CN103045589A - Degenerate primer combination for amplifying soil nematode CO I genes and applications thereof - Google Patents
Degenerate primer combination for amplifying soil nematode CO I genes and applications thereof Download PDFInfo
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Abstract
The invention provides a degenerate primer combination for amplifying soil nematode CO I genes. The primer combination comprises 7COX1JB-F1 and 7COX1JB-R1, and 7COX1JB-F2 and 7COX1JB-R2 (as shown in Seq ID No.1-4). The degenerate primer combination utilizes a nested PCR (Polymerase Chain Reaction) method to amplify a part of CO I gene segment of soil nematodes, and the segment can be utilized to effectively classify and identify the soil nematodes. The requirement on operators is not high, only the operators are proficient in PCR operation. Even if a person does not have the nematode morphology classification and identification knowledge, the person can also easily complete the operation. The classification and identification of a lot of samples can be completed at the same time through one-time nested PCR, and the identification time is saved. Compared with the traditional method, the degenerate primer combination selects the CO I genes, the resolution ratio is higher, the soil nematodes can be more effectively distinguished in terms of species, and meanwhile the blank of the field of soil nematode classification and identification is filled.
Description
Technical field
The present invention relates to Nested PCR Technique, specifically, relate to a kind of degenerated primer combination and application thereof for amplification soil nematodes CO I gene.
Background technology
Almost in all types of ecosystems, from the ocean to fresh water, soil, from the torrid zone to the arctic regions, be up to the existence that there is nematode in the minimum zone of height above sea level from height above sea level.Nematode can be divided into free living and parasitic life two large classes, and the scope that takes food of free-living nematode comprises bacterium, fungi, algae, protozoon and soil ulmin etc., and parasitic nematode then all is found in animal and plant.Soil nematodes is a key link of soil food chain, basic energy pathway (plant tissue at three soil, bacterium and fungi) in, pathogenic nematode has participated in the plant tissue path, food bacterium and fungivorous nematode have participated in bacterium and fungi path, heterophagous nematode has then been contained all paths, this is so that nematode more and more is applied to the indicator organism (Yeates. of soil ecosystem, G.W., Bongers, T., de Goede, R.G.M., Freckman, D.W.and Georgieva, S.S.1993.Feeding habits in soil nematode families and genera-an outline for soil ecologists.Journal of Nematology, 25:315 – 31).Compare with other soil animal, nematode is as the advantage of indicator organism: 1) the large and abundant species of quantity, and protozoic quantity is also very abundant, be difficult to the classification evaluation but lack morphological feature, and unicellular structure is too simple; 2) habitat is extensive, and the existence of nematode is arranged at a lot of extreme environments; 3) cultivate easily and the method for research various (Andr é, H.M., Ducarme, X.and Lebrun, biodiversity:myth P.2002.Soil, reality or conning Oikos, 96 (1): 3-24).
Nematode is at occurring in nature One's name is legion and abundant species, and the line insect types of only living in the ocean may just surpass 1,000,000 kinds, and wherein only has general 1000 kinds to obtain identifying and describing.This is to identify and have very large difficulty because nematode classified from the morphology: nematode is individual little and lack the feature of identifying, in the situation that relies on conventional optical microscope, also be difficult to identification mark is quantized (De Ley, P., De Ley, I.T., Morris, K.et al.2005.An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding.Philosophical Transactions of the Royal Society B:Biological Sciences, 360 (1462): 1945-1958).In order to address this problem, increasing investigator begins to select to utilize conservative molecular sequences to make up Molecular Phylogeny tree (molecular phylogenetic tree) and establishes molecule manipulation taxon (molecular operational taxonomic units, MOTU).The gene order that is used for analyzing just is called " DNA bar code (DNA barcode) ", and MOTU " kind " this concept (Blaxter on corresponding the traditional taxonomy just, M.L.2004.The promise of a DNA taxonomy.Philosophical Transactions of the Royal Society of London.SeriesB:Biological Sciences, 359 (1444): 669-679).Using the MOTU system can fast and effeciently identify most of species, therefore has a lot of nematode taxonomists to bring into use the method for DNA bar code nematode to be identified the research of classification and evolutionary process.
At present, the research that utilizes the DNA barcode technology that nematode is classified also is nowhere near with respect to the huge kind number of nematode.The conserved sequence fragment of comparatively commonly using at present in the research of most nematode DNA bar codes is 18S rDNA, but 18S rRNA is multiplex taxon in analyzing more than belonging in the achievement process, at the not (Meldal of resolving power that concerning the nearer species of some sibships, will seem, B.H.M., Debenham, N.J., De Ley, P.et al.2007.An improved molecular phylogeny of the Nematoda with special emphasis on marine taxa.Molecular Phylogenetics and Evolution, 42 (3): 622-636).Although some investigator begins to attempt to use another commonly used to be positioned at mitochondrial cytochrome C oxidase subunit base I gene (cytochrome oxidase csubunit I, CO I) comes the nematode (Derycke that classifies, S., Vanaverbeke, J., Rigaux, A., Backeljau, T.and Moens, T.2010.Exploring " PLoS ONE; 5 (10): e13716); Yu Haiyang nematode and parasitic nematode but this part research focuses mostly on does not almost have for the classification of soil nematodes for the use of cytochrome oxidase c subunit I (COI) for DNA barcoding of free-living marine nematodes..Because chondriogen the putting in order and nucleotide sequence on plastosome of sea nematode and parasitic nematode, widely different with the soil nematodes of free living, the universal primer of the amplification CO I that these institutes provide also is not suitable for the amplification of soil nematodes CO I gene.
Summary of the invention
The purpose of this invention is to provide a kind of degenerated primer combination for amplification soil nematodes CO I gene.
In order to realize the object of the invention, a kind of degenerated primer combination for amplification soil nematodes CO I gene of the present invention comprises:
First pair of primer:
7COX1JB-F1:5’-ATACCTWSWWTAATYGGDGGKTTTGG-3’，
7COX1JB-R1:5 '-AYACCHGTTAAMCCRCCHAHAGTAAA-3 '; And
Second pair of primer:
7COX1JB-F2:5’-GGDGCHCCTGATATRAGNTTYCCHCG-3’，
7COX1JB-R2:5’-ACYTTHACHCCNGTHGGNACAGCAAT-3’；
Wherein, Y represents base C or T, and W represents base A or T, and K represents bases G or T, and D represents bases G, A or T, and R represents base A or G, and M represents base A or C, and H represents A, T or C, and S represents G or C, and N represents A, T, C or G.Wherein, first pair of primer has 2*2*2*2*2*3*2*2*3*2*2*3*3=41472 kind array configuration, and second pair of primer has 3*3*2*4*2*2*3*3*4*3*4=124416 kind array configuration.When carrying out nest-type PRC, what first round PCR used is the mixture of 41472 kinds of permutation and combination of first pair of primer, and second what take turns that PCR uses is the mixture of 124416 kinds of permutation and combination of second pair of primer.
The present invention also provide contain the combination of described degenerated primer for detection of or carry out the test kit that taxonomy is identified soil nematodes.Described test kit also comprises one or more in dNTPs, LA Taq archaeal dna polymerase, the PCR reaction buffer etc.Preferably, described test kit also comprises standard positive template.
The present invention also provides the combination of described degenerated primer or described test kit detecting or carrying out taxonomy and identify and may further comprise the steps application in the soil nematodes: 1) extract the DNA in the sample; 2) DNA that extracts in the step 1) carries out the nest-type PRC amplified reaction as template; 3) analyze the PCR product.
The nest-type PRC amplified reaction is as follows:
First round PCR reaction system is 25 μ L, comprise: 5 ' end primer 7COX1JB-F1(10 μ M) 2 μ L, 3 ' end primer 7COX1JB-R1(10 μ M) 2 μ L, 10 * LA Taq damping fluid, 2.5 μ L, dNTP(2.5mM) 2.5 μ L, LA Taq enzyme (5U/ μ L) 0.3 μ L, nematode DNA2 μ L, ddH
2O13.7 μ L.
First round PCR reaction conditions is: 94 ℃ of 5min; 94 ℃ of 20s, 60 ℃-50 ℃ (every circulation reduces by 0.5 ℃) 20s, 72 ℃ of 1min, 20 circulations; 94 ℃ of 20s, 50 ℃ of 20s, 72 ℃ of 1min, 40 circulations; 72 ℃ of 10min.
Second to take turns the PCR reaction system be 50 μ L, comprise: 5 ' end primer 7COX1JB-F2(10 μ M) 4 μ L, 3 ' end primer 7COX1JB-R2(10 μ M) 4 μ L, 10 * LA Taq damping fluid, 5 μ L, dNTP(2.5mM) 5 μ L, LA Taq enzyme (5U/ μ L) 0.5 μ L, first round PCR product 2 μ L, ddH
2O29.5 μ L.
Second takes turns the PCR reaction conditions is: 94 ℃ of 5min; 94 ℃ of 20s, 56 ℃ of 20s, 72 ℃ of 1min, 40 circulations; 72 ℃ of 10min.
Particularly, the present invention utilizes the CO I gene of several known nematodes to carry out sequence alignment, and according to conservative region design universal primer, the increase CO I fragment of unknown kind soil nematodes of recycling universal primer is verified.Flow process is as follows:
1) universal primer design
Download seven kinds of known nematode (beautiful rhabditis axei Caenorhabditis briggsae from the NCBI website, Caenorhabditis elegans Caenorhabditis elegans, have a liking for bacterium heterorhabditis indica Heterorhabditis bacteriophora, strongyloides intestinalis Strongyloides stercoralis, nematode Steinernema carpocapsae Steinernema carpocapsae, Strelkovimermis spiculatus and Pristionchuspacificus) CO I gene order, its gene I/D is respectively 5666636,2565700,4421891,37805768,326200256,2846681 and 4078882.Utilize Clustal X2 software that these seven sequences are compared, find out conservative zone, design two pairs of degenerated primers (Seq ID No.1-4):
First pair of primer (amplification length is about 860bp):
7COX1JB-F1:5’-ATACCTWSWWTAATYGGDGGKTTTGG-3’；
7COX1JB-R1:5’-AYACCHGTTAAMCCRCCHAHAGTAAA-3’。
Second pair of primer (amplification length is about 695bp):
7COX1JB-F2:5’-GGDGCHCCTGATATRAGNTTYCCHCG-3’；
7COX1JB-R2:5’-ACYTTHACHCCNGTHGGNACAGCAAT-3’。
Wherein, Y represents base C or T, and W represents base A or T, and K represents bases G or T, and D represents bases G, A or T, and R represents base A or G, and M represents base A or C, and H represents A, T or C, and S represents G or C, and N represents A, T, C or G.Wherein, first pair of primer has 41472 kinds of array configurations, and second pair of primer has 124416 kinds of array configurations.When carrying out nest-type PRC, what first round PCR used is the mixture of 41472 kinds of permutation and combination of first pair of primer, and second what take turns that PCR uses is the mixture of 124416 kinds of permutation and combination of second pair of primer.
2) separation of soil nematodes
Be that the glass funnel end of 12cm connects one section rubber tubing at diameter, the rubber tubing end clamps with iron clamp, is fixed on the iron stand, adds a certain amount of deionized water in funnel.The 200g pedotheque respectively with one deck paper handkerchief and one deck gauze parcel, is positioned in the funnel, and the deionized water that fills into capacity makes the complete submergence of soil, place under the room temperature condition and separate.Through behind 24h, 48h, the 72h, open iron clamp respectively, emit in the rubber tubing end water about 2mL in the 50mL centrifuge tube.After last the collection, the centrifugal 5min of 8000g abandons supernatant, and the nematode suspension at the pipe end is transferred in the 1.5mL centrifuge tube, adds isopyknic frozen storing liquid (NaCl5.85g, K
2HPO
46.8g, glycerine 300g, 1mol/L NaOH5.6mL is settled to 1L with deionized water, behind the high-temperature sterilization, adds 0.1mol/L MgSO
43mL), put into immediately liquid nitrogen and suddenly freeze, change again-80 ℃ of Refrigerator stores over to.
3) extraction of soil nematode DNA
Use TIANGEN TIANamp Genomic DNA Kit(blood/cell/tissue genome DNA extracting reagent kit) the part reagent in.
A. picking wall scroll nematode adds 20 μ L Proteinase K solution in 200 μ L damping fluid GA, and mixing is at 56 ℃ of digestion 3h.
B. add 200 μ L damping fluid GB, fully put upside down mixing, place 10min for 70 ℃.
C. add isopyknic phenol/chloroform/primary isoamyl alcohol (25:24:1), mixing, in 12,000rpm centrifugal 10 minutes.
D. the upper strata water is sucked in the new 1.5ml EP pipe, add the two volumes dehydrated alcohol ,-20 ℃ of precipitation 30min.
E.12, the centrifugal 10min of 000rpm abandons supernatant, adds 600 μ L rinsing liquid PW, and mixing is in the centrifugal 10min of 12,000rpm.
F. discard the rinsing liquid on upper strata, and thoroughly dry in room temperature.
G. add 50 μ L deionized water dissolving DNA.
4) pcr amplification and order-checking
Utilize nest-type PRC, carry out respectively the two-wheeled PCR purpose fragment that increases.
First round PCR:
First round PCR reaction system is 25 μ L, comprise: 5 ' end primer 7COX1JB-F1(10 μ M) 2 μ L, 3 ' end primer 7COX1JB-R1(10 μ M) 2 μ L, 10 * LA Taq damping fluid, 2.5 μ L, dNTP(2.5mM) 2.5 μ L, LA Taq enzyme (5U/ μ L) 0.3 μ L, nematode DNA2 μ L, ddH
2O13.7 μ L.
PCR condition: 94 ℃ of denaturation 5min; 94 ℃ of 20s, 60 ℃-50 ℃ (every circulation reduces by 0.5 ℃) 20s, 72 ℃ of 1min, 20 circulations; 94 ℃ of 20s, 50 ℃ of 20s, 72 ℃ of 1min, 40 circulations; 72 ℃ are extended 10min.
Second takes turns PCR:
Second to take turns the PCR reaction system be 50 μ L, comprise: 5 ' end primer 7COX1JB-F2(10 μ M) 4 μ L, 3 ' end primer 7COX1JB-R2(10 μ M) 4 μ L, 10 * LA Taq damping fluid, 5 μ L, dNTP(2.5mM) 5 μ L, LATaq enzyme (5U/ μ L) 0.5 μ L, first round PCR product 2 μ L, ddH
2O29.5 μ L.
PCR condition: 94 ℃ of denaturation 5min; 94 ℃ of 20s, 56 ℃ of 20s, 72 ℃ of 1min, 40 circulations; 72 ℃ are extended 10min.
Take turns the PCR product with second and identify clip size with 1% agarose gel electrophoresis, and send company's order-checking, Blast identify sequence information on NCBI.
The present invention utilizes labelled by nested-PCR method to amplify the part CO I gene fragment of soil nematodes by two pairs of universal primers of design, utilizes this fragment that soil nematodes is classified.Degenerated primer combination for amplification soil nematodes CO I gene provided by the invention can be used for the amplification of the soil nematodes CO I gene fragment of free living, utilize this section sequence can be effectively to the soil nematodes evaluation of classifying.
The present invention has the following advantages:
(1) simplification: tradition utilizes opticmicroscope to do the typoiogical classification evaluation of soil nematodes, and is very high to identifier's requirement, do not have the people of a large amount of soil nematodes identification of morphology experiences to be difficult to competent.And utilize universal primer of the present invention, only need skilled PCR operation to get final product, even there is not the nematode typoiogical classification to identify that the people of knowledge also can easily finish fully.
(2) high efficiency: traditional identification of morphology need to spend the plenty of time and identify one by one sample, utilizes universal primer of the present invention, and the classification that nest-type PRC just can be finished a large amount of samples is simultaneously identified, has greatly saved qualification time.
(3) practicality: compare with other methods of utilizing 18S rna gene design primer to be nematode barcoding, the present invention has selected CO I gene, resolving power is higher, can more effectively carry out the differentiation of kind to soil nematodes, has filled up simultaneously the blank in soil nematodes classification evaluation field.
Description of drawings
Fig. 1 is the PCR product electrophoresis result of utilizing the CO I gene of described degenerated primer combination and 20 nematodes of labelled by nested-PCR method amplification in the embodiment of the invention 2; Wherein, leftmost side swimming lane is DNA Marker, and 1 ~ 20 swimming lane is 1 ~ No. 20 sample, and 21 swimming lanes are blank.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is all according to the normal experiment condition, such as Sambrook equimolecular cloning experimentation handbook (New York:Gold Spring Harbor Laboratory Press, 1989).The raw materials used commercial goods that is.
Download seven kinds of known nematode (beautiful rhabditis axei Caenorhabditis briggsae from the NCBI website, Caenorhabditis elegans Caenorhabditis elegans, have a liking for bacterium heterorhabditis indica Heterorhabdtis bacteriophora, strongyloides intestinalis Strongyloides stercoralis, nematode Steinernema carpocapsae Steinernema carpocapsae, Strelkovimermis spiculatus and Pristionchus pacificus) CO I gene order, its gene I/D is respectively 5666636,2565700,4421891,37805768,326200256,2846681 and 4078882.Utilize Clustal X2 software that these seven sequences are compared, find out conservative zone, design two pairs of degenerated primers (Seq ID No.1-4):
First pair of primer (amplification length is about 860bp):
7COX1JB-F1:5’-ATACCTWSWWTAATYGGDGGKTTTGG-3’；
7COX1JB-R1:5’-AYACCHGTTAAMCCRCCHAHAGTAAA-3’。
Second pair of primer (amplification length is about 695bp):
7COX1JB-F2:5’-GGDGCHCCTGATATRAGNTTYCCHCG-3’；
7COX1JB-R2:5’-ACYTTHACHCCNGTHGGNACAGCAAT-3’。
Wherein, Y represents base C or T, and W represents base A or T, and K represents bases G or T, and D represents bases G, A or T, and R represents base A or G, and M represents base A or C, and H represents A, T or C, and S represents G or C, and N represents A, T, C or G.Wherein, first pair of primer has 2*2*2*2*2*3*2*2*3*2*2*3*3=41472 kind array configuration, and second pair of primer has 3*3*2*4*2*2*3*3*4*3*4=124416 kind array configuration.When carrying out nest-type PRC, what first round PCR used is the mixture of 41472 kinds of permutation and combination of first pair of primer, and second what take turns that PCR uses is the mixture of 124416 kinds of permutation and combination of second pair of primer.
The classification of embodiment 2 soil nematodess is identified
1. the separation of soil nematodes
Pedotheque derives from Earthquake of Anyang station in Henan.Be that the glass funnel end of 12cm connects one section rubber tubing at diameter, the rubber tubing end clamps with iron clamp, is fixed on the iron stand, adds a certain amount of deionized water in funnel.The 200g pedotheque respectively with one deck paper handkerchief and one deck gauze parcel, is positioned in the funnel, and the deionized water that fills into capacity makes the complete submergence of soil, place under the room temperature condition and separate.After 24 hours, open iron clamp, emit the water about 2mL in the rubber tubing end, dissect Microscopic observation, draw at random 20 soil nematodess with liquid-transfering gun, put into respectively 20 1.5mL centrifuge tubes.
2. the DNA extraction of soil nematodes
Use TIANGEN TIANamp Genomic DNA Kit(blood/cell/tissue genome DNA extracting reagent kit) the part reagent in.
1) contain at each and add 200 μ L damping fluid GA in the 1.5mL centrifuge tube of nematode, add 20 μ L Proteinase K solution, mixing is at 56 ℃ of digestion 3h.
2) add 200 μ L damping fluid GB, fully put upside down mixing, place 10min for 70 ℃.
3) add isopyknic phenol/chloroform/primary isoamyl alcohol (25:24:1), mixing is in the centrifugal 10min of 12,000rpm.
4) the upper strata water is sucked in the new 1.5mL centrifuge tube, add the two volumes dehydrated alcohol ,-20 ℃ of precipitation 30min.
5) 12, the centrifugal 10min of 000rpm abandons supernatant, adds 600 μ L rinsing liquid PW, and mixing is in the centrifugal 10min of 12,000rpm.
6) discard the rinsing liquid on upper strata, and thoroughly dry in room temperature.
7) add 50 μ L deionized water dissolving DNA.
3. nest-type PRC amplification purpose fragment
Use deionized water as blank during the PCR reaction.
First round PCR:
First round PCR reaction system is 25 μ L, comprise: 5 ' end primer 7COX1JB-F1(10 μ M) 2 μ L, 3 ' end primer 7COX1JB-R1(10 μ M) 2 μ L, 10 * LA Taq damping fluid, 2.5 μ L, dNTP(2.5mM) 2.5 μ L, LA Taq enzyme (5U/ μ L) 0.3 μ L, nematode DNA2 μ L, ddH
2O13.7 μ L.
PCR condition: 94 ℃ of denaturation 5min; 94 ℃ of 20s, 60 ℃-50 ℃ (every circulation reduces by 0.5 ℃) 20s, 72 ℃ of 1min, 20 circulations; 94 ℃ of 20s, 50 ℃ of 20s, 72 ℃ of 1min, 40 circulations; 72 ℃ are extended 10min.
Second takes turns PCR:
Second to take turns the PCR reaction system be 50 μ L, comprise: 5 ' end primer 7COX1JB-F2(10 μ M) 4 μ L, 3 ' end primer 7COX1JB-R2(10 μ M) 4 μ L, 10 * LA Taq damping fluid, 5 μ L, dNTP(2.5mM) 5 μ L, LA Taq enzyme (5U/ μ L) 0.5 μ L, first round PCR product 2 μ L, ddH
2O29.5 μ L.
PCR condition: 94 ℃ of denaturation 5min; 94 ℃ of 20s, 56 ℃ of 20s, 72 ℃ of 1min, 40 circulations; 72 ℃ are extended 10min.
Second takes turns the PCR product detects with 1% agarose gel electrophoresis, identifies clip size, the result as shown in Figure 1, all the nematode samples all can amplify band, clip size meets expection.
4.BLAST result for retrieval
Send company's order-checking with the PCR product, obtain to carry out Blast identify sequence information in the NCBI website after the sequence.The result shows, 20 nematodes of from soil, separating at random, belong to 5 kinds of nematodes, comprise Caenorhabditis elegans Caenorhabditis elegans, with nematode Cooperia oncophora similarity be a kind of nematode of 90%, with nematode Stronngyloides mirzai similarity be a kind of nematode of 89%, with nematode Stronngyloides mirzai similarity be 89% another kind of nematode, with nematode Ancylostoma caninum similarity be a kind of nematode of 81%.
Above result shows, according to isolated 20 nematodes at random from soil, utilizes degenerated primer combination of the present invention, adopts the method for nest-type PRC all can amplify band, can be divided into 5 kinds, high specificity through Sequence Identification.Even the DNA sample of wall scroll nematode also can be realized accurate detection, and is highly sensitive.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (9)
1. be used for the degenerated primer combination of amplification soil nematodes CO I gene, it is characterized in that, comprising:
First pair of primer:
7COX1JB-F1:5’-ATACCTWSWWTAATYGGDGGKTTTGG-3’，
7COX1JB-R1:5 '-AYACCHGTTAAMCCRCCHAHAGTAAA-3 '; And
Second pair of primer:
7COX1JB-F2:5’-GGDGCHCCTGATATRAGNTTYCCHCG-3’，
7COX1JB-R2:5’-ACYTTHACHCCNGTHGGNACAGCAAT-3’；
Wherein, Y represents base C or T, and W represents base A or T, and K represents bases G or T, and D represents bases G, A or T, and R represents base A or G, and M represents base A or C, and H represents A, T or C, and S represents G or C, and N represents A, T, C or G.
2. contain the combination of the described degenerated primer of claim 1 for detection of or carry out the test kit that taxonomy is identified soil nematodes.
3. test kit according to claim 2 is characterized in that, described test kit also comprises one or more in dNTPs, LA Taq archaeal dna polymerase, the PCR reaction buffer.
4. according to claim 2 or 3 described test kits, it is characterized in that described test kit also comprises standard positive template.
5. the combination of the described degenerated primer of claim 1 or each described test kit of claim 2-4 are detecting or are carrying out taxonomy and identify application in the soil nematodes.
6. application according to claim 5 is characterized in that, may further comprise the steps:
1) DNA in the extraction sample;
2) DNA that extracts in the step 1) carries out the nest-type PRC amplified reaction as template;
3) analyze the PCR product.
7. according to claim 5 or 6 described application, it is characterized in that, first round PCR reaction system is counted with 25 μ L: 10 μ M5 ' end primer 7COX1JB-F12 μ L, 10 μ M3 ' end primer 7COX1JB-R12 μ L, 10 * LA Taq damping fluid, 2.5 μ L, 2.5mM dNTP2.5 μ L, 5U/ μ L LA Taq enzyme 0.3 μ L, nematode DNA2 μ L, ddH
2O13.7 μ L;
Second takes turns the PCR reaction system counts with 50 μ L: 10 μ M5 ' end primer 7COX1JB-F24 μ L, 10 μ M3 ' end primer 7COX1JB-R24 μ L, 10 * LA Taq damping fluid, 5 μ L, 2.5mM dNTP5 μ L, 5U/ μ L LA Taq enzyme 0.5 μ L, first round PCR product 2 μ L, ddH
2O29.5 μ L.
8. application according to claim 7 is characterized in that, first round PCR reaction conditions is: 94 ℃ of 5min; 94 ℃ of 20s, 60 ℃-50 ℃ (every circulation reduces by 0.5 ℃) 20s, 72 ℃ of 1min, 20 circulations; 94 ℃ of 20s, 50 ℃ of 20s, 72 ℃ of 1min, 40 circulations; 72 ℃ of 10min.
9. application according to claim 7 is characterized in that, second takes turns the PCR reaction conditions is: 94 ℃ of 5min; 94 ℃ of 20s, 56 ℃ of 20s, 72 ℃ of 1min, 40 circulations; 72 ℃ of 10min.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110106172A (en) * | 2010-03-22 | 2011-09-28 | 한국해양연구원 | Identifying method of marine fishes in southern sea of korea, polynucleotide probe, dna chip and kit for identifying the same |
| CN102373292A (en) * | 2011-12-19 | 2012-03-14 | 云南师范大学 | Molecular-biological method for quickly distinguishing noctuidae pests |
-
2012
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110106172A (en) * | 2010-03-22 | 2011-09-28 | 한국해양연구원 | Identifying method of marine fishes in southern sea of korea, polynucleotide probe, dna chip and kit for identifying the same |
| CN102373292A (en) * | 2011-12-19 | 2012-03-14 | 云南师范大学 | Molecular-biological method for quickly distinguishing noctuidae pests |
Non-Patent Citations (1)
| Title |
|---|
| DERYCKE，S.等: "Exploring the Use of Cytochrome Oxidase c Subunit 1 (COI) for DNA Barcoding of Free-Living Marine Nematodes", 《PLOS ONE》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107557478A (en) * | 2017-09-15 | 2018-01-09 | 中国长江三峡集团公司中华鲟研究所 | A kind of method and its application that amplification the Changjiang river fish chondriogen total order is combined based on degenerate primer |
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| CN103045589B (en) | 2014-02-19 |
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