CN104164426A - Primer for amplifying COI sequence of DNA code bar of viper species, PCR method and kit - Google Patents
Primer for amplifying COI sequence of DNA code bar of viper species, PCR method and kit Download PDFInfo
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Abstract
The invention belongs to the technical field of viper species and traditional Chinese herbal identification, and discloses a primer for amplifying the COI sequence of DNA code bar of viper species, a PCR method, and a kit; wherein the method comprises the steps of DNA extraction, PCR amplification, and electrophoresis detection. The identification method has the characteristics of convenience, little DNA using amount, and good universality; and overcomes the shortages of COI sequence universal primers (LCO1490 and HCO2198) in viper gene amplification and traditional Chinese herbal identification in the prior art. The primer can be applied to the amplification of COI sequence of viper, and establishes a foundation for molecular identification of viper.
Description
Technical field
The invention belongs to Viperidae snake class species and Materia Medica Identification technical field, be specifically related to a kind of universal primer and PCR method and test kit of the Viperidae snake class species COI bar code sequence that can increase.
Background technology
Snake class medicinal material uses with a long history in China, can trace back to the B.C. the Spring and Autumn and the Warring States Periods, and the successive dynasties book on Chinese herbal medicine particularly Compendium of Material Medica of LI Shi-Zhen is extensively received rich holding especially, becomes and in book on Chinese herbal medicine in ancient times, records the medicine work that snake class medicine is maximum." Chinese Pharmacopoeia " (version in 2010) recorded 5 kinds such as long-nosed pit viper (Agkistrodon), Zaocys, mone snake (coral snake) and snake slough (Zaocys, rat snake, Elaphe taeniurus Cope), 4 taste medicinal materials.
Snake be an ecologic facies to fragile population, the one, fecundity own is not strong, the 2nd, ecotope easily goes to pot.Due to the larger pressure that seizure and the snake class resource of various objects face, the commercially available of Medicinal Snakes held at high price.In this situation that supply falls short of demand and interests are maximized order about under, some illegal businessmans adulterate, and Medicinal Snakes is faked.Along with molecular biological development, DNA molecular technology is more and more for the research of various snake class medicinal material qualifications.
DNA barcode (DNA barcoding) is proposed first by Canadian zoologist Herbert, and its scientific theory is by using a segment standard DNA fragmentation, species being identified fast and accurately.2003, Herbert etc. analyzed right mitochondrial cytochrome C oxidase subunit I (COI) gene order of 13320 species of 11 doors in animal kingdom, found that between this sequence kind, variation can be distinguished the species except Cnidaria preferably.Research subsequently shows, between COI sequence kind variation large and in planting variation little, its DNA sequence dna itself seldom exists and inserts and lack, and is acknowledged as the DNA barcode gene of animal kingdom's Plays.At present, recommending for the universal primer of COI sequence amplification is LCO1490 and HCO2198, but the inventor finds under study for action, and universal primer LCO1490 and HCO2198 can not obtain good amplified production in the qualification of Viperidae snake class species.
Summary of the invention
The above-mentioned situation existing for prior art, the inventor's research and design primer of the Viperidae snake class species COI sequence that can increase, for various Viperidae snake class species COI sequence amplifications, make up the deficiency of COI sequence universal primer in snake class species qualifications.The invention provides PCR primer and the method thereof of Viperidae snake class species COI sequence amplification.The inventive method is simple and efficient, versatility good, can increase rapidly and accurately and obtain the COI sequence of these Viperidae snake class species, for Viperidae snake class species and the Molecular Identification that derives from the medicinal material of undergraduate course animal are laid a good foundation.
The primer that the present invention proposes a pair of Viperidae species COI gene that can be used for increasing, described primer is DK1-CO1 and DK1-CO2, its sequence is:
DK1-CO1：5’-CAACTAACCACAAAGACATCGG-3’(SEQ?ID?NO.1)
DK1-CO2：5’-CTTCTGGGTGGCCGAAAAATCA-3’(SEQ?ID?NO.2)
The PCR method that the invention allows for a kind of Viperidae species COI gene that increases, comprises the following steps:
1) extraction of DNA sample;
2) carry out PCR reaction with primer of the present invention, PCR reaction parameter comprises: 93 DEG C of denaturation 5min, 55 DEG C of 2min; 93 DEG C of sex change 30s, 55 DEG C of renaturation 45s, 70 DEG C are extended 45s, 35 circulations; 70 DEG C are extended 5min.Described primer DK1-CO1 and DK1-CO2, its sequence is:
DK1-CO1：5’-CAACTAACCACAAAGACATCGG-3’(SEQ?ID?NO.1)
DK1-CO2：5’-CTTCTGGGTGGCCGAAAAATCA-3’(SEQ?ID?NO.2)
3) amplified production of the aforementioned gained of electrophoresis detection: in the electrophoretogram of described amplified production, be the single clear DNA band of 700bp if there is molecular weight, determine and increase successfully, gained PCR product can be used for subsequent analysis.
In the step 1 of authentication method of the present invention) in, adopt the test kit of prior art to extract the method for DNA, do not need to prepare any reagent, extraction time is shortened greatly, sample size only needs 30mg, can extract the DNA of any tissue of described snake body.
The present invention also proposes a kind of test kit, for the PCR method of described amplification Viperidae species COI gene.This test kit comprises: primer DK1-CO1 (10 μ M); Primer DK1-CO2 (10 μ M); 2 × Taq archaeal dna polymerase cocktail buffer, it comprises: Taq archaeal dna polymerase (0.1U/ μ L), four kinds of deoxyribonucleoside triphosphates (the each 500 μ M of dATP, dTTP, dCTP and dGTP), Tris-HCl (20 μ M, pH8.3), KCl (100 μ M), MgCl
2(3 μ M) etc.; Sterilizing distilled water.
In sum, the invention provides primer and PCR method and the test kit of Viperidae snake class species COI gene amplification, taking each Viperidae snake class DNA as template, can by the COI gene efficient of this Viperidae snake class sample, exactly amplification out, for the Molecular Identification of Viperidae snake class medicinal material is laid a good foundation.The features such as that the present invention has is simple and efficient, DNA consumption is few, versatility is good, have made up the deficiency of COI sequence universal primer (LCO1490 and HCO2198) in the amplification of Viperidae snake genoid and medicinal material qualification in prior art.
Brief description of the drawings
Fig. 1 is 10 kinds of snake class DNA profiling electrophoresis result of COI sequence universal primer (LCO1490 and HCO2198) amplification: 1. agkistrodon acutus 2. mountain Trimeresurus mucrosquamatus 3. circle spot adder 4. former spearhead pallas pit viper 5. Vietnam's Trimeresurus mucrosquamatus 6. Trimeresurus albolabris (Gray) snake 7. Fujian Trimeresurus stejnegeri 8. Yunnan Trimeresurus stejnegeri 9. Gang Shi Trimeresurus stejnegeri 10. short-tail pallas pit viper 11. negative controls (other reaction conditions is constant, water alternate template DNA); MK.DNA molecular weight standard: be followed successively by from top to bottom 1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, 100bp.
Fig. 2 is by the primer described in this patent and 10 kinds of snake class DNA profiling electrophoresis result of method amplification: 1. 2. agkistrodon acutus 3. mountain Trimeresurus mucrosquamatus 4. circle spot adder 5. former spearhead pallas pit viper 6. Vietnam's Trimeresurus mucrosquamatus 7. Trimeresurus albolabris (Gray) snake 8. Fujian Trimeresurus stejnegeri 9. Yunnan Trimeresurus stejnegeri 10. Gang Shi Trimeresurus stejnegeri 11. short-tail pallas pit vipers of negative control (other reaction conditions is constant, water alternate template DNA); MK.DNA molecular weight standard: be followed successively by from top to bottom 1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, 100bp.
Embodiment
In conjunction with following specific embodiments and the drawings, the present invention is described in further detail, and protection content of the present invention is not limited to following examples.Do not deviating under the spirit and scope of inventive concept, variation and advantage that those skilled in the art can expect are all included in the present invention, and taking appending claims as protection domain.Implement process of the present invention, condition, reagent, experimental technique etc., except the content of mentioning specially below, be universal knowledege and the common practise of this area, the present invention is not particularly limited content.
1, material
10 parts of former animals of Viperidae, (table 1) referred in its source.All sample standard deviation carries out macroscopical identification by the Zhang Liang of south China animals on the brink of extinction institute.
Table 1 Viperidae snake class sample
2, universal primer design
Genbank searches for and downloads Viperidae snake class plastochondria gene order (Genbank accession number is EU913476, KF311102, FJ752492 and NC011390) completely, DNAMAN software is to above-mentioned all fronts plastochondria gene order and carry out contraposition by the sequence that COI universal primer (LCO1490 and HCO2198) amplifies and arrange comparison, and finds out the conservative region between each species COI sequence.With Primer software at can increase from the each snake class species universal primer pair of certain length fragment of conservative region design, i.e. upstream primer DK1-CO1 and downstream primer DK1-CO2.Primer sequence is as follows:
DK1-CO1?5’-CAACTAACCACAAAGACATCGG-3’(SEQ?ID?NO.1)
DK1-CO2?5’-CTTCTGGGTGGCCGAAAAATCA-3’(SEQ?ID?NO.2)
3, DNA extraction
Get each sample tissue, extract according to TIANGEN company's T IANamp genome DNA extracting reagent kit operational manual, concrete operation step is as follows:
1) cut no more than 30mg snake muscle tissue, by liquid nitrogen grinding, add 200 μ L damping fluid GA, vibration is to thoroughly suspending.
2) add 20 μ L Proteinase K solution, mix.56 DEG C of placements, until histolysis, the globule of brief centrifugal removal cap wall.(organization material is fresh, and pyrolysis time is short, conventionally needs within 1 hour, can complete.)
3) add 200 μ L damping fluid GB, fully put upside down and mix, place 10 minutes for 70 DEG C, solution strain is limpid, the globule of brief centrifugal removal cap wall.(while adding damping fluid GB, may produce white precipitate, one is placed 70 DEG C time and can disappear, and as solution does not become limpid, illustrate that lysis is not thorough, may cause extracting that DNA measures less and the DNA extracting is impure.)
4) add 200 μ L dehydrated alcohols, fully vibration mixes 15 seconds, now may occur flocks, the globule of brief centrifugal removal cap wall.
5) previous step gained solution and flocks are all added in an adsorption column CB3 (adsorption column is put into collection tube), centrifugal 30 seconds of 12000rpm, outwells waste liquid, and adsorption column CB3 is put back in collection tube.
6) in adsorption column CB3, add 500 μ L damping fluid GD, centrifugal 30 seconds of 12000rpm, outwells waste liquid, and adsorption column CB3 is put into collection tube.
7) in adsorption column CB3, add 600 μ L rinsing liquid PW, centrifugal 30 seconds of 12000rpm, outwells waste liquid, and adsorption column CB3 is put into collection tube.
8) in adsorption column CB3, add 600 μ L rinsing liquid PW, centrifugal 30 seconds of 12000rpm, outwells waste liquid.
9) adsorption column CB3 is put back in collection tube, centrifugal 2 minutes of 12000rpm, outwells waste liquid.Adsorption column CB3 is placed in to room temperature and places several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.
10) adsorption column CB3 is proceeded in a clean centrifuge tube, to the unsettled dropping in the middle part 50-200 μ L elution buffer TE of adsorption film, room temperature is placed 2-5 minute, and centrifugal 2 minutes of 12000rpm, collects solution in centrifuge tube.
11) for increasing the yield of genomic dna, the centrifugal solution obtaining can be added in adsorption column CB3 again, room temperature is placed 2 minutes, and centrifugal 2 minutes of 12000rpm, collects freezing preservation in centrifuge tube by solution.
4, pcr amplification
PCR reaction is carried out in 200 μ L PCR reaction tubess, and reaction cumulative volume 25 μ L, comprise following reagent:
For preventing that false positive results from appearring in experiment, experiment arranges negative control, with aseptic double-distilled water alternate template DNA, carries out DNA extraction and pcr amplification by the method that sample is same.
After PCR reaction solution has been prepared, the centrifugal 10s of 4000rpm, inserts PCR pipe in PCR instrument, reacts as follows: 93 DEG C of denaturation 5min, 55 DEG C of 2min; 93 DEG C of sex change 30s, 55 DEG C of renaturation 45s, 70 DEG C are extended 45s, 35 circulations; 70 DEG C are extended 5min, obtain amplified production.
Get 5 μ L PCR products o'clock to 1.2% agarose electrophoresis hole, 100V electrophoresis 25min, ultraviolet gel analysis detects, imaging.
10 kinds of snake class DNA profiling electrophoresis result of COI sequence universal primer (LCO1490 and HCO2198) amplification as shown in Figure 1, all there is primer dimer in all snake class samples and negative control, only 1,4,6 there is lighter band at 700bp place, show that this is not high to primer pair Viperidae snake class species amplification efficiency.
Increase 10 kinds of snake class DNA profiling electrophoresis result as shown in Figure 2 by primer of the present invention and method, all snake class samples are that 700bp place all has clear unique DNA band at molecular weight, there is not target stripe in negative control, shows the amplification system of the present invention above-mentioned Viperidae snake class species COI gene that can successfully increase.
Test kit and application thereof
Test kit used in authentication method of the present invention, comprising: primer DK1-CO1 (10 μ M); Primer DK1-CO2 (10 μ M); 2 × Taq archaeal dna polymerase cocktail buffer, it comprises: Taq archaeal dna polymerase (0.1U/ μ L), four kinds of deoxyribonucleoside triphosphates (the each 500 μ M of dATP, dTTP, dCTP and dGTP), Tris-HCl (20 μ M, pH8.3), KCl (100 μ M), MgCl
2(3 μ M) etc.; Sterilizing distilled water.Test kit using method is as follows:
1) extraction of testing sample DNA profiling;
2) pcr amplification: PCR reaction is carried out in 200 μ L PCR reaction tubess, and reaction cumulative volume 25 μ L, comprise following reagent:
After PCR reaction solution has been prepared, the centrifugal 10s of 4000rpm, inserts PCR pipe in PCR instrument, reacts as follows: 93 DEG C of denaturation 5min, 55 DEG C of 2min; 93 DEG C of sex change 30s, 55 DEG C of renaturation 45s, 70 DEG C are extended 45s, 35 circulations; 70 DEG C are extended 5min.
Pcr amplification the primer sequence is:
DK1-CO1：5’-CAACTAACCACAAAGACATCGG-3’，
DK1-CO2：5’-CTTCTGGGTGGCCGAAAAATCA-3’。
3) electrophoresis detection: get 5 μ L PCR products, point sample in 1.2% agarose gel electrophoresis hole, 100V electrophoresis 25min, ultra-violet analysis detect.In this amplified production electrophoretogram, if detect that molecular weight is the unique DNA band of 700bp, determine that the testing sample of examining increases successfully.
Claims (3)
1. the primer of pair for amplification Viperidae snake class species COI sequence, is characterized in that, described primer comprises DK1-CO1 and DK1-CO2, and its sequence is:
DK1-CO1：5’-CAACTAACCACAAAGACATCGG-3’；
DK1-CO2：5’-CTTCTGGGTGGCCGAAAAATCA-3’。
2. a PCR method for the Viperidae that increases snake class species COI sequence, is characterized in that, said method comprising the steps of:
1) extraction of DNA sample;
2) carry out PCR reaction with primer claimed in claim 1, obtain amplified production; Described PCR reaction parameter comprises: 93 DEG C of denaturation 5min, 55 DEG C of 2min; 93 DEG C of sex change 30s, 55 DEG C of renaturation 45s, 70 DEG C are extended 45s, 35 circulations; 70 DEG C are extended 5min;
3) amplified production of the aforementioned gained of electrophoresis detection: in the electrophoretogram of described amplified production, be the single clear DNA band of 700bp if there is molecular weight, determine and increase successfully, obtain target amplification product, i.e. COI gene fragment.
3. for a test kit for the PCR method of the Viperidae snake class species COI sequence that increases, it is characterized in that, described test kit comprises: 10 μ M primer DK1-CO1; 10 μ M primer DK1-CO2; 2 × Taq archaeal dna polymerase cocktail buffer, it comprises: 0.1U/ μ L Taq archaeal dna polymerase, dATP, dTTP, the each 500 μ M of dCTP, dGTP, 20 μ M Tris-HCl (pH8.3), 100 μ M KCl, 3 μ M MgCl
2; Sterilizing distilled water.
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Cited By (5)
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| CN104988228A (en) * | 2015-07-09 | 2015-10-21 | 国家海洋局第三海洋研究所 | Primer group and method for detecting fish CO I genes through nested PCR method |
| CN106086228A (en) * | 2016-08-26 | 2016-11-09 | 中山市中智药业集团有限公司 | A kind of primer analyzing mixing Chinese medicinal powder composition species for DGGE |
| CN110387370A (en) * | 2019-08-13 | 2019-10-29 | 上海赛伦生物技术股份有限公司 | It is a kind of to extract micro adder gene and its method for detecting specificity |
| CN111455022A (en) * | 2019-10-22 | 2020-07-28 | 上海赛伦生物技术股份有限公司 | Extraction method of trace pallas pit viper venom gene, specificity detection method and kit thereof |
| CN114657255A (en) * | 2020-12-23 | 2022-06-24 | 广东一方制药有限公司 | Primer combination for PCR identification of agkistrodon medicinal materials, standard decoction and traditional Chinese medicine formula granules, application and identification method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104988228A (en) * | 2015-07-09 | 2015-10-21 | 国家海洋局第三海洋研究所 | Primer group and method for detecting fish CO I genes through nested PCR method |
| CN106086228A (en) * | 2016-08-26 | 2016-11-09 | 中山市中智药业集团有限公司 | A kind of primer analyzing mixing Chinese medicinal powder composition species for DGGE |
| CN110387370A (en) * | 2019-08-13 | 2019-10-29 | 上海赛伦生物技术股份有限公司 | It is a kind of to extract micro adder gene and its method for detecting specificity |
| CN111455022A (en) * | 2019-10-22 | 2020-07-28 | 上海赛伦生物技术股份有限公司 | Extraction method of trace pallas pit viper venom gene, specificity detection method and kit thereof |
| CN114657255A (en) * | 2020-12-23 | 2022-06-24 | 广东一方制药有限公司 | Primer combination for PCR identification of agkistrodon medicinal materials, standard decoction and traditional Chinese medicine formula granules, application and identification method thereof |
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Application publication date: 20141126 |