CN103525935A - Identification primers for penis cervi of New Zealand wapiti and penis cervi of sika deer as well as PCR (Polymerase Chain Reaction) identification method - Google Patents
Identification primers for penis cervi of New Zealand wapiti and penis cervi of sika deer as well as PCR (Polymerase Chain Reaction) identification method Download PDFInfo
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- CN103525935A CN103525935A CN201310495476.2A CN201310495476A CN103525935A CN 103525935 A CN103525935 A CN 103525935A CN 201310495476 A CN201310495476 A CN 201310495476A CN 103525935 A CN103525935 A CN 103525935A
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- 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
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Abstract
The invention relates to identification primers for penis cervi of New Zealand wapiti and penis cervi of sika deer as well as a PCR (Polymerase Chain Reaction) identification method, wherein an upstream primer is 5'-TCTTTGCCGATGGTATAGGT-3', and a downstream primer is 5'-CGGACGAAACTTTGTGAGATTAA-3'. The identification method comprises the following steps: extracting genomic DNAs (Deoxyribonucleic Acid) of penis cervi of New Zealand wapiti and penis cervi of sika deer by using a phenol-chloroform-isoamylol extraction method, and the reaction system is that the PCR reaction system is 25 microlitres with components being the upstream and downstream primers, 10*Buffer, dNTP and rTaqDNA polymerase for PCR amplification; carrying out electrophoresis on a PCR amplification product on sepharose gel; determining the authenticity of the penis cervi of New Zealand wapiti and penis cervi of sika deer through electrophoretogram. The identification method is strong in resolving ability and good in repeatability, ensures the medication safety of people, remedies the weakness of animalian traditional Chinese medicine identification in conventional traditional Chinese medicine identification, and provides a scientific basis for clinical rational drug use.
Description
Technical field
The present invention relates to diagnostic primers and the PCR discrimination method thereof of a kind of New Zealand wapiti deer whip and spotted deer deer whip.
Technical background
Deer whip has another name called deer kidney, deer punching, primary treatment impotence, tinnitus due to deficiency of the kidney, married woman's cold uterus, fail to be impregnated for a long time, the disease such as chronic testis inflammation.Spotted deer deer whip pharmaceutical use is high but market value expensive, New Zealand's wapiti deer whip is similar with shape and cheap to spotted deer deer whip outward appearance, but the pharmaceutical use of New Zealand's wapiti deer whip is far away not as good as spotted deer deer whip, because spotted deer Lu Bianhe New Zealand wapiti deer whip is difficult to differentiate by naked eyes, on market, higher economic interests are sought by some illegal businessmans, with New Zealand's wapiti deer whip, pretend to be spotted deer deer whip to sell, have a strong impact on the drug effect of deer whip, and caused the impaired and financial loss of patient health.Composition and complicated component due to Chinese patent medicine, interfering factors is many, by traditional pharmacognosy method, identify that its true and false and purity have larger difficulty, therefore, also do not have at present a kind of relatively rapid sensitive, accurately and reliably, strong, the New Zealand's wapiti deer whip of favorable reproducibility of resolving power and the discrimination method of spotted deer deer whip.
Summary of the invention
The technical problem to be solved in the present invention be to provide a kind of rapid sensitive, accurately and reliably, strong, the New Zealand's wapiti deer whip of favorable reproducibility of resolving power and diagnostic primers and the PCR discrimination method thereof of spotted deer deer whip.
Technical solution of the present invention is:
A diagnostic primers for New Zealand's wapiti deer whip and spotted deer deer whip, primer sequence is:
Upstream primer: 5'-TCTTTGCCGATGGTATAGGT-3';
Downstream primer: 5'-CGGACGAAACTTTGTGAGATTAA-3'.
A PCR discrimination method for wapiti deer whip and spotted deer deer whip, its concrete steps are as follows:
The genome DNA extracting method of 1.1, New Zealand wapiti deer whip and spotted deer deer whip
New Zealand's wapiti deer whip and spotted deer deer whip are processed totally with alcohol respectively, adopted phenol-chloroform-primary isoamyl alcohol extraction process to extract New Zealand's wapiti deer whip genomic dna and spotted deer deer whip genomic dna;
1.2, for the PCR primer of the sample that increases
Upstream primer: 5'-TCTTTGCCGATGGTATAGGT-3';
Downstream primer: 5'-CGGACGAAACTTTGTGAGATTAA-3';
1.3, PCR reaction system and the amplification condition for increasing
PCR reaction system: New Zealand's wapiti deer whip genomic dna and the spotted deer deer whip genomic dna of getting 50ng/ μ L are 2 μ L, the Buffer of 10 * PCR is 2 μ L, the dNTP of 2.5mM is 2 μ L, each 1 μ L of the upstream primer of 10pM/ μ L and downstream primer, the rTaqDNA polysaccharase of 5U/ μ L is 0.2 μ L, with sterilizing deionized water, is supplemented to 25 μ L;
Amplification condition: 94.0 ℃ of denaturation 8min; 94.0 ℃ of sex change 1min, 56.2 ℃ are extended 1min, 72.0 ℃ of annealing 2min, totally 35 circulations; 72.0 ℃ are extended 10min, obtain pcr amplification product, 4 ℃ of preservations;
1.4, the detection method of pcr amplification product
The sepharose that preparation quality percentage concentration is 1.5%～1.8% in Horizontal electrophoresis tank; Pcr amplification product in step 1.3 5 μ l are added to point sample hole, in contrast hole, add 5 μ l Marker DL 2000 as a comparison, wherein, electrophoretic voltage is 120V, and electrophoresis time is 40 min～60min;
1.5, by electrophoretogram, judge spotted deer Lu Bianhe New Zealand wapiti deer whip
The electrophoresis detection result of pcr amplification product has three specific bands at 250bp～500bp, at 1000bp, there is no specific band, what at 100bp～250bp, have two specific bands is spotted deer deer whip;
The electrophoresis detection result of pcr amplification product has two specific bands at 250bp～500bp, at 1000bp, has a specific band, what at 100bp～250bp, have a specific band is New Zealand's wapiti deer whip.
beneficial effect of the present invention:
With DNA molecular marker, differentiate that the Lu Bianyu New Zealand wapiti deer whip operation steps of spotted deer is simple, rapid sensitive, the sample that only need get minute quantity just can raise the DNA profiling of polymerase chain reaction (PCR) amplification of enough consumptions, then differentiates judgement.This discrimination method resolving power is strong, favorable reproducibility, can clarify the true and false and the quality of spotted deer deer whip and pulvis thereof, financial loss and healthy adverse consequences that Chinese medicinal materials New Zealand wapiti deer whip of poor quality causes to patient have been reduced, ensure that masses' drug safety has Chinese medicine meaning, make up the weakness that in traditional Chinese medicine evaluation, animal tcm is differentiated, for clinical rational drug use provides scientific basis.
Accompanying drawing explanation
Fig. 1 is the electrophoresis result figure of spotted deer Lu Bianhe of the present invention New Zealand wapiti deer whip.
In figure: 1-3Wei New Zealand wapiti deer whip pcr amplification product, 4-6 is spotted deer deer whip pcr amplification product, M-is Marker DL 2000.
Embodiment
The diagnostic primers of New Zealand's wapiti deer whip and spotted deer deer whip, the design of primer is that the 18SrRNA gene order (GenBank accession number is AY225108) according to spotted deer is designed primer, primer sequence is:
Upstream primer: 5'-TCTTTGCCGATGGTATAGGT-3';
Downstream primer: 5'-CGGACGAAACTTTGTGAGATTAA-3'.
The PCR discrimination method of New Zealand's wapiti deer whip and spotted deer deer whip, its concrete steps are as follows:
The genome DNA extracting method of 1.1, New Zealand wapiti deer whip and spotted deer deer whip
New Zealand's wapiti deer whip and spotted deer deer whip are processed respectively clean with alcohol, get spotted deer deer whip and organize 2g, get New Zealand's wapiti whip and organize 2g, adopt phenol-chloroform-primary isoamyl alcohol extraction process to extract New Zealand's wapiti deer whip genomic dna and spotted deer deer whip genomic dna;
1.2, for the PCR primer of the sample that increases
Upstream primer: 5'-TCTTTGCCGATGGTATAGGT-3';
Downstream primer: 5'-CGGACGAAACTTTGTGAGATTAA-3';
1.3, PCR reaction system and the amplification condition for increasing
PCR reaction system: New Zealand's wapiti deer whip genomic dna and the spotted deer deer whip genomic dna of getting 50ng/ μ L are 2 μ L, the Buffer of the Buffer(10 of 10 * PCR PCR doubly) be 2 μ L, the dNTP of 2.5mM is 2 μ L, each 1 μ L of the upstream primer of 10pM/ μ L and downstream primer, the rTaqDNA polysaccharase of 5U/ μ L is 0.2 μ L, with sterilizing deionized water, is supplemented to 25 μ L;
Amplification condition: 94.0 ℃ of denaturation 8min; 94.0 ℃ of sex change 1min, 56.2 ℃ are extended 1min, 72.0 ℃ of annealing 2min, totally 35 circulations; 72.0 ℃ are extended 10min, obtain pcr amplification product, 4 ℃ of preservations;
1.4, the detection method of pcr amplification product
The sepharose that preparation quality percentage concentration is 1.5% in Horizontal electrophoresis tank; Pcr amplification product in step 1.3 5 μ l are added to point sample hole, in contrast hole, add 5 μ l Marker DL2000 as a comparison, wherein, electrophoretic voltage is 120V, and electrophoresis time is 40 min;
1.5, by electrophoretogram, judge spotted deer Lu Bianhe New Zealand wapiti deer whip
As shown in Figure 1, the electrophoresis detection result of spotted deer deer whip pcr amplification product has three specific bands at 250bp～500bp to electrophoresis result, and the electrophoresis detection result of New Zealand's wapiti deer whip pcr amplification product has two specific bands at 250bp～500bp;
The electrophoresis detection result of spotted deer deer whip pcr amplification product does not have specific band at 1000bp, and the electrophoresis detection result of New Zealand's wapiti deer whip pcr amplification product has a specific band at 1000bp;
The electrophoresis detection result of spotted deer deer whip pcr amplification product has two specific bands at 100bp～250bp, and the electrophoresis detection result of New Zealand's wapiti deer whip pcr amplification product has a specific band at 100bp～250bp.
The diagnostic primers of New Zealand's wapiti deer whip and spotted deer deer whip, primer sequence is:
Upstream primer: 5'-TCTTTGCCGATGGTATAGGT-3';
Downstream primer: 5'-CGGACGAAACTTTGTGAGATTAA-3'.
The PCR discrimination method of New Zealand's wapiti deer whip and spotted deer deer whip, its concrete steps are as follows:
The genome DNA extracting method of 1.1, New Zealand wapiti deer whip and spotted deer deer whip
New Zealand's wapiti deer whip and spotted deer deer whip are processed respectively clean with alcohol, get spotted deer deer whip and organize 2g, get New Zealand's wapiti whip and organize 2g, adopt phenol-chloroform-primary isoamyl alcohol extraction process to extract New Zealand's wapiti deer whip genomic dna and spotted deer deer whip genomic dna;
1.2, for the PCR primer of the sample that increases
Upstream primer: 5'-TCTTTGCCGATGGTATAGGT-3';
Downstream primer: 5'-CGGACGAAACTTTGTGAGATTAA-3';
1.3, PCR reaction system and the amplification condition for increasing
PCR reaction system: New Zealand's wapiti deer whip genomic dna and the spotted deer deer whip genomic dna of getting 50ng/ μ L are 2 μ L, the Buffer of 10 * PCR is 2 μ L, the dNTP of 2.5mM is 2 μ L, each 1 μ L of the upstream primer of 10pM/ μ L and downstream primer, the rTaqDNA polysaccharase of 5U/ μ L is 0.2 μ L, with sterilizing deionized water, is supplemented to 25 μ L;
Amplification condition: 94.0 ℃ of denaturation 8min; 94.0 ℃ of sex change 1min, 56.2 ℃ are extended 1min, 72.0 ℃ of annealing 2min, totally 35 circulations; 72.0 ℃ are extended 10min, obtain pcr amplification product, 4 ℃ of preservations;
1.4, the detection method of pcr amplification product
The sepharose that preparation quality percentage concentration is 1.8% in Horizontal electrophoresis tank; Pcr amplification product in step 1.3 5 μ l are added to point sample hole, in contrast hole, add 5 μ l Marker DL 2000 as a comparison, wherein, electrophoretic voltage is 120V, and electrophoresis time is 50min;
1.5, by electrophoretogram, judge spotted deer Lu Bianhe New Zealand wapiti deer whip
The electrophoresis detection result of spotted deer deer whip pcr amplification product has three specific bands at 250bp～500bp, and the electrophoresis detection result of New Zealand's wapiti deer whip pcr amplification product has two specific bands at 250bp～500bp;
The electrophoresis detection result of spotted deer deer whip pcr amplification product does not have specific band at 1000bp, and the electrophoresis detection result of New Zealand's wapiti deer whip pcr amplification product has a specific band at 1000bp;
The electrophoresis detection result of spotted deer deer whip pcr amplification product has two specific bands at 100bp～250bp, and the electrophoresis detection result of New Zealand's wapiti deer whip pcr amplification product has a specific band at 100bp～250bp.
Embodiment 3
The diagnostic primers of New Zealand's wapiti deer whip and spotted deer deer whip, primer sequence is:
Upstream primer: 5'-TCTTTGCCGATGGTATAGGT-3';
Downstream primer: 5'-CGGACGAAACTTTGTGAGATTAA-3'.
The PCR discrimination method of New Zealand's wapiti deer whip and spotted deer deer whip, its concrete steps are as follows:
The genome DNA extracting method of 1.1, New Zealand wapiti deer whip and spotted deer deer whip
New Zealand's wapiti deer whip and spotted deer deer whip are processed respectively clean with alcohol, get spotted deer deer whip and organize 2g, get New Zealand's wapiti whip and organize 2g, adopt phenol-chloroform-primary isoamyl alcohol extraction process to extract New Zealand's wapiti deer whip genomic dna and spotted deer deer whip genomic dna;
1.2, for the PCR primer of the sample that increases
Upstream primer: 5'-TCTTTGCCGATGGTATAGGT-3';
Downstream primer: 5'-CGGACGAAACTTTGTGAGATTAA-3';
1.3, PCR reaction system and the amplification condition for increasing
PCR reaction system: New Zealand's wapiti deer whip genomic dna and the spotted deer deer whip genomic dna of getting 50ng/ μ L are 2 μ L, the Buffer of 10 * PCR is 2 μ L, the dNTP of 2.5mM is 2 μ L, each 1 μ L of the upstream primer of 10pM/ μ L and downstream primer, the rTaqDNA polysaccharase of 5U/ μ L is 0.2 μ L, with sterilizing deionized water, is supplemented to 25 μ L;
Amplification condition: 94.0 ℃ of denaturation 8min; 94.0 ℃ of sex change 1min, 56.2 ℃ are extended 1min, 72.0 ℃ of annealing 2min, totally 35 circulations; 72.0 ℃ are extended 10min, obtain pcr amplification product, 4 ℃ of preservations;
1.4, the detection method of pcr amplification product
The sepharose that preparation quality percentage concentration is 1.6% in Horizontal electrophoresis tank; Pcr amplification product in step 1.3 5 μ l are added to point sample hole, in contrast hole, add 5 μ l Marker DL2000 as a comparison, wherein, electrophoretic voltage is 120V, and electrophoresis time is 50min;
1.5, by electrophoretogram, judge spotted deer Lu Bianhe New Zealand wapiti deer whip
The electrophoresis detection result of spotted deer deer whip pcr amplification product has three specific bands at 250bp～500bp, and the electrophoresis detection result of New Zealand's wapiti deer whip pcr amplification product has two specific bands at 250bp～500bp;
The electrophoresis detection result of spotted deer deer whip pcr amplification product does not have specific band at 1000bp, and the electrophoresis detection result of New Zealand's wapiti deer whip pcr amplification product has a specific band at 1000bp;
The electrophoresis detection result of spotted deer deer whip pcr amplification product has two specific bands at 100bp～250bp, and the electrophoresis detection result of New Zealand's wapiti deer whip pcr amplification product has a specific band at 100bp～250bp.
No matter the electrophoresis detection result of the electrophoresis detection result of spotted deer deer whip pcr amplification product and New Zealand's wapiti deer whip pcr amplification product the size of banding pattern and fragment, all exists difference between sample, this is the polymorphism foundation of identifying the sample true and false and kind.
Claims (2)
- The diagnostic primers of 1.Yi Zhong New Zealand wapiti deer whip and spotted deer deer whip, is characterized in that:Primer sequence is:Upstream primer: 5'-TCTTTGCCGATGGTATAGGT-3';Downstream primer: 5'-CGGACGAAACTTTGTGAGATTAA-3'.
- The PCR discrimination method of 2.Yi Zhong New Zealand wapiti deer whip and spotted deer deer whip, is characterized in that:Concrete steps are as follows:The genome DNA extracting method of 1.1, New Zealand wapiti deer whip and spotted deer deer whipNew Zealand's wapiti deer whip and spotted deer deer whip are processed totally with alcohol respectively, adopted phenol-chloroform-primary isoamyl alcohol extraction process to extract New Zealand's wapiti deer whip genomic dna and spotted deer deer whip genomic dna;1.2, for the PCR primer of the sample that increasesUpstream primer: 5'-TCTTTGCCGATGGTATAGGT-3';Downstream primer: 5'-CGGACGAAACTTTGTGAGATTAA-3';1.3, PCR reaction system and the amplification condition for increasingPCR reaction system: New Zealand's wapiti deer whip genomic dna and the spotted deer deer whip genomic dna of getting 50ng/ μ L are 2 μ L, the Buffer of 10 * PCR is 2 μ L, the dNTP of 2.5mM is 2 μ L, each 1 μ L of the upstream primer of 10pM/ μ L and downstream primer, the rTaqDNA polysaccharase of 5U/ μ L is 0.2 μ L, with sterilizing deionized water, is supplemented to 25 μ L;Amplification condition: 94.0 ℃ of denaturation 8min; 94.0 ℃ of sex change 1min, 56.2 ℃ are extended 1min, 72.0 ℃ of annealing 2min, totally 35 circulations; 72.0 ℃ are extended 10min, obtain pcr amplification product, 4 ℃ of preservations;1.4, the detection method of pcr amplification productThe sepharose that preparation quality percentage concentration is 1.5%～1.8% in Horizontal electrophoresis tank; Pcr amplification product in step 1.3 5 μ l are added to point sample hole, in contrast hole, add 5 μ l Marker DL 2000 as a comparison, wherein, electrophoretic voltage is 120V, and electrophoresis time is 40 min～60min;1.5, by electrophoretogram, judge spotted deer Lu Bianhe New Zealand wapiti deer whipThe electrophoresis detection result of pcr amplification product has three specific bands at 250bp～500bp, at 1000bp, there is no specific band, what at 100bp～250bp, have two specific bands is spotted deer deer whip;The electrophoresis detection result of pcr amplification product has two specific bands at 250bp～500bp, at 1000bp, has a specific band, what at 100bp～250bp, have a specific band is New Zealand's wapiti deer whip.
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| CN201310495476.2A CN103525935A (en) | 2013-10-22 | 2013-10-22 | Identification primers for penis cervi of New Zealand wapiti and penis cervi of sika deer as well as PCR (Polymerase Chain Reaction) identification method |
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| CN201310495476.2A CN103525935A (en) | 2013-10-22 | 2013-10-22 | Identification primers for penis cervi of New Zealand wapiti and penis cervi of sika deer as well as PCR (Polymerase Chain Reaction) identification method |
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Cited By (1)
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| CN106399521A (en) * | 2016-10-09 | 2017-02-15 | 中国农业科学院特产研究所 | Multiple-PCR primer combination for authenticating sika deer and product genders thereof through one-step method, authentication method of multiple-PCR primer combination and kit |
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| CN106399521B (en) * | 2016-10-09 | 2019-07-30 | 中国农业科学院特产研究所 | One-step method identifies primer combination and its identification method, kit of sika deer and products thereof the multiplex PCR of gender |
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Application publication date: 20140122 |