CN105603107A - PCR (polymerase chain reaction) method and kit for identifying spina date seeds and counterfeits thereof on basis of ITS sequence site - Google Patents
PCR (polymerase chain reaction) method and kit for identifying spina date seeds and counterfeits thereof on basis of ITS sequence site Download PDFInfo
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- CN105603107A CN105603107A CN201610161689.5A CN201610161689A CN105603107A CN 105603107 A CN105603107 A CN 105603107A CN 201610161689 A CN201610161689 A CN 201610161689A CN 105603107 A CN105603107 A CN 105603107A
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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
- C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
Abstract
The invention discloses a PCR (polymerase chain reaction) method and kit for identifying spina date seeds and counterfeits thereof on the basis of ITS sequence site. The invention provides primers for identifying spina date seeds, particularly a primer pair capable of amplifying amplification products of nucleotide disclosed as Sequence 1 in the sequence table and nucleotide disclosed as Sequence 2 in the sequence table. The experiment proves that screening is performed on the basis of the nuclear gene ITS sequence of the original plant to obtain the site specific primers for identifying the spina date seeds and counterfeits thereof, and the PCR reaction system and conditions are regulated to establish a site-specific PCR process. The site-specific primer PCR is simple to operate, and has the advantages of low cost, favorable repetitiveness, single identification strip, low sample consumption and the like. The specific primer PCR can greatly lower the false positive rate of PCR and ensure the identification accuracy.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of discriminating spina date seed and puppet thereof based on ITS sequence siteThe PCR method of product and kit.
Background technology
Spina date seed is Rhamnaceae plant wild jujube ZiziphusjujubaMill.var.spinosa (Bunge) HuexH.The dry mature seed of F.Chou. Originate from the provinces such as Hebei, Shaanxi, Liaoning, Shandong, Henan, Anhui. Spina date seedFlat, taste is sweet, sour, the effect of there is the tonifying liver of nourishing heart, antitoxic heart-soothing and sedative, arrest sweating, promoting the production of body fluid, be used for the treatment of restlessness of asrhenia type and insomnia,The diseases such as the empty hidrosis of palpitation with fear dreaminess, body, Tianjin wound are thirsty. Modern pharmacology research shows, this product has tranquilizing soporific, townAnticonvulsion, the step-down of pain, reducing blood lipid, enhancing immunologic function, anti-anoxic, anti-arrhythmia, anti-ageing, radioresistance andThe effects such as platelet aggregation-against.
Because spina date seed wild resource reduces, market consumption is large, and supply falls short of demand for the market source of goods, goes out at present on medicinal material marketExisting main mixed adulterant has seed reason jujube kernel, the hoveniae semoveniae semen of congener indian jujube ZiziphusmauritianaHoveniadulcis (having another name called honey raisin tree benevolence), Lens culinaris Lensculinaris and the sub-Cercischinensis of cercis etc.Wherein, reason jujube kernel is that product are commonly used in place, and market consumption is less, but very similar to spina date seed profile, and proterties is differentiated notEasily distinguish.
At present for the discriminating research of spina date seed adopt Medicinal Materials Characters, microscopic features, thin-layer chromatography, ultraviolet spectra,Traditional discrimination method such as electrophoresis, ESEM, but because these 5 kind appearance characters are close, microstructure characteristicNot obvious, complex chemical composition, above these method specificities are not strong, and are easily subject to the interference of additional factor. WithCompare, the feature that locus specificity round pcr has is stable, be not subject to such environmental effects, at present about spina date seedThe research of molecule discriminating aspect is less.
Summary of the invention
An object of the present invention is to provide a kind of primer of identifying spina date seed.
Primer provided by the invention, can amplify the primer pair of DNA fragmentation A;
Described DNA fragmentation A is taking spina date seed as template, increases and obtains with primer pair shown in sequence 1 and sequence 2Product.
In practical application, may be because of the difference of template, there is the difference of indivedual bases or several bases in the product expanding,But this all belongs to the category of DNA fragmentation A.
In above-mentioned primer, described primer pair is made up of primer 1 and primer 2;
Described primer 1 is following 1) or 2):
1) be the single strand dna shown in sequence 1;
2) for sequence 1 is deleted and/or increase and/or change the single strand dna that one or several nucleotides obtains;
Described primer 2 is following 3) or 4):
3) be the single strand dna shown in sequence 2;
4) for sequence 2 is deleted and/or increase and/or change the single strand dna that one or several nucleotides obtains.
In above-mentioned primer, the mol ratio of described primer 1 and described primer 2 is 1:1.
Another object of the present invention be to provide a kind of for the identification of or the PCR reagent of assistant identification spina date seed.
PCR reagent provided by the invention, comprises the primer that power is above-mentioned;
In described primer, every primer final concentration in described PCR reagent is 0.2 μ molL-1。
The 3rd object of the present invention is to provide the kit of a kind of qualification or assistant identification spina date seed.
Kit provided by the invention, comprises above-mentioned primer or above-mentioned PCR reagent.
Above-mentioned primer or above-mentioned PCR reagent or above-mentioned kit are in following (A1)-(A4) in anyApplication be also the scope of protection of the invention:
(A1) qualification or assistant identification spina date seed;
(A2) differentiate or assist and differentiate spina date seed
(A3) differentiate or assist and differentiate spina date seed and adulterant thereof;
(A4) identify or whether assistant identification testing sample is spina date seed;
(A5) qualification or assistant identification testing sample are spina date seed or its adulterant;
Described adulterant is reason jujube kernel, hoveniae semoveniae semen, cercis or Lens culinaris.
The 4th object of the present invention is to provide a kind of detection or auxiliary detection testing sample is spina date seed or its adulterantMethod.
Method provided by the invention, comprises the steps: to detect in testing sample, whether to contain DNA fragmentation A; IfContain, testing sample is or candidate is spina date seed; If do not contained, testing sample is or candidate is spina date seed puppetProduct;
Described DNA fragmentation A is taking spina date seed as template, increases and obtains with primer pair shown in sequence 1 and sequence 2Product.
In said method, the method that whether contains DNA fragmentation A in described detection testing sample comprises the steps: to useAbove-mentioned primer pair testing sample carries out pcr amplification, obtains DNA fragmentation A.
In said method, the size of described DNA fragmentation A is 60-70bp.
In said method, the genomic DNA that the template of described pcr amplification is testing sample;
The annealing temperature of described pcr amplification is 57 DEG C.
The karyogene ITS sequence of this research based on former plant that experiment showed, of the present invention, screening obtains differentiates spina date seedAnd the site-specific primer of mixed adulterant, and by regulating PCR reaction system and condition to set up site-specific PCR sideMethod; That locus specificity primer PCR has is simple to operate, cost is low, reproducible, qualification band is single, sample is usedThe advantages such as amount is few, Auele Specific Primer PCR can greatly reduce the false positive rate of PCR, ensures the accuracy of qualification;It is high, easy and simple to handle that site-specific PCR discrimination method has the degree of accuracy, identifies fireballing advantage; In addition for discriminatingPersonnel's experimental implementation requires neither be very high, and therefore, the foundation of the method will be the quick identification reagent box of exploitation spina date seedLay the first stone.
Brief description of the drawings
Fig. 1 is different DNA profiling amount PCR reaction result figure.
Fig. 2 is different annealing temperature PCR reaction result figure.
Fig. 3 is the result of carrying out PCR experiment with two kinds of enzymes.
Fig. 4 is the NJ tree of spina date seed based on ITS sequence construct and mixed adulterant thereof.
Detailed description of the invention
The experimental technique using in following embodiment if no special instructions, is conventional method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
Instrument electrophoresis system (Beijing Liuyi Instrument Factory, model DYY-6D), PCR instrument (LifeTechnologiesCompany, model Veriti; Germany EppendorfAG22331Hamburg; Germany EppendorfMastercyclerPro), desk centrifuge (German SiGMR company, model 1-14), VilberLourmat gel imaging instrument (France),Plant genes group DNA rapid extraction kit (Beijing Bo Ling section is bio tech ltd), PlantGenomicDNAkit50Preps plant genome DNA extracts kit (TianGen).
Reagent 2xPCRSuperMix，2xPhusionHigh-FidelityPCRMMW/HFBuffer，TransFastTaqDNAPolymerase(5U·μL-1)，TaKaRadNTPMixture2.5mmeach,Trans10xFastTaqBuffer(Mg2+)，DNAMarker (Beijing Quanshijin Biotechnology Co., Ltd), fine jadeLipolysaccharideRegularAgaroseG-10(Strength(1％)750gr·cm2)，Tiangen10000xGeneGreenNucleicAcidDye, (raw work bioengineering (Shanghai) share has 10xMops electrophoretic bufferLimit company)
20 parts of materials that the medicinal material market of Anguo, Bozhou is collected in this experiment of medicinal material comprise 11 parts of spina date seeds, 5 portions of reason jujubesBenevolence, 2 parts of hoveniae semoveniae semens, 1 part of cercis, 1 portion of Lens culinaris.
The design of primers of embodiment 1, qualification spina date seed and synthetic
Search the ITS sequence of spina date seed, reason jujube kernel, Lens culinaris, hoveniae semoveniae semen, cercis from Genbank, according to ITSPhylogenetic tree construction, is shown in Fig. 4, and method adopts Kimura2-parameter distance, and letter is put by the each branch of genealogical treeBootstrapmethod inspection for degree, carries out 1000 circulations altogether. From scheming, spina date seed and reason jujube kernelAffiliation is nearest, is secondly hoveniae semoveniae semen, far away with the relation of cercis and Lens culinaris, can distinguish confidence levelMore than 98%, meet plant classification result, reason jujube kernel, Lens culinaris, hoveniae semoveniae semen, cercis become the mixed puppet of spina date seedProduct. Result shows that ITS sequence can be for differentiating spina date seed and adulterant thereof.
Increase with the universal primer in table 1, obtain the ITS sequence of spina date seed and adulterant thereof. By spina date seed andAdulterant ITS sequence with BioEdit analysis software analyze, multiple sequence contrast, find the variation position of stablizing differencePoint, according to variant sites with PrimerPremier5.0 Software for Design for the identification of special primer. Primer sequence byBeijing Bioisystech Co., Ltd of farsighted Boxing section is synthetic. Primer sequence is in table 1.
Table 1 primer and PCR reaction condition
Embodiment 2, the application of primer in qualification spina date seed
One, groping of special primer
The fruit genomic DNA that extracts spina date seed, reason jujube kernel, hoveniae semoveniae semen, cercis and Lens culinaris, adds respectively as followsIn reaction system, carry out pcr amplification,
Reaction system is 25 μ L, comprises 12.5 μ L2xPCRSuperMix buffer solution (TRAN), justEach 0.5 μ L (the 0.2 μ molL of anti-primer-1), genomic DNA concentration is 35ng μ L-1Totally 1.5 μ L, asepticWater 10 μ L.
PCR reaction condition is: 94 DEG C of 5min, and 35 circulations (94 DEG C of 30s, 57 DEG C of 15s, 72 DEG C of 30s),72℃7min。
The positive anti-primer of ZmITS3 carries out amplification as Fig. 2, under above-mentioned reaction condition, only has spina date seed sample to expandIncrease the fragment that 66bp, and adulterant reason jujube kernel, hoveniae semoveniae semen, cercis and Lens culinaris can not amplify any band; DrawThing ZmITS3 can be used as the special diagnostic primers of spina date seed and mixed adulterant thereof.
The PCR reagent of qualification spina date seed is by 2xPCRSuperMix buffer solution, the positive anti-primer of ZmITS3 andWater composition.
The kit of the PCR reagent that contains the positive anti-primer of ZmITS3 or qualification spina date seed can be used to identify spina date seed.
Two, sensitivity detects
By the genomic DNA of spina date seed, reason jujube kernel, hoveniae semoveniae semen, cercis and Lens culinaris dilute respectively add as followsIn reaction system, carry out pcr amplification,
Reaction system is 25 μ L, comprises 12.5 μ L2xPCRSuperMix buffer solution (TRAN), ZmITS3Positive each 0.5 μ L (the 0.2 μ molL of anti-primer-1), genomic DNA concentration is respectively 10.5,17.5,24.5,31.5,38.5、45.5、52.5、59.5、66.5ng·μL-1Totally 1.5 μ L, sterilized water 10 μ L.
PCR reaction condition is: 94 DEG C of 5min, and 35 circulations (94 DEG C of 30s, 57 DEG C of 15s, 72 DEG C of 30s),72℃7min。
As shown in Figure 1, M is 2000bpDNAMarker to result, and 3,7,8 is certified products spina date seed, and 1,5 is reasonJujube kernel, 13,14 is hoveniae semoveniae semen, and 15 is cercis, and 16 is Lens culinaris; From top to bottom DNA profiling amount be respectively 10.5,17.5、24.5、31.5、38.5、45.5、52.5、59.5、66.5ng·μL-1, can find out 10.5ng μ L-1Time positive adulterant all there is not band, and 52.5ng μ L-1And the above band, all the other template Liang Junneng districts of all occurringSeparately spina date seed and mixed adulterant thereof, therefore determines that this method DNA profiling amount ranges should be 17.5-42.5ng μ L-1，Sensitivity can reach 17.5ng μ L-1DNA profiling amount.
Three, the investigation of annealing temperature
The genomic DNA of spina date seed, reason jujube kernel, hoveniae semoveniae semen, cercis and Lens culinaris is added respectively to following reaction systemIn carry out pcr amplification,
Reaction system is 25 μ L, comprises 12.5 μ L2xPCRSuperMix buffer solution (TRAN), ZmITS3Positive each 0.5 μ L (the 0.2 μ molL of anti-primer-1), genomic DNA concentration is 35ng μ L-1Totally 1.5 μ L, asepticWater 10 μ L.
Under the PCR reaction condition of following different annealing temperature, react, annealing temperature is respectively 53,55,56,57,59、61、63℃。
As shown in Figure 2, M is 2000bpDNAMarker to result; Be followed successively by from top to bottom annealing temperature 53,55,56,57,59,61,63 DEG C; 3,4,7,8,9,10,11,12,17,18,19 is certified products spina date seed, 1,2,5,6,20 is reason jujube kernel, and 13,14 is hoveniae semoveniae semen, and 15 is cercis, and 16 is Lens culinaris; Can find out annealingWhen temperature is 53,55 DEG C, adulterant has false positive band, and 56 DEG C to 59 DEG C adulterants are without band, and certified products spina date seedThere is specific amplification band clearly, and along with the rising of annealing temperature, distinguish effect further clear. For ensureing PCRThe repeatability of reaction, this research adopts 57 DEG C of annealing temperatures.
Four, the broad spectrum activity of archaeal dna polymerase
The genomic DNA of spina date seed, reason jujube kernel, hoveniae semoveniae semen, cercis and Lens culinaris is added respectively to following 2 kinds of reactionsIn system, carry out pcr amplification,
1: reaction system is 25 μ L, comprise 12.5 μ L2xPCRSuperMix buffer solution (TRAN),Each 0.5 μ L (the 0.2 μ molL of the positive anti-primer of ZmITS3-1), genomic DNA concentration is 17.5ng μ L-1Totally 1.5 μ L,Sterilized water 10 μ L.
2: reaction system is 25 μ L, TransFastTaqDNAPolymerase (5U μ L-1)0.2μL(TRAN)，TaKaRadNTPMixture(TaKaRaD4030A)2.5mmeach2μL，Trans10xFastTaqBuffer(Mg2+)) 2.5 μ L, each 1 μ L (the 0.4 μ molL of the positive anti-primer of ZmITS3-1), DNA profiling 35ng μ L-1(1μ L), sterilized water 17.3 μ L
Above-mentioned pcr amplification reaction condition is: 94 DEG C of 5min, 35 circulations (94 DEG C of 30s, 57 DEG C of 15s, 72 DEG C30s)，72℃7min。
As shown in Figure 3, M is 2000bpDNAMarker to result; 3,4,7,8,9,10,11,12,17,18,19 is certified products spina date seed, and 1,2,5,6,20 is reason jujube kernel, and 13,14 is hoveniae semoveniae semen, and 15 is cercis,16 is Lens culinaris; Upper is enzyme TransFastTaqDNAPolymerase (5U μ L-1), lower is enzyme 2x PCRSuperMix, can find out, adulterant is without band, and certified products all has 66bp specific amplification band, explanationAuele Specific Primer can be tested by plurality of enzymes, without single limitation.
Five, the broad spectrum activity of PCR instrument
The genomic DNA of spina date seed, reason jujube kernel, hoveniae semoveniae semen, cercis and Lens culinaris is added respectively to following 2 kinds of reactionsIn system, carry out pcr amplification,
Reaction system is 25 μ L, comprises 12.5 μ L2xPCRSuperMix buffer solution (TRAN), ZmITS3Positive each 0.5 μ L (the 0.2 μ molL of anti-primer-1), genomic DNA concentration is 17.5ng μ L-1Totally 1.5 μ L, nothingBacterium water 10 μ L.
Above-mentioned pcr amplification reaction condition is: 94 DEG C of 5min, 35 circulations (94 DEG C of 30s, 57 DEG C of 15s, 72 DEG C30s)，72℃7min。
Pcr amplification has been selected three different PCR instrument (LifeTechnologies company, model Veriti; MoralState EppendorfAG22331Hamburg; Germany EppendorfMastercyclerpro) and same instrument manyInferior experiment.
Result all can distinguish spina date seed and mixed adulterant thereof, shows that PCR instrument does not affect the qualification of spina date seed and mixed adulterant thereof.
Claims (10)
1. identify a primer for spina date seed, for amplifying the primer pair of DNA fragmentation A;
Described DNA fragmentation A is taking spina date seed as template, increases and obtains with primer pair shown in sequence 1 and sequence 2Product.
2. primer according to claim 1, is characterized in that:
Described primer pair is made up of primer 1 and primer 2;
Described primer 1 is following 1) or 2):
1) be the single strand dna shown in sequence 1;
2) for sequence 1 is deleted and/or increase and/or change the single strand dna that one or several nucleotides obtains;
Described primer 2 is following 3) or 4):
3) be the single strand dna shown in sequence 2;
4) for sequence 2 is deleted and/or increase and/or change the single strand dna that one or several nucleotides obtains.
3. primer according to claim 1 and 2, is characterized in that: described primer 1 and described primer 2Mol ratio is 1:1.
For the identification of or the PCR reagent of auxiliary acid jujube kernel, comprise arbitrary described drawing in claim 1 ?3Thing;
In described primer, every primer final concentration in described PCR reagent is 0.2 μ molL-1。
5. qualification or the kit of assistant identification spina date seed, comprise in claim 1-3 arbitrary described primer orPCR reagent claimed in claim 4.
Claim 1 ?arbitrary described primer or PCR reagent claimed in claim 4 or claim 5 in 3Described kit is the application in any in following (A1)-(A4):
(A1) qualification or assistant identification spina date seed;
(A2) differentiate or assist and differentiate spina date seed
(A3) differentiate or assist and differentiate spina date seed and adulterant thereof;
(A4) identify or whether assistant identification testing sample is spina date seed;
(A5) qualification or assistant identification testing sample are spina date seed or its adulterant;
Described adulterant is specially reason jujube kernel, hoveniae semoveniae semen, cercis or Lens culinaris.
7. whether detection or auxiliary detection testing sample are a method for spina date seed, comprise the steps:
Detect in testing sample and whether contain DNA fragmentation A; If contained, testing sample is or candidate is spina date seed;If do not contained, testing sample be not or candidate for spina date seed;
Described DNA fragmentation A is taking spina date seed as template, increases and obtains with primer pair shown in sequence 1 and sequence 2Product.
8. method according to claim 7, is characterized in that:
The method that whether contains DNA fragmentation A in described detection testing sample comprises the steps: to use claim 1-3In arbitrary described primer pair testing sample carry out pcr amplification, obtain DNA fragmentation A.
9. according to the method described in claim 7 or 8, it is characterized in that:
The size of described pcr amplification product is 60-70bp.
10. according to arbitrary described method in claim 7-9, it is characterized in that:
The template of described pcr amplification is the genomic DNA of testing sample;
The annealing temperature of described pcr amplification is 57 DEG C.
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| TWI660044B (en) * | 2017-11-22 | 2019-05-21 | 統一企業股份有限公司 | Nucleic acid molecule, diagnostic kit, biochip and method for the detection of plant of ziziphus genus |
| WO2020212190A1 (en) | 2019-04-16 | 2020-10-22 | Indena S.P.A. | Method and kit for the identification of vaccinium myrtillus |
| CN114487180A (en) * | 2022-01-20 | 2022-05-13 | 广西壮族自治区食品药品检验所 | Adulteration detection method of jujube kernels in Tianwang heart tonifying preparation |
| CN117625834A (en) * | 2024-01-04 | 2024-03-01 | 中国农业科学院特产研究所 | SNP molecular marker based on senecio scandens ITS2 sequence and application thereof |
| CN117625834B (en) * | 2024-01-04 | 2024-04-16 | 中国农业科学院特产研究所 | SNP molecular marker based on senecio scandens ITS2 sequence and application thereof |
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