CN102344953A - Primer for detecting peach-derived component in sample, method and kit - Google Patents
Primer for detecting peach-derived component in sample, method and kit Download PDFInfo
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Abstract
The present invention relates to oligonucleotide primer for detecting a peach-derived component in a sample, and further relates to a real time fluorescence PCR detection method for detecting the peach-derived component in the sample, wherein the method comprises using the specific oligonucleotide primer for the peach-derived component in the sample. The invention further relates to a PCR detection kit for rapidly detecting the peach-derived component in the sample, wherein the kit comprises the specific oligonucleotide primer for detecting the peach-derived component in the sample through the real time fluorescence PCR detection. The invention further relates to an application of the specific oligonucleotide for the peach-derived component in detection of the peach-derived component in the sample. With adopting the PCR detection method and the PCR detection kit provided by the present invention, the peach-derived component in fruit juice, food, medicine, nourishments and other samples can be simply, rapidly, specifically and sensitively detected.
Description
Technical field
The invention belongs to biological technical field; Particularly; The present invention relates to be used for the Oligonucleolide primers of sample Land of Peach Blossoms property composition detection; The real-time fluorescence PCR detection method that is used for working sample Land of Peach Blossoms property composition; The PCR detection kit that is used for rapid detection sample Land of Peach Blossoms property composition, and specific oligonucleotide primer and probe application in the Land of Peach Blossoms property composition in test sample of Land of Peach Blossoms property composition in the sample.
Background technology
China has abundant fruit resource, and fruit juice processing industry development in recent years is rapid.Along with the raising of people's living standard and health perception, the nectar product becomes more diversified, and the kind of various nectars, type and brand emerge in an endless stream.Yet be subjected to ordering about of economic interests, some illegal manufacturers are marked with at fruit juice kind, content and play tricks, and some are fallen short of the reality even the fruit juice of passing a fake product off as a genuine one floods 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; Cause serious negative social effect; Upset social sincere system, and influence Chinese commodity image in the international market, infringement China foreign trade interests.
Owing to contain a kind of important allergen-class monellin Thaumatin-LikeProtein (TLP) in the fruit such as peach, become one of main fruit that causes allergic reaction at present, and received increasing concern gradually.TLP extracts from multiple food; Like pears, apple, peach, tomato and potato etc.; One of important member of pathogenesis-related proteins (PRs) family of its to be fruit induce in the body when receiving pathogenic micro-organism infringement generation; Participating in multiple antimycotic reaction process, also is part crowd's important allergen.Class monellin composition in the peach juice beverage maybe be because sign be unclear or mix pseudo-the fraud in the market, causes the part crowd irritated and invade consumer rights to it.Therefore, the allergen constituent class monellin in the nectar detects and has theory and realistic meaning.
Along with development of science and technology, peach juice and concerned drink thereof to mingle means also more and more hidden.Initial mingling only is to add water and sweeting agents such as sucrose and syrup; The composition that has now developed into according to various fruit juice carries out very fine interpolation; Even the fruit juice that food false distinguishing expert sets up formed database as " prescription " mingled, make various traditional Land of Peach Blossoms property composition detection more and more difficult that becomes.Current, be badly in need of a kind ofly not influenced by adding means the authentication detection technology that detects in essence from the peach raw material.
The process of using to multiple characters, special component, ad hoc analysis and mathematical statistics method from single traits, common component, routine analysis has been experienced in the research of differentiating about fruit juice constituents both at home and abroad.Along with the development of modern biotechnology, the applied molecular biology method is carried out the food discriminating and is just being become the research direction that receives much attention in the world.Protocols in Molecular Biology is with its convenient, accurate, rapid, succinct characteristics; Analyze food raw material and characteristics of product and source from gene level; Sensitivity is higher, specificity is stronger; Its result is for the true and false of proof food provides truly, reliable foundation; Fresh blood has been injected in food false distinguishing researchs such as feeding article kind is differentiated, product is traced to the source, and has become many mechanisms of countries in the world and scholar's research direction in recent years.
And the birth of real-time fluorescence PCR technology has remedied many deficiencies of conventional chemical detection technique, for new field has been opened up in the development of detection technique.Real-time fluorescence PCR is in the PCR reaction system, to add fluorescence labeling probe, utilizes the fluorescent signal whole PCR process of monitoring in real time, through typical curve template concentrations is carried out quantitative analysis at last, makes round pcr develop into quantitative level from qualitative level.In recent years, in molecular biology research, utilize quantitative PCR that gene expression amount and transgenic product content are detected, obtained widespread use.Because it has improved the sensitivity, specificity and the accuracy that detect greatly, also can effectively reduce the danger that produces pollution in the experimentation, the composition that has been widely used in fruit juice and other food is at present differentiated the field.
The birth of real-time fluorescence PCR technology has remedied many deficiencies of conventional chemical detection technique, for new field has been opened up in the development of detection technique.Real-time fluorescence PCR is in the PCR reaction system, to add fluorescence labeling probe, utilizes the fluorescent signal whole PCR process of monitoring in real time, through typical curve template concentrations is carried out quantitative analysis at last, makes round pcr develop into quantitative level from qualitative level.In recent years, in molecular biology research, utilize quantitative PCR that gene expression amount and transgenic product content are detected, obtained widespread use.Because it has improved the sensitivity, specificity and the accuracy that detect greatly, also can reduce the danger that produces pollution in the experimentation effectively, has been widely used in every field at present.
At present, domestic and international not appearing in the newspapers as yet can be detected the method and the test kit of property composition in the Land of Peach Blossoms in the samples such as fruit juice, food, medicine and nutritious prod quick, simple, special and delicately.
Therefore; The detection method of Land of Peach Blossoms property composition and the test kit that is used for rapid detection sample Land of Peach Blossoms property composition carry out the detection of property composition in the Land of Peach Blossoms in the samples such as fruit juice, food, medicine and nutritious prod in the sample that this area needs are a kind of fast, specificity is good, highly sensitive.
Summary of the invention
One object of the present invention is, the specific oligonucleotide primer of property composition in the Land of Peach Blossoms in the rapid detection sample is provided.
Another object of the present invention is, the PCR detection method of property composition in the Land of Peach Blossoms in the rapid determination sample is provided.
A further object of the present invention is, is provided for the PCR detection kit of property composition in the Land of Peach Blossoms in the rapid detection sample.
Also purpose of the present invention is the specific oligonucleotide primer of Land of Peach Blossoms property composition and the probe application in the Land of Peach Blossoms property composition in test sample in the sampling.
To the foregoing invention purpose, the present invention provides following technical scheme:
According to one embodiment of the invention; It is right that the present invention is provided for the specific oligonucleotide primer of property composition in the Land of Peach Blossoms in the real time fluorescent PCR method test sample, and said primer is to being that ITS region sequence according to class monellin gene intron and rDNA has an otherness in different plant species characteristics design.In one embodiment, wherein said primer is to being selected from No2-F:5 ' GAGCCAGCAGCTCTCCTCG 3 ' (SEQ ID NO:1) and No2-R:5 ' GCACCTGCATATTCAAGAACG 3 ' (SEQ ID NO:2); No3-F:5 ' GAATTGCGCCAAGGAAATTG 3 ' (SEQ ID NO:3) and No3-R:5 ' GAGAGCCGAGATATCCGTTG 3 ' (SEQ ID NO:4); And Tao-F:TGATCGACCCTCAATCTTACTGTA (SEQ ID NO:5) and Tao-R:CCCAAGAGCTTCTTATACTTTTACT (SEQ ID NO:6).
In a preferred embodiment, the said specific oligonucleotide primer that is used for real time fluorescent PCR method test sample Land of Peach Blossoms property composition is to being SEQ ID NO:1 and SEQ ID NO:2.In another preferred embodiment, the said specific oligonucleotide primer that is used for real time fluorescent PCR method test sample Land of Peach Blossoms property composition is to being SEQ ID NO:3 and SEQ ID NO:4.In another preferred embodiment, the said specific oligonucleotide primer that is used for real time fluorescent PCR method test sample Land of Peach Blossoms property composition is to being SEQ ID NO:5 and SEQ ID NO:6.
According to another embodiment of the invention; The real-time fluorescence PCR detection method of Land of Peach Blossoms property composition in the sampling of the present invention; Said method comprises that the specific oligonucleotide primer that uses to property composition in the Land of Peach Blossoms in the sample is right, and said primer is to being that ITS region sequence according to class monellin gene intron and rDNA has an otherness in different plant species characteristics design.In one embodiment, in sample of the present invention in the real-time fluorescence PCR detection method of Land of Peach Blossoms property composition employed Auele Specific Primer to being selected from SEQ ID NO:1 and SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4; And SEQ ID NO:5 and SEQ ID NO:6.
In an embodiment preferred of the inventive method, said real time fluorescent PCR method is a SYBR fluorescence dye method.In another embodiment preferred of the inventive method, said real time fluorescent PCR method is a Taqman fluorescent probe method.In one embodiment, in test sample of the present invention, in the Taqman fluorescent probe method of Land of Peach Blossoms property composition, also use specific probe.In a preferred embodiment, employed probe sequence is No3-P:5 ' FAM-CGGCGTCGTCATCTTCAAATATG-TAMAR 3 ' (SEQ ID NO:7).In one embodiment, Land of Peach Blossoms property composition PCR detection method also further comprises the step of extracting sample total DNA in the sample of the present invention.In one embodiment, said DNA extraction step is through detecting the endogenous reference gene chloroplast(id) trn gene that plant itself is had, the extraction quality of coming the total DNA of specimen.In a preferred embodiment; The universal primer sequence of said internal control gene plant chloroplast trn gene is No1-F:5 ' CTTGATTTTACCAAAGATGATGA3, (SEQ ID NO:8) and No1-R:5 ' TTCTTCGCATGTACCCGCAG 3 ' (SEQ IDNO:9).In the Land of Peach Blossoms property composition PCR detection method, employed control sample comprises Sucus Mali pumilae, pear juice, orange juice, Fructus actinidiae chinensis juice, tomato juice etc. in sample of the present invention.In one embodiment, the specific oligonucleotide primer that uses Land of Peach Blossoms property composition is to carrying out the double PCR amplification with plant universal amplification primer.In a preferred embodiment, the specific oligonucleotide primer of employed Land of Peach Blossoms property composition is to being SEQ ID NO:1 and SEQ ID NO:2, and employed plant universal amplification primer is SEQ ID NO:8 and SEQ ID NO:9.In another preferred embodiment, the specific oligonucleotide primer of employed Land of Peach Blossoms property composition is to being SEQ ID NO:3 and SEQ ID NO:4, and employed plant universal amplification primer is SEQ ID NO:8 and SEQ ID NO:9.In another preferred embodiment, the specific oligonucleotide primer of employed Land of Peach Blossoms property composition is to being SEQ ID NO:5 and SEQ ID NO:6, and employed plant universal amplification primer is SEQ ID NO:8 and SEQ ID NO:9.
According to another embodiment of the invention, the real-time fluorescence PCR detection method of Land of Peach Blossoms property composition in the sampling of the present invention, said method comprises uses the right combination of specific oligonucleotide primer to property composition in the Land of Peach Blossoms in the 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 internal control gene plant chloroplast trn gene universal primer sequence SEQ ID NO:8 and SEQ ID NO:9; Through detecting the endogenous reference gene chloroplast(id) trn gene that plant itself is had, the extraction quality of coming the total DNA of specimen.In sample of the present invention, in the PCR detection method of Land of Peach Blossoms property composition, 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, said pcr amplification condition is 95 ℃, 10min; 95 ℃ of 15s; 60 ℃, 1min.
According to another embodiment of the invention; The present invention provides the test kit of property composition in the Land of Peach Blossoms in the rapid detection sample, said test kit comprise the specific oligonucleotide primer that is used for real time fluorescent PCR method test sample Land of Peach Blossoms property composition of the present invention to and working instructions.In a preferred embodiment, said test kit also comprises reagent that is used for the sample DNA extraction and the reagent that is used for the PCR reaction.In a preferred embodiment, comprise description in the working instructions of said test kit to the condition that is used for sample Land of Peach Blossoms property composition pcr amplification.In a preferred embodiment, the pcr amplification condition that provides in the specification sheets of said test kit is 95 ℃, 10min; 95 ℃ of 15s; 60 ℃, 1min.
According to another embodiment of the present invention, the present invention provides the specific oligonucleotide primer that is used for real time fluorescent PCR method test sample Land of Peach Blossoms property composition of the present invention to the application in the Land of Peach Blossoms property composition in test sample.In a preferred embodiment, in the sampling of the present invention the specific oligonucleotide primer of Land of Peach Blossoms property composition to SEQ ID NO:1 and SEQ ID NO:2; SEQ IDNO:3 and SEQ ID NO:4; Or SEQ ID NO:5 and SEQ ID NO:6 application in the Land of Peach Blossoms property composition in test sample.
The present invention detects the basis with the DNA of peach; In different plant species, have the characteristics of otherness according to the ITS region sequence of class monellin gene intron and rDNA, cloned the ITS1-5.8S-ITS2 region sequence of peach class monellin gene intron and rDNA thereof.According to these sequences Design primers, utilize the Land of Peach Blossoms property composition in the real-time fluorescence PCR method test sample.
Real-time fluorescence quantitative PCR is promptly 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 fully synchronously.Therefore can monitor whole PCR reaction process in real time, and finally detect the initial copy number of testing sample, thereby can detect contained Land of Peach Blossoms property composition in the 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 accurate advantage such as sensitivity.The test kit of processing 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, the result is reliable and stable and avoid crossed contamination to cause false-positive advantage.Detection method of the present invention and test kit not only are fit to detect the Land of Peach Blossoms property composition in the fruit juice, can also detect other article, for example the Land of Peach Blossoms property composition in food, the nutritious prod etc.Use PCR detection method of the present invention and PCR detection kit; Both can be used for qualitative detection; Can be used for detection by quantitative again, its simple, quick, special and sensitive characteristics is suitable for peach juice 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 following: M:DNA molecular weight marker (2000bp); 1: Chinese pear; 2: Huiyuan Pear Juice; 3: the sea is pear juice too; 4: Huiyuan's pear flesh beverage; 5: eat dream board pear juice; 6: carefree pear juice; 7: Guoguang apple; 8: bright Sucus Mali pumilae; 9: Huiyuan's Sucus Mali pumilae; 10: the big lake Sucus Mali pumilae; 11: happy living grown 100% Sucus Mali pumilae; 12: honey peach; 13: Huiyuan Peach Juice; 14: Huiyuan's peach fruit squash; 15: eat dream board peach juice; 16: the sea is peach juice too; 17: orange juice; 18: Fructus actinidiae chinensis juice; 19: tomato juice; 20: blank (sterilized water).
Fig. 2 is the result who shows through the class monellin gene of peach in the real-time fluorescence PCR test sample, and wherein fluorescence curve 1-11 is followed successively by positive control (honey peach), Huiyuan Peach Juice, eats dream board peach juice, sea too peach juice, Huiyuan's peach fruit squash, Sucus Mali pumilae, pear juice, orange juice, Fructus actinidiae chinensis juice, tomato juice and blank.
Fig. 3 is the result who shows the ITS region sequence of peach in the real-time fluorescence PCR test sample, and wherein fluorescence curve 1-11 is followed successively by positive control (honey peach), Huiyuan Peach Juice, Huiyuan Peach Juice, eats dream board peach juice, sea too peach juice, Sucus Mali pumilae, pear juice, orange juice, Fructus actinidiae chinensis juice, tomato juice and blank.
Embodiment
The present invention is further illustrated for mode through embodiment, but the present invention is not limited only to following examples.
Embodiment 1
Present embodiment is the extraction quality through the total DNA of use plant universal primer specimen.
Through detecting the endogenous reference gene chloroplast(id) trn gene that plant itself is had, extraction quality that can the total DNA of specimen.
The employed plant chloroplast trn gene universal primer sequence that is used for the plant-derived composition of test sample is SEQ ID NO:8 and SEQ ID NO:9 in the present embodiment.
In the present embodiment; Detected Chinese pear; Huiyuan Pear Juice (Beijing Huiyuan Food & Beverage Co., Ltd.; China); The sea too pear juice (sea too; Korea S); Pear flesh beverage (Beijing Huiyuan Food & Beverage Co., Ltd. of Huiyuan; China); Eat dream board pear juice (big lake (Tianjin) fresh provisions fruit juice company limited; China); Carefree pear juice is (carefree; Korea S); Guoguang apple; Bright Sucus Mali pumilae (Shanghai Bright Dairy & Food Co., Ltd.'s dairy factory; China); Sucus Mali pumilae (Beijing Huiyuan Food & Beverage Co., Ltd. of Huiyuan; China); Big lake Sucus Mali pumilae (big lake (Tianjin) fresh provisions fruit juice company limited; China); Happy living grown 100% Sucus Mali pumilae (the happy development in science and technology company limited of growing alive in Beijing; China); Honey peach; Huiyuan Peach Juice (Beijing Huiyuan Food & Beverage Co., Ltd.; China); Peach fruit squash (Beijing Huiyuan Food & Beverage Co., Ltd. of Huiyuan; 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 juice; Fructus actinidiae chinensis juice; The extraction quality of total DNA of tomato juice.
As shown in Figure 1, all can band occur during with the DNA of plant universal primer amplification testing sample, show that all testing sample DNA extraction successfully at about 159bp place.
Embodiment 2
The present inventor is the class monellin gene intron sequence through the PCR clone and the peach of having checked order first.
Present embodiment is for obtaining the class monellin gene intron sequence of peach through the PCR cloning and sequencing.
In different plant varieties, have the characteristics of otherness according to class monellin gene intron, adopt apple class monellin gene order (Genbank No.AY792604) design upstream and downstream primer amplification peach.Operation instructions according to Wizard Gel Extraction Kit (Promega, the U.S.) is carried out purifying, recovery to peach PCR product.The specification sheets of pressing TaKaRa pMD19-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 are provided with the positive and negative control simultaneously.Linked system is placed room temperature (22-37 ℃) reaction 30min, and reaction places 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 place 2min on ice immediately, add gone out the brain heart infusion of bacterium of 800 μ L then, 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 handle, 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, each 0.5 μ L of upstream and downstream primer, 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 through agarose gel electrophoresis.
(2) order-checking: after confirming 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 peach class monellin gene intron partial sequence that the PCR cloning and sequencing obtains is following:
TGTAAGAAGATGAGAGCCAGCAGCTCTCCTCGGCCTCACCACCTTGGCCATCCTCTTCTTCTCAGGTATAATATTTTACAATTTTTGTTAGTCGATCAGATATCGATCAAAAGTTTATTTTTACAATTGTCTCTGTAATCTGTATTCTTATGATCACGTACGTTCTTGAATATGCAGGTGCACATGCAGCGAAAATCAC(SEQ?ID?NO：10)。
Embodiment 3
Present embodiment is the class monellin gene order through primer sequence property composition in the Land of Peach Blossoms in real-time fluorescence PCR specific detection sample of the class monellin gene that uses peach.SYBR fluorescence dye method: SYBR Green is high-sensitive DNA fluorescence dye, in various analyses, can detect 20pgDNA at least.The avidity of SYBR Green dyestuff and double-stranded DNA is very high, in PCR reaction, combines with product dsDNA the back fluorescent signal can strengthen 800-1000 doubly and the enzyme of using always in to molecular biology do not have restraining effect.SYBR Green PCR reaction system is: Faststart Universal SYBRGreen 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 (about 50ng), using sterilized water to mend extremely total system is 25 μ L.Adopt Bio-Rad iQTM5 multicolor real time PCR appearance to carry out pcr amplification and interpretation of result, amplification condition is following: 95 ℃ of preparatory sex change 10min; 95 ℃ of 10s, 58 ℃ of 30s, 40 circulations are carried out the melting curve analysis after the PCR reaction.
Primer sequence is: SEQ ID NO:1 and SEQ ID NO:2.
Employed detection key instrument:
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) (Takara) give birth to worker company available from Shanghai; 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: Huiyuan Peach Juice, eat dream board peach juice, sea too peach juice, Huiyuan's peach fruit squash, Sucus Mali pumilae, pear juice, orange juice, Fructus actinidiae chinensis juice and tomato juice.
Get in the clean culture dish of 30mL sample to, vacuumize freeze-drying; Take by weighing in the clean 50mL centrifuge tube of the freezing dry-matter to of 0.2g, add the 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 the 1 clean 2.0mL centrifuge tube; Add 700 μ L chloroforms; Violent mixing 30s, 14500rpm 10min gets respectively in 650 μ L supernatant liquors to the clean 2.0mL centrifuge tube; Add 1300 μ L CTAB precipitated liquid (0.5%CTAB (W/V); 0.04mol/LNaCl), violent mixing 30s, room temperature leaves standstill 1h; 14500rpm 20min abandons supernatant liquor, adds 350 μ L 1.2M NaCl, and thermal agitation 30s adds 350 μ L chloroforms again, violent mixing 30s, 14500rpm 10min; Get supernatant liquor 320 μ L respectively, add 0.8 times of volume Virahol, behind the mixing ,-20 ℃ of 1h, 14500rpm 20min abandons supernatant liquor, adds 500 μ L, 70% ethanol, and behind the 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:
Faststart?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
Template DNA 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; With the peach juice is example; When utilizing the class monellin gene of peach in the real-time fluorescence PCR test sample; Honey peach, Huiyuan Peach Juice, eat dream board peach juice, sea too the melting curve peak value of peach juice, the peach fruit squash sample P CR of Huiyuan amplified fragments all at 73.20 ± 1.0 ℃; And Sucus Mali pumilae, pear juice, orange juice, Fructus actinidiae chinensis juice, tomato juice and blank all do not have this melting curve peak value, show that this primer can effectively detect the Land of Peach Blossoms property composition in the sample.
Embodiment 4
With identical according to embodiment 3 described methods; Be template just with testing sample DNA; Adopt type primer sequence SEQ ID NO:1 of monellin gene and SEQ ID NO:2 and plant universal amplification primer to SEQ ID NO:8 and SEQ ID NO:9, carry out the double PCR amplification.
Embodiment 5
Present embodiment is the sequence through primer sequence property composition in the Land of Peach Blossoms in real-time fluorescence PCR specific detection sample of the ITS gene that uses peach.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 one and the two fluorescence labeling probes of the special complementary of target-gene sequence; Utilize the fluorescent signal accumulation whole PCR process of monitoring in real time, through typical curve unknown template is carried out quantitative analysis at last.Quantitative step: confirm that 1. (C representes cycle number (Cycle) to the CT value, and T representes fluorescence thresholding (Threshold), the cycle number that is experienced when promptly the fluorescent signal in each reaction tubes arrives the thresholding of setting; 2. utilize typical curve that unknown sample is carried out quantitative assay.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 promptly initial copy number is many more, and the CT value is more little.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.
The ITS gene primer sequence of Land of Peach Blossoms property composition is in the employed test sample: 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 with 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 5 ' end of probe, and quencher TAMAR (6-carboxyl rhodamine (6-carboxy tetramethyl rhodamine)) is then at 3 ' end.When the pairing of complete probe and target sequence, the fluorophor emitted fluorescence because of with the quencher of 3 ' end near cancellation.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.
Employed detection key instrument:
Micropipet (10 μ L, 100 μ L, 1000 μ L Eppendorf), quantitative real time PCR Instrument (ABI 7700 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) (Takara) give birth to worker company available from Shanghai; 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.) synthetic etc. by Shanghai AudioCodes bio tech ltd.
Detect key step:
1DNA extracts
Samples of juice to be measured is: Huiyuan Peach Juice, eat dream board peach juice, sea too peach juice, Huiyuan's peach fruit squash, Sucus Mali pumilae, pear juice, orange juice, Fructus actinidiae chinensis juice and tomato juice.
Get in the clean culture dish of 30mL sample to, vacuumize freeze-drying; Take by weighing 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 the 1 clean 2.0mL centrifuge tube, adds 700 μ L chloroforms; Violent mixing 30s, 14500rpm 10min gets respectively in 650 μ L supernatant liquors to the clean 2.0mL centrifuge tube; Add 1300 μ L CTAB precipitated liquid, violent mixing 30s, room temperature leaves standstill 1h; 14500rpm 20min abandons supernatant liquor, adds 350 μ L 1.2M NaCl, and thermal agitation 30s adds 350 μ L chloroforms again, violent mixing 30s, 14500rpm 10min; Get supernatant liquor 320 μ L respectively, add 0.8 times of volume Virahol, behind the mixing ,-20 ℃ of 1h, 14500rpm 20min abandons supernatant liquor, adds 500 μ L, 70% ethanol, and behind the 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 M) 0.5 μ L
Upstream primer (10 μ mol/L) 0.5 μ L
Downstream primer (10 μ mol/L) 0.5 μ L
Template DNA 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: with the peach juice is example; When utilizing the ITS region sequence of peach in the real-time fluorescence PCR test sample; Honey peach, Huiyuan Peach Juice, too pcr amplification all appears in peach juice, Huiyuan's peach fruit squash sample to eat dream board peach juice, sea; And Sucus Mali pumilae, pear 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 Land of Peach Blossoms property composition in the sample.
Embodiment 6
With identical according to embodiment 5 described methods; Be template just with testing sample DNA; Primer sequence SEQ ID NO:3, SEQ ID NO:4 and the probe SEQ ID NO:7 of employing ITS gene and plant universal amplification primer carry out the double PCR amplification to SEQ ID NO:8, SEQ ID NO:9.
Embodiment 7
The present inventor clones and the chloroplast(id) Trnk gene of the peach of having checked order and the sequence of matK intergenic region first.
Present embodiment is for obtaining the chloroplast(id) Trnk gene of peach and the sequence of matK intergenic region through the PCR cloning and sequencing.
DNA according to plant Trnk gene and matK intergenic region design upstream and downstream primer amplification peach.According to the operation instructions of Wizard Gel Extraction Kit the PCR product of peach is carried out purifying, recovery.The specification sheets of pressing TaKaRa pMD19-T Vector test kit is connected purified product with pMD19-TVector, 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 are provided with the positive and negative control simultaneously.Linked system is placed room temperature (22-37 ℃) reaction 30min, and reaction places 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 place 2min on ice immediately, add gone out the brain heart infusion of bacterium of 800 μ L then, 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 handle, 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, each 0.5 μ L of upstream and downstream primer, 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 through agarose gel electrophoresis.
(2) order-checking: after confirming 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) Trnk gene and matK intergenic region that the PCR cloning and sequencing obtains peach is following:
CTCAGT
TGATCGACCCTCAATCTTACTGTATGAACATTTCATAATAGAAATAAATGCAAATTTTTTGTTATCTCTTCATTATTTAAAGATAATTCCATTTCTACGATCGCATAACCAATTATTCATAATTGATTAGATCATTGATGCAAAAAATATCCAAATACCAAATCCGACCTCTATAAAGTTTCTTAAA
AGTAAAAGTATAAGAAGCTCTTGGGAAGACC(SEQ?ID?NO：11)。
Through peach juice relative content in the PCR primer detection by quantitative sample of chloroplast(id) Trnk gene and matK intergenic region, be used for the chloroplast(id) Trnk gene of detection by quantitative fruit juice blends peach juice composition relative content and the PCR primer of matK intergenic region and be:
SEQ?ID?NO：5
SEQ?ID?NO：6。
With this DNA to the primer amplification fruit juice blends, it is 212bp that peach becomes branch amplification PCR products length; Through on 2.5% agarose, carrying out electrophoretic separation, can confirm PCR product length, thereby judge whether contain the peach juice composition in the mixing juice.
Embodiment 8
Present embodiment provides the test kit of property composition in the Land of Peach Blossoms in the rapid detection sample.
Said test kit comprise primer to SEQ ID NO:5, SEQ ID NO:6 and primer to SEQ IDNO:8, SEQ ID NO:9; And working instructions; Wherein primer is that to be used for the specific oligonucleotide primer of real-time fluorescence PCR test sample Land of Peach Blossoms property composition right to SEQ ID NO:5, SEQID NO:6; SEQ ID NO:8, SEQ ID NO:9 are plant universal amplification primers; Provided the pcr amplification condition in the said working instructions; This condition is 95 ℃, 10min; 95 ℃, 15s; 60 ℃, 1min.For different instruments, reaction parameter is done suitable adjustment.
Embodiment 9
Present embodiment provides the test kit of property composition in the Land of Peach Blossoms in the rapid detection sample.
Said test kit comprise primer to SEQ ID NO:1, SEQ ID NO:2 and primer to SEQ IDNO:8, SEQ ID NO:9; And working instructions; Wherein primer is that to be used for the specific oligonucleotide primer of real-time fluorescence PCR test sample Land of Peach Blossoms property composition right to SEQ ID NO:1 and SEQID NO:2; SEQ ID NO:8, SEQ ID NO:9 are plant universal amplification primers; Provided the pcr amplification condition in the said working instructions; This condition is 95 ℃, 10min; 95 ℃, 15s; 58 ℃, 30s.For different instruments, reaction parameter is done suitable adjustment.
Embodiment 10
Present embodiment provides the test kit of property composition in the Land of Peach Blossoms in the rapid detection sample.
Said test kit comprises that primer SEQ ID NO:3 and SEQ ID NO:4 and probe SEQ IDNO:7 and primer are to SEQ ID NO:8, SEQ ID NO:9; And working instructions; Wherein primer is that to be used for the specific oligonucleotide primer of real-time fluorescence PCR test sample Land of Peach Blossoms property composition right to SEQ ID NO:3 and SEQ ID NO:4 and probe SEQ ID NO:7; SEQ ID NO:8, SEQ ID NO:9 are plant universal amplification primers; Provided the pcr amplification condition in the said working instructions; This condition is 95 ℃, 10min; 95 ℃, 15s; 60 ℃, 1min.For different instruments, reaction parameter is done suitable adjustment.
Though 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 claims and the coordinator scope thereof.
Claims (9)
1. the specific oligonucleotide primer that is used for real time fluorescent PCR method test sample Land of Peach Blossoms property composition is right, and wherein said primer is to being selected from one or more of following primer centering: SEQ ID NO:1 and SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4; And SEQ ID NO:5 and SEQ ID NO:6.
2. Oligonucleolide primers according to claim 1 is right, and wherein said primer is to being SEQ ID NO:5 and SEQ ID NO:6.
3. Oligonucleolide primers according to claim 1 is right, and wherein said real time fluorescent PCR method is a SYBR fluorescence dye method, and said primer is to being SEQ ID NO:1 and SEQ ID NO:2.
4. Oligonucleolide primers according to claim 1 is right, and wherein said real time fluorescent PCR method is a Taqman fluorescent probe method, and said primer is to being SEQ ID NO:3 and SEQ ID NO:4, and employed probe is SEQ ID NO:7.
5. the real-time fluorescence PCR detection method of Land of Peach Blossoms property composition in the sample, said method comprise that to use among the claim 1-4 each described specific oligonucleotide primer right.
6. real-time fluorescence PCR detection method according to claim 5 also comprises and uses plant chloroplast trn gene universal primer sequence SEQ ID NO:8 and SEQ ID NO:9.
7. the test kit of Land of Peach Blossoms property composition in the rapid detection sample, said test kit comprise among the claim 1-4 each described specific oligonucleotide primer to and working instructions.
8. test kit according to claim 7, wherein said test kit also comprise plant chloroplast trn gene universal primer sequence SEQ ID NO:8 and SEQ ID NO:9.
Among the claim 1-4 each described specific oligonucleotide primer to the application in the Land of Peach Blossoms property composition in test sample.
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| CN108998567A (en) * | 2018-05-24 | 2018-12-14 | 内蒙古农业大学 | Molecular marker primer and method for identifying amygdalus pedunculata pall, amygdalus mongolicus and prunus ulmaria |
| CN110241252A (en) * | 2019-07-30 | 2019-09-17 | 中国农业科学院郑州果树研究所 | SNP marker for constructing peach DNA fingerprinting combines and application and method |
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| CN106701907A (en) * | 2015-11-18 | 2017-05-24 | 中国检验检疫科学研究院 | Primer, probe, method and kit for detecting cassava-derived ingredients |
| CN108998567A (en) * | 2018-05-24 | 2018-12-14 | 内蒙古农业大学 | Molecular marker primer and method for identifying amygdalus pedunculata pall, amygdalus mongolicus and prunus ulmaria |
| CN108998567B (en) * | 2018-05-24 | 2022-05-13 | 内蒙古农业大学 | Molecular marker primer and method for identifying amygdalus pedunculata pall, amygdalus mongolicus and prunus ulmaria |
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| CN110628939A (en) * | 2019-11-04 | 2019-12-31 | 中国检验检疫科学研究院 | Primer probe, method and kit for detecting camellia oleifera derived components |
| CN110734998A (en) * | 2019-11-04 | 2020-01-31 | 中国检验检疫科学研究院 | Primers, method and kit for identifying NFC orange juice and FC orange juice |
| CN110734998B (en) * | 2019-11-04 | 2022-04-12 | 中国检验检疫科学研究院 | Primers, method and kit for identifying NFC orange juice and FC orange juice |
| CN110628939B (en) * | 2019-11-04 | 2022-04-22 | 中国检验检疫科学研究院 | Primer probe, method and kit for detecting camellia oleifera derived components |
| CN114350831A (en) * | 2020-10-12 | 2022-04-15 | 谱尼测试集团上海有限公司 | Primer probe and method for detecting cocoa-derived components in cocoa powder |
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