CN108300717A - The specific primer pair of vegetable-derived components and its application in a kind of detection cigarette - Google Patents
The specific primer pair of vegetable-derived components and its application in a kind of detection cigarette Download PDFInfo
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- CN108300717A CN108300717A CN201810141854.XA CN201810141854A CN108300717A CN 108300717 A CN108300717 A CN 108300717A CN 201810141854 A CN201810141854 A CN 201810141854A CN 108300717 A CN108300717 A CN 108300717A
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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
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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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
- C12Q1/6869—Methods for sequencing
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Abstract
The invention discloses a species-specific primer pair, the upstream primer sequence of the specific primer pair is as shown in SEQ No.1, and downstream primer sequence is as shown in SEQ No.2.The application and its application process that the present invention also provides a kind of primer pairs in identifying cigarette plant derived component.The method includes extracting food cigarette total DNA to be measured, PCR amplification, clone and sequencing determine ingredient.Using specific primer pair of the present invention and round pcr, can quickly, it is accurate, delicately detect cigarette plant derived component, and can be applied in production scene.
Description
Technical field
The invention belongs to genetic engineering technology fields, further belong to cigarette constituent analysis technical field, and in particular to one
The specific primer pair of vegetable-derived components and its application in kind detection cigarette.
Background technology
Industrial crops are important in tobacco, contain alkaloid about 1%~9% and rutin sophorin, organic acid, fat, tree in tobacco
The substances such as fat, protein, sugar, fragrance.There is very extensive purposes in chemical industry, pesticide and medicine and other fields.Tobacco has very strong
Physiological activity, there are many records cured the disease using tobacco for China's traditional medicine.Tobacco business is also that country has paid greatly simultaneously
Important contribution is made in the profits tax of amount, the economic construction for country, especially to remote districts such as Yunnan, the development in the area such as Guizhou
The contribution made is more prominent.The income of outlying mountain area peasant can greatly be improved especially by the cultivating and growing of tobacco,
Society can be made to keep stablizing.
Because interests drive, currently there are a large amount of false smoke, the inside may adulterate other vegetable materials, pole
The big health for compromising consumer, exists simultaneously some behaviors for illegally selling private cigarette, these behaviors be it is illegal,
Great loss is caused to state tax revenue, needs whether a kind of product the inside that effective method detection is discovered and seized contains tobacco at present
Composition then illustrates that the product violates Law on Monopoly of Tabacoo once containing tobacco composition.
Tobacco product check system is mainly organoleptic examination and physical and chemical inspection at present.These traditional identification methods mainly with
By means of the experience accumulation of insider, there is also the disadvantages that empiricism cannot be qualitative.With the development of science and technology, molecule is given birth to
Object identification technology gradually penetrates into each biological field, but also seldom in the application of tobacco product ingredient analysis field.
For this purpose, the present invention has developed a kind of method detecting plant derived component in food using Protocols in Molecular Biology.
Invention content
The first object of the present invention is to provide a species-specific primer pair, and the sequence of the primer pair sense primer is such as
Shown in SEQ No.1, the sequence of downstream primer is as shown in SEQ No.2.
The second object of the present invention is to provide a kind of specific primer to the plant derived component in identifying cigarette
In application.
The third object of the present invention be to provide a kind of application specific primer in identification cigarette it is plant-derived at
The method divided, described method includes following steps：
（1）Extract cigarette total DNA to be measured；
（2）PCR amplification：Using total DNA as template, according to plant all in NCBIrbclGene order designs universal primer, so
After carry out PCR amplification, recycling and purifying pcr amplification product；
（3）Clone and sequencing：The PCR product is inserted into recombinant vector, the recombinant vector is then transferred to Escherichia coli, is expanded
Plasmid is extracted after numerous, and plasmid is sequenced；
（4）Determine ingredient：The sequencing result is subjected to blast alignments on NCBI, determines the plant in the food
Derived component.
The fourth object of the present invention is a kind of plant derived component detection kit containing the specific primer pair.
Description of the drawings
Fig. 1 TPS methods extract Shanghai and Yunnan tobacco DNA；
In figure, M- molecular weight markers, the Shanghai SH- brand cigarette, the Yunnan YN- brand cigarette；
The Shanghai Fig. 2 and Yunnan tobacco pcr amplification product；
In figure, M- molecular weight markers, the Shanghai SH- brand cigarette, the Yunnan YN- brand cigarette.
Specific implementation mode
The present invention will be further described below with reference to the drawings, but is not limited in any way to the present invention, base
In present invention teach that done it is any transform or replace, all belong to the scope of protection of the present invention.
The sequence of the sense primer of a species-specific primer pair of the present invention as shown in SEQ No.1, downstream primer
Sequence is as shown in SEQ No.2.The universal primer can be applied to plant derived component in identification food cigarette, using described
Specific primer includes the following steps the method for plant derived component in identification cigarette：
（1）Extract cigarette total DNA to be measured；
（2）PCR amplification：Using total DNA as template, expanded carrying out PCR amplification, recycling and purifying PCR using the specific primer
Increase production object；
（3）Clone and sequencing：The PCR product is inserted into recombinant vector, the recombinant vector is then transferred to Escherichia coli, is expanded
Plasmid is extracted after numerous, and plasmid is sequenced；
（4）Determine ingredient：The sequencing result is subjected to Blast alignments on NCBI, determines the plant in the cigarette
Derived component.
Wherein, total DNA is extracted using conventional method, such as CTAB methods or TPS methods.
Preferably, the PCR amplification system is 50 μ L systems to one kind as the present invention：1 μ L of DNA profiling, upstream and downstream are drawn
Object（10 μmol/L）Each 1 μ L, 5 × buffer 10 μ L, dNTP（10 mmol/L）1 μ L, DNA polymerase, 0.5 μ L, most
Afterwards plus ddH2O to 50 μ L；Pcr amplification reaction condition is：98 DEG C of pre-degeneration 5min；98 DEG C of denaturation 30s, 58 DEG C of 30s that anneal, 72
DEG C extend 30s, 30 cycle；72 DEG C of 5 min of extension；4 DEG C of preservations.Archaeal dna polymerase can use Phusion, EasyTaq PCR
The high fidelity enzymes such as SuperMix.
The PCR product of acquisition is inserted into recombinant vector, then is transferred to Escherichia coli.The Escherichia coli clones of acquisition are expanded numerous
And extract plasmid.It is as follows：
The bacterium colony for carrying out conversion is put into 37 DEG C of shake cultures in the LB culture medium solutions of 2 mL to stay overnight.The Escherichia coli that will have been shaken
Bacterium solution is poured into 2 mL centrifuge tubes, and 2 min are then centrifuged under the revolution of 12000 r/min.Supernatant is removed, precipitation is left.To
The P1 solution of 250 μ L is added in the precipitation of centrifuge tube, then fully shaking.Next the P2 of 250 μ L is added into centrifuge tube
Solution, then concussion gently.Then 350 μ L N1 solution are added into centrifuge tube, spins upside down 7-8 times and is uniformly mixed, this
Step not shake acutely, in order to avoid plasmid is become into open loop structure.
Centrifuge tube is centrifuged into 10 min at 12000 r/min, then carries the careful immigration kit of supernatant
In purification column, purification column is then put into centrifuge, 1min is centrifuged at 12000 r/min.Liquid phase is discarded supernatant, then pure
Change the PE solution that 500 μ L are added on column, purification column is put into centrifuge centrifuges 1 min at 12000 r/min, repeats above-mentioned
Step.It discards supernatant, purification column is centrifuged into 1 min at 12000 r/min.At this time by purification column be re-applied to one it is new from
On heart pipe, purification column nozzle is opened, static 5 min fully dries filter membrane, in order to avoid ethanol pollution.Add 30 μ L on filter membrane
Water, then static 1 min, allows plasmid to be adequately dissolved in water.Then purification column is centrifuged 1 at 12000 r/min
min.The liquid phase obtained at this time is plasmid.
The method of the sequencing is the sequencing of Sanger methods or high-flux sequence.Then the data such as GenBank or BOLD are utilized
Library carries out Blast analyses.
In addition, kit is made using the universal primer, the kit can be used for detecting the plant in food cigarette
Object derived component.
The present invention synthesizes universal primer by design, and plant derived component in cigarette is detected using round pcr.The user
Method is quick, accurate, sensitive, can be applied in production scene.
Embodiment 1:CTAB methods extract cigarette total DNA
Two kinds of cigarette for choosing Shanghai and Yunnan production are detected.The total DNA of two kinds of cigarette is extracted using CTAB methods：It weighs
0.2 g face powders more than ten end, is added the CTAB extracting solutions of 600 μ L（Table 1）, it is ground into homogenate；Homogenate is transferred to 1.5 mL
Eppendorf is managed, 60 DEG C~65 DEG C water-bath 1h；Isometric phenol-chloroform-isoamyl alcohol is added（25：24：1）, overturn mixing；Often
Under temperature, 12000 r/min centrifuge 15min；Carefully supernatant is transferred in new centrifuge tube with rifle, is careful not among inhaling
Protein layer；12000 r/min centrifuge 10min, supernatant are transferred in new pipe, still it is noted that not touch intermediate egg
Lamina albae；0.6 times is added into supernatant to isometric isopropanol, thoroughly overturns mixing, DNA is made to be precipitated from solution, form wadding
Shape precipitates；Choose DNA precipitations with glass bar or pipette tips, be put into the 1.5 mL Eppendorf pipes for having been added to 70% ethyl alcohol,
Reverse pipe several times, to wash DNA, dissolves the substances such as the pigment wherein contained；Centrifugation, pours out the alcohol in pipe, is used in combination rifle will
Remaining liquid feed exhaustion, is concentrated in vacuo after instrument drying, appropriate TE buffer solutions DNA is added into pipe.Electrophoresis detection
The DNA of extraction（Fig. 1）.
1 CTAB buffer solution configuration methods of table
Embodiment 2：PCR amplification
Using the DNA of acquisition as template, the universal primer that design is added carries out PCR amplification.PCR amplification system is 50 μ L systems：
1 μ L of DNA profiling, upstream and downstream primer（10 μmol/L）Each 1 μ L, 5 × buffer 10 μ L, dNTP（10 mmol/L） 1 μL,
0.5 μ L of Phusion DNA Polymerase finally add ddH2O to 50 μ L；Pcr amplification reaction condition is：98 DEG C of pre-degenerations
5min；98 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 30s, 30 recycle；72 DEG C of 5 min of extension；4 DEG C of preservations.It will obtain
The PCR product obtained carries out electrophoresis detection, it can be seen that electrophoresis can obtain clear band（Fig. 2）.Wherein, primer sequence is
Rbcl- detections-F：CTGCCGAATCTTCTACTGGTACATGGAC；
Rbcl- detections-R：AGACATTCATAAACAGCTCTACCGTAG.
Embodiment 3：Clone and sequencing
The PCR product of acquisition is inserted into recombinant vector, then is transferred to Escherichia coli.The Escherichia coli clones of acquisition are expanded numerous and are carried
Take plasmid.It is as follows：
The bacterium colony for carrying out conversion is put into 37 DEG C of shake cultures in the LB culture medium solutions of 2 mL to stay overnight.The Escherichia coli that will have been shaken
Bacterium solution is poured into 2 mL centrifuge tubes, and 2 min are then centrifuged under the revolution of 12000 r/min.Supernatant is removed, precipitation is left.To
The P1 solution of 250 μ L is added in the precipitation of centrifuge tube, then fully shaking.Next the P2 of 250 μ L is added into centrifuge tube
Solution, then concussion gently.Then 350 μ L N1 solution are added into centrifuge tube, spins upside down 7-8 times and is uniformly mixed, this
Step not shake acutely, in order to avoid plasmid is become into open loop structure.
Centrifuge tube is centrifuged into 10 min at 12000 r/min, then carries the careful immigration kit of supernatant
In purification column, purification column is then put into centrifuge, 1min is centrifuged at 12000 r/min.Liquid phase is discarded supernatant, then pure
Change the PE solution that 500 μ L are added on column, purification column is put into centrifuge centrifuges 1 min at 12000 r/min, repeats above-mentioned
Step.It discards supernatant, purification column is centrifuged into 1 min at 12000 r/min.At this time by purification column be re-applied to one it is new from
On heart pipe, purification column nozzle is opened, static 5 min fully dries filter membrane, in order to avoid ethanol pollution.Add 30 μ L on filter membrane
Water, then static 1 min, allows plasmid to be adequately dissolved in water.Then purification column is centrifuged 1 at 12000 r/min
min.The liquid phase obtained at this time is plasmid.
It selects Shanghai cigarette and each 10 clones of Yunnan tobacco is sequenced, these results are subjected to blast on NCBI
Analysis.The result shows that：Main composition in Shanghai and Yunnan tobacco product is all made of tobacco, is not adulterated other
Plant source composition.
Should the result shows that：The present invention can efficiently and accurately detect the plant derived component in cigarette.
Sequence table
<110>Yunnan Academy of Tobacco Agricultural Science
<120>The specific primer of plant derived component and its application in a kind of detection cigarette
<130> 2018
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 28
<212> DNA
<213>Artificial sequence
<400> 1
ctgccgaatc ttctactggt acatggac 28
<210> 2
<211> 27
<212> DNA
<213>Artificial sequence
<400> 2
agacattcat aaacagctct accgtag 27
Claims (7)
1. a species-specific primer pair, which is characterized in that the sequence of the primer pair sense primer is as shown in SEQ No.1, downstream
The sequence of primer is as shown in SEQ No.2.
2. specific primer described in a kind of claim 1 is to the application in the plant derived component in identifying cigarette.
3. a kind of method using specific primer described in claim 1 to identifying plant derived component in cigarette, feature exists
In described method includes following steps：
（1）Extract cigarette total DNA to be measured；
（2）PCR amplification：Using total DNA as template, specific primer is designed according to plant rbcl genes, and carry out PCR amplification, returned
Receive and purify pcr amplification product；
（3）Clone and sequencing：The PCR product is inserted into recombinant vector, the recombinant vector is then transferred to Escherichia coli, is expanded
Plasmid is extracted after numerous, and plasmid is sequenced；
（4）Determine ingredient：The sequencing result is subjected to blast alignments on NCBI, determines the plant in the cigarette
Derived component.
4. the method for identifying plant derived component in cigarette according to claim 1, which is characterized in that the PCR amplification body
System is 50 μ L systems：1 μ L of DNA profiling, upstream and downstream primer（10 μmol/L）Each 1 μ L, 5 × buffer 10 μ L, dNTP（10
mmol/L）1 μ L, 0.5 μ L of archaeal dna polymerase finally add ddH2O to 50 μ L；Pcr amplification reaction condition is：98 DEG C of pre- changes
Property 5min；98 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 30s, 30 recycle；72 DEG C of 5 min of extension；4 DEG C of preservations.
5. the method for identifying plant derived component in cigarette according to claim 4, which is characterized in that the archaeal dna polymerase
For phusion, KOD Plus or EasyTaq PCR SuperMix.
6. a kind of method for identifying plant derived component in cigarette according to claim 3, which is characterized in that the sequencing side
Method is Sanger methods.
7. a kind of plant derived component detection kit containing specific primer pair described in claim 1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108192897A (en) * | 2018-02-11 | 2018-06-22 | 云南省烟草农业科学研究院 | One grows tobacco rbcl genetic fragments and its application |
| CN110452905A (en) * | 2019-08-22 | 2019-11-15 | 云南省烟草农业科学研究院 | A kind of extracting method and its application improving tobacco DNA deposition efficiency |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108192897A (en) * | 2018-02-11 | 2018-06-22 | 云南省烟草农业科学研究院 | One grows tobacco rbcl genetic fragments and its application |
| CN110452905A (en) * | 2019-08-22 | 2019-11-15 | 云南省烟草农业科学研究院 | A kind of extracting method and its application improving tobacco DNA deposition efficiency |
| CN110452905B (en) * | 2019-08-22 | 2023-06-02 | 云南省烟草农业科学研究院 | Extraction method for improving tobacco DNA precipitation efficiency and application thereof |
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Application publication date: 20180720 |