CN101942510A - Multiple PCR method for rapidly identifying four varieties of sea cucumbers - Google Patents
Multiple PCR method for rapidly identifying four varieties of sea cucumbers Download PDFInfo
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Abstract
The invention relates to a multiple PCR method for rapidly identifying four varieties of sea cucumbers and belongs to the field of molecular biology. The method adopts mitochondria CO I genes of four varieties of sea cucumbers as target DNA, and by sequence comparison, species specificity primers of the four varieties of sea cucumbers are designed respectively. The CO I genes of the sea cucumbers are extracted, multiple-PCR detection is carried out, thus judging the varieties of the sea cucumbers according to the molecular weight of PCR products. After the multiple-PCR detection is carried out on apostichopus japonicus, thelenota ananas, California apostichopus japonicus and Iceland sea cucumbers, the molecular weights of characteristic electrophoretic bands are 202bp, 301bp, 261bp and 358bp sequentially. The identification method has simple operation, low cost, short inspection period, good specificity and repeatability and high sensitivity, overcomes the defects of large workload, long consumed time, high cost and the like of the traditional sea cucumber morphology identification method and can realize rapid and accurate identification of the varieties of the sea cucumbers.
Description
Technical field
The present invention relates to identify in a kind of molecular biology the method for sea cucumber kind, particularly relate to the multiple PCR method of four kinds of sea cucumber kinds of a kind of Rapid identification.
Background technology
Sea cucumber is the general name of Echinodermata (Echinodermata) Holothuroidea (Holothrioider) animal, and there is the about kind more than 900 of sea cucumber in the whole world, China's kind more than 140 of having an appointment.The whole world has 40 kinds of sea cucumbers can be edible approximately.In recent years, along with the physiology and biochemistry of domestic and international utilization modern science and technology to sea cucumber, deepening continuously of researchs such as the separation of sea cucumber bio active substance, evaluation and biological medical action thereof, it is found that sea cucumber contains many materials that important biomolecule is learned active and pharmacologically active that have, this makes that also beche-de-mer products is more and more paid close attention to.Occurred various beche-de-mer products in recent years on the domestic market, malpractice such as huge commercial benefits expedited the emergence of some frauds, mingle is for example forged the place of production, is adulterated or the like.Therefore, being badly in need of setting up a kind of method fast and effectively differentiates and quality control beche-de-mer products.
Morphological feature, physiological character and ecogeography distribution situation by means of species are the main means of research classification of organisms, and traditional sea cucumber is differentiated main formalness and anatomical features according to sea cucumber.Because the beche-de-mer products of selling on the market all passes through a series of processing treatment, formalness changes greatly, utilizes the formalness feature to be difficult to accurately identification sea cucumber kind.Osteocomma is the sea cucumber endoskeleton that is positioned at sea cucumber corium top layer, has difference between species, is the key character that carries out the sea cucumber classification and differentiate.Liao Yulin etc. once carried out morphologic classification to 134 kinds of sea cucumbers of China, and its osteocomma is described in detail.Can be with this foundation of identifying as the sea cucumber classification.But, utilize osteocomma to identify and to recognize and add up that to the osteocomma type of every sea cucumber and ratio thereof the qualification process workload is very big, and is consuming time also long, human factor is apparent in view, can not be as a kind of method of the kind of sea cucumber fast and accurately evaluation.
Mitochondrial DNA (mitochondrial DNA; MtDNA), simple, the strict matrilinear inheritance of molecular structure is arranged, do not have reorganization, do not have with the nuclear gene group that common sequences, evolutionary rate are fast, advantages such as multiple copied and molecular clock theory as the theoretical basis of molecule marker.Therefore carry out method that kind identifies with the Mitochondrial DNA technology and have that specificity is good, can detect after processing can't be with the advantages such as sample of morphological method evaluation.Increase to mitochondrial cytochrome oxydase subunit I (CO I) sequence of multiple sea cucumber in existing report employing polymerase chain reaction (PCR).Through clone, order-checking and the online comparison of sequence, be used for the evaluation of sea cucumber kind.But this PCR sequencing cost is also grown (5-7 days) than higher, required time.The kind numerous and complicated of sea cucumber wastes time and energy if adopt simple sequence measurement that a large amount of sea cucumber converted productss is carried out the kind evaluation, and cost is also very high.
Summary of the invention
The object of the invention is to provide the multiple PCR method of four kinds of sea cucumber kinds of a kind of Rapid identification, and it can overcome the above-mentioned shortcoming of prior art, realizes the Rapid identification of sea cucumber kind.
The multiple PCR method of four kinds of sea cucumber kinds of a kind of Rapid identification is characterized in that earlier the dry Stichopus japonicus bubble being sent out; The dry Stichopus japonicus that bubble is sent out carries out the dna profiling extraction again; Specific fragment to CO I gene carries out the multiplex PCR amplification then; At last the multiplex PCR amplified production is carried out electrophoresis detection, obtain the purpose band of 202bp, 301bp, 261bp and 358bp, corresponding sea cucumber is respectively imitative stichopus japonicus, Thelenota ananas (Jaeger), the California intends stichopus japonicus and Iceland is joined.
Advantage of the present invention: 1, multiplex PCR is identified the sea cucumber kind, and it is big to have overcome the workload that exists when traditional morphological method is identified sea cucumber, shortcomings such as length consuming time.Adopt the inventive method, only need a PCR reaction and an agarose gel electrophoresis, can tell four kinds of sea cucumbers, have advantages such as saving time, reduce cost, raise the efficiency; 2, has higher specificity and sensitivity preferably; 3, has circulation ratio preferably.
Description of drawings
Fig. 1 identifies the specificity experimental result of four kinds of sea cucumbers: M-DNA Marker (50bp) for multiplex PCR; 1-Iceland ginseng; The 2-Thelenota ananas (Jaeger); Stichopus japonicus is intended in the 3-California; 4-Japan stichopus japonicus; The dark-coloured stichopus japonicus of 5-; The 6-hojothuria leucospilota; The 7-trepang; 8-Mexico stichopus japonicus; The 9-Bohadschia argus Jaeger; 10-water.
Fig. 2 is the multiplex PCR sensitivity determination: M-DNA Marker (50bp); 1-4 is that Iceland's ginseng template DNA dilutes 10,100,1000 and 10000 times successively; 5-8 is that the Thelenota ananas (Jaeger) template DNA dilutes 10,100,1000 and 10000 times successively; 9-12 is that California plan stichopus japonicus template DNA dilutes 10,100,1000 and 10000 times successively; 13-16 dilutes 10,100,1000 and 10000 times successively for imitative stichopus japonicus template DNA; 17 is water.The sensitivity of detected result proof multiplex PCR is the ng level.
Fig. 3 is the multiplex PCR repeated experiment: figure A is Iceland's ginseng experimental result; Figure B is the Thelenota ananas (Jaeger) experimental result; Figure C is that the stichopus japonicus experimental result is intended in the California; Figure D is imitative stichopus japonicus experimental result.1-10 is 10 different sea cucumber individualities; The negative contrast of Y; M:DNAMarker (50bp).
Embodiment
The multiple PCR method of four kinds of sea cucumber kinds of Rapid identification of the present invention is characterized in that earlier the dry Stichopus japonicus bubble being sent out; The dry Stichopus japonicus that bubble is sent out carries out the dna profiling extraction again; Specific fragment to CO I gene carries out the multiplex PCR amplification then; At last the multiplex PCR amplified production is carried out electrophoresis detection, obtain the purpose band of 202bp, 301bp, 261bp and 358bp, corresponding sea cucumber is respectively imitative stichopus japonicus, Thelenota ananas (Jaeger), the California intends stichopus japonicus and Iceland is joined.
Multiplex PCR amplified reaction of the present invention totally is 50 μ L, comprising: water 6.5 μ L; 10 * PCR Buffer, 5 μ L; 25mM MgCl
22 μ L; 10mM dNTP 1 μ L; AjapEF (10 μ mol/L) 0.5 μ L; AjapER (10 μ mol/L) 0.5 μ L; TnasEF (10 μ mol/L) 0.5 μ L; TnasER (10 μ mol/L) 0.5 μ L; PcalEF (10 μ mol/L) 0.5 μ L; PcalER (10 μ mol/L) 0.5 μ L; CfroEF (10 μ mol/L) 0.5 μ L; CfroER (10 μ mol/L) 0.5 μ L; Template DNA 1 μ L; Tap archaeal dna polymerase 0.5 μ L; The Auele Specific Primer that uses is respectively: imitative stichopus japonicus (Apostichopus japonicus) primer is: AjapEF:5 '-CTCCCTCCTTCATTCTTCTTCTTG-3 '; AjapER:5 '-TATCCC[T/C] GGAGTCCGCATTTTA-3 '; Thelenota ananas (Jaeger) (Thelenota ananas) primer is: TnasEF:5 '-GGAACAGGATGACCATCTACCA-3 '; TnasER:5 '-CAGGGTCGAAGAAGGTTGTTTTT-3 '; Stichopus japonicus (Parastichopuscalifornicus) primer is intended in the California: PcalEF:5 '-TGAACAATCTACCCTCCCCTCTCG-3 '; PcalER:5 '-GTCCTTAATAACATTGTTATTGCACCG-3 '; Iceland's ginseng (Cucumaria frondosa) primer is: CfroEF:5 '-AGTAGAAAAAGGGGCAGGAACC-3 '; CfroER:5 '-GCAGAATAGGTGTTGGAAGAGTATAGA-3 '.
Embodiment
The evaluation of dry Stichopus japonicus kind
(1) sea cucumber The pretreatment: dry Stichopus japonicus is rinsed well with tap water, and 55 ℃ of distillation bubbly waters are sent out and spent the night;
(2) extraction of dna profiling: 1. take by weighing 100mg sea cucumber muscle post, rinse well, blot, shred or put liquid nitrogen and grind with aseptic paper.Add 400 μ L lysate (50mM Tris-HCl pH 7.5 then; 1.5%CTAB; 1M NaCl; 15mM EDTA) and 12 μ L 20mg/mL Proteinase Ks, place 55 ℃ 5-8 hour until sample dissolution.The muscle post that 2. will spend the night adds 600 μ L chloroforms, mixes 3min, leaves standstill 7min, the centrifugal 2min of 12000g.3. get supernatant, add 500 μ L chloroforms, put upside down and mix 2min, the centrifugal 2min of 12000g.4. get supernatant, add 500 μ L precipitated liquid (1%CTAB; 50mMTris-HCl PH 7.5; 10mM EDTA) mixing leaves standstill 2min.The centrifugal 10-20min of 12000g abandons supernatant, stays precipitation.5. add 200 μ L 1.2M NaCl, mixing dissolves fully to DNA gently, adds 6 μ L RNase again, places 37 ℃ of following 30-60min.6. add 1mL ice ethanol and 100 μ L 3M NaAc, place 10min on ice, middle mixing once, the centrifugal 10min of 15000g, the gained precipitation is DNA.7. the reject supernatant adds 1mL 70% ice ethanol again and cleans the centrifugal 5min of 12000g.8. DNA is deposited in the sterile air and dries, add 20 μ L sterilized water dissolution precipitations, place 4 ℃ standby.
(3) specific fragment of multiplex PCR amplification CO I gene is identified the sea cucumber kind, and concrete steps are as follows: reaction system: total system 50 μ L comprise: water 6.5 μ L; 10 * PCR Buffer, 5 μ L; 25mM MgCl
22 μ L; 10mM dNTP 1 μ L; AjapEF (10 μ mol/L) 0.5 μ L; AjapER (10 μ mol/L) 0.5 μ L; TnasEF (10 μ mol/L) 0.5 μ L; TnasER (10 μ mol/L) 0.5 μ L; PcalEF (10 μ mol/L) 0.5 μ L; PcalER (10 μ mol/L) 0.5 μ L; CfroEF (10 μ mol/L) 0.5 μ L; CfroER (10 μ mol/L) 0.5 μ L; Template DNA 1 μ L; Tap archaeal dna polymerase 0.5 μ L; Auele Specific Primer is respectively: imitative stichopus japonicus (Apostichopus japonicus) primer is: AjapEF:5 '-CTCCCTCCTTCATTCTTCTTCTTG-3 '; AjapER:5 '-TATCCC[T/C] GGAGTCCGCATTTTA-3 '; Thelenota ananas (Jaeger) (Thele-nota ananas) primer is: TnasEF:5 '-GGAACAGGATGACCATCTACCA-3 '; TnasER:5 '-CAGGGTCGAAGAAGGTTGTTTTT-3 '; Stichopus japonicus (Parastichopus californicus) primer is intended in the California: PcalEF:5 '-TGAACAATCTACCCTCCCCTCTCG-3 '; PcalER:5 '-GTCCTTAATAACATTGTTATTGCACCG-3 '; Iceland's ginseng (Cucumaria frondosa) primer is: CfroEF:5 '-AGTAGAAAAAGGGGCAGGAACC-3 '; CfroER:5 '-GCAGAATAGGTGTTGGAAGAGTATAGA-3 '.PCR reaction cycle parameter is as follows: 94 ℃ of pre-sex change 5min; After through 25 circulations, each circulation comprises: 94 ℃ of 50sec, 60 ℃ of 1min, 72 ℃ of 45sec; Last 72 ℃ are extended 10min; 4 ℃ of preservations.
(4) electrophoresis detection is identified: the multi-PRC reaction product is carried out electrophoresis detection.With 1 * tbe buffer liquid preparation, 2% (w/v) sepharose solution, heating for dissolving is cooled to 50-60 ℃, pours in the glue groove, treat that gelling is solid after, remove comb, glue is put into the electrophoresis chamber that contains 1 * tbe buffer liquid; With 5 μ L PCR products and 1 μ L sample loading buffer mixing, add in the well successively again; Control voltage remains on the 100V electrophoresis, when the swimming of tetrabromophenol sulfonphthalein band is arrived apart from the about 2cm in gel forward position, stops electrophoresis; Take out sepharose, place the ethidium bromide of 1 μ g/mL to soak 10-15min, take out and place ultraviolet gel imaging system observation analysis result.The purpose band that contains 202bp, 301bp, 261bp and 358bp in the electrophorogram, corresponding sea cucumber are respectively imitative stichopus japonicus, Thelenota ananas (Jaeger), the California intends stichopus japonicus and Iceland is joined.
Sea cucumber involved in the present invention is available from market, adopt morphological method to identify through professor Liao Yulin of the Institute of Oceanology of the Chinese Academy of Sciences, determine its kind: stichopus japonicus (Parastichopus californicus) and Iceland's ginseng (Cucumaria frondosa) are intended in imitative stichopus japonicus (Apostichopus japonicus), Thelenota ananas (Jaeger) (Thelenota ananas), California.
The used main agents of the present invention: TapDNA polysaccharase, dNTP, 50bp DNA Ladder (the precious biological company limited in Dalian), primer (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd), agarose (BBI).
The used key instrument of the present invention: Mastercycler personal PCR instrument (Eppendorf AG, Germany), day can digital gel imaging instrument (Tanon science and technology Shanghai company limited), ultraviolet reflectance projectoscope (going up Industrial Co., Ltd. of Nereid section), DYY-6B type voltage stabilization and current stabilization electrophoresis apparatus (Beijing Liuyi Instrument Factory).
The present invention is according to the specificity of four kinds of sea cucumber plastosome CO I genes, and four pairs of Auele Specific Primers have been synthesized in design, and primer information sees Table 1.
Table 1 Auele Specific Primer information
Multiplex PCR is identified the specificity experiment of four kinds of sea cucumbers
9 kinds of sea cucumber individualities of identifying through morphology are carried out pcr amplification, the specificity of four pairs of primers of checking.PCR reaction cumulative volume is 50 μ L, comprises 25mM MgCl
22 μ L, 10mM dNTP 1 μ L, each 0.5 μ L (every primer final concentration 0.1 μ M) of 8 primers, Taq archaeal dna polymerase (precious biotechnology company limited) 0.5 μ L, 10 * PCR Buffer, 5 μ L and template DNA 1 μ L, water 36.5 μ L.On the pcr amplification instrument behind 94 ℃ of pre-sex change 5min through 25 circulations, each circulation comprises: 94 ℃ of 50sec, 60 ℃ of 1min, 72 ℃ of 45sec, last 72 ℃ are extended 10min.The PCR product behind the electrophoresis, places the ethidium bromide of 1 μ g/mL to soak 10-15min in 2% sepharose, observes and photographic recording under the UV-light.Experimental result such as Fig. 1, the result shows, 4 couples of Auele Specific Primers (AjapEF and AjapER of design, TnasEF and TnasER, CfroEF and CfroE, PcalEF and PcalER) can amplify the gene fragment of respective length to the purpose sea cucumber, and other sea cucumbers are not had non-specific amplification, illustrate that the specificity of 4 pairs of species-specific primers is better.
Multiplex PCR is identified the sensitivity experiment of four kinds of sea cucumbers
The template DNA that extracts with 4 kinds of sea cucumbers is a stoste respectively, as template, carries out multiplex PCR, reaction system such as embodiment 2 behind 10 times, 100 times, 1000 times, 10000 times of the gradient dilutions.Experimental result such as Fig. 2, result show that multiplex PCR still had band clearly after the dna profiling of extraction diluted 100 times, and the sensitivity of detected result proof multiplex PCR is the ng level.
Multiplex PCR is identified the repeated experiment of four kinds of sea cucumbers
10 individualities of every kind of sea cucumber picked at random extract DNA as template, adopt the multiplex PCR amplification respectively, reaction system such as embodiment 2, the repeatability of checking multi-PRC reaction.Experimental result such as Fig. 3, the result shows: the amplification of 10 sea cucumber individualities does not have obviously difference, proves that the method repeatability of this invention is better.
Claims (2)
1. the multiple PCR method of four kinds of sea cucumber kinds of a Rapid identification is characterized in that earlier the dry Stichopus japonicus bubble being sent out; The dry Stichopus japonicus that bubble is sent out carries out the dna profiling extraction again; Specific fragment to CO I gene carries out the multiplex PCR amplification then; At last the multiplex PCR amplified production is carried out electrophoresis detection, obtain the purpose band of 202bp, 301bp, 261bp and 358bp, corresponding sea cucumber is respectively imitative stichopus japonicus, Thelenota ananas (Jaeger), the California intends stichopus japonicus and Iceland is joined.
2. multiple PCR method as claimed in claim 1 is characterized in that the Auele Specific Primer that described multiplex PCR amplification is used is respectively: imitative stichopus japonicus primer AjapEF:5 '-CTCCCTCCTTCATTCTTCTTCTTG-3 '; AjapER:5 '-TATCCC[T/C] GGAGTCCGCATTTTA-3 '; Thelenota ananas (Jaeger) primer TnasEF:5 '-GGAACAGGATGAACCATCTACCA-3 '; TnasER:5 '-CAGGGTCGAAGAAGGTTGTTTTT-3 '; Stichopus japonicus primer PcalEF:5 '-TGAACAATCTACCCTCCCCTCTCG-3 ' is intended in the California; PcalER:5 '-GTCCGTTAATAACATTGTTATTGCACCG-3 '; Iceland reference thing CfroEF:5 '-AGTAGAAAAAGGGGCAGGAACC-3 '; CfroER:5 '-GCAGAATAGGTGTTGGAAGAGTATAGGA-3 '.
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Cited By (6)
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| CN102911934A (en) * | 2012-11-06 | 2013-02-06 | 大连工业大学 | DNA (Deoxyribonucleic Acid) extraction method of sea cucumber body surface bacteria |
| CN103333966A (en) * | 2013-07-02 | 2013-10-02 | 曹际娟 | Apostichopus japonicus detection primer, kit and real-time fluorescent PCR (polymerase chain reaction) detection method |
| CN103484553A (en) * | 2013-10-12 | 2014-01-01 | 湛江师范学院 | Species-specific PCR method for rapidly identifying ten sea cucumber species |
| CN106701989A (en) * | 2017-02-10 | 2017-05-24 | 牡丹江友搏药业有限责任公司 | DNA (Deoxyribonucleic Acid) barcode molecular identification method of holothuria fuscopunctata |
| CN109234424A (en) * | 2017-07-06 | 2019-01-18 | 大连长生岛集团有限公司 | For identifying the real-time fluorescence PCR primer detection method and application of the Liao Dynasty's ginseng |
| CN110699461A (en) * | 2019-09-30 | 2020-01-17 | 浙江省检验检疫科学技术研究院 | Fluorescent PCR detection method of thelenota ananas, primers and probes thereof |
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| CN102911934A (en) * | 2012-11-06 | 2013-02-06 | 大连工业大学 | DNA (Deoxyribonucleic Acid) extraction method of sea cucumber body surface bacteria |
| CN102911934B (en) * | 2012-11-06 | 2014-06-25 | 大连工业大学 | DNA (Deoxyribonucleic Acid) extraction method of sea cucumber body surface bacteria |
| CN103333966A (en) * | 2013-07-02 | 2013-10-02 | 曹际娟 | Apostichopus japonicus detection primer, kit and real-time fluorescent PCR (polymerase chain reaction) detection method |
| CN103484553A (en) * | 2013-10-12 | 2014-01-01 | 湛江师范学院 | Species-specific PCR method for rapidly identifying ten sea cucumber species |
| CN106701989A (en) * | 2017-02-10 | 2017-05-24 | 牡丹江友搏药业有限责任公司 | DNA (Deoxyribonucleic Acid) barcode molecular identification method of holothuria fuscopunctata |
| CN109234424A (en) * | 2017-07-06 | 2019-01-18 | 大连长生岛集团有限公司 | For identifying the real-time fluorescence PCR primer detection method and application of the Liao Dynasty's ginseng |
| CN110699461A (en) * | 2019-09-30 | 2020-01-17 | 浙江省检验检疫科学技术研究院 | Fluorescent PCR detection method of thelenota ananas, primers and probes thereof |
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