CN110229873A - A kind of method in quick detection field the Yellow River carp spawning ground - Google Patents
A kind of method in quick detection field the Yellow River carp spawning ground Download PDFInfo
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- 241000316729 Tor douronensis Species 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000001514 detection method Methods 0.000 title claims abstract description 19
- 238000005070 sampling Methods 0.000 claims abstract description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 38
- 241000252233 Cyprinus carpio Species 0.000 claims abstract description 20
- 239000012528 membrane Substances 0.000 claims abstract description 14
- 230000014509 gene expression Effects 0.000 claims abstract description 10
- 238000010222 PCR analysis Methods 0.000 claims abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 8
- 238000012360 testing method Methods 0.000 claims abstract description 7
- 238000004458 analytical method Methods 0.000 claims abstract description 4
- 238000000605 extraction Methods 0.000 claims abstract description 4
- 230000013011 mating Effects 0.000 claims abstract description 4
- 230000003321 amplification Effects 0.000 claims description 8
- 238000000137 annealing Methods 0.000 claims description 8
- 230000004087 circulation Effects 0.000 claims description 8
- 238000004925 denaturation Methods 0.000 claims description 8
- 230000036425 denaturation Effects 0.000 claims description 8
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 8
- 238000004321 preservation Methods 0.000 claims description 8
- 238000004153 renaturation Methods 0.000 claims description 8
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 8
- 239000012498 ultrapure water Substances 0.000 claims description 8
- 239000000463 material Substances 0.000 abstract description 4
- 238000009395 breeding Methods 0.000 description 6
- 235000019688 fish Nutrition 0.000 description 6
- 241000251468 Actinopterygii Species 0.000 description 5
- 238000007400 DNA extraction Methods 0.000 description 5
- 230000001488 breeding effect Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000010410 layer Substances 0.000 description 5
- 230000001850 reproductive effect Effects 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 239000002344 surface layer Substances 0.000 description 5
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 235000013527 bean curd Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 210000004681 ovum Anatomy 0.000 description 2
- 241001519451 Abramis brama Species 0.000 description 1
- 241000271510 Agkistrodon contortrix Species 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241001139305 Channa asiatica Species 0.000 description 1
- 241001372210 Gobioides broussonnetii Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001282110 Pagrus major Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000269799 Perca fluviatilis Species 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention discloses a kind of methods in quickly detection field the Yellow River carp spawning ground, specifically includes the following steps: step 1: finding the place in doubtful spawning ground in Yellow River Region as test object, and carry out water sampling to test object site in carp mating period；Step 2: the water sample of acquisition being filtered, DNA is extracted to filtered filter membrane；Step 3: quantitative fluorescent PCR analysis being carried out to the DNA of extraction, based on the analysis results the expression contents of the target gene of more each sampling position, the position in the high as spawning ground of the Yellow River carp of destination gene expression content.The present invention can quick and precisely identify the position in the Yellow River carp spawning ground by the content of the target gene in detection water sample, and the method that the present invention compares traditional searching spawning ground saves a large amount of manpower and material resources and financial resources.
Description
Technical field
The invention belongs to Animal molecular breeding method and technology fields, and in particular to a kind of quickly detection field the Yellow River carp production
The method of ovum field.
Background technique
The Yellow River is the second largest river in China, 5464 kilometers of overall length, 750,000 square kilometres of drainage area, is known as " copperhead, iron
Tail, bean curd waist " title, pass through domestic 692 kilometers in Henan, be predominantly located on bean curd waist.River starts to relax to the east of Mengjin, and two
Bunding is away from general 10 kilometers or so, and for the widest part up to 20 kilometers, riverbed is wide and shallow, 11.3-25.0 centimetres of non-flood period silt content, water
In rich in aquatile growth needed for various nutrient salts.The basin (Henan Section) has a moderate climate, sunshine-duration in year and fish
Growth period is long, and the Yellow River beach, which grows a large amount of ruderal, can be used as fish feed, these are all the superior of development fish production
Condition.And the main reason for domestic section of our province abounds with high-quality the Yellow River carp in history.The Yellow River carp is the same as the Songjiang River perch, Xinkaihu Lake
Fish, Song Hua River salmon are described as four big, China fish altogether.The Yellow River carp just has from ancient times " its ichthyophagy, must river carp ", " Lip river carp she
It is triangular bream, expensive such as cattle and sheep " say, to the top grade for food.The Yellow River carp is also delicious with its fine and tender taste, long excellent of the red tail of golden squama, figure shuttle
U.S. form, has won fame both at home and abroad, and is the valuable stock of fish in our province and China.The legend succeeded in the civil service examination in old times, it is almost widely known.It is white
The easily equal ancient times poet Zeng Weiqi in residence, which write the poem, to be assigned, and " dragonfish " is called.Folklore has " three ruler carp of the Yellow River, this in Meng Jinju,
Point volume not Cheng Long, comes back with all fishes " etc. fine verse.Since the Yellow River is very huge, the spawning ground of annual the Yellow River carp is not solid
It is fixed indefinite, so scientific research personnel need to spend a large amount of man power and material to go to the spawning ground for accurately finding the Yellow River carp.
Summary of the invention
The object of the present invention is to provide a kind of methods in quickly detection field the Yellow River carp spawning ground, specifically include following step
It is rapid:
1. a kind of method in quickly detection field the Yellow River carp spawning ground, which is characterized in that specifically includes the following steps:
Step 1: the place in doubtful spawning ground is found in Yellow River Region as test object, and in carp mating period pair
Test object site carries out water sampling；
Step 2: the water sample of acquisition being filtered, DNA is extracted to filtered filter membrane；
Step 3: quantitative fluorescent PCR analysis being carried out to the DNA of extraction, based on the analysis results the purpose of more each sampling position
The expression contents of gene, the position in the high as spawning ground of the Yellow River carp of destination gene expression content.
The features of the present invention also characterized in that
Quantitative fluorescent PCR reaction primer sequence used in step 3 are as follows:
F:GGTCGTATCGGAATCGTG
R:TCCAAGCGTACCAGAACTT。
In step 3 quantitative fluorescent PCR react response procedures are as follows: 94 DEG C initial denaturation 3 minutes；94 DEG C are denaturalized 30 seconds, annealing
Temperature renaturation 30 seconds, 72 DEG C extended 30 seconds, 40 circulations；72 DEG C extend 5 minutes；4 DEG C of preservations, solubility curve are with 0.2 DEG C
Amplification is incremented to 95 DEG C from 65 DEG C.
In step 3 quantitative fluorescent PCR react reaction system are as follows: each 1ul of DNA 2ul, primer, ultrapure water 6ul,
SYBR Taq is 10ul.
The beneficial effects of the present invention are:
A kind of method in quickly detection field the Yellow River carp spawning ground of the invention, passes through the target gene in detection water sample
Content can quick and precisely identify the position in the Yellow River carp spawning ground.The method that the present invention compares traditional searching spawning ground is saved
A large amount of manpower and material resources and financial resources.
Detailed description of the invention
Fig. 1 is a kind of embodiment of the method fluorescent quantitation expression of results signal in quickly detection field the Yellow River carp spawning ground
Figure；
Specific embodiment
The following describes the present invention in detail with reference to the accompanying drawings and specific embodiments.
A kind of method in quickly detection field the Yellow River carp spawning ground of the invention, specifically includes the following steps:
Step 1: the place in doubtful spawning ground is found in Yellow River Region as test object, and in carp mating period pair
Test object site carries out water sampling；
Step 2: the water sample of acquisition being filtered, DNA is extracted to filtered filter membrane；
Step 3: quantitative fluorescent PCR analysis being carried out to the DNA of extraction, based on the analysis results the purpose of more each sampling position
The expression contents of gene, the position in the high as spawning ground of the Yellow River carp of destination gene expression content.
Quantitative fluorescent PCR reaction primer sequence used in step 3 are as follows:
In step 3 quantitative fluorescent PCR react response procedures are as follows: 94 DEG C initial denaturation 3 minutes；94 DEG C are denaturalized 30 seconds, annealing
Temperature renaturation 30 seconds, 72 DEG C extended 30 seconds, 40 circulations；72 DEG C extend 5 minutes；4 DEG C of preservations, solubility curve are with 0.2 DEG C
Amplification is incremented to 95 DEG C from 65 DEG C.
In step 3 quantitative fluorescent PCR react reaction system are as follows: each 1ul of DNA 2ul, primer, ultrapure water 6ul,
SYBR Taq is 10ul.
Embodiment 1
Step 1: according to the Yellow River carp reproductive habit, in the season of carp in March breeding, screening doubtful carp spawning ground position
It sets, 2 sampled points of every 6 kilometers of settings, 4 sampling locations is set in the Yellow River cross section of each sampled point.In each sample bits
When setting sampling, the Yellow River surface layer, middle layer and lower water are taken respectively three times.It must be sampled simultaneously when sampling.Each sampled point palpus
Continuous 3 samplings.
Step 2: the Yellow River water samples adopted back being filtered and (according to machine, the air pressure in bottle,suction are lower, water sample passes through
Filter in paper water inlet bottle,suction, but the DNA in water sample is stayed on paper), and filtered filter membrane is subjected to DNA extraction.
Step 3: filter membrane DNA being extracted into result and carries out quantitative fluorescent PCR analysis, standard curve is established, according to fluorescent quantitation
The CT value that PCR is obtained, the experimental result of more each sampling position calculate carp DNA concentration highest (the higher the Yellow River carp of concentration
Activity it is more active) sampling position be spawning ground position.
Wherein quantitative fluorescent PCR reaction primer sequence used are as follows:
In step 3 quantitative fluorescent PCR react response procedures are as follows: 94 DEG C initial denaturation 3 minutes；94 DEG C are denaturalized 30 seconds, annealing
Temperature renaturation 30 seconds, 72 DEG C extended 30 seconds, 40 circulations；72 DEG C extend 5 minutes；4 DEG C of preservations, solubility curve are with 0.2 DEG C
Amplification is incremented to 95 DEG C from 65 DEG C.
In step 3 quantitative fluorescent PCR react reaction system are as follows: each 1ul of DNA 2ul, primer, ultrapure water 6ul,
SYBR Taq is 10ul.
Embodiment 2
Step 1: according to the Yellow River carp reproductive habit, in the season of carp in April breeding, screening doubtful carp spawning ground position
It sets, 3 sampled points of every 6 kilometers of settings, 4 sampling locations is set in the Yellow River cross section of each sampled point.In each sample bits
When setting sampling, the Yellow River surface layer, middle layer and lower water are taken respectively three times.It must be sampled simultaneously when sampling.Each sampled point palpus
Continuous 4 samplings.
Step 2: the Yellow River water samples adopted back being filtered and (according to machine, the air pressure in bottle,suction are lower, water sample passes through
Filter in paper water inlet bottle,suction, but the DNA in water sample is stayed on paper), and filtered filter membrane is subjected to DNA extraction.
Step 3: filter membrane DNA being extracted into result and carries out quantitative fluorescent PCR analysis, standard curve is established, according to fluorescent quantitation
The CT value that PCR is obtained, the experimental result of more each sampling position calculate carp DNA concentration highest (the higher the Yellow River carp of concentration
Activity it is more active) sampling position be spawning ground position.
Wherein quantitative fluorescent PCR reaction primer sequence used are as follows:
In step 3 quantitative fluorescent PCR react response procedures are as follows: 94 DEG C initial denaturation 3 minutes；94 DEG C are denaturalized 30 seconds, annealing
Temperature renaturation 30 seconds, 72 DEG C extended 30 seconds, 40 circulations；72 DEG C extend 5 minutes；4 DEG C of preservations, solubility curve are with 0.2 DEG C
Amplification is incremented to 95 DEG C from 65 DEG C.
In step 3 quantitative fluorescent PCR react reaction system are as follows: each 1ul of DNA 2ul, primer, ultrapure water 6ul,
SYBR Taq is 10ul.
Embodiment 3
Step 1: according to the Yellow River carp reproductive habit, in the season of carp in May breeding, screening doubtful carp spawning ground position
It sets, 4 sampled points of every 6 kilometers of settings, 5 sampling locations is set in the Yellow River cross section of each sampled point.In each sample bits
When setting sampling, the Yellow River surface layer, middle layer and lower water are taken respectively three times.It must be sampled simultaneously when sampling.Each sampled point palpus
Continuous 3 samplings.
Step 2: the Yellow River water samples adopted back being filtered and (according to machine, the air pressure in bottle,suction are lower, water sample passes through
Filter in paper water inlet bottle,suction, but the DNA in water sample is stayed on paper), and filtered filter membrane is subjected to DNA extraction.
Step 3: filter membrane DNA being extracted into result and carries out quantitative fluorescent PCR analysis, standard curve is established, according to fluorescent quantitation
The CT value that PCR is obtained, the experimental result of more each sampling position calculate carp DNA concentration highest (the higher the Yellow River carp of concentration
Activity it is more active) sampling position be spawning ground position.
Wherein quantitative fluorescent PCR reaction primer sequence used are as follows:
In step 3 quantitative fluorescent PCR react response procedures are as follows: 94 DEG C initial denaturation 3 minutes；94 DEG C are denaturalized 30 seconds, annealing
Temperature renaturation 30 seconds, 72 DEG C extended 30 seconds, 40 circulations；72 DEG C extend 5 minutes；4 DEG C of preservations, solubility curve are with 0.2 DEG C
Amplification is incremented to 95 DEG C from 65 DEG C.
In step 3 quantitative fluorescent PCR react reaction system are as follows: each 1ul of DNA 2ul, primer, ultrapure water 6ul,
SYBR Taq is 10ul.
Embodiment 4
Step 1: according to the Yellow River carp reproductive habit, in the season of carp in May breeding, screening doubtful carp spawning ground position
It sets, 4 sampled points of every 6 kilometers of settings, 5 sampling locations is set in the Yellow River cross section of each sampled point.In each sample bits
When setting sampling, the Yellow River surface layer, middle layer and lower water are taken respectively three times.It must be sampled simultaneously when sampling.Each sampled point palpus
Continuous 4 samplings.
Step 2: the Yellow River water samples adopted back being filtered and (according to machine, the air pressure in bottle,suction are lower, water sample passes through
Filter in paper water inlet bottle,suction, but the DNA in water sample is stayed on paper), and filtered filter membrane is subjected to DNA extraction.
Step 3: filter membrane DNA being extracted into result and carries out quantitative fluorescent PCR analysis, standard curve is established, according to fluorescent quantitation
The CT value that PCR is obtained, the experimental result of more each sampling position calculate carp DNA concentration highest (the higher the Yellow River carp of concentration
Activity it is more active) sampling position be spawning ground position.
Wherein quantitative fluorescent PCR reaction primer sequence used are as follows:
In step 3 quantitative fluorescent PCR react response procedures are as follows: 94 DEG C initial denaturation 3 minutes；94 DEG C are denaturalized 30 seconds, annealing
Temperature renaturation 30 seconds, 72 DEG C extended 30 seconds, 40 circulations；72 DEG C extend 5 minutes；4 DEG C of preservations, solubility curve are with 0.2 DEG C
Amplification is incremented to 95 DEG C from 65 DEG C.
In step 3 quantitative fluorescent PCR react reaction system are as follows: each 1ul of DNA 2ul, primer, ultrapure water 6ul,
SYBR Taq is 10ul.
Embodiment 5
Step 1: according to the Yellow River carp reproductive habit, in the season of carp in June breeding, screening doubtful carp spawning ground position
It sets, 3 sampled points of every 6 kilometers of settings, 4 sampling locations is set in the Yellow River cross section of each sampled point.In each sample bits
When setting sampling, the Yellow River surface layer, middle layer and lower water are taken respectively three times.It must be sampled simultaneously when sampling.Each sampled point palpus
Continuous 3 samplings.
Step 2: the Yellow River water samples adopted back being filtered and (according to machine, the air pressure in bottle,suction are lower, water sample passes through
Filter in paper water inlet bottle,suction, but the DNA in water sample is stayed on paper), and filtered filter membrane is subjected to DNA extraction.
Step 3: filter membrane DNA being extracted into result and carries out quantitative fluorescent PCR analysis, standard curve is established, according to fluorescent quantitation
The CT value that PCR is obtained, the experimental result of more each sampling position calculate carp DNA concentration highest (the higher the Yellow River carp of concentration
Activity it is more active) sampling position be spawning ground position.
Wherein quantitative fluorescent PCR reaction primer sequence used are as follows:
In step 3 quantitative fluorescent PCR react response procedures are as follows: 94 DEG C initial denaturation 3 minutes；94 DEG C are denaturalized 30 seconds, annealing
Temperature renaturation 30 seconds, 72 DEG C extended 30 seconds, 40 circulations；72 DEG C extend 5 minutes；4 DEG C of preservations, solubility curve are with 0.2 DEG C
Amplification is incremented to 95 DEG C from 65 DEG C.
In step 3 quantitative fluorescent PCR react reaction system are as follows: each 1ul of DNA 2ul, primer, ultrapure water 6ul,
SYBR Taq is 10ul.
Fig. 1 is a kind of embodiment of the method fluorescent quantitation expression of results signal in quickly detection field the Yellow River carp spawning ground
Figure；It can be seen that the water sample that B, D, F, G point are got contains a large amount of target gene, illustrate that the place has a large amount of the Yellow River carp
Aggregation, this four places are spawning ground, and excess-three is not spawning ground.
By the above-mentioned means, a kind of method in quickly detection field the Yellow River carp spawning ground of the present invention, by detecting water sample
In the content of target gene can quick and precisely identify the position in the Yellow River carp spawning ground.The present invention compares traditional searching and produces
The method of ovum field saves a large amount of manpower and material resources and financial resources.
SEQUENCE LISTING
<110>Xi'an University of Technology
<120>a kind of method in quickly detection field the Yellow River carp spawning ground
<130> 2
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>artificial sequence
<400> 1
GGTCGTATCGGAATCGTG 18
<210> 2
<211> 19
<212> DNA
<213>artificial sequence
<400> 2
TCCAAGCGTACCAGAACTT
Claims (4)
1. a kind of method in quickly detection field the Yellow River carp spawning ground, which is characterized in that specifically includes the following steps:
Step 1: the place in doubtful spawning ground is found in Yellow River Region as test object, and in carp mating period to detection
Object site carries out water sampling；
Step 2: the water sample of acquisition being filtered, DNA is extracted to filtered filter membrane；
Step 3: quantitative fluorescent PCR analysis being carried out to the DNA of extraction, based on the analysis results the target gene of more each sampling position
Expression contents, destination gene expression content it is high be the Yellow River carp spawning ground position.
2. a kind of method in quickly detection field the Yellow River carp spawning ground according to claim 1, which is characterized in that described
Quantitative fluorescent PCR reaction primer sequence used in step 3 are as follows:
F:GGTCGTATCGGAATCGTG
R:TCCAAGCGTACCAGAACTT。
3. a kind of method in quickly detection field the Yellow River carp spawning ground according to claim 1, which is characterized in that described
In step 3 quantitative fluorescent PCR react response procedures are as follows: 94 DEG C initial denaturation 3 minutes；94 DEG C are denaturalized 30 seconds, annealing temperature renaturation
30 seconds, 72 DEG C extended 30 seconds, 40 circulations；72 DEG C extend 5 minutes；4 DEG C of preservations, solubility curve are with 0.2 DEG C of amplification from 65
DEG C it is incremented to 95 DEG C.
4. a kind of method in quickly detection field the Yellow River carp spawning ground according to claim 1, which is characterized in that described
The reaction system that quantitative fluorescent PCR reacts in step 3 are as follows: each 1ul of DNA 2ul, primer, ultrapure water 6ul, SYBR Taq are
10ul。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107099595A (en) * | 2017-05-12 | 2017-08-29 | 中国长江三峡集团公司中华鲟研究所 | Fish natural propagation monitoring method based on environment DNA technology |
| CN109486915A (en) * | 2018-12-29 | 2019-03-19 | 中国水产科学研究院长江水产研究所 | One kind detecting Spawning of The Grass Carp, ctenopharyngodon Idellus field method based on environment DNA |
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- 2019-05-31 CN CN201910467948.0A patent/CN110229873A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107099595A (en) * | 2017-05-12 | 2017-08-29 | 中国长江三峡集团公司中华鲟研究所 | Fish natural propagation monitoring method based on environment DNA technology |
| CN109486915A (en) * | 2018-12-29 | 2019-03-19 | 中国水产科学研究院长江水产研究所 | One kind detecting Spawning of The Grass Carp, ctenopharyngodon Idellus field method based on environment DNA |
Non-Patent Citations (2)
| Title |
|---|
| 梁恺龙等: ""青岛文昌鱼NADH脱氢酶亚基I基因片段的克隆"", 《山东大学学报》, vol. 37, no. 1, 31 March 2002 (2002-03-31), pages 89 * |
| 白俊杰等: "《鱼类种质分子鉴定技术》", vol. 1, 31 January 2011, 海洋出版社, pages: 116 - 118 * |
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