CN102344952B - Primer, method and kit for detecting apple-derived materials in sample - Google Patents
Primer, method and kit for detecting apple-derived materials in sample Download PDFInfo
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Abstract
The invention relates to an oligonucleotides primer for detecting apple-derived materials in a sample, and also relates to a realtime fluorescence PCR (Polymerase Chain Reaction) detection method for detecting the apple-derived materials in the sample, wherein the method comprises the step of using the specific oligonucleotides primer aiming at the apple-derived materials in the sample. The invention also relates to a PCR detection kit for quickly detecting the apple-derived materials in the sample, wherein the kit comprises the specific oligonucleotides primer for detecting the apple-derived materials in the sample by using the realtime fluorescence PCR detection method. In addition, the invention relates to application of the specific oligonucleotides primer aiming at the apple-derived materials in the sample in detecting the apple-derived materials in the sample. Due to the use of the PCR detection method and the PCR detection kit, whether the apple-derived materials are contained in the samples such as fruit juice, food, drug, nourishment and the like can be simply, quickly, specifically and sensitively detected.
Description
Technical field
The invention belongs to biological technical field, particularly, the present invention relates to the Oligonucleolide primers for the apple-derived materials in sample detection, the real-time fluorescence PCR detection method that is used for the working sample apple-derived materials, the PCR detection kit that is used for the rapid detection apple-derived materials in sample, and the specific oligonucleotide primer of apple-derived materials in sample and probe application in apple-derived materials in test sample.
Background technology
China is Apple Culture state the biggest in the world, is also Sucus Mali pumilae production and big export country.Sucus Mali pumilae as the second-biggest-in-the-world nectar that is only second to orange juice, is one of fastest-rising fruit juice of present global consumption.In recent years, along with the raising of living standards of the people, the sales volume of China's Sucus Mali pumilae constantly increases, and kind and type are on the increase, and brand is multifarious.But, being subjected to ordering about of economic interests, some illegal manufacturers are marked with at fruit juice constituents and content and practise fraud, and counterfeit and shoddy goods flood market.As in nineteen ninety-five, due to the shortage of Sucus Mali pumilae on the world market, the Sucus Mali pumilae price is increaseing, and pear juice even the price such as syrup is lower, therefore mix pear juice even the Sucus Mali pumilae of syrup etc. flood market.This has not only directly damaged human consumer's economic interests, and very likely endanger the consumer health, the legitimate rights and interests that trade mark is held enterprise have simultaneously also been invaded, finally not only destroy the China market economic order, upset social credit system, and affect Chinese commodity image in the international market, damage China's foreign trade interests.
In addition, along with the change of people life style, the sickness rate of China's food allergy day by day increases, food allergy be subjected to attention degree more and more higher, the detection of all kinds of foods sources property allergen is also seemed more and more important.Class Sweet protein thaumatin-Like Protein (TLP) conduct is a kind of important allergen wherein, receives gradually increasing concern.TLP extracts from multiple food at present, as pears, apple, peach, wheat, tomato and potato etc., one of important member of pathogenesis-related proteins (PRs) family of its to be plant induce in body when being subject to pathogenic micro-organism infringement generation, participate in multiple fungus resistant reaction process, or the important anaphylactogen of part population.Class monellin composition in apple juice possibility because identify unclear or mix pseudo-the fraud, causes part population to invade consumer rights to its allergy in the market.Therefore, the allergen constituent class monellin in nectar detects and has theory and realistic meaning.
The adulterated mode of Sucus Mali pumilae mainly contains two kinds at present: the first is to mix the more honest and cleaner fruit juice of some prices in Sucus Mali pumilae, such as Fructus Vins juice or pear juice; The second is to add the compositions such as entry and syrup in Sucus Mali pumilae, increases its volume.And existing Sucus Mali pumilae distinguishing method between true and false has: instrumental method such as the special component discriminance such as carbohydrate discriminance, organic acid discriminance, pectin substance identification method, aldehydes matter identification method, letones identification method, amino acid differential method, inorganic elements discriminance and high-efficient liquid phase chromatogram technology, gas-chromatography, stable isotope mass spectroscopy, MALDI-TOMS method, thermo-cracking mass spectroscopy, near-infrared spectrum technique, ultraviolet spectral technique, artificial neural network etc.The application of above-mentioned these methods has been played certain effect to supervision and the management of fruit juice market.But above-mentioned technology can be subject to the impact of the several factors such as kind, the place of production, harvest season, raw material environment, processing conditions, storing packaging means to a great extent, has certain limitation.In addition, the factor that affects physics and chemistry or instrument authentication detection technology sample representation is very many, guarantee the reliability of its method, just needs a large amount of detection samples and the mathematical model analytical technology of science, makes aforesaid method be very restricted in practical application.
In international Sucus Mali pumilae processing industry, more and more, the new technology of deep processed product emerges in an endless stream, and China's apple processing industry also exists the residual problem that exceeds standard etc. and to still need and to solve of insufficient raw material, agriculture, and it is also one of restraining factors that affect China's Sucus Mali pumilae industry healthy development that Sucus Mali pumilae is mixed pseudo-discrimination method.Current, be badly in need of a kind ofly not affected by adding means the authentication detection technology that is detected in essence from fruit juice raw material.Along with the development of modern biotechnology, molecular biology method carries out the food discriminating and is just becoming the research direction that receives much attention in the world.Protocols in Molecular Biology is with its convenient, accurate, rapid, succinct characteristics, analyze characteristic and the source of food raw material and product from gene level, sensitivity is higher, specificity is stronger, its result has been for the true and false of proof food provides truly, reliable foundation, is used for gradually in recent years the false distinguishing that food variety discriminating, product are traced to the source etc.
At present, there is not yet report both at home and abroad can be quick, simple, special and detect delicately method and the test kit of the apple-derived materials in samples such as fruit juice, food, medicine and nutritious prod.
Therefore, the detection method of the apple-derived materials in sample that this area needs are a kind of fast, specificity is good, highly sensitive and the test kit that is used for the rapid detection apple-derived materials in sample carry out the detection of the apple-derived materials in samples such as fruit juice, food, medicine and nutritious prod.
Summary of the invention
One object of the present invention is, the specific oligonucleotide primer of rapid detection apple-derived materials in sample is provided.
Another object of the present invention is, the PCR detection method of Fast Measurement apple-derived materials in sample is provided.
A further object of the present invention is, is provided for the PCR detection kit of rapid detection apple-derived materials in sample.
Also purpose of the present invention is, the specific oligonucleotide primer of apple-derived materials and the probe application in apple-derived materials in test sample in sampling.
For the foregoing invention purpose, the invention provides following technical scheme:
According to one embodiment of the invention, the invention provides the specific oligonucleotide primer pair for real time fluorescent PCR method test sample apple-derived materials, described primer pair is that the ITS region sequence according to class sweet protein gene intron and rDNA has an otherness in different plant species characteristics design.In one embodiment, wherein said primer pair is selected from No2-F:TTCAATGTCGATGATGAAGAG (SEQ ID NO:1) and No2-R:AGACTTCCTACTTTCACGTTTAAC (SEQ ID NO:2); No3-F:GGAATATGAACGAAAGAGCG (SEQ ID NO:3) and No3-R:AACGACTCTCGGCAACGGAT (SEQ ID NO:4); And Rp116-F 5 ' AATCTATGGAATCGTGGGATTC 3 ' (SEQ ID NO:5) and Rpl 16-R 5 ' CATTATAGCGTTCTACCAAAACG 3 ' (SEQ ID NO:6).
In a preferred embodiment, described specific oligonucleotide primer pair for real time fluorescent PCR method test sample apple-derived materials is SEQ ID NO:1 and SEQ ID NO:2.In another preferred embodiment, described specific oligonucleotide primer pair for real time fluorescent PCR method test sample apple-derived materials is SEQ ID NO:3 and SEQ ID NO:4.In another preferred embodiment, described specific oligonucleotide primer pair for real time fluorescent PCR method test sample apple-derived materials is SEQ ID NO:5 and SEQ ID NO:6.
According to another embodiment of the invention, the invention provides the real-time fluorescence PCR detection method of apple-derived materials in sample, described method comprises the specific oligonucleotide primer pair that uses for apple-derived materials in sample, and described primer pair is that the ITS region sequence according to class sweet protein gene intron and rDNA has an otherness in different plant species characteristics design.In one embodiment, the Auele Specific Primer that uses in the real-time fluorescence PCR detection method of apple-derived materials in sample of the present invention is to being selected from SEQ ID NO:1 and SEQ ID NO:2; SEQ IDNO:3 and SEQ ID NO:4; And SEQ ID NO:5 and SEQ ID NO:6.
In a preferred embodiment of the inventive method, described real time fluorescent PCR method is SYBR fluorescence dye method.In another preferred embodiment of the inventive method, described real time fluorescent PCR method is Taqman fluorescent probe method.In one embodiment, in the Taqman fluorescent probe method of apple-derived materials, also use specific probe in test sample of the present invention.In a preferred embodiment, the probe sequence that uses is No3-P:FAM-TGCGTCGTCGTCTTCGATAA-TAMAR (SEQ ID NO:7).In one embodiment, apple-derived materials in sample PCR detection method of the present invention also further comprises the step of extracting sample total DNA.In one embodiment, described DNA extraction step by the endogenous reference gene chloroplast(id) trn gene that detection plant itself has, is come the extraction quality of the total DNA of specimen.In a preferred embodiment, the universal primer sequence of described reference gene plant chloroplast trn gene is No1-F:CTTGATTTTACCAAAGATGATGA (SEQ IDNO:8) and No 1-R:TTCTTCGCATGTACCCGCAG (SEQ ID NO:9).In apple-derived materials in sample PCR detection method of the present invention, the control sample that uses comprises pear juice, peach juice, orange, Kiwifruit, tomato etc.In one embodiment, use the specific oligonucleotide primer pair of apple-derived materials and plant universal amplification primer to carry out the double PCR amplification.In a preferred embodiment, the specific oligonucleotide primer pair of the apple-derived materials that uses is SEQ ID NO:1 and SEQ ID NO:2, and the plant universal amplification primer that uses is SEQ ID NO:8 and SEQ ID NO:9.In another preferred embodiment, the specific oligonucleotide primer pair of the apple-derived materials that uses is SEQ ID NO:3 and SEQ ID NO:4, and the plant universal amplification primer that uses is SEQ ID NO:8 and SEQ ID NO:9.In another preferred embodiment, the specific oligonucleotide primer pair of the apple-derived materials that uses is SEQ ID NO:8 and SEQ IDNO:9 as SEQ ID NO:5 and SEQ ID NO:6, the plant universal amplification primer that uses.
According to another embodiment of the invention, the invention provides the real-time fluorescence PCR detection method of apple-derived materials in sample, described method comprises the combination of using the specific oligonucleotide primer pair for apple-derived materials in sample of the present invention.In a real-time scheme, according to real-time fluorescence PCR detection method of the present invention, also comprise and use reference gene plant chloroplast trn gene universal primer sequence SEQ ID NO:8 and SEQ ID NO:9, by the endogenous reference gene chloroplast(id) trn gene that detection plant itself has, come the extraction quality of the total DNA of specimen.In the PCR of apple-derived materials in sample of the present invention detection method, also further comprise the step that the pcr amplification condition is further optimized.In a preferred embodiment, in the step of pcr amplification condition optimizing, use different annealing temperatures to increase.In a preferred embodiment, described pcr amplification condition is 95 ℃, 10min; 95 ℃ of 15s; 60 ℃, 1min.
According to another embodiment of the invention, the invention provides the test kit of rapid detection apple-derived materials in sample, described test kit comprises specific oligonucleotide primer pair and the working instructions for real time fluorescent PCR method test sample apple-derived materials of the present invention.In a preferred embodiment, described test kit also comprises the reagent that extracts for sample DNA and the reagent that is used for the PCR reaction.In a preferred embodiment, the working instructions of described test kit comprise the description to the condition that is used for the apple-derived materials in sample pcr amplification.In a preferred embodiment, the pcr amplification condition that provides in the specification sheets of described test kit is 95 ℃, 10min; 95 ℃ of 15s; 60 ℃, 1min.
According to another embodiment of the present invention, the invention provides the specific oligonucleotide primer pair application in apple-derived materials in test sample for real time fluorescent PCR method test sample apple-derived materials of the present invention.In a preferred embodiment, the invention provides specific oligonucleotide primer pair SEQ ID NO:1 and the SEQ ID NO:2 of apple-derived materials in sample; SEQ ID NO:3 and SEQ ID NO:4; Or SEQ ID NO:5 and SEQ ID NO:6 application in apple-derived materials in test sample.
The present invention is basic as detecting take the DNA of apple, between transcribing according to class sweet protein gene intron and rDNA, district's (ITS) sequence has the characteristics of otherness in different plant species, has cloned the class sweet protein gene intron of apple and the ITS 1-5.8S-ITS2 region sequence of rDNA thereof.According to these primers, utilize the apple-derived materials in real-time fluorescence PCR method test sample.
Real-time fluorescence quantitative PCR is namely on the basis of conventional PCR method, add fluorescently-labeled probe or fluorescence dye, accumulation along with the PCR product, the fluorescent signal that probe or dyestuff send strengthens, and the fluorescence monitoring system can receive fluorescent signal, be DNA chain of every generation, just have a fluorescence molecule to form, realized that the accumulation of fluorescent signal and PCR product form Complete Synchronization.Therefore can the whole PCR reaction process of Real Time Monitoring, and finally detect the initial copy number of testing sample, thus can detect contained apple-derived materials in testing sample.
Real-time fluorescence PCR detection method of the present invention adopts complete stopped pipe to detect, and need not the PCR aftertreatment, has avoided crossed contamination and false positive.Method of the present invention has used dexterously that the DNA of round pcr efficiently increases, the specificity of nucleic acid hybridization and detection technique of fluorescence fast and susceptibility, have simple to operate, time saving and energy saving, reliable results and the accurate advantage such as sensitive.The test kit of making according to primer sequence of the present invention is used for the qualitative and quantitative analysis of this series products, has highly sensitive, high specificity, result is reliable and stable and avoid crossed contamination to cause false-positive advantage.Use PCR detection method of the present invention and PCR detection kit, both can be used for qualitative detection, can be used for again detection by quantitative, its simple, quick, special and sensitive characteristics are suitable for Sucus Mali pumilae and the real and fake discrimination of concerned drink and the detection of allergen composition etc. on the domestic and international market.
Description of drawings
Fig. 1 is the gel electrophoresis figure that shows testing sample DNA extraction effect, wherein uses the plant universal primer to detect, and the sample of each swimming lane is as follows: M:DNA molecular weight marker (2000bp); 1: Chinese pear; 2: Huiyuan Pear Juice; 3: eat dream board pear juice; 4: the sea is pear juice too; 5: Huiyuan's pear flesh beverage; 6: juice drinks mixes in Huiyuan; 7: Guoguang apple; 8: bright fruit lures 100%; 9: Huiyuan's Sucus Mali pumilae; 10: happy living grows 100%; 11: Huiyuan's apple pulp beverage; 12: honey peach; 13: Huiyuan Peach Juice; 14: eat dream board peach juice; 15: the sea is peach juice too; 16: Huiyuan's peach fruit squash; 17: orange; 18: Kiwifruit; 19: tomato; 20: blank (sterilized water).
Fig. 2 shows the result that detects the class sweet protein gene of apple in Sucus Mali pumilae by real-time fluorescence PCR, and wherein fluorescence curve 1-11 is followed successively by that positive control (Guoguang apple), bright fruit lure 100%, Huiyuan's Sucus Mali pumilae, the happy taste 100% of living, Huiyuan's apple pulp beverage, Huiyuan Pear Juice, eats dream board pear juice, peach juice, orange juice, Fructus actinidiae chinensis juice, tomato juice and blank.
Fig. 3 shows that real-time fluorescence PCR detects the result of the ITS region sequence of apple in Sucus Mali pumilae, wherein fluorescence curve 1-11 be followed successively by that positive control (Guoguang apple), bright fruit lure 100%, Huiyuan's Sucus Mali pumilae, happy live grow 100%, Huiyuan's apple pulp beverage, pear juice, peach juice, orange juice, Fructus actinidiae chinensis juice, tomato juice and blank.
Embodiment
The present invention is further illustrated for mode by embodiment, but the present invention is not limited only to following examples.
The present embodiment is the extraction quality by the total DNA of use plant universal primer specimen.
The endogenous reference gene chloroplast(id) trn gene that has by detecting plant itself, extraction quality that can the total DNA of specimen.
The plant chloroplast trn gene universal primer sequence for detection of plant derived component in sample of using in the present embodiment is SEQ ID NO:8 and SEQ ID NO:9.
in the present embodiment, detected Chinese pear, Huiyuan Pear Juice (Beijing Huiyuan Food ﹠ Beverage Co., Ltd., China), the sea is pear juice (sea too, Korea S) too, Huiyuan's pear flesh beverage (Beijing Huiyuan Food ﹠ Beverage Co., Ltd., China), eat dream board pear juice (big lake (Tianjin) fresh provisions fruit juice company limited, China), carefree pear juice (carefree, Korea S), Guoguang apple, bright Sucus Mali pumilae (Shanghai Bright Dairy ﹠ Food Co., Ltd.'s dairy factory, China), Huiyuan's Sucus Mali pumilae (Beijing Huiyuan Food ﹠ Beverage Co., Ltd., China), big lake Sucus Mali pumilae (big lake (Tianjin) fresh provisions fruit juice company limited, China), happy living grown 100% Sucus Mali pumilae (Beijing is happy live grow development in science and technology company limited, China), honey peach, Huiyuan Peach Juice (Beijing Huiyuan Food ﹠ Beverage Co., Ltd., China), Huiyuan's peach fruit squash (Beijing Huiyuan Food ﹠ Beverage Co., Ltd., China), eat dream board peach juice (big lake (Tianjin) fresh provisions fruit juice company limited, China), the sea is peach juice (sea too, Korea S) too, orange, Kiwifruit, the extraction quality of total DNA of tomato, and use duck as negative control.As shown in Figure 1, all can band occur at about 159bp place during with the DNA of plant universal primer amplification testing sample, show that all testing sample DNA extraction successfully.
The present inventor's first passage PCR clones and the class sweet protein gene partial sequence of the apple that checked order.
The present embodiment is for obtaining the class sweet protein gene partial sequence of apple by the PCR cloning and sequencing.
Have the characteristics of otherness according to class sweet protein gene intron in different plant varieties, adopt apple class sweet protein gene sequence (Genbank No.AY792604) design upstream and downstream primer amplification apple.Operation instructions according to Wizard Gel Extraction Kit (Promega, the U.S.) is carried out purifying, recovery to apple PCR product.The specification sheets of pressing TaKaRa pMD 19-T Vector test kit (TaKaRa, Japan) is connected purified product with pMD19-T Vector, linked system 10 μ L, and its reactive component is: pMD19-T Vector 1 μ L, PCR product 2 μ L, ddH
2O 2 μ L, Solution I 5 μ L arrange the positive and negative control simultaneously.Linked system is placed in room temperature (22-37 ℃) reaction 30min, and reaction is placed on ice after finishing immediately.Add and connect product in 50 μ LTOP competent cells, flick mixing, ice bath 30min, 42 ℃ of accurate heat shock 90s are placed in 2min on ice immediately, then add the gone out brain heart infusion of bacterium of 800 μ L, 37 ℃, 200r/min shaking culture 1h.5000r/min low-speed centrifugal 1min abandons 600 μ L supernatants, will precipitate mixing, gets 100 μ L mixed solutions and is applied on the nutrient agar plate that Amp+, X-Gal and IPTG process, and 37 ℃ of incubated overnight are placed on 4h in 4 ℃ of refrigerators.Picking list bacterium colony hickie is cultivated at 37 ℃, 200r/min shaken overnight in the brain heart infusion of the Amp that contains final concentration 200 μ g/mL.
(1) positive colony is identified: the single white clone of picking, carry out bacterium colony PCR reaction, system is 25 μ L, and its component is: reaction 10 * Buffer 2.5 μ L, dNTPs 1 μ L, upstream and downstream primer each 0.5 μ L, Taq enzyme 0.2 μ L, picking list bacterium colony adds water and mends to 25 μ L as template.Response procedures is: 94 ℃ of 5min, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min, 40 circulations.The PCR product is identified by agarose gel electrophoresis.
(2) order-checking: after determining positive colony, this bacterium colony of picking is cultivated at 37 ℃, 200r/min shaken overnight in the 5 μ L brain heart infusions that contain final concentration 200 μ g/mL Amp.Get 1mL bacterium liquid and send biotech firm's evaluation of checking order.
The apple class sweet protein gene partial sequence that the PCR cloning and sequencing obtains is as follows:
GCAAACAGGCAATTAAGACATATTCAATGTCGATGATGAAGAGCCAAGTAGCTTCCCTCCTCGGCCTCACCTTGGCCATCCTCTTCTTCTCAGGTAATATTATACAAACTTCTCATTATATTTTTTGTTCTTTTTGTTTCCTATTAGTCTCTAGTATATTGTATGCATGCCTGCCTGATGCAAATATGAGAGGGGTTAGTCCGCACTATATTGTAATACTGTTAAACGTGAAAGTAGGAAGTCTAAGAGAGAATAACGATGATGGATAATTCTGTGACGTCCACTAATAATTTTATTAAAACAAGGAGTACTAGTACTTTTTAAAAGGATACCGTAAAAGTTCTCAATCCGGCCACATATAAGTGAACTAACCAATGCGGCTAACATGAGATTAGATATATTTATAATTGTATGTAGATTTTTCAGTATCAATATTCTTAGTATCTAGTTCTATGTTGAACATAAATGCAGGTGCACATGCAGCGAAAATCACTTTCACAAACAACTGCCCCAACACTGTCTGGCCAGGAACCTTAACC(SEQ ID NO：10)。
The present embodiment is the primer sequence of the class sweet protein gene by the using apple class sweet protein gene sequence through real-time fluorescence PCR specific detection apple-derived materials in sample.SYBR fluorescence dye method: SYBR Green is high-sensitive DNA fluorescence dye, can detect 20pg DNA at least in various analyses.The avidity of SYBR Green dyestuff and double-stranded DNA is very high, and the rear fluorescent signal of being combined with product dsDNA in the PCR reaction can strengthen 800-1000 doubly and the enzyme of commonly using in to molecular biology does not have restraining effect.SYBR Green PCR reaction system is: Faststart UniversalSYBR Green Master (Roche) 12.5 μ L; Each 0.5 μ L of upstream and downstream primer (10 μ mol/L); DNA profiling 5 μ L (approximately 50ng), mending with sterilized water is totally extremely 25 μ L.Adopt Bio-RadiQTM5 multicolor real time PCR instrument to carry out pcr amplification and interpretation of result, amplification condition is as follows: 95 ℃ of denaturation 10min; 95 ℃ of 10s, 58 ℃ of 30s, 40 circulations are carried out melting curve analysis after the PCR reaction.
Primer sequence is: SEQ ID NO:1 and SEQ ID NO:2.
The detection key instrument that uses:
micropipet (10 μ L, 100 μ L, 1000 μ L Eppendorf), quantitative real time PCR Instrument (Mastercycler ep realplex4 Eppendorf), high speed tabletop centrifuge (Pico17 Thermo), high speed disintegrator (IKA-WEARKE GERMANY), gel imaging system (Gene Genius), electrophoresis apparatus (DYY22C type, Liuyi Instruments Plant, Beijing), nucleic acid-protein analyser (DYY-6C, Liuyi Instruments Plant, Beijing), freeze drier (Modulyod Freeze Dryer Thermo), ice-making machine (XB70GRANT) etc.
Detect main agents:
Taq enzyme, dNTPs, 10 * PCR Buffer, ethidium bromide, DNA Ladder Marker (2000) are (Takara) available from Shanghai living work company; TaqMan Universal SYBR Green Master is available from Roche company; Primer (SEQ ID NO:1 and SEQ ID NO:2) is synthetic etc. by Shanghai AudioCodes bio tech ltd.
Detect key step:
1DNA extracts
Samples of juice to be measured is: bright fruit lures 100%, Huiyuan's Sucus Mali pumilae, happy living are grown 100%, Huiyuan's apple pulp beverage, Huiyuan Pear Juice, eaten dream board pear juice, peach juice, orange juice, Fructus actinidiae chinensis juice and tomato juice.
Get in the clean culture dish of 30mL sample to, vacuumize freeze-drying; Take in the clean 50mL centrifuge tube of the freezing dry-matter to of 0.2g, add 5mLCTAB lysate (2%CTAB (W/V), 0.1mol/LTris-HCl, 20mmol/L EDTA, 1.4mol/LNaCl), 65 ℃ of 2h, interval continuous mixing several times; 8000rpm 15min, get in 1mL supernatant liquor to 1 a clean 2.0mL centrifuge tube, add 700 μ L chloroforms, violent mixing 30s, 14500rpm 10min gets respectively 650 μ L supernatant liquors to clean 2.0mL centrifuge tube, add 1300 μ L CTAB precipitated liquid (0.5%CTAB (W/V), 0.04mol/LNaCl), violent mixing 30s, the standing 1h of room temperature; 14500rpm 20min abandons supernatant liquor, adds 350 μ L 1.2M NaCl, thermal agitation 30s, then add 350 μ L chloroforms, violent mixing 30s, 14500rpm 10min; Get respectively supernatant liquor 320 μ L, add 0.8 times of volume Virahol, after mixing ,-20 ℃ of 1h, 14500rpm 20min abandons supernatant liquor, adds 500 μ L 70% ethanol, and after mixing, 14500rpm 20min abandons supernatant liquor, dries in the air to air-dry, adds 30 μ L ddH
2The O dissolving, 4 ℃ store for future use.
2 real-time fluorescence PCRs detect the primer
SEQ ID NO:1 and SEQ ID NO:2.
3 real-time fluorescence PCR reaction systems:
F aststart Universal SYBR Green Master 12.5μL
Upstream primer (10 μ mol/L) 0.5 μ L
Downstream primer (10 μ mol/L) 0.5 μ L
Add ddH
2O to cumulative volume be 25 μ L
Annotate: each PCR detects and all sets up corresponding blank (ultrapure water with the preparation reaction system replaces DNA profiling, and whether detection reagent is polluted);
4 real-time fluorescence PCR reaction parameters:
95℃ 10min
95℃ 10s
58℃ 30s
40 circulating reactions.
Annotate: different instruments should be done suitable adjustment with each reagent of PCR and reaction parameter.
As shown in Figure 2, when utilizing the class sweet protein gene sequence of apple in the real-time fluorescence PCR test sample, Guoguang apple, bright fruit lure 100%, Huiyuan's Sucus Mali pumilae, happy live grow 100%, the melting curve peak value of Huiyuan's apple pulp beverage pcr amplified fragment is all at 74.82 ± 1.0 ℃, and pear juice, peach juice, orange juice, Fructus actinidiae chinensis juice, tomato juice and blank show that all without this melting curve peak value this primer can effectively detect the apple-derived materials in sample.
Identical with method described according to embodiment 3, just take testing sample DNA as template, adopt primer sequence SEQ ID NO:1 and SEQ ID NO:2 and plant universal amplification primer pair SEQ ID NO:8 and the SEQ ID NO:9 of class sweet protein gene, carry out the double PCR amplification.
The present embodiment is the primer sequence of the ITS gene by the using apple sequence through real-time fluorescence PCR specific detection apple-derived materials in sample.Taqman fluorescent probe method: the TaqMan technology is a kind of technology of single tube PCR product being carried out the real time fluorescent quantitative detection, in the regular-PCR amplification system, add a double fluorescence labeling probe with the special complementation of target-gene sequence, utilize the whole PCR process of fluorescent signal accumulation Real-Time Monitoring, by typical curve, unknown template is carried out quantitative analysis at last.Quantitative step: determine that 1. (C represents cycle number (Cycle) to the CT value, and T represents fluorescence thresholding (Threshold), the cycle number that experiences when namely the fluorescent signal in each reaction tubes arrives the thresholding of setting; 2. utilize typical curve to carry out quantitative assay to unknown sample.After obtaining the CT value of unknown sample, calculate the initial copy number of this sample from typical curve.There is linear relationship in the logarithm of the CT value of each template and the initial copy number of this template, and namely initial copy number is more, and the CT value is less.Reaction system is: TaqMan Universal probe Master 12.5 μ L; Probe (10 μ mol/L) 0.5 μ L; Each 0.5 μ L of upstream and downstream primer (10 μ mol/L); Template DNA 5 μ L; Add ddH
2O to cumulative volume be 25 μ L.Response procedures is 95 ℃ of 10min; 95 ℃ of 15s; 60 ℃ of 1min.
In the test sample of using, the ITS gene primer sequence of apple-derived materials is: upstream primer SEQ ID NO:3; Downstream primer SEQ ID NO:4; Probe SEQ ID NO:7.The TaqMan probe is a kind of oligonucleotide probe, and its fluorescence is relevant to the amplification of aim sequence.It is designed to and target sequence upstream primer and downstream primer between sequence pairing.Fluorophor FAM (6-Fluoresceincarboxylic acid (6-carboxy fluo-rescein)) and TAMRA are connected to 5 ' end of probe, and quencher TAMAR (6-carboxyl rhodamine (6-carboxy tetramethyl rhodamine)) is at 3 ' end.When complete probe and target sequence pairing, the fluorescence of fluorophor emission approaches cancellation because of the quencher with 3 ' end.But when carrying out extension, 5 ' 5 prime excision enzyme activity of polysaccharase carries out enzyme with probe to be cut, and makes fluorophor separate with quencher.
The detection key instrument that uses:
micropipet (10 μ L, 100 μ L, 1000 μ L Eppendorf), quantitative real time PCR Instrument (ABI7700 Applied Biosystems, USA)), high speed tabletop centrifuge (Pico17 Thermo), high speed disintegrator (IKA-WEARKE GERMANY), gel imaging system (Gene Genius), electrophoresis apparatus (DYY22C type Liuyi Instruments Plant, Beijing), nucleic acid-protein analyser (DYY-6C Liuyi Instruments Plant, Beijing), freeze drier (Modulyod Freeze Dryer Thermo), ice-making machine (XB70GRANT) etc.
Detect main agents:
Taq enzyme, dNTPs, 10 * PCR Buffer, ethidium bromide, DNA LadderMarker (2000) are (Takara) available from Shanghai living work company; TaqMan Universal probe Master is available from ABI company; Primer (upstream primer SEQ ID NO:3; Downstream primer SEQ ID NO:4; Probe SEQ ID NO:7.) synthesized by Shanghai AudioCodes bio tech ltd etc.
Detect key step:
1DNA extracts
Samples of juice to be measured is: bright fruit lures 100%, Huiyuan's Sucus Mali pumilae, happy live grow 100%, Huiyuan's apple pulp beverage, pear juice, peach juice, orange juice, Fructus actinidiae chinensis juice and tomato juice.
Get in the clean culture dish of 30mL sample to, vacuumize freeze-drying; Take in the clean 50mL centrifuge tube of the freezing dry-matter to of 0.2g, add the 5mLCTAB lysate, 65 ℃ of 2h, interval continuous mixing several times; 8000rpm 15min gets in 1mL supernatant liquor to 1 a clean 2.0mL centrifuge tube, adds 700 μ L chloroforms, violent mixing 30s, 14500rpm 10min gets respectively 650 μ L supernatant liquors to clean 2.0mL centrifuge tube, add 1300 μ L CTAB precipitated liquid, violent mixing 30s, the standing 1h of room temperature; 14500rpm 20min abandons supernatant liquor, adds 350 μ L 1.2M NaCl, thermal agitation 30s, then add 350 μ L chloroforms, violent mixing 30s, 14500rpm 10min; Get respectively supernatant liquor 320 μ L, add 0.8 times of volume Virahol, after mixing ,-20 ℃ of 1h, 14500rpm 20min abandons supernatant liquor, adds 500 μ L 70% ethanol, and after mixing, 14500rpm 20min abandons supernatant liquor, dries in the air to air-dry, adds 30 μ L ddH
2The O dissolving, 4 ℃ store for future use.
2 real-time fluorescence PCRs detect the primer and probe
Primer sequence is: upstream primer SEQ ID NO:3; Downstream primer SEQ ID NO:4; Probe SEQ ID NO:7:
3 real-time fluorescence PCR reaction systems:
TaqMan Universal probe Master 12.5μL
Probe (10 μ mol/L) 0.5 μ L
Upstream primer (10 μ mol/L) 0.5 μ L
Downstream primer (10 μ mol/L) 0.5 μ L
Add ddH
2O to cumulative volume be 25 μ L
Annotate: each PCR detects and all sets up corresponding blank (ultrapure water with the preparation reaction system replaces DNA profiling, and whether detection reagent is polluted);
4 real-time fluorescence PCR reaction parameters:
95℃ 10min
95℃ 15s
60℃ 1min
Annotate: different instruments should be done suitable adjustment with each reagent of PCR and reaction parameter.
As shown in Figure 3, when utilizing the ITS gene order of apple in the real-time fluorescence PCR test sample, Guoguang apple, bright fruit lure 100%, Huiyuan's Sucus Mali pumilae, happy live grow 100%, pcr amplification all appears in Huiyuan's apple pulp beverage, and pear juice, peach juice, orange juice, Fructus actinidiae chinensis juice, tomato juice and blank all do not occur, and show that this primer probe can effectively detect the apple-derived materials in sample.
Identical with method described according to embodiment 5, just take testing sample DNA as template, adopt primer sequence SEQ ID NO:3, SEQ ID NO:4 and probe SEQ ID NO:7 and plant universal amplification primer pair SEQ ID NO:8, the SEQ ID NO:9 of ITS gene, carry out the double PCR amplification.
The present inventor clones and the partial sequence of chloroplast(id) rpl 16 genes of checked order apple and pears first.
DNA according to plant chloroplast rpl16 gene order design upstream and downstream primer amplification apple and pears.According to the operation instructions of Wizard Gel Extraction Kit, the PCR product of apple and pears is carried out purifying, recovery.The specification sheets of pressing TaKaRa pMD19-T Vector test kit is connected purified product with pMD19-T Vector, linked system 10 μ L, and its reactive component is: pMD19-T Vector1 μ L, PCR product 2 μ L, ddH
2O 2 μ L, Solution I 5 μ L arrange the positive and negative control simultaneously.Linked system is placed in room temperature (22-37 ℃) reaction 30min, and reaction is placed on ice after finishing immediately.Add and connect product in 50 μ LTOP competent cells, flick mixing, ice bath 30min, 42 ℃ of accurate heat shock 90s are placed in 2min on ice immediately, then add the gone out brain heart infusion of bacterium of 800 μ L, 37 ℃, 200r/min shaking culture 1h.5000r/min low-speed centrifugal 1min abandons 600 μ L supernatants, will precipitate mixing, gets 100 μ L mixed solutions and is applied on the nutrient agar plate that Amp+, X-Gal and IPTG process, and 37 ℃ of incubated overnight are placed on 4h in 4 ℃ of refrigerators.Picking list bacterium colony hickie is cultivated in 37 ℃ of brain heart infusions, the 200r/min shaken overnight of the Amp that contains final concentration 200 μ g/mL.
(1) positive colony is identified: the single white clone of picking, carry out bacterium colony PCR reaction, system is 25 μ L, and its component is: reaction 10 * Buffer 2.5 μ L, dNTPs 1 μ L, upstream and downstream primer each 0.5 μ L, Taq enzyme 0.2 μ L, picking list bacterium colony adds water and mends to 25 μ L as template.Response procedures is: 94 ℃ of 5min, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min, 40 circulations.The PCR product is identified by agarose gel electrophoresis.
(2) order-checking: after determining positive colony, this bacterium colony of picking, 37 ℃, the cultivation of 200r/min shaken overnight in the 5 μ L brain heart infusions that contain final concentration 200 μ g/mLAmp.Get 1mL bacterium liquid and send biotech firm's evaluation of checking order.
The partial sequence of chloroplast(id) rpl16 gene that the PCR cloning and sequencing obtains apple and pears is as follows:
Pears 159
TTAA
TAATCTATGGAATCGTGGGATTCTTTGAAATTTGATCTAATCGATTATAAATTAGAAATTTTTTGTGTTTTAATATATAATTTAACACGTATTTATATAATTTAACACGTATTCTATTTATAGATAATGTCGTTTT
GGTAGAACGCTATAATGAATCTTTATTT(SEQ ID NO：11)。
Apple 139
TTAA
TAATATATGGAATCGTGGGATTCTTTGAAATTCGATCTAATCGATTATAAATTAGAAATTTTTTGTGTTTTAATATAGAATTTAACACGTATTCTATTTATAGATAATGTCGTTTT
GGTAGAACGCTATAATGA ATCTTTATTT(SEQ ID NO：12)。
Sucus Mali pumilae relative content in PCR primer detection by quantitative apple by chloroplast(id) rpl16 gene and pears fruit juice blends, the PCR primer that is used for the chloroplast(id) rpl16 gene of detection by quantitative fruit juice blends Sucus Mali pumilae relative content is:
SEQ ID NO：5
SEQ ID NO：6。
With this DNA to the primer amplification fruit juice blends, the PCR product length that the apple composition increases is 159bp, and the PCR product length that the pear juice composition increases is 139bp.By carry out electrophoretic separation on 2.5% agarose or 5% polyacrylamide gel, can distinguish two kinds of PCR fragments, and quantitatively determine apple and the shared ratio of pears in mixing juice according to the relative abundance of two kinds of PCR fragments.
The present embodiment provides the test kit of rapid detection apple-derived materials in sample.
Described test kit comprises primer pair SEQ ID NO:5, SEQ ID NO:6 and primer pair SEQ IDNO:8, SEQ ID NO:9, and working instructions, wherein primer pair SEQ ID NO:5, SEQID NO:6 are the specific oligonucleotide primer pairs for real-time fluorescence PCR test sample apple-derived materials, SEQ ID NO:8, SEQ ID NO:9 are plant universal amplification primers, provided the pcr amplification condition in described working instructions, this condition is 95 ℃, 10min; 95 ℃, 15s; 58 ℃, 30s.For different instruments, reaction parameter is done suitable adjustment.
The present embodiment provides the test kit of rapid detection apple-derived materials in sample.
Described test kit comprises primer pair SEQ ID NO:1, SEQ ID NO:2 and primer pair SEQ IDNO:8, SEQ ID NO:9, and working instructions, wherein primer pair SEQ ID NO:1 and SEQID NO:2 are the specific oligonucleotide primer pairs for real-time fluorescence PCR test sample apple-derived materials, SEQ ID NO:8, SEQ ID NO:9 are plant universal amplification primers, provided the pcr amplification condition in described working instructions, this condition is 95 ℃, 10min; 95 ℃, 15s; 60 ℃, 1min.For different instruments, reaction parameter is done suitable adjustment.
The present embodiment provides the test kit of rapid detection apple-derived materials in sample.
Described test kit comprises primer SEQ ID NO:3 and SEQ ID NO:4 and probe SEQ IDNO:7 and primer pair SEQ ID NO:8, SEQ ID NO:9, and working instructions, wherein primer pair SEQ ID NO:3 and SEQ ID NO:4 and probe SEQ ID NO:7 are the specific oligonucleotide primer pairs for real-time fluorescence PCR test sample apple-derived materials, SEQ ID NO:8, SEQ ID NO:9 are plant universal amplification primers, provided the pcr amplification condition in described working instructions, this condition is 95 ℃, 10min; 95 ℃, 15s; 60 ℃, 1min.For different instruments, reaction parameter is done suitable adjustment.
Although specific embodiments of the present invention is described, those skilled in the art will appreciate that under the prerequisite that does not depart from scope of the present invention or spirit and can carry out multiple change and modification to the present invention.Thereby, this invention is intended to contain all these changes and modification of dropping in appended claims book and coordinator scope thereof.
Claims (5)
1. for detection of the real-time fluorescence PCR detection method of apple-derived materials in sample, wherein said real time fluorescent PCR method is SYBR fluorescence dye method, and its primer pair that uses is SEQ ID NO:1 and SEQ ID NO:2; Perhaps, described real time fluorescent PCR method is Taqman fluorescent probe method, and its primer pair that uses is SEQ ID NO:3 and SEQ ID NO:4, and the probe that uses is SEQ IDNO:7.
2. real-time fluorescence PCR detection method according to claim 1, also comprise and use plant chloroplast trn gene universal primer sequence SEQ ID NO:8 and SEQ ID NO:9.
3. the test kit of rapid detection apple-derived materials in sample, described test kit comprises primer pair SEQ ID NO:1 and SEQ ID NO:2; Perhaps described test kit comprises primer pair and probe, and described primer pair is SEQ ID NO:3 and SEQ ID NO:4, and described probe is SEQ ID NO:7.
4. test kit according to claim 3, wherein said test kit also comprises plant chloroplast trn gene universal primer sequence SEQ ID NO:8 and SEQ ID NO:9.
5. the described test kit of claim 3 or 4 application in apple-derived materials in test sample.
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| CN106701908A (en) * | 2015-11-18 | 2017-05-24 | 中国检验检疫科学研究院 | Primers, probe and method for detecting lotus root-sourced component and kit |
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