CN101130815A - PCR-RFLP identification method for seven crassostrea oyster on south China coast - Google Patents
PCR-RFLP identification method for seven crassostrea oyster on south China coast Download PDFInfo
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- 238000007894 restriction fragment length polymorphism technique Methods 0.000 title claims abstract description 9
- 238000000034 method Methods 0.000 title abstract description 9
- 241000237501 Crassostrea Species 0.000 title description 4
- 241000237502 Ostreidae Species 0.000 claims abstract description 64
- 235000020636 oyster Nutrition 0.000 claims abstract description 64
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- 239000012634 fragment Substances 0.000 claims abstract description 33
- 238000012408 PCR amplification Methods 0.000 claims abstract description 7
- 238000000246 agarose gel electrophoresis Methods 0.000 claims abstract description 7
- 238000001514 detection method Methods 0.000 claims abstract description 4
- 241000548230 Crassostrea angulata Species 0.000 claims description 42
- 102100030878 Cytochrome c oxidase subunit 1 Human genes 0.000 claims description 39
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 35
- 238000001228 spectrum Methods 0.000 claims description 25
- 238000006243 chemical reaction Methods 0.000 claims description 24
- 241000411788 Crassostrea iredalei Species 0.000 claims description 23
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- 108091008146 restriction endonucleases Proteins 0.000 claims description 15
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- 102100031780 Endonuclease Human genes 0.000 claims description 8
- 108010042407 Endonucleases Proteins 0.000 claims description 8
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- 238000012850 discrimination method Methods 0.000 claims description 6
- 229910052751 metal Inorganic materials 0.000 claims description 6
- 239000002184 metal Substances 0.000 claims description 6
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- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 4
- 239000012295 chemical reaction liquid Substances 0.000 claims description 4
- 230000004087 circulation Effects 0.000 claims description 4
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Abstract
The present invention relates to a PCR-RFLP identification method for 7 kinds of Chinese south inshore oysters. Said identification method includes the following several main steps: making genome DNA extraction, PCR amplification of object fragment, selecting proper incision enzyme combination, utilizing selected incision enzyme to enzyme-cut the PCR product and utilizing agarose gel electrophoresis to make detection.
Description
Technical field
The present invention relates to a kind of novel molecular biology method, further relate to a kind of PCR-RFLP discrimination method of seven crassostrea oyster on south China coast.
Technical background
Oyster is one of domestic and international topmost economic shellfish, and its meat flavour deliciousness have the title of " marine milk ", has nutritive value and economic worth.Oyster culture is an output maximum in the shellfish culture industry, the widest industry that distributes, and the ultimate production of oyster culture and yield per unit all rank first at all breed variety metas.Just having more than the 6-7 kind that China cultures, the huge oyster of The South China coastal belongs to oyster and mainly contains Pacific oyster (C.gigas), Portuguese oyster (C.angulata), the huge oyster in Hong Kong (C.hongkongensis), bright huge oyster (C.ariakensis), Kumamoto oyster (C.sikamea), C.iredalei and C.iredalei sp are arranged.Yet, because the shell morphological plasticity of oyster is very strong, very easily be subjected to residing environmental influence, rely on the formalness of shell to be difficult to sometimes distinguish concrete kind merely, especially all the more so to the oyster young and juvenile mollusk; This investigation to China's oyster resource, research and utilization are very unfavorable, and simultaneously, this also is a problem that always perplexs the shellfish splitter.Protocols in Molecular Biology provides good terms of settlement for this problem, and plastosome 16S rDNA and COI sequence are very conservative in living species, are widely used in the classification and the Study on Evolution of species.Utilize the difference of its sequence nucleic acid that between each species, exists, use restriction enzyme that the 16S rDNA and the COI sequence enzyme of amplification are cut, can produce the nucleic acid fragment of different lengths and quantity, carry out common agarose aggegation electrophoresis and can tell different sorts.This method yet there are no open report in the evaluation of China's oyster kind, this evaluation that will belong to kind for the huge oyster that The South China coastal distributes provides scientific methods, selects the development of breeding and aquaculture that technical support is provided to oyster.
Summary of the invention
The purpose of this invention is to provide a kind of PCR-RFLP discrimination method that seven kinds of huge oysters of The South China coastal is belonged to oyster.It is applied widely, and is simple to operate, low to the plant and instrument requirement, detects fast, more accurate, compares morphological method reliability height.
The objective of the invention is to realize by the following technical solutions: at first utilize the 16S rDNA and the COI sequence of South China's oyster kind of a large amount of order-checkings,, choose suitable restriction endonuclease combination by the MapDraw software analysis among the DNAstar; Subsequently, carry out the segmental pcr amplification of extracting genome DNA and purpose; Utilize the restriction endonuclease of choosing that PCR product enzyme is cut with agarose gel electrophoresis at last and detect, concrete steps are as follows:
(1) chooses the restriction endonuclease combination
The huge oyster of The South China coastal is belonged to kind, according to its acquired 16S rDNA and COI sequence, carry out the difference that sequence between different sorts was found and detected to sequence alignment by ClustalW, use the MapDraw software analysis, select the restriction endonuclease combination, wherein 16S rDNA fragment is picked out Dde I/Dra I and two groups of double digestion combinations of Alu I/Mse I, the COI sequence fragment is picked out Nis I/Alu I and two groups of double digestion combinations of Nis I/Dde I;
(2) extracting genome DNA
A. get closed shell muscular tissue 0.1 gram, add under 37 ℃, to digest in the 700ul extraction buffer and spend the night;
B. with the phenol-chloroform-primary isoamyl alcohol mixed solution of equal-volume proportional volume than 25: 24: 1, the mixing that fully vibrates, 12000g/ branch, centrifugal 10-15 minute;
C. get supernatant liquor, repeating step B;
D. add isopyknic chloroform, mixing, 12000g/ branch, centrifugal 5-10 minute;
E. get supernatant liquor, add the dehydrated alcohol of 2 times of volumes and the sodium acetate of 1/10 volume 3M;
F.12000g/ divide, centrifugal 12-20 minute, remove supernatant liquor, 70% washing with alcohol 1-2 time;
G. allow ethanol thoroughly volatilize, the genomic dna precipitation is dissolved in the 50ul sterile purified water, promptly get genomic dna, 4 ℃ of preservations are standby;
(3) purpose fragment PCR amplification
The PCR reaction system is 50ul, comprises 5ul 10 * buffer, 0.6ul 2.5U/ul Taq enzyme, and 100ng genomic dna, primer are 0.2uM, dNTP is 0.2mM, Mg
2+Be 2mM; The PCR reaction conditions is, 94 ℃ of sex change 45 seconds are then carried out in 94 ℃ of earlier pre-sex change 2 minutes, to 48 ℃ of COI or to 50 ℃ of renaturation of 16S 1 minute and 72 ℃ 1 minute, carries out 35 circulations altogether, last 72 ℃ of extensions 7 minutes, and sample must increase; Amplification 16S and two pairs of general primers of COI gene fragment are respectively:
16SarL/16SarH：5`-CGGTGTTTATCAAAAACAT-3`/5`-CCGGTCTGAACTCAGATCACGT-3`；
COIL1/COIH：5`-GGTCAACAATCATAAAGATATTGG-3`/5`-TAAACTTCAGGGTGACCAAAAAA?TCA-3`；
(4) endonuclease reaction
Use Dde I/Dra I and two groups of enzymes of Alu I/Mse I 16S rDNA amplified fragments is carried out the double digestion reaction respectively, reaction volume is 20ul, comprise 2ul 10 * buffer, to Dde I/Dra I composite reaction Dde I and each 0.5ul of Dra I enzyme, to Alu I/Mse I composite reaction Alu I and each 0.5ul of Mse I enzyme, the 16S rDNA of the 17ul sample that increases; On the metal thermostatic bath 37 ℃ respectively, reacted 2 hours;
Use Nis I/Alu I and two groups of enzymes of Nis I/Dde I the COI amplified fragments is carried out the double digestion reaction respectively, reaction volume is all 20ul, comprise 2ul 10 * buffer, to Nis I/Alu I composite reaction Nis I and each 1ul of Alu I enzyme, the COI of the 16ul sample that increases; Nis I/Dde I composite reaction Nis I and Dde I enzyme are respectively 1ul and 0.5ul, the COI amplification sample of 16.5ul; On the metal thermostatic bath 37 ℃ respectively, reacted 2 hours;
Enzyme is cut result's standard band spectrum: Pacific oyster 16S rDNA is at Dde I/Dra I and Alu I/Mse I, and the COI amplified fragments is respectively at the band spectrum of Nis I/Alu I and the cutting-out of Nis I/Dde I enzyme: 316bp+117bp+100bp, 203bp+150b, 167bp+161bp+134bp+125bp, 284bp+160bp+142bp+115bp; By same order, the Portuguese oyster band spectrum is respectively: 316bp+117b+100bp, 242bp+150bp, 167bp+161bp+134bp+125bp, 426bp+160bp+115bp; The huge oyster band spectrum in Hong Kong is respectively: 316bp+117bp+100bp, 150bp, 269bp+157bp+111bp, 426bp+111bp; There is bright oyster band spectrum to be respectively: 355bp+100bp+78bp, 150bp+82bp, 157bp+145bp+122bp+111bp, 284bp+142bp+111bp; Kumamoto oyster band spectrum is respectively: 217bp+216bp+100bp, 242bp+150bp, 268bp+145bp+134bp+122bp, 535bp+164bp; The C.iredalei band spectrum is respectively: 216bp+117bp+100bp, 150bp+82bp, 259bp+194b+167bp, 426bp+153bp+115bp; C.iredalei sp band spectrum is respectively: 216bp+100bp+71bp, 125bp+99bp, 259bp+145bp+111bp, 272bp+153bp+111bp.Shown in Figure 1A, 1B, 2A, 2B.
More particularly, the result that 16S rDNA downcuts at Dde I/Dra I and Alu I/Mse I enzyme: under the Dde I/Dra I double digestion, Pacific oyster (C.gigas), Portuguese oyster (C.angulata) and the huge oyster in Hong Kong (C.hongkongensis) produce identical band spectrum, and three stripe size are followed successively by 315bp, 117bp and 102bp; Other oyster then produces various types of unique band spectrum, has bright huge oyster (C.ariakensis) three stripe size to be followed successively by 352bp, 102bp and 78bp; Kumamoto oyster (C.sikamea) three stripe size are followed successively by 217bp, 216bp and 102bp; C.iredalei three stripe size are followed successively by 216bp, 117bp and 102bp; C.iredalei sp three stripe size are followed successively by 216bp, 102bp and 70bp (Figure 1A, 1B with reference to the accompanying drawings);
For the undistinguishable Pacific oyster of Dde I/Dra I double digestion (C.gigas), the huge oyster of Portuguese oyster (C.angulata) and Hong Kong (C.hongkongensis), under Alu I/Mse I double digestion, Pacific oyster (C.gigas), Portuguese oyster (C.angulata) has presented different band spectrums with Hong Kong huge oyster (C.hongkongensis), Pacific oyster (C.gigas) has 207bp and 150bp two bands, Portuguese oyster (C.angulata) has 242bp and 150bp two bands, and Hong Kong huge oyster (C.hongkongensis) only has 150bp one band; And that other four seed oysters enzyme is cut type is as follows: Kumamoto oyster (C.sikamea) is identical with Portuguese oyster (C.angulata), there are bright huge oyster (C.ariakensis) and C.iredalei to have more the band of a 82bp than Hong Kong huge oyster (C.hongkongensis), and C.iredalei sp two bands are less, are 125bp and 98bp; Cut the result of 16S rDNA in conjunction with Dde I/Dra I and Alu I/Mse I enzyme, utilize 16S rDNA to be easy to identify different types of oyster.
The COI amplified fragments is in the result of Nis I/Alu I and the cutting-out of Nis I/Dde I enzyme, under the Nis I/Alu I double digestion, 7 seed oysters have produced 6 kinds of band spectrums: Pacific oyster (C.gigas) is identical with Portuguese oyster (C.angulata) band, is respectively 167bp, 161bp, 134bp and 125bp; Close with the two is to have bright huge oyster (C.ariakensis) also to have four bands, and disperse phase is respectively evenly a little sizes on the banding pattern: 157bp, 145bp, 122bp and 111bp; Hong Kong huge oyster (C.hongkongensis) has three bands to be respectively 269bp, 157bp and 111bp; The close C.iredaleisp of being three bands of banding pattern are respectively 259bp, 145bp and 111bp with it; Kumamoto oyster (C.sikamea) and C.iredalei have the feature band, Kumamoto oyster (C.sikamea) four stripe size are respectively: 268bp, 145bp, 134bp and 122bp, C.iredalei three bands are respectively 259bp, 194bp and 167bp (Fig. 2 A, 2B with reference to the accompanying drawings).
Nis I/Dde I enzyme downcuts, and 7 seed oysters have produced 7 kinds of different band spectrums: Pacific oyster (C.gigas) four stripe size are respectively: 284bp, 160bp, 142bp and 115bp; Three of Portuguese oysters (C.angulata) are respectively 426bp, 160bp and 115bp; Close with it is that three of C.iredalei are respectively 426bp, 153bp and 115bp; The huge oyster in Hong Kong (C.hongkongensis) two bands are respectively 426bp and 111bp; Kumamoto oyster (C.sikamea) two bands are respectively 535bp and 164bp; There are bright huge oyster (C.ariakensis) and C.iredalei sp to have three bands that size is close, are respectively 284bp, 142bp and 111bp; And C.iredalei sp is respectively 272bp, 153bp and 111bp.Therefore in conjunction with the result of Nis I/Alu I and the cutting-out of Nis I/Dde I enzyme, utilize the COI amplified fragments to be easy to identify different oyster kinds.Only consider the fragment of clip size more than 70bp in more than analyzing, be difficult for differentiating for littler fragment band when the electrophoresis.
Enzyme is cut also free list 1 expression of band spectrum
The enzyme of table 1 seven seed oyster 16S rDNA and COI amplified fragments is cut band spectrum data (fragment of size below 70bp disregarded)
(5) agarose gel electrophoresis detects
Get 16S rDNA and COI sequence endonuclease reaction liquid that the 7ul enzyme was cut, under 120V, detected in electrophoresis 2-3 hour, up to the dna segment sharp separation with 2.5% sepharose; Add DNA Ladder simultaneously, and the 16S rDNA that does not cut or COI amplified fragments are done contrast; Result and band spectrum Figure 1A, 1B, 2A and 2B or table 1 compare result of determination.
The present invention uses 16S rDNA and compares with other discrimination method with COI PCR-RFLP method discriminating seven crassostrea oyster on south China coast, and the present invention has following advantage and beneficial effect:
1. the present invention can differentiate the oyster kind more accurately, and extreme is conservative between each kind of 16S rDNA and COI, compares morphological method reliability height;
2. the present invention is applied widely, is not subjected to the restriction of the individual size of oyster, and is suitable equally to very little oyster juvenile mollusk;
3. the present invention is simple to operate, detects fast, can finish in general 1-2 days, compares with the complete sequence order-checking discrimination method of conserved sequences such as 16S rDNA and COI and not only can save time but also can reduce expense;
4. the present invention does not require too many classification expertise to the staff, and is low to the plant and instrument requirement, and general personnel can operate through training, and general Molecular Biology Lab all meets the requirement to plant and instrument.
Description of drawings
Figure 1A is the Dde I/Dra I restriction enzyme mapping of seven seed oyster 16S rDNA.Line1:100bp ladder, line2: the 16S rDNA amplified fragments cut of enzyme not, line3-4: Pacific oyster, line5-6: Portuguese oyster; Line7-8: the huge oyster in Hong Kong, line9-10: bright huge oyster is arranged, line11-12: Kumamoto oyster, line13-14:C.iredalei, line15-16:C.iredalei sp.
Figure 1B is the Alu I/Mse I restriction enzyme mapping of seven seed oyster 16S rDNA.Line1:100bp ladder, line2: the 16S rDNA amplified fragments cut of enzyme not, line3-4: Pacific oyster, line5-6: Portuguese oyster; Line7-8: the huge oyster in Hong Kong, line9-10: bright huge oyster is arranged, line11-12: Kumamoto oyster, line13-14:C.iredalei, line15-16:C.iredalei sp.
The segmental Nis I/Alu of Fig. 2 A seven seed oyster COI I restriction enzyme mapping.Line1:100bp ladder, line2: the enzyme COI amplification segment of cutting not, line3-4: Pacific oyster, line5-6: Portuguese oyster; Line7-8: the huge oyster in Hong Kong, line9-10: bright huge oyster is arranged, line11-12: Kumamoto oyster, line13-14:C.iredalei, line15-16:C.iredalei sp.line17:50bp ladder.
Fig. 2 B is the segmental Nis I/Dde of seven a seed oyster COI I restriction enzyme mapping.Line1:100bp ladder, line2: the enzyme COI amplification segment of cutting not, line3-4: Pacific oyster, line5-6: Portuguese oyster; Line7-8: the huge oyster in Hong Kong, line9-10: bright huge oyster is arranged, line11-12: Kumamoto oyster, line13-14:C.iredalei, line15-16:C.iredalei sp.line17:50bp ladder.
Embodiment
The present invention is further illustrated below in conjunction with embodiment, but not should be understood to limitation of the present invention.
(1) extracting genome DNA
Get Hong Kong huge oyster closed shell muscular tissue 0.1 gram, add under 37 ℃, to digest in the 700ul extraction buffer and spend the night; With isopyknic phenol-chloroform-primary isoamyl alcohol (25: 24: 1) mixed solution, the mixing that fully vibrates, 12000g/ branch, centrifugal 10 minutes; Get supernatant, the step once on the repeating step; Add isopyknic chloroform, mixing, 12000g/ branch, centrifugal 5 minutes; Get supernatant liquor, add the dehydrated alcohol of 2 times of volumes and the sodium acetate of 1/10 volume 3M; The 12000g/ branch centrifugal 12 minutes, removes supernatant liquor, 70% washing with alcohol 1-2 time; Treat that ethanol thoroughly volatilizees, the genomic dna precipitation is dissolved in the 50ul sterile purified water, promptly get genomic dna, 4 ℃ of preservations are standby.
(2) 16S rDNA fragment PCR amplification
The PCR reaction system is 50ul, comprises 5ul 10 * buffer, 0.6ul 2.5U/ul Taq enzyme, and 100ng genomic dna, primer are 0.2uM, dNTP is 0.2mM, Mg
2+Be 2mM; The PCR reaction conditions is, 94 ℃ of earlier pre-sex change 2 minutes, then carry out 94 ℃ 45 seconds, 50 ℃ of 1 minute and 72 ℃ 1 minute carry out 35 circulations altogether, last 72 ℃ of extensions 7 minutes promptly get the sample that increases.
(3) endonuclease reaction
Use Dde I/Dra I and two groups of enzymes of Alu I/Mse I 16S rDNA amplified fragments is carried out the double digestion reaction respectively, reaction volume is 20ul, comprise 2ul 10 * buffer, to Dde I/Dra I composite reaction Dde I and each 0.5ul of Dra I enzyme, to Alu I/Mse I composite reaction Alu I and Mse I enzyme 0.5ul, the 16S rDNA of the 17ul sample that increases; On the metal thermostatic bath 37 ℃ respectively, reacted 2 hours.
(4) agarose gel electrophoresis detects
Get the 16S rDNA sequence endonuclease reaction liquid that the 7ul enzyme was cut, carry out electrophoresis detection, under 120V, ran 2-3 hour, up to the dna fragmentation sharp separation with 2.5% sepharose.Add 100bp DNA Ladder simultaneously, and the 16S rDNA amplified fragments of not cutting is done contrast.
According to the restriction enzyme digestion and electrophoresis collection of illustrative plates, contrast Figure 1A, 1B or table 1 can be accredited as the huge oyster in Hong Kong.
(1) extracting genome DNA
Get closed shell muscular tissue 0.1 gram, add under 37 ℃, to digest in the 700ul extraction buffer and spend the night; With isopyknic phenol-chloroform-primary isoamyl alcohol (25: 24: 1) mixed solution, the mixing that fully vibrates, 12000g/ branch, centrifugal 10 minutes; Get supernatant, the step once on the repeating step; Add isopyknic chloroform, mixing, 12000g/ branch, centrifugal 5 minutes; Get supernatant liquor, add the dehydrated alcohol of 2 times of volumes and the sodium acetate of 1/10 volume 3M; The 12000g/ branch centrifugal 12 minutes, removes supernatant liquor, 70% washing with alcohol 1-2 time; Treat that ethanol thoroughly volatilizees, the genomic dna precipitation is dissolved in the 50ul sterile purified water, promptly get genomic dna, 4 ℃ of preservations are standby.
(2) COI fragment PCR amplification
The PCR reaction system is 50ul, comprises 5ul 10 * buffer, 0.6ul 2.5U/ul Taq enzyme, and 100ng genomic dna, primer are 0.2uM, dNTP is 0.2mM, Mg
2+Be 2mM; The PCR reaction conditions is, 94 ℃ of earlier pre-sex change 2 minutes, then carry out 94 ℃ 45 seconds, 48 ℃ of 1 minute and 72 ℃ 1 minute carry out 35 circulations altogether, last 72 ℃ of extensions 7 minutes promptly get the sample that increases
(3) endonuclease reaction
Use Nis I/Alu I and two groups of enzymes of Nis I/Dde I the COI amplified fragments is carried out the double digestion reaction respectively, reaction volume is all 20ul, comprise 2ul 10 * buffer, to the COI amplification sample of Nis I/Alu I composite reaction Nis I and each 1ul of Alu I enzyme and 16ul; To Nis I/Dde I composite reaction Nis I with Dde I enzyme is respectively 1ul and 0.5ul is right and the COI of 16.5ul amplification sample; On the metal thermostatic bath 37 ℃ respectively, reacted 2 hours.
(4) agarose gel electrophoresis detects
Get the COI sequence endonuclease reaction liquid that the 7ul enzyme was cut, carry out electrophoresis detection, under 120V, ran 2-3 hour, up to the dna fragmentation sharp separation with 2.5% sepharose.Add 100bp DNA Ladder simultaneously, and the COI amplified fragments of not cutting is done contrast.
According to the restriction enzyme digestion and electrophoresis collection of illustrative plates, comparison diagram 2A, 2B or table 1 can be accredited as bright huge oyster.
Claims (1)
1. PCR-RFLP discrimination method that the huge oysters of seven kinds of The South China coastal is belonged to oysters, it is characterized in that at first utilizing the 16S rDNA and the COI sequence of South China's oyster kind of a large amount of order-checkings, by the MapDraw software analysis among the DNAstar, choose suitable restriction endonuclease combination; Subsequently, carry out the segmental pcr amplification of extracting genome DNA and purpose; Utilize the restriction endonuclease of choosing that PCR product enzyme is cut with the agarose gel electrophoresis detection, confirmed that concrete steps are as follows at last:
(1) chooses the restriction endonuclease combination
The huge oyster of The South China coastal is belonged to kind, according to its acquired 16S rDNA and COI sequence, carry out the difference that sequence between different sorts was found and detected to sequence alignment by ClustalW, use the MapDraw software analysis, select the restriction endonuclease combination, wherein 16S rDNA fragment is picked out Dde I/Dra I and two groups of double digestion combinations of Alu I/Mse I, the COI sequence fragment is picked out Nis I/Alu I and two groups of double digestion combinations of Nis I/Dde I;
(2) extracting genome DNA
A. get closed shell muscular tissue 0.1 gram, add under 37 ℃, to digest in the 700ul extraction buffer and spend the night;
B. with isopyknic ratio phenol-chloroform-primary isoamyl alcohol mixed solution of 25: 24: 1, the mixing that fully vibrates, 12000g/ branch, centrifugal 10-15 minute;
C. get supernatant liquor, repeating step B;
D. add isopyknic chloroform, mixing, 12000g/ branch, centrifugal 5-10 minute;
E. get supernatant liquor, add the dehydrated alcohol of 2 times of volumes and the sodium acetate of 1/10 volume 3M;
F.12000g/ divide, centrifugal 12-20 minute, remove supernatant liquor, 70% washing with alcohol 1-2 time;
G. allow ethanol thoroughly volatilize, the genomic dna precipitation is dissolved in the 50ul sterile purified water, promptly get genomic dna, 4 ℃ of preservations are standby;
(3) purpose fragment PCR amplification
The PCR reaction system is 50ul, comprises 5ul 10 * buffer, 0.6ul 2.5U/ul Taq enzyme, and 100ng genomic dna, primer are 0.2uM, dNTP is 0.2mM, Mg
2+Be 2mM; The PCR reaction conditions is, 94 ℃ of sex change 45 seconds are then carried out in 94 ℃ of earlier pre-sex change 2 minutes, extend 1 minute in 1 minute and 72 ℃ to 48 ℃ of COI or to 50 ℃ of renaturation of 16S, carry out 35 circulations altogether, last 72 ℃ of extensions 7 minutes, and sample must increase; Amplification 16S and two pairs of general primers of COI gene fragment are respectively:
16SarL/16SarH：5`-CGGTGTTTATCAAAAACAT-3`/5`-CCGGTCTGAACTCAGATCACGT-3`；
COIL1/COIH：5`-GGTCAACAATCATAAAGATATTGG-3`/5`-TAAACTTCAGGGTGACCAAAAAA?TCA-3`；
(4) endonuclease reaction
Use Dde I/Dra I and two groups of enzymes of Alu I/Mse I 16S rDNA amplified fragments is carried out the double digestion reaction respectively, reaction volume is 20ul, comprise 2ul 10 * buffer, to Dde I/Dra I composite reaction Dde I and each 0.5ul of Dra I enzyme, to Alu I/Mse I composite reaction Alu I and each 0.5ul of Mse I enzyme, the 16S rDNA of the 17ul sample that increases; On the metal thermostatic bath 37 ℃ respectively, reacted 2 hours;
Use Nis I/Alu I and two groups of enzymes of Nis I/Dde I the COI amplified fragments is carried out the double digestion reaction respectively, reaction volume is all 20ul, comprise 2ul 10 * buffer, to Nis I/Alu I composite reaction Nis I and each 1ul of Alu I enzyme, the COI of the 16ul sample that increases; Nis I/Dde I composite reaction Nis I and Dde I enzyme are respectively 1ul and 0.5ul, the COI amplification sample of 16.5ul; On the metal thermostatic bath 37 ℃ respectively, reacted 2 hours;
Enzyme is cut result's standard band spectrum: Pacific oyster 16S rDNA is at Dde I/Dra I and Alu I/Mse I, and the COI amplified fragments is respectively at the band spectrum of Nis I/Alu I and the cutting-out of Nis I/Dde I enzyme: 316bp+117bp+100bp, 203bp+150b, 167bp+161bp+134bp+125bp, 284bp+160bp+142bp+115bp; By same order, the Portuguese oyster band spectrum is respectively: 316bp+117b+100bp, 242bp+150bp, 167bp+161bp+134bp+125bp, 426bp+160bp+115bp; The huge oyster band spectrum in Hong Kong is respectively: 316bp+117bp+100bp, 150bp, 269bp+157bp+111bp, 426bp+111bp; There is bright oyster band spectrum to be respectively: 355bp+100bp+78bp, 150bp+82bp, 157bp+145bp+122bp+111bp, 284bp+142bp+111bp; Kumamoto oyster band spectrum is respectively: 217bp+216bp+100bp, 242bp+150bp, 268bp+145bp+134bp+122bp, 535bp+164bp; The C.iredalei band spectrum is respectively: 216bp+117bp+100bp, 150bp+82bp, 259bp+194b+167bp, 426bp+153bp+115bp; C.iredalei sp band spectrum is respectively: 216bp+100bp+71bp, 125bp+99bp, 259bp+145bp+111bp, 272bp+153bp+111bp;
(5) agarose gel electrophoresis detects
Get 16S rDNA and COI sequence endonuclease reaction liquid that the 7ul enzyme was cut, under 120V, detected in electrophoresis 2-3 hour, up to the dna segment sharp separation with 2.5% sepharose; Add DNA Ladder simultaneously, and the 16S rDNA that does not cut or COI amplified fragments are done contrast; Result and standard band spectrum compare result of determination.
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