CN110172518A - A kind of method in quick detection field Lanzhou Huanghe catfish spawning ground - Google Patents
A kind of method in quick detection field Lanzhou Huanghe catfish spawning ground Download PDFInfo
- Publication number
- CN110172518A CN110172518A CN201910467717.XA CN201910467717A CN110172518A CN 110172518 A CN110172518 A CN 110172518A CN 201910467717 A CN201910467717 A CN 201910467717A CN 110172518 A CN110172518 A CN 110172518A
- Authority
- CN
- China
- Prior art keywords
- catfish
- spawning ground
- dna
- lanzhou
- huanghe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241001233037 catfish Species 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000001514 detection method Methods 0.000 title claims abstract description 19
- 238000005070 sampling Methods 0.000 claims abstract description 39
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 38
- 239000012528 membrane Substances 0.000 claims abstract description 14
- 230000014509 gene expression Effects 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 8
- 238000012360 testing method Methods 0.000 claims abstract description 5
- 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
- 230000004044 response Effects 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
- 238000010222 PCR analysis Methods 0.000 abstract description 6
- 239000000463 material Substances 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract 1
- 238000009395 breeding Methods 0.000 description 10
- 230000001488 breeding effect Effects 0.000 description 9
- 238000004153 renaturation Methods 0.000 description 7
- 230000001850 reproductive effect Effects 0.000 description 7
- 238000007400 DNA extraction Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000010410 layer Substances 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 239000002344 surface layer Substances 0.000 description 5
- 241000251468 Actinopterygii Species 0.000 description 3
- 241000416916 Cephalocassis jatia Species 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 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
- 241000252496 Siluriformes Species 0.000 description 2
- 235000013527 bean curd Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000017448 oviposition Effects 0.000 description 2
- 210000004681 ovum Anatomy 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 241000271510 Agkistrodon contortrix Species 0.000 description 1
- 241000208340 Araliaceae Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 241000347488 Silurus Species 0.000 description 1
- 241000902878 Silurus lanzhouensis Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000003911 water pollution Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of methods in quickly detection field Lanzhou Huanghe catfish 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 catfish 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 Lanzhou Huanghe catfish of destination gene expression content.A kind of method in quickly detection field Lanzhou Huanghe catfish spawning ground of the present invention, the content for the target gene crossed in detection water sample can quick and precisely identify the position in Lanzhou Huanghe catfish spawning ground；The method for comparing 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 Lanzhou Huanghe catfish
The method in spawning ground.
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 (Lanzhou 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 Lanzhou catfish in history.Lanzhou catfish (Silurus
Lanzhouensis) also known as the Yellow River Nian, it is under the jurisdiction of siluriformes (Siluriformes), catfish section (Siluridea), catfish category
(Silurus), nutritive value is high, and custom has the title of " the Yellow River catfish living person ginseng ", has very wide breeding prospect.The Yellow River Ningxia
Section is its exemplary distribution area, but due to the factors such as water pollution, overfishing, hydraulic engineering construction, Lanzhou catfish wild stocks day
Become decline, is classified as endangered species by " Chinese species Red List ".Since the Yellow River is very huge, the production of annual Lanzhou Huanghe catfish
Ovum field is not fixed indefinite, so scientific research personnel need to spend a large amount of man power and material to go the production for accurately finding Lanzhou Huanghe catfish
Ovum field.
Summary of the invention
The object of the present invention is to provide a kind of methods in quickly detection field Lanzhou Huanghe catfish spawning ground, solve existing side
Method detects spawning ground difficulty, the inconvenient problem of Lanzhou Huanghe catfish.
The technical scheme adopted by the invention is that a kind of method in quick detection field Lanzhou Huanghe catfish spawning ground, specifically
The following steps are included:
Step 1: the place in doubtful spawning ground is found in Yellow River Region as test object, and in catfish mating period to inspection
It surveys object site and 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 reaction being carried out to the DNA of extraction, more each sampling site is analyzed according to PCR reaction result
The expression contents of the target gene of point, the position in the high as spawning ground of Lanzhou Huanghe catfish 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:AGGCCCAGACTCACTTCA
R:CCGTAACCGACCTAAACC。
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: DNA is 2 μ l, each 1 μ l of primer, ultrapure water be 6 μ l,
SYBR Taq is 10 μ l.
The beneficial effects of the present invention are:
The object of the present invention is to provide a kind of methods in quickly detection field Lanzhou Huanghe catfish spawning ground, by detecting water sample
In the content of target gene can quick and precisely identify the position in Lanzhou Huanghe catfish spawning ground.Compare traditional searching spawning ground
Method save 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 Lanzhou Huanghe catfish 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 Lanzhou Huanghe catfish 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 catfish mating period to inspection
It surveys object site and 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 reaction being carried out to the DNA of extraction, more each sampling site is analyzed according to PCR reaction result
The expression contents of the target gene of point, the position in the high as spawning ground of Lanzhou Huanghe catfish of destination gene expression content.
Quantitative fluorescent PCR reaction primer sequence used are as follows:
Quantitative fluorescent PCR reaction 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.
The reaction system of quantitative fluorescent PCR reaction are as follows: DNA is 2 μ l, each 1 μ l of primer, ultrapure water are that 6 μ l, SYBR Taq are
10μl。
Embodiment 1
Step 1: according to Lanzhou Huanghe catfish reproductive habit, in the season of catfish in March breeding, screening doubtful catfish 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 catfish DNA concentration highest (the higher Lanzhou Huanghe catfish 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: DNA is 2 μ l, each 1 μ l of primer, ultrapure water be 6 μ l,
SYBR Taq is 10 μ l.
Embodiment 2
Step 1: according to Lanzhou Huanghe catfish reproductive habit, in the season of catfish in April breeding, screening doubtful catfish 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 catfish DNA concentration highest (the higher Lanzhou Huanghe catfish 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: DNA is 2 μ l, each 1 μ l of primer, ultrapure water be 6 μ l,
SYBR Taq is 10 μ l.
Embodiment 3
Step 1: according to Lanzhou Huanghe catfish reproductive habit, in the season of catfish in May breeding, screening doubtful catfish 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 catfish DNA concentration highest (the higher Lanzhou Huanghe catfish 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: DNA is 2 μ l, each 1 μ l of primer, ultrapure water be 6 μ l,
SYBR Taq is 10 μ l.
Embodiment 4
Step 1: according to Lanzhou Huanghe catfish reproductive habit, in the season of catfish in May breeding, screening doubtful catfish 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 catfish DNA concentration highest (the higher Lanzhou Huanghe catfish 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: DNA is 2 μ l, each 1 μ l of primer, ultrapure water be 6 μ l,
SYBR Taq is 10 μ l.
Embodiment 5
Step 1: according to Lanzhou Huanghe catfish reproductive habit, in the season of catfish in June breeding, screening doubtful catfish 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 catfish DNA concentration highest (the higher Lanzhou Huanghe catfish 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: DNA is 2 μ l, each 1 μ l of primer, ultrapure water be 6 μ l,
SYBR Taq is 10 μ l.
Embodiment 6
Doubtful catfish spawning ground position is screened in the season of catfish in May breeding according to Lanzhou Huanghe catfish reproductive habit, remaining
Step is same as Example 1.
Embodiment 7
Doubtful catfish spawning ground position is screened in the season of catfish in June breeding according to Lanzhou Huanghe catfish reproductive habit, remaining
Step is same as Example 2.
Fig. 1 is a kind of embodiment of the method 1-7 fluorescent quantitation expression of results in quickly detection field Lanzhou Huanghe catfish spawning ground
Schematic diagram.Abscissa respectively indicates each sampling position A, B, C, D, E, F, G on figure, and ordinate indicates fluorescence content, it can be seen that
B, the water sample that C, E point are got contains a large amount of target gene, illustrates that the place is assembled with a large amount of the Yellow River catfish, these three places
For spawning ground, remaining four are not spawning grounds.Finally, truly having a large amount of catfish oviposition breeding by site inspection B, C, E point.
By the above-mentioned means, a kind of method in quickly detection field Lanzhou Huanghe catfish spawning ground of the present invention, by detecting water
The content of target gene in sample can quick and precisely identify the position in Lanzhou Huanghe catfish spawning ground, compare traditional searching oviposition
The method of 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 catfish spawning ground
<130> 2
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>artificial sequence
<400> 1
AGGCCCAGACTCACTTCA 18
<210> 2
<211> 18
<212> DNA
<213>artificial sequence
<400> 2
CCGTAACCGACCTAAACC 18
Claims (4)
1. a kind of method in quickly detection field Lanzhou Huanghe catfish 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 catfish mating period to detection pair
As 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 reaction being carried out to the DNA of extraction, more each sampling position is analyzed according to PCR reaction result
The expression contents of target gene, the position in the high as spawning ground of Lanzhou Huanghe catfish of destination gene expression content.
2. a kind of method in quickly detection field Lanzhou Huanghe catfish spawning ground according to claim 1, which is characterized in that institute
State the primer sequence that quantitative fluorescent PCR reaction is used in step 3 are as follows:
F:AGGCCCAGACTCACTTCA
R:CCGTAACCGACCTAAACC。
3. a kind of method in quickly detection field Lanzhou Huanghe catfish spawning ground according to claim 1, which is characterized in that institute
State the response procedures that quantitative fluorescent PCR in step 3 reacts are as follows: 94 DEG C initial denaturation 3 minutes；94 DEG C are denaturalized 30 seconds, and annealing temperature is multiple
Property 30 seconds, 72 DEG C extend 30 seconds, 40 circulation；72 DEG C extend 5 minutes；4 DEG C preservation, solubility curve be with 0.2 DEG C of amplification from
65 DEG C are incremented to 95 DEG C.
4. a kind of method in quickly detection field Lanzhou Huanghe catfish spawning ground according to claim 1, which is characterized in that institute
State the reaction system that quantitative fluorescent PCR reacts in step 3 are as follows: DNA is 2 μ l, each 1 μ l of primer, ultrapure water are 6 μ l, SYBR Taq
For 10 μ l.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910467717.XA CN110172518A (en) | 2019-05-31 | 2019-05-31 | A kind of method in quick detection field Lanzhou Huanghe catfish spawning ground |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910467717.XA CN110172518A (en) | 2019-05-31 | 2019-05-31 | A kind of method in quick detection field Lanzhou Huanghe catfish spawning ground |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110172518A true CN110172518A (en) | 2019-08-27 |
Family
ID=67696107
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910467717.XA Pending CN110172518A (en) | 2019-05-31 | 2019-05-31 | A kind of method in quick detection field Lanzhou Huanghe catfish spawning ground |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110172518A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2019
- 2019-05-31 CN CN201910467717.XA patent/CN110172518A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 2 * |
| 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 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110951838B (en) | Primer, probe and kit for detecting meloidogyne incognita based on RPA-LFD technology and application | |
| CN106318939B (en) | Produce aflatoxin fungi multiplex PCR detection primer sets, kit and detection method | |
| CN101603080A (en) | The quick identification reagent box of 5 kinds of rare fish of Cypriniformes | |
| CN106318938B (en) | Produce substance PCR detection primer pair combination, detection method and the purposes of aflatoxin fungi | |
| CN101619360B (en) | Molecular mark detection method of downy mildew resistance of celery cabbage and primer used by same | |
| CN103525814B (en) | A kind of special SCAR mark of Cynoglossus semilaevis sex and application method | |
| CN112176074A (en) | Real-time fluorescent PCR primer probe and method for detecting patinopecten yessoensis | |
| CN105176989A (en) | Primers and method for identifying takifugu obscurus fry and takifugu xanthopterus fry | |
| CN109628608A (en) | A kind of investigation method releasing Penaeus Chinensis Resources | |
| CN105087567A (en) | Duplex PCR (polymerase chain reaction) primer and method for identifying tomato chlorosis viruses and tomato yellow leaf curl viruses | |
| CN110172518A (en) | A kind of method in quick detection field Lanzhou Huanghe catfish spawning ground | |
| CN103911444B (en) | Primer for discriminating anguilla marmorata and anguilla bicolor pacifica fry and method thereof | |
| CN104962660B (en) | A kind of mottle clam species real-time fluorescence PCR specific detection system and application | |
| CN106868117A (en) | Rare aquatile water sample environment DNA Optimal culture water sample pre-detection method | |
| CN103131759A (en) | Molecular biological method of identifying sparus latus | |
| CN106119384A (en) | A kind of Aeromonas hydrophila method for nucleic acid analysis and the application in legal medical expert detects | |
| CN110229873A (en) | A kind of method in quick detection field the Yellow River carp spawning ground | |
| CN105624295A (en) | Minutocellus polymorphus fluorescent quantitative PCR detection method | |
| CN113337621A (en) | Molecular monitoring method for starfish larvae in coral reef area | |
| CN106868198A (en) | It is a kind of at the same detect SILURIFORMES four kinds of pathogenic bacteria of fish multiple PCR primer group and detection method | |
| CN103131769A (en) | Molecular biological method of identifying trachinotus ovatus | |
| CN103131762A (en) | Molecular biological method of identifying rhabdosargus sarba | |
| CN112695105A (en) | Real-time fluorescence PCR identification method of chlamys farreri | |
| CN106011270A (en) | Detecting method of vibrio parahemolyticus in coastal seawater | |
| CN111593129A (en) | Primer for identifying zander family and method for identifying zander family |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190827 |
|
| RJ01 | Rejection of invention patent application after publication |