PDFInfo
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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
- C12Q1/6876—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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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/02—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation containing fruit or vegetable juices
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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
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a preparation method and system for a toxin accumulation type obese gene individualized intervention beverage. The preparation method comprises four steps as follows: establishment of combined gene type risk levels; individual gene detection; beverage formula determination on the basis of the risk level which a detection result belongs to; beverage preparation. According to the method and the system, the targeted diet beverage can be prepared according to the combined gene type of an obese individual, and a better diet effect is realized; with the adoption of a combined detection technology, the combined gene type of the individual is acquired through one-time detection procedure; with the adoption of the preparation system of the individualized intervention beverage, the integrating process from sample detection to beverage preparation is realized; the prepared individualized intervention beverage has good taste and is highly popular.
Description
Technical field
The present invention relates to a kind of ob gene Individual treatment drink, particularly relate to a kind of preparation method and system thereof of toxin accumulation type ob gene Individual treatment drink.
Background technology
Fat of a kind of disease, the health of the positive serious threat mankind.WHO expert committee proposes using constitutional index (Body-MassIndex, BMI) as the overweight or fat standard of health, and namely BMI be overweight between 25 ~ 28, and 28 ~ 32 is obesity, is very fat higher than 32.Obesity is a kind of and inherited genetic factors and the closely-related chronic disease of mode of life, it can cause blood fat and raise, easily cause fatty liver, arteries pathology, heart trouble, diabetes, fat children also can induced functional development bad and cause the various disease incidence of adulthood and mortality ratio to rise.
Clinical treatment mainly diet and the kinesitherapy of current obesity, but the non-individualized treatment mode also existing that carbohydrate, fat and protein intake control clean cut and blindness employing slimming medicine.Owing to having at least the difference of 40% body fat quantity to be caused by inherited genetic factors, so be easy to clear and definite heredity, obesity is had a significant impact.The different people of genetic constitution should take different fat-reducing strategies when suffering from obesity, utilize modern scientific research achievement, develop a Weight management Molecular Detection product based on Molecular and genetic basis and prepare corresponding diet food to provide personalized Weight management suggestion, there are stronger social effect and market outlook.
After exogenous toxic chemical substance is rapidly absorbed into body by different approaches, a series of chemical transformation will be there is and form some degradation productions or derivative, be i.e. bio-transformation, forming carcinogenic substance.There are some enzymes can be combined by series reaction and these materials or its metabolic reaction product in human body, reduce the toxicity of these materials, excrete external with certain form thus make cell from infringement.Due to the difference of genetic constitution, the deciphering toxin expelling ability of different body also difference to some extent, and this species diversity can have influence on body to the susceptibility of many diseases as diseases such as obesity, cancers.
In body, the permanent accumulation of waste cannot be discharged, and is also the arch-criminal causing stature too fat to move.Toxin is all materials worked the mischief to health, or can cause that the rejection of health is reacted, allergic symptom.If toxin is piled up for a long time in vivo, cell and tissue injury can be made, cause the disorder of body metabolism system and vivotoxin is piled up, cause human body taste transporting metabolism declines, blood toxicity is many, viscosity is high, cause that whole body obesity, hypomnesis, complexion are obscure, the disease such as constipation and hemorrhoid.
Glutathione S-transferase (GlutathioneS-transferase, GSTs) belongs to II phase metabolic enzyme super families, is Chlorogenic Acid important in body.It can directly and nucleophilie electronic compound reaction, increases that it is water-soluble and be conducive to getting rid of external, thus reach the object of removing toxic substances.GSTM1, GSTT1, GSTP1 are three kinds of important hypotypes of glutathione S-transferase.Find that GSTM1, T1, P1 have Polymorphic Population, and had racial difference.Wherein GSTM1 and GSTT1 gene pleiomorphism is encoding gene and lacks completely, causes having this genotype individuals enzymic activity extremely low, and GSTP1 gene shows as GSTP1Val105Ile the genetics of 5 exons is polymorphic, also causes GSTP1 enzymic activity to reduce.GSTs transgenation has very important impact to clinical.Combined by gsh and chemical substance or its metabolic reaction product, the toxicity of these materials can be reduced, thus make cell from infringement.After gsh and multiple oxidative chain meta-bolites react, the attenuation binding substances of generation can make it be easy to enter next step metabolism, and finally excretes external with the form of mercapturic acids.Encode the gene of this class of enzymes once the variation of its normal function that has an impact, body will be affected to the normal detoxification of hazardous and noxious substances.
Cytochrome P 450 enzymes (CYP1A1, CYP1B1 etc.) be one group containing heme, the isozyme of structurally and functionally related superfamily genes encoding, very important effect is played in source property and the endogenous compound metabolism outside of this fermentoid, CYP450 is extensively present in body, but be mainly present on hepatocyte endoplasmic reticulum, participate in endogenous material and (comprise cholesterol, lipid acid etc.) biosynthesizing and degraded and the metabolism of exogenous material (comprising medicine and environmental pollutant), wherein CYP1A1 is one of important member in CYP450 enzyme system, not only take part in the metabolism of medicine, and can also the reactivation process of the many procarcinogen of catalysis and front poisonous substance, can highly be induced at liver.Existing experiment confirms, hepatomicrosome CYP1A1 express strengthen to increase with the generation of hydroxy radical qiao and lipid peroxidation relevant.Hydroxy radical qiao is the startup agent of peroxidatic reaction of lipid, and high fat diet can increase plastosome or microsome causes oxidative stress to the oxidation of lipid acid, therefore lipid acid can induce the generation of CYP1A1.Along with the rise that CYP1A1 expresses, POS generates increase, and peroxidatic reaction of lipid aggravates, and forms vicious cycle.
Summary of the invention
The object of this invention is to provide a kind of preparation method and system thereof of toxin accumulation type ob gene Individual treatment drink, make different intervention drinks for the gene physique that individual is different, thus reach better fat-reducing effect.
For achieving the above object, the invention provides a kind of preparation method of toxin accumulation type ob gene Individual treatment drink, described method is by carrying out joint-detection to individual multiple gene pleiomorphisms fat relevant to toxin accumulation type, and determine that risk class of toxin accumulation type obesity occurs for it according to detected result, then the risk class for individual Polymorphism type generation toxin accumulation type obesity prepares fat-reducing drink targetedly, said method comprising the steps of:
The foundation of step one, Polymorphism type risk class
(1) select the gene polymorphism sites fat relevant to toxin accumulation type, the single-gene risk of the different genotype in each genetic risk factors related polymorphic site is determined in the data provided according to document or voluntarily sampling;
(2) by each genotype permutation and combination of all pleomorphism sites of employing, then Polymorphism type risk class is set up according to each single-gene risk product.
Wherein, the concrete grammar determining the single-gene risk of the different genotype in each genetic risk factors related polymorphic site of sampling voluntarily in step (1) is:
A. case-control study method is utilized, adopt chi square test or Fisher precise probabilistic method, credibility interval CI gets 95%, and significant difference gets p<0.05, and the related locus of the fat genetic risk factors in specific crowd of contratoxin accumulation type screens;
B. utilize online SPSS statistics computed in software relative risk OR, CI gets 95%, p<0.05, as the single-gene risk of each related polymorphic site different genotype.
Preferably, the gene polymorphism sites fat relevant to toxin accumulation type is selected can to select according to the genetic risk factors that toxin accumulation type in documents searching Chinese population is fat common in step (1).
Preferably, fat relevant to toxin accumulation type gene polymorphic position property point is selected from least two in following table 1:
Table 1
| Gene Name | Site title | NCBI Ref ID |
| GSTP1(SEQ 1) | Val105Ile | rs1695 |
| CYP1A1(SEQ 2) | Val462Ile | rs1048943 |
| CYP1A1(SEQ 3) | MspI | rs4646903 |
In the preferred embodiment of the present invention, consult according to the molecular epidemiology correlative study achievement in extensive Chinese population, in the upper table adopted, the risk in each related polymorphic site is as shown in table 2 below:
Table 2
| Gene polymorphism sites | Genotype | Risk (OR) |
| GSTP1 Val105Ile | Hypotype 1 (AA) | 1 |
| Hypotype 2 (AG) | 1 | |
| Hypotype 3 (GG) | 3.26 | |
| CYP1A1 Val462Ile | Hypotype 1 (AA) | 1 |
| Hypotype 2 (AG) | 1 | |
| Hypotype 3 (GG) | 1.89 | |
| CYP1A1 MspI | Hypotype 1 (CC) | 3.12 |
| Hypotype 2 (CT) | 1 | |
| Hypotype 3 (TT) | 1 |
Further, the concrete grammar of step (2) is:
Each gene polymorphism sites fat relevant to toxin accumulation type has three kinds of hypotypes respectively, and what all for selected site hypotypes occurred may carry out permutation and combination; Then the risk of these hypotypes is multiplied and obtains the risk product of different permutation and combination; The risk product of acquisition is arranged from small to large or from big to small, merges the group that risk product is equal; The final height determining each group of risk by the size of risk product.
Each site is weighted process as independent factor by aforesaid method, and risk product is larger, and evaluation and grading is larger; The Polymorphism type heredity excessive risk individuality calculated with aforesaid method can be expressed as that the genetic risk factors himself causing toxin accumulation type obesity is more or result determinacy is stronger.
Further, be a, b, c by different related polymorphic site-tag ..., different hypotypes is labeled as 1,2,3, and the haplotype namely carrying hypotype 1+ site 3, hypotype 1+ site 2, site 1 hypotype 1 is expressed as a1b1c1; In a preferred embodiment of the invention, the related polymorphic site chosen be rs1695 (
gSTP1), rs1048943 (
cYP1A1) and rs4646903 (
cYP1A1), they are labeled as a, b, c respectively, as shown in table 3 according to single-gene risk calculation risk degree result of product listed in above table:
Table 3
| Haplotype | Risk product | Haplotype | Risk product | Haplotype | Risk product |
| a1b1c1 | 3.12 | a2b1c1 | 3.12 | a3b1c1 | 10.171 |
| a1b1c2 | 1 | a2b1c2 | 1 | a3b1c2 | 3.26 |
| a1b1c3 | 1 | a2b1c3 | 1 | a3b1c3 | 3.26 |
| a1b2c1 | 3.12 | a2b2c1 | 3.12 | a3b2c1 | 10.171 |
| a1b2c2 | 1 | a2b2c2 | 1 | a3b2c2 | 3.26 |
| a1b2c3 | 1 | a2b2c3 | 1 | a3b2c3 | 3.26 |
| a1b3c1 | 5.897 | a2b3c1 | 5.897 | a3b3c1 | 19.224 |
| a1b3c2 | 1.89 | a2b3c2 | 1.89 | a3b3c2 | 6.161 |
| a1b3c3 | 1.89 | a2b3c3 | 1.89 | a3b3c3 | 6.161 |
Preferably, risk product adopts PHP to program, and then inputs the laggard row of each single-gene risk angle value in a computer and calculates.
Then the haplotype in upper table 3 arranged from low to high by risk product and divide into groups, as shown in table 4; Risk class is higher, and risk product is larger, and namely the genetic risk of self initiation toxin accumulation type obesity individual is higher.
Table 4
Step 2, genes of individuals detect
The Polymorphism type of gene polymorphism sites selected in carry out step one to the DNA obtained from individuality detects.
Preferably, described Polymorphism type detects and adopts TaqMan
?-MCB technology is carried out; Further preferably, same amplification program is adopted to different risk sites, in different reacting holes, carry out joint-detection simultaneously.After quantitative fluorescent PCR reaction, the sample in each reacting hole is read fluorescence volume data on quantitative real time PCR Instrument, and the sample for each individuality can obtain the figure in corresponding different risk site, is combined the Polymorphism type obtaining this individuality.
Preferably, the risk site of joint-detection be rs1695 (
gSTP1), rs1048943 (
cYP1A1) and rs4646903 (
cYP1A1).
Preferably, said gene pleomorphism site joint-detection adopt primer and probe design as shown in table 5 below:
Table 5
Preferably, the amplification condition of quantitative fluorescent PCR reaction is, first preheating: 50 DEG C, 2 minutes, 95 DEG C, 10 minutes, 95 DEG C, 30 seconds that then carry out 60 circulations, 60 DEG C, 1 minute.
In another better embodiment of the present invention, the means that Polymorphism type detects also can adopt gene chip sequencing technologies.
Step 3, risk class determination beverage formulation belonging to detected result
Determine individual risk class according to the result that Polymorphism type detects, and press nutrient demand and the means of intervention of different risk class, determine the addition of Different Nutrition element in drink.
Preferably, the drinking method that drink of the present invention is used in toxin accumulation type ob gene Individual treatment process is: fixing weekly 1-3 days for going on a diet day, drinking drink 5-8 cup prepared by the present invention each day of going on a diet, and every 6-8 week is one and intervenes the course for the treatment of.
Preferably, as shown in table 6 below for go on a diet recommended intake every day of day each nutrient substance of the individuality of high-risk grade (the V-VIII level as in table 4):
Table 6
| Heat | 1364.94 kilocalorie | VitB1 | 0.12 milligram | Calcium | 196.26 milligram |
| Protein | 8.88 gram | Riboflavin | 0.06 milligram | Magnesium | 112.14 milligram |
| Fat | 1.74 gram | Nicotinic acid | 2.7 milligram | Iron | 5.88 milligram |
| Carbohydrate | 78.84 grams | Vitamins C | 140.1 milligram | Manganese | 0.9 milligram |
| Food fibre | 10.74 grams | Vitamin-E | 10.02 milligrams | Zinc | 2.4 milligram |
| Vitamin A | 879.72 microgram | Cholesterol | 0 milligram | Copper | 1.02 milligram |
| Carotene | 5259.84 microgram | Potassium | 1182.54 milligram | Phosphorus | 198.6 milligram |
| Vogan-Neu | 0 microgram | Sodium | 134.64 milligram | Selenium | 4.26 microgram |
Go on a diet recommended intake every day of day each nutrient substance of individuality for low risk level (the I-IV level as in table 4) is as shown in table 7 below:
Table 7
| Heat | 1206.66 kilocalorie | VitB1 | 0.12 milligram | Calcium | 120.42 milligram |
| Protein | 5.82 gram | Riboflavin | 0.01 milligram | Magnesium | 77.22 milligrams |
| Fat | 1.26 gram | Nicotinic acid | 1.80 milligram | Iron | 4.14 milligram |
| Carbohydrate | 71.58 grams | Vitamins C | 82.2 milligrams | Manganese | 0.48 milligram |
| Food fibre | 8.94 gram | Vitamin-E | 8.7 milligram | Zinc | 1.74 milligram |
| Vitamin A | 451.56 microgram | Cholesterol | 0 milligram | Copper | 0.9 milligram |
| Carotene | 2711.88 microgram | Potassium | 921.6 milligram | Phosphorus | 141.36 milligram |
| Vogan-Neu | 0 microgram | Sodium | 80.1 milligrams | Selenium | 3.06 microgram |
In a preferred embodiment of the invention, two site Polymorphism type risk class III and IV are defined as toxin accumulation type ob gene type, and risk class I and II is defined as non-toxin accumulation type ob gene type; In another preferred embodiment of the present invention, three site Polymorphism type risk class V-VIII are defined as toxin accumulation type ob gene type, and risk class I-IV is defined as non-toxin accumulation type ob gene type.
One embodiment of the present invention are beverage formulations corresponding with the diet Design with Rule of day of going on a diet according to the nutrient demand of above-mentioned day of going on a diet.
Fruits and vegetables are the food of modal rich vitamin and various nutrient substance, after fruits and vegetables make juice and mud, nutrition more easily absorbs, delicious and easily prepare, a kind of preferred implementation of the present invention is: adopt the mode of fresh squeezing fruits and vegetables to prepare to meet the drink that formula Middle nutrition element requires.
In a preferred embodiment of the invention, individuality is divided into toxin accumulation type ob gene type and non-toxin accumulation type ob gene type, the formula of the drink that the individuality for toxin accumulation type ob gene type makes is as follows:
Containing apple 62.5g, pears 20g, purple cabbage 30g, Caulis et Folium Brassicae capitatae 7.5g, spinach 12.5g, tomato 12.5g, Chinese yam 20g, lemon 2g in every 300mL drink.
The formula of the drink made for the individuality of non-toxin accumulation type ob gene type is as follows:
Containing apple 62.5g, pears 20g, purple cabbage 15g, Caulis et Folium Brassicae capitatae 3.8g, spinach 6.3g, tomato 6.3, Chinese yam 20g, lemon 2g in every 300mL drink.
Step 4, make drink according to the formula determined in step 3
Adopt above-mentioned fruits and vegetables formula to make drink, come by artificial or automatization draining device.
Preferably, the drinking method that drink of the present invention is used in toxin accumulation type ob gene Individual treatment process is: fixing weekly 1-3 days for going on a diet day, drinking drink 5-8 cup prepared by the present invention each day of going on a diet, and every 6-8 week is one and intervenes the course for the treatment of.
Present invention also offers a kind of preparation system of toxin accumulation type ob gene Individual treatment drink, this system comprises control unit, Polymorphism type detecting unit and drink production unit, and wherein control unit is electrically connected with Polymorphism type detecting unit, drink production unit respectively; Be preset with the single-gene risk angle value in multiple related polymorphic site in toxin accumulation type ob gene risk factors in described control unit, and the pleomorphism site can selected according to user sets up risk class automatically; Described Polymorphism type detecting unit is for detecting the Polymorphism type of individual DNA sample, export detected result to described control unit simultaneously, described control unit can export the corresponding risk class of this sample according to the detected result of Polymorphism type detecting unit, then export drink production unit to according to risk class or risk product value determination beverage formulation, last drink production unit makes Individual treatment drink according to the instruction of control unit.
Preferably, described drink production unit comprises automatic juice extractor, described automatic juice extractor has hyperchannel feed(raw material)inlet, be equipped with in described passage and be cut into block fruit and vegetable materials, valve is provided with in each passage, the formula that described automatic juice extractor can provide according to control unit, by the opening and closing of different channel valve, the different fruit and vegetable materials of certain volume proportioning are dropped in juice extractor inner chamber, described fruit and vegetable materials flows out from outlet be squeezed into fruit juice and puree mixture in the inner chamber of this juice extractor after, collect this mixture, mix suitable quantity of water, be toxin accumulation type ob gene Individual treatment drink of the present invention.Wherein, the fruit and vegetable materials that by volume proportioning drops into is considered as acceptable error in the scope of ± 0.5g.
Preferably, described bulk refers to that length is all not more than the fritter of 3cm.
Preferably, described automatic juice extractor also has self-cleaning function; A kind of optimal way is, water or scavenging solution is injected in a-road-through road, when juice extractor starts clean, the valve open of this water filling or scavenging solution passage, water or scavenging solution flow into juice extractor inner chamber, start function cleaning inner chamber of squeezing the juice, cleaned rear water or scavenging solution flows out from fruit juice export, completed a self-stip.
The present invention has following technique effect:
1, the present invention can prepare fat-reducing drink targetedly according to the Polymorphism type of obese individuals, has better fat-reducing effect than existing clean cut diet products;
2, the present invention adopts associated detection technique to obtain Polymorphism type by first order fluorescence quantitative PCR program, can determine individual genotype more comprehensively, rapidly;
3, the present invention adopts the preparation system of Individual treatment drink, achieves the integrated process of the preparation from sample detection to drink;
4, the drink that prepared by the present invention adopts commercially available fruits and vegetables to be raw material, and preparation process is simple, and nectar has good mouthfeel and distinct color and luster, is a kind of very popular and have the drink of good fat-reducing effect.
Be described further below with reference to the technique effect of accompanying drawing to design of the present invention, concrete structure and generation, to understand object of the present invention, characteristic sum effect fully.
Accompanying drawing explanation
Fig. 1 is the workflow diagram of the preparation system of toxin accumulation type ob gene Individual treatment drink of the present invention;
Fig. 2 is the structural representation of automatic juice extractor in a preferred embodiment of the present invention.
Embodiment
The foundation of embodiment 1 Polymorphism type risk class
The pleomorphism site detected in the present embodiment is: rs1695 (
gSTP1), rs1048943 (
cYP1A1) and rs4646903 (
cYP1A1), they are labeled as a, b, c respectively, and their hypotype risk is as shown in table 8 below:
Table 8
| Gene polymorphism sites | Genotype | Risk (OR) |
| a | 1(AA) | 1 |
| 2(AG) | 1 | |
| 3(GG) | 3.26 | |
| b | 1(AA) | 1 |
| 2(AG) | 1 | |
| 3(GG) | 1.89 | |
| c | 1(CC) | 3.12 |
| 2(CT) | 1 | |
| 3(TT) | 1 |
Listed by table 8, single-gene risk calculates shown in the risk grade table 4 described above of acquisition.
Embodiment 2 genes of individuals detects
3 polymorphic sites in embodiment 1 are adopted to carry out genes of individuals detection.
Gather individual mouth epithelial cells sample, with silica gel adsorption extracting genomic dna, after electrophoresis detection checking, carry out quantitative fluorescent PCR reaction.
Individual specimen is put into respectively 3 reacting holes, simultaneously detect 3 sites, namely rs13306517 (
lEP), rs1805094 (
lEPR), rs1800795 (
iL-6), experimentally need to set up not containing the NTC blank control wells of DNA profiling in addition;
In each reacting hole, add reagent is quantitative fluorescent PCR reaction system, and cumulative volume is 10 μ L, and namely concentration is DNA profiling 2 μ L, 10 × quantitative fluorescent PCR reaction buffer ABITaqman of 20ng/ μ L
?four kinds of deoxynucleotide substrate 0.1 μ L, 25mMMgCl of MGB1 μ L, 25mMdNTP synthetic DNA
2solution 0.5 μ L, (5units/ μ L) Taq DNA polymerase 0.02 μ L, deionized water 4.98 μ L, 3 sites adopt different forward primer (20 μMs respectively, 0.225 μ L), reverse primer (20 μMs, 0.225 μ L), VIC fluorescent probe (10 μMs, 0.25 μ L) and FAM fluorescent probe (10 μMs, 0.25 μ L); 3 sites use primer and probe as shown in Table 5.
Reacting hole and blank are reacted in PCR amplification instrument, first preheating: 50 DEG C, 2 minutes, 95 DEG C, 10 minutes, then 95 DEG C, 30 seconds that carry out 60 circulations, 60 DEG C, 1 minute, reaction terminates rear taking-up reaction system and puts on quantitative real time PCR Instrument again and read fluorescent amount, obtains three figure of three genes.
Figure and the NTC blank of the final sample fluorescent signal that quantitative real time PCR Instrument shows is compared, the one in three kinds of different signals may be there is in each site, namely pure VIC fluorescent signal, VIC and FAM heterozygosis fluorescent signal and pure FAM fluorescent signal, represent the genotype that three kinds, this site is different respectively.
Embodiment 3 risk class and beverage formulation
Adopt the risk class set up in embodiment 1, wherein risk class V-VIII is defined as toxin accumulation type ob gene type, risk class I-IV is defined as non-toxin accumulation type ob gene type.
Formula for toxin accumulation type ob gene type individuality:
Containing apple 62.5g, pears 20g, purple cabbage 30g, Caulis et Folium Brassicae capitatae 7.5g, spinach 12.5g, tomato 12.5g, Chinese yam 20g, lemon 2g in every 300mL drink.
Regular convention formula for non-toxin accumulation type ob gene type individuality:
Containing apple 62.5g, pears 20g, purple cabbage 15g, Caulis et Folium Brassicae capitatae 3.8g, spinach 6.3g, tomato 20g, Chinese yam 20g, lemon 2g in every 300mL drink.
Embodiment 4 Individual treatment drink preparation system
The workflow of the toxin accumulation type ob gene Individual treatment drink preparation system in the present embodiment as shown in Figure 1, this system comprises control unit, Polymorphism type detecting unit and drink production unit, and wherein control unit is electrically connected with Polymorphism type detecting unit, drink production unit respectively; Be preset with the single-gene risk angle value in multiple related polymorphic site in toxin accumulation type ob gene risk factors in described control unit, and the pleomorphism site can selected according to user sets up risk class automatically; Described Polymorphism type detecting unit is for detecting the Polymorphism type of individual DNA sample, export detected result to described control unit simultaneously, described control unit can calculate the corresponding risk class of this sample according to the detected result of Polymorphism type detecting unit, then export drink production unit to according to risk class or risk product value determination beverage formulation, last drink production unit produces Individual treatment drink according to the instruction of control unit.
Described Polymorphism type detecting unit comprises quantitative real time PCR Instrument, adopts TaqMan
?the DNA sample of-MCB technology to individuality detects.
Described drink production unit comprises automatic juice extractor as shown in Figure 2, described automatic juice extractor has 8 passage feed(raw material)inlets (in figure 1-8), can be used for depositing 8 kinds of different fruits and vegetables, the fruits and vegetables in each passage all adopt commercially available fresh fruit of vegetables, are cut into the fritter that length, width and height are all not more than 3cm, valve is provided with in each passage, the controlled Systematical control of opening and closing of described valve, the time length of all right by-pass valve control unlatching of Controlling System simultaneously, the volume ratio of fruits and vegetables not of the same race in juice extractor inner chamber is controlled to enter according to the time length of valve opening, the formula that described automatic juice extractor can provide according to control unit, by the opening and closing of different channel valve, the different fruit and vegetable materials of certain volume proportioning are dropped in juice extractor inner chamber 9, described fruit and vegetable materials flows out from outlet 10 be squeezed into fruit juice and puree in the inner chamber 9 of this juice extractor after, collect this juice puree mixture, toxin accumulation type ob gene Individual treatment drink of the present invention is after allocating suitable quantity of water again.
This automatic juice extractor also has self-cleaning function, in 8 paths Zhong mono-tunnels, inject water, when juice extractor needs clean, the valve open of this waterflood path, water flows into juice extractor inner chamber, starts function cleaning inner chamber of squeezing the juice, clean rear water to flow out from fruit juice export 10, complete a self-stip.
Embodiment 7 Clinic intervention study on urogenital
According to the risk class set up in embodiment 1, risk class V-VIII is defined as toxin accumulation type ob gene type, risk class I-IV is defined as non-toxin accumulation type ob gene type.Choose 40 routine BMI index >=28, and the individuality that risk class is judged as toxin accumulation type ob gene carries out fat Intervention trial, the age of test individuality is all between 25 ~ 50 years old.Wherein 20 examples (10 men, 10 female) are test group, adopt the formula of the toxin accumulation type ob gene type individuality in embodiment 5; Another 20 examples (10 men, 10 female) are control group, adopt the regular convention formula of the non-toxin accumulation type ob gene type in embodiment 5.
In test experimenter weekly 2 days for going on a diet day (Monday and Thursday), select to select to 18 from 8 and drank one bottle of individuation drink 300ml(totally 6 bottles every 2 hours), and at not other food of re-uptake on the same day; A non-day normal diet of going on a diet, and food unifies dispensing by test organization person, sendout slightly adjusts by individual difference, and all do not produce abnormal sensation of hunger with all experimenters and be as the criterion, the test period amounts to eight weeks.
Before intervention, the BW(body weight of test group and control group) and BMI(weight index), WC(waistline), HC(hip circumference), WHR(waist-to-hipratio) all there is no significant difference (P value >0.1); After intervention, the These parameters of experimenter all be improved significantly, but the improvement result of test group is better than control group, the difference of its weight index and waist-to-hipratio has statistical significance (the equal ﹤ 0.05 of P value), in table 9 and 10.
The change of the indices such as table 9 patients before and after intervention body weight with compare (male sex's group)
* P<0.05 is represented
The change of the indices such as table 10 patients before and after intervention body weight with compare (women's group)
* P<0.05 is represented.
More than describe preferred embodiment of the present invention in detail.Should be appreciated that the ordinary skill of this area just design according to the present invention can make many modifications and variations without the need to creative work.Therefore, all technician in the art, all should by the determined protection domain of claims under this invention's idea on the basis of existing technology by the available technical scheme of logical analysis, reasoning, or a limited experiment.
Sequence table
The upper marine eugenic object height science and technology limited Company of <110>
The preparation method of <120> toxin accumulation type ob gene Individual treatment drink and system thereof
<130>2015
<160>15
<170>PatentInversion3.3
<210>1
<211>643
<212>DNA
<213> people
<220>
<221>mutation
<222>(369)
<223>n=a or g
<400>1
ccccgcagccctctggagtggaggaaactgagacccactgaggttacgtagtttgcccaa60
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cctcccaggctggggctcacagacagccccctggttggcccatccccagtgactgtgtgt240
tgatcaggcgcccagtcacgcggcctgctcccctccacccaaccccagggctctatggga300
aggaccagcaggaggcagccctggtggacatggtgaatgacggcgtggaggacctccgct360
gcaaatacntctccctcatctacaccaactatgtgagcatctgcaccagggttgggcact420
gggggctgaacaaagaaaggggcttcttgtgccctcaccccccttacccctcaggtggct480
tgggctgaccccttcttgggtcagggtgcaggggctgggtcagctctgggccaggggccc540
aggggcctgggacaagacacaacctgcacccttattgcctgggacatcaaccagccaagt600
aacgggtcatgggggcgagtgcaaggacagagacctccagcaa643
<210>2
<211>601
<212>DNA
<213> people
<220>
<221>mutation
<222>(301)
<223>n=a or g
<400>2
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acacttctgagccctgaactgccacttcagctgtctccctctggttacaggaagctatgg180
gtcaacccatctgagttcctacctgaacggtttctcacccctgatggtgctatcgacaag240
gtgttaagtgagaaggtgattatctttggcatgggcaagcggaagtgtatcggtgagacc300
nttgcccgctgggaggtctttctcttcctggctatcctgctgcaacgggtggaattcagc360
gtgccactgggcgtgaaggtggacatgacccccatctatgggctaaccatgaagcatgcc420
tgctgtgagcacttccaaatgcagctgcgctcttaggtgcttgagagccctgaggcctag480
actctgtctacctggtctggttgggcagccagaccagcaggctggcctatgtggtctaag540
gttcagcctgaaactcatagacactgatctggctgcagttttgctatctgggctgtgggc600
a601
<210>3
<211>1000
<212>DNA
<213> people
<220>
<221>mutation
<222>(501)
<223>n=c or t
<400>3
aggactagggctggagtgaggggggggtatttcaattaccttctattggtctcccttctc60
tacactcttgtaataaaatgtctatttttaatgtttgtacacaacaatccttctattcta120
gcctgcattgagcttgcatgcttgcataagagcttaagaaccattgatttaatgtaatag180
ggaaaattctaacccaggtatccaaaaatgtgtaagaacaactacctgagctaaataaag240
atattgttcagaaatcctataggtggagattttttgaatcataaatgattcatcactcgt300
ctaaatactcaccctgaaccccattctgtgttgggttttactgtagggaggaagaagagg360
aggtagcagtgaagaggtgtagccgctgcacttaagcagtctgtttgagggacaagactc420
tattttttgagacagggtccccaggtcatccaggctggagtgcactggtaccattttgtt480
tcactgtaacctccacctccngggctcacacgattctcccacctcagcctctgagtagtt540
ggggccgcaggcacacgccaccacagcttttttttttttttttttttttttttgtagaga600
tggggtttcaccatgttgcccaggctggtctcaaactcctgagctcaagtgatccacctg660
cctcagcctcccaaagtgctgggattacaggcatgagacaagactcctaatcactgtgct720
gacttacgccctctctaacttatcacaaattgatgaagcgtattctccgaattaggcaat780
aagggtatcaagtgaatatagtacagtccctgctttcaagaacctcccaaaatggttgag840
ggaactgacacataaacacatcattcaattcattgtggtcagaactctaccagacagatg900
catacgggaggtggggtccagaggctgggttggggagtgggcaccgcgagatttccaggt960
ctttttttttttgagacggagtctcactgtgtcgcccagg1000
<210>4
<211>16
<212>DNA
<213> artificial sequence
<220>
<223> is with VIC fluorescence A type probe, is combined with GSTP1rs1695SNP site
<400>4
tgcaaatacgtctccc16
<210>5
<211>16
<212>DNA
<213> artificial sequence
<220>
<223> is with FAM fluorescence G type probe, is combined with GSTP1rs1695SNP site
<400>5
tgcaaatacatctccc16
<210>6
<211>22
<212>DNA
<213> artificial sequence
<220>
<223> is used for the forward primer of GSTP1rs1695SNP sequence amplification
<400>6
tctatgggaaggaccagcag20
<210>7
<211>23
<212>DNA
<213> artificial sequence
<220>
<223> is used for the reverse primer of GSTP1rs1695SNP sequence amplification
<400>7
gaagcccctttctttgttca20
<210>8
<211>12
<212>DNA
<213> artificial sequence
<220>
<223> is with VIC fluorescence A type probe, is combined with CYP1A1rs1048943SNP site
<400>8
agaccattgccc12
<210>9
<211>11
<212>DNA
<213> artificial sequence
<220>
<223> is with FAM fluorescence G type probe, is combined with CYP1A1rs1048943SNP site
<400>9
gaccgttgccc11
<210>10
<211>20
<212>DNA
<213> artificial sequence
<220>
<223> is used for the forward primer of CYP1A1rs1048943SNP sequence amplification
<400>10
gtgttaagtgagaaggtgat20
<210>11
<211>20
<212>DNA
<213> artificial sequence
<220>
<223> is used for the reverse primer of CYP1A1rs1048943SNP sequence amplification
<400>11
aggatagccaggaagagaaa20
<210>12
<211>13
<212>DNA
<213> artificial sequence
<220>
<223> is with VIC fluorescence C type probe, is combined with CYP1A1rs4646903SNP site
<400>12
cctcccgggctca13
<210>13
<211>13
<212>DNA
<213> artificial sequence
<220>
<223> is with the T-shaped probe of FAM fluorescence, is combined with CYP1A1rs4646903SNP site
<400>13
cctcctgggctca13
<210>14
<211>19
<212>DNA
<213> artificial sequence
<220>
<223> is used for the forward primer of CYP1A1rs4646903SNP sequence amplification
<400>14
aggctggagtgcactggta19
<210>15
<211>20
<212>DNA
<213> artificial sequence
<220>
<223> is used for the reverse primer of CYP1A1rs4646903SNP sequence amplification
<400>15
acatggtgaaaccccatctc20
Claims (10)
1. a preparation method for toxin accumulation type ob gene Individual treatment drink, is characterized in that described preparation method comprises:
The foundation of step one Polymorphism type risk class:
(1) select the gene polymorphism sites fat relevant to toxin accumulation type, the single-gene risk of the different genotype in each genetic risk factors related polymorphic site is determined in the data provided according to document or voluntarily sampling;
(2) by each genotype permutation and combination of all pleomorphism sites of employing, then Polymorphism type risk class is set up according to each single-gene risk product;
Step 2 genes of individuals detects:
The Polymorphism type of gene polymorphism sites selected in carry out step one to the DNA obtained from individuality detects;
The risk class determination beverage formulation of step 3 belonging to detected result:
Determine individual risk class according to the result that Polymorphism type detects, and press nutrient demand and the means of intervention of different risk class, determine the addition of Different Nutrition element in drink;
Step 4 makes drink according to the formula determined in step 3.
2. the preparation method of toxin accumulation type ob gene Individual treatment drink as claimed in claim 1, to is characterized in that described in step (1) that the concrete grammar that the single-gene risk of the different genotype in each genetic risk factors related polymorphic site is determined in sampling is voluntarily:
A. case-control study method is utilized, adopt chi square test or Fisher precise probabilistic method, credibility interval CI gets 95%, and significant difference gets p<0.05, and the related locus of the fat genetic risk factors in specific crowd of contratoxin accumulation type screens;
B. utilize online SPSS statistics computed in software relative risk OR, CI gets 95%, p<0.05, as the single-gene risk of each related polymorphic site different genotype.
3. the preparation method of toxin accumulation type ob gene Individual treatment drink as claimed in claim 1, is characterized in that the concrete grammar of the gene polymorphism sites that selection described in step (1) is fat relevant to toxin accumulation type is: select according to the genetic risk factors that toxin accumulation type in documents searching Chinese population is fat common.
4. the preparation method of toxin accumulation type ob gene Individual treatment drink as claimed in claim 1, is characterized in that the described gene polymorphic position property point fat relevant to toxin accumulation type is selected from least two: the rs1695 in following three sites, rs1048943, rs4646903.
5. the preparation method of toxin accumulation type ob gene Individual treatment drink as claimed in claim 1, it is characterized in that the concrete grammar of step (2) is: each gene polymorphism sites fat relevant to toxin accumulation type has three kinds of hypotypes respectively, and what all for selected site hypotypes occurred may carry out permutation and combination; Then the risk of these hypotypes is multiplied and obtains the risk product of different permutation and combination; The risk product of acquisition is arranged from small to large or from big to small, merges the group that risk product is equal; The final height determining each group of risk by the size of risk product.
6. the preparation method of toxin accumulation type ob gene Individual treatment drink as claimed in claim 1, is characterized in that the described Polymorphism type in step 2 detects employing TaqMan
?-MCB technology detects simultaneously.
7. the preparation method of toxin accumulation type ob gene Individual treatment drink as claimed in claim 1, it is characterized in that adopting the mode of fresh squeezing fruits and vegetables to make drink for the individuality of high-risk grade in step 3, its formula is as follows: containing apple 62.5g, pears 20g, purple cabbage 30g, Caulis et Folium Brassicae capitatae 7.5g, spinach 12.5g, tomato 12.5g, Chinese yam 20g, lemon 2g in every 300mL drink.
8. the preparation system of a toxin accumulation type ob gene Individual treatment drink, it is characterized in that comprising control unit, Polymorphism type detecting unit and drink production unit, wherein control unit is electrically connected with Polymorphism type detecting unit, drink production unit respectively; Be preset with the single-gene risk angle value in multiple related polymorphic site in toxin accumulation type ob gene risk factors in described control unit, and the pleomorphism site can selected according to user sets up risk class automatically; Described Polymorphism type detecting unit is for detecting the Polymorphism type of individual DNA sample, export detected result to described control unit simultaneously, described control unit can export the corresponding risk class of this sample according to the detected result of Polymorphism type detecting unit, then export drink production unit to according to risk class or risk product value determination beverage formulation, last drink production unit makes Individual treatment drink according to the instruction of control unit.
9. the preparation system of toxin accumulation type ob gene Individual treatment drink as claimed in claim 8, it is characterized in that described drink production unit comprises automatic juice extractor, described automatic juice extractor has hyperchannel feed(raw material)inlet, be equipped with in described passage and be cut into block fruit and vegetable materials, valve is provided with in each passage, the formula that described automatic juice extractor can provide according to control unit, by the opening and closing of different channel valve, the different fruit and vegetable materials of certain volume proportioning are dropped in juice extractor inner chamber, described fruit and vegetable materials flows out from outlet be squeezed into fruit juice and puree mixture in the inner chamber of this juice extractor after, collect described mixture, mix certain water gaging, be toxin accumulation type ob gene Individual treatment drink of the present invention.
10. the preparation system of toxin accumulation type ob gene Individual treatment drink as claimed in claim 8, is characterized in that the concrete grammar that the described pleomorphism site selected according to user sets up risk class is automatically: what all for selected site hypotypes occurred may carry out permutation and combination; Then the risk of these hypotypes is multiplied and obtains the risk product of different permutation and combination; The risk product of acquisition is arranged from big to small, merges the group that risk product is equal; The final height determining each group of risk by the size of risk product.
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Cited By (2)
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| CN105624288A (en) * | 2016-03-16 | 2016-06-01 | 上海中优生物高科技有限责任公司 | Toxin accumulation type obese gene individual treatment composition and preparing method and system thereof |
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