CN110106283A - A kind of high-throughput quantification detection kit of aquiculture animal virus - Google Patents
A kind of high-throughput quantification detection kit of aquiculture animal virus Download PDFInfo
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Abstract
The invention discloses a kind of high-throughput quantification detection kits of aquiculture animal virus, feature is the LAMP detection primer group for including 14 kinds of aquiculture animal virus, feature is gene order as shown in SEQ ID NO.1~NO.56, further includes sample template, 10 × reaction buffer, MgSO4、dNTP、Bst Archaeal dna polymerase and distilled water, advantage are that high specificity, high sensitivity, flux are high, highly reliable, at low cost and without false negative result.
Description
Technical field
The present invention relates to a kind of quick detection kits for causing aquiculture animal virus, support more particularly, to a kind of aquatic products
Grow the high-throughput quantification detection kit of animal virus.
Background technique
The pathogenic conditions for endangering aquiculture animal mainly have viral disease, bacteriosis and parasitic diseases
Deng.Wherein virosis due to its pathogen it is small, replicated in host cell, so be difficult to be controlled using drug, once
Wild virus disease will suffer heavy losses.The virosis morbidity that is mainly characterized by of aquiculture animal virosis has with kind and water temperature
It closes, virosis is difficult to control viral disease with drug different from bacteriosis, since virus is unable to own amplification, it is necessary to according to
Energy and substance proliferation in host cell are obligate cytozoicus biologies.Therefore it can treat bacteriosis
Antibiotic etc does not act on viral disease substantially.Mostly without effective drug therapy measure, mainly with prevention
Based on.Isolation and control difficulty are big, these animals live in water, and virus is propagated by medium of water body, once there is individual morbidity
It can all infect quickly, it is also difficult to control.Clinical symptoms are complicated and changeable, infectiousness is strong, spread speed is fast, the death rate is high, serious to endanger
Evil culture fishery, can result in heavy economic losses.Currently, aquiculture animal virus includes pancreas necrosis virus
(Infectious pancreatic necrosis virus, IPNV), Taura syndrome (Taura syndrome virus,
TSV), Nodavirus (Macrobrachium rosenbergii nodavirus, MrNV), giant salamander irido virus (Chinese
Giant salamander iridovirus, CGSIV), lymphocystis disease virus (Lymphosystis Disease Virus
China, LCDVcn), bright and beautiful lithium herpesviral (Koi herpesvirus, KHV), infectious spleen and kidney necrosis virus (Infectious
Spleen and kidney necrosis virus, ISKNV), spring viremia of carp virus disease (Spring viraemia of carp,
SVC), penaeus monodon baculovirus (Penaeus monodon-type baeulovirus, MBV), hepatopancreatic parvovirus shape
Virus (Hepatopancreatic parvovirus, HPV), yellow head virus (Yellow head virus, YHV), infectiousness
Subcutaneous and haematopoietic necrosis virus (Infctious hypodernmm and haematopoietic necrosis
Virus, IHHNV), steal dead nodavirus (Covert mortality nodavirus, CMNV), white spot virus (White
Spot syndrome virus, WSSV), minimum virus (Extra Small Virus, XSV), viral nervous necrosis
(Viral nervous necrosis, VNN), infectivity muscle necrosis virus (Infectious myonecrosis virus,
It) etc. IMNV is to cause the dead very important pathogenic microorganisms of aquiculture animal.Spread speed is fast, and the death rate is high, so, it is early
Phase is quickly detected extremely important.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of high specificity, high sensitivity, flux it is high, it is highly reliable,
The high-throughput quantification detection kit of aquiculture animal virus at low cost, without false negative result.
The technical scheme of the invention to solve the technical problem is: a kind of high throughput of aquiculture animal virus
Immue quantitative detection reagent box, the LAMP detection primer group including 14 kinds of aquiculture animal virus, gene order such as the following table 1 institute
Show:
Table 1
。
The LAMP reaction system of the detection kit and the final concentration of each ingredient are as follows: 1 μ L of sample template DNA,
2.5 μ 10 × reaction buffers of L, 1.5 μ L concentration are the MgSO of 100mM4, 3.5 μ L concentration are the dNTP of 10mM, 1 μ L concentration
For 8 U/ μ l Bst DNA polymerase, 1 μ L FIP/BIP primer sets solution, 1 μ L F3/B3 primer sets solution and 4 μ L glycine betaines,
Then 25 μ L are supplied with distilled water；Wherein the FIP/BIP primer sets solution be respectively pancreas necrosis virus described in table 1,
Giant salamander irido virus, lymphocystis disease virus, bright and beautiful lithium herpesviral, infectious spleen and kidney necrosis virus, spring viremia of carp virus disease, Penaeus monodon
Baculoviral, hepatopancreatic parvovirus shape virus, yellow head virus, infectious subcutaneous and haematopoietic necrosis virus steal dead wild field
Village's virus, Leucodermia virus, minimum viral and viral nervous necrosis FIP/BIP primer sets solution, each primer final concentration
It is 40 μM, the F3/B3 primer sets solution is respectively pancreas necrosis virus described in table 1, giant salamander irido virus, lymph
Tumour virus, bright and beautiful lithium herpesviral, infectious spleen and kidney necrosis virus, spring viremia of carp virus disease, penaeus monodon baculovirus, hepatopancrease are thin
Small virus shape virus, yellow head virus, infectious subcutaneous and haematopoietic necrosis virus, steal dead nodavirus, Leucodermia virus,
The F3/B3 primer sets solution of minimum virus and viral nervous necrosis, each primer final concentration is 5 μM.
The detection kit further includes color developing agent, and the color developing agent is calcein/manganese chloride solution or hydroxyl
Base naphthol blue solution or SYBR-Green I, wherein calcein concentration is 75 μM in calcein/manganese chloride solution, chlorination
Manganese concentration is 500 μM；The hydroxynaphthol blue solution concentration be 150 μM, the calcein/manganese chloride solution or
The additive amount of the hydroxynaphthol blue solution is 1 hole μ L/, and the additive amount of the SYBR-Green I color developing agent is 5 μ L/
Hole.
Compared with the prior art, the advantages of the present invention are as follows: a kind of high throughput of aquiculture animal virus of the present invention is fixed
The primer specific for measuring detection kit design is strong, can carry out rapid amplifying, by fluorescent dye according to double-stranded DNA number come it is true
Determine the amount of cause of disease template, to reach quantitative detection, the time just completes entire detection in 2-3h, has high specificity, sensitive
The advantages of degree is high, flux is high, highly reliable, at low cost, without false negative result.
Detailed description of the invention
Fig. 1 is 14 kinds of aquiculture animal virus LAMP specific detection results；Wherein note: A: pancreas necrosis virus inspection
Survey result；B: giant salamander irido virus testing result；C: lymphocystis disease virus testing result；D: bright and beautiful lithium herpesviral testing result；
E: infectious spleen and kidney necrosis virus testing result；F: spring viremia of carp virus disease testing result；G: penaeus monodon baculovirus testing result；
H: hepatopancreatic parvovirus shape viral diagnosis result；I: yellow head virus testing result；J: infectious subcutaneous and hematopoietic tissue necrosis
Viral diagnosis result；K: dead nodavirus testing result is stolen；L: Leucodermia virus testing result；M: minimum viral diagnosis knot
Fruit；N: viral nervous necrosis testing result；0:DNA Marker；1: pancreas necrosis virus；2: giant salamander irido virus；3: leaching
Bar tumour virus；4: bright and beautiful lithium herpesviral；5: infectious spleen and kidney necrosis virus；6: spring viremia of carp virus disease；7: the rod-shaped disease of Penaeus monodon
Poison；8: hepatopancreatic parvovirus shape virus；9: yellow head virus；10: infectious subcutaneous and haematopoietic necrosis virus；11: stealing dead
Nodavirus；12: Leucodermia virus；13: minimum virus；14: viral nervous necrosis；15: negative control；
Fig. 2 is 14 kinds of aquiculture animal virus LAMP sensitivity technique results；Wherein note: A: pancreas necrosis virus detection knot
Fruit；B: giant salamander irido virus testing result；C: lymphocystis disease virus testing result；D: bright and beautiful lithium herpesviral testing result；E: it passes
Metachromia spleen and kidney necrosis virus testing result；F: spring viremia of carp virus disease testing result；G: penaeus monodon baculovirus testing result；H: liver
Pancreas parvovirus shape viral diagnosis result；I: yellow head virus testing result；J: infectious subcutaneous and haematopoietic necrosis virus
Testing result；K: dead nodavirus testing result is stolen；L: Leucodermia virus testing result；M: minimum viral diagnosis result；N:
Viral nervous necrosis testing result；0:DNA Marker；1: negative control；8-2: being respectively that 10 times of concentration gradients are diluted
Plasmid template；
Fig. 3 is sample detection instance graph, wherein note: A is hydroxynaphthol blue as the microflow controlled biochip of indicator and detects knot
Fruit, B are microflow controlled biochip testing result of the hydroxynaphthol blue as indicator；1: pancreas necrosis virus；2: giant salamander iris
Virus；3: lymphocystis disease virus；4: bright and beautiful lithium herpesviral；5: infectious spleen and kidney necrosis virus；6: spring viremia of carp virus disease；7: spot section pair
Shrimp baculoviral；8: hepatopancreatic parvovirus shape virus；9: yellow head virus；10: infectious subcutaneous and haematopoietic necrosis virus；
11: stealing dead nodavirus；12: Leucodermia virus；13: minimum virus；14: viral nervous necrosis；15: negative control.
Specific embodiment
The present invention will be described in further detail below with reference to the embodiments of the drawings.
Specific embodiment one
A kind of high-throughput quantification detection kit of aquiculture animal virus, the LAMP including 14 kinds of aquiculture animal virus
Detection primer group, gene order are as shown in table 1 below:
Table 1
Said gene target position point design: gene design primer special between seed selection, conservative in kind is because of external primers F3
Equally have an opportunity in conjunction with the F3c in template and extend, displaces the complementary single strand of complete FIP connection.At this point, on FIP
F1c can be achieved with it is complementary with this single-stranded upper Fl, pass through self base pairing formed cyclic structure.Likewise, downstream primer BIP
There is the synthesis similar to primers F IP and F3 with B3, this just provides possibility to form the single-stranded structure of dumbbell shaped.At this point, 3 '
The Fl section at end will carry out DNA synthesis using itself as template, be formed under the archaeal dna polymerase effect with strand displacement capability
Stem loop structure.
It is target according to the above-mentioned specific gene segment for selecting 14 kinds of aquiculture animal viruses, application
The synthesis of PRIMEREXPLORER V5 software Design primers, primer is completed by Shanghai Sheng Gong bioengineering Co., Ltd.By primer
It is compared with target, carries out a large amount of screening tests and analysis, obtain special, sensitive, stable above-mentioned F3, B3, FIP, BIP
LAMP detection primer group；Will design synthesis 4 primers carry out the reaction time and temperature optimization experiment after, determine amplification efficiency and
The best reaction condition of specificity.
Specific embodiment two
The LAMP reaction system of the high-throughput quantification detection kit of aquiculture animal virus and the final concentration of each ingredient are as follows:
1 μ L of sample template DNA, 2.5 μ 10 × reaction buffers of L, 1.5 μ L concentration are the MgSO of 100mM4, 3.5 μ L concentration are
The dNTP of 10mM, 1 μ L concentration are 8 U/ μ l Bst DNA polymerase, 1 μ L FIP/BIP primer sets solution, 1 μ L F3/B3 draw
Object group solution and 4 μ L glycine betaines, then supply 25 μ L with distilled water；Wherein FIP/BIP primer sets solution is respectively embodiment 1
Pancreas necrosis virus, giant salamander irido virus, lymphocystis disease virus in table, bright and beautiful lithium herpesviral, infectious spleen and kidney necrosis virus,
Spring viremia of carp virus disease, penaeus monodon baculovirus, hepatopancreatic parvovirus shape virus, yellow head virus, infectious subcutaneous and hematopoiesis group
It knits necrosis virus, steal dead nodavirus, Leucodermia virus, minimum viral and viral nervous necrosis FIP/BIP primer
Group solution, each primer final concentration is 40 μM, F3/B3 primer sets solution be respectively pancreas necrosis virus in 1 table of embodiment,
Giant salamander irido virus, lymphocystis disease virus, bright and beautiful lithium herpesviral, infectious spleen and kidney necrosis virus, spring viremia of carp virus disease, Penaeus monodon
Baculoviral, hepatopancreatic parvovirus shape virus, yellow head virus, infectious subcutaneous and haematopoietic necrosis virus steal dead wild field
Village's virus, Leucodermia virus, minimum viral and viral nervous necrosis F3/B3 primer sets solution, each primer final concentration are equal
It is 5 μM.
Above-mentioned detection kit further includes color developing agent, and color developing agent is calcein/manganese chloride solution or hydroxynaphthol blue
Solution or SYBR-Green I, wherein calcein concentration is 75 μM in calcein/manganese chloride solution, and manganese chloride concentration is
500μM；The hydroxynaphthol blue solution concentration is 150 μM, calcein/manganese chloride solution or hydroxynaphthol blue solution
Additive amount is 1 hole μ L/, and the additive amount of SYBR-Green I color developing agent is 5 holes μ L/.Being additionally provided with negative control is water, positive right
According to the DNA for double-strand.
Specific embodiment three
It is a kind of using above-mentioned two detection kit of specific embodiment carry out aquiculture animal virus high-throughput quantification detect
Method, comprising the following steps:
Measuring samples are taken, sample template DNA is extracted according to commercially available DNA of bacteria extracts kit, takes 1 μ L of sample template DNA, press
It is formed according to LAMP reaction system and 2.5 μ 10 × reaction buffers of L, 1.5 μ L MgSO4,3.5 μ L dNTP, 1 μ L Bst DNA is added
Polymerase, 1 μ L FIP/BIP primer sets solution, 1 μ L F3/B3 primer sets solution and 4 μ L glycine betaines, are then supplied with distilled water
It is mixed after 25 μ L, taking 20 μ L of mixed liquor to add to each well of sample detection chip, (wherein primer sets solution is respectively pancreas
Necrosis virus, giant salamander irido virus, lymphocystis disease virus, bright and beautiful lithium herpesviral, infectious spleen and kidney necrosis virus, spring viremia of carp virus
Disease, penaeus monodon baculovirus, hepatopancreatic parvovirus shape virus, yellow head virus, infectious subcutaneous and hematopoietic tissue necrosis disease
It is malicious, to steal dead nodavirus, Leucodermia virus, minimum virus and the malicious 14 viral primer sets of viral nervous necrosis molten
The primer sets solution of liquid, each virus is accordingly added in a well, in addition adding the well of negative control, participates in reaction
Well to have altogether be 15 reaction chamber caps, chip is put into instrument reaction chamber, covers reaction chamber cap, clicks " beginning " on screen
Button, instrument carry out automatically, and reaction is waited to terminate, and detection menu interface are entered after instrument prompt, selection detection, instrument can be automatic
(positive is blue to the color data of reading chip reaction chamber when using hydroxynaphthol blue as color developing agent, and feminine gender is pansy；
Using calcein be color developing agent when, green for the positive, yellow be feminine gender) and according to rgb value carry out concentration conversion, finally provide
The copy number of contained bacterial nucleic acid in each hole sample.
If when using SYBR-Green I developing solution, 20 μ L of mixed liquor is taken to add to each sample-adding of sample detection chip
Hole.Power switch device is opened, is completed to screen self-test, reaction chamber cap is opened, chip is put into instrument reaction chamber, is covered anti-
Chamber cap is answered, START button is clicked on screen, instrument carries out automatically, and reaction is waited to terminate, and takes out chip, and each well adds 5
The working solution of the SYBR-Green I of μ L.Menu interface, selection detection are detected into instrument, instrument can read chip reaction automatically
The color data (green is the positive, orange for feminine gender) of room simultaneously carries out concentration conversion according to rgb value, finally provides in each sample
The copy number of contained bacterial nucleic acid.
Fig. 1 is used for the electrophorogram after the LAMP reaction of above-mentioned 14 kinds of aquiculture animals virus and using the above method
The LAMP reaction result of 14 kinds of aquiculture animal virus after SYBR-Green I dyestuff, it is green for the positive, it is orange to be
It is negative.As shown in Figure 1, there was only corresponding aquiculture animal virus LAMP detection primer in sample detection chip is the positive, empty
White control and remaining non-purpose bacterium testing result are feminine gender.
Specific embodiment four
Sensitivity test: 10 times of gradient dilutions of plasmid respectively containing 14 kinds of aquiculture animal virus specific fragments take each
Dilution carries out LAMP amplified reaction, and the LAMP method detection of 14 kinds of different virus is limited to 10-80 copies/μ L.Such as Fig. 2
Shown, 8-2 is respectively 10 times of diluted templates, and 8 be original concentration, after carrying out LAMP reaction, uses electrophoresis respectively and uses SYBR-
Green I dyeing observes result.Initial concentration A: 1.13 × 107Copies/μ L, B:2.56 × 107 copies /μ
L, C:4.09 × 107Copies/μ L, D:3.54 × 107Copies/μ L, E:2.43 × 107Copies/μ L, F:5.91
×107Copies/μ L, G:3.98 × 107Copies/μ L, H:6.35 × 107Copies/μ L, I:6.44 × 107
Copies/μ L, J:5.15 × 107Copies/μ L, K:3.68 × 107Copies/μ L, L:4.77 × 107 copies /μ
L, M:5.22 × 107Copies/μ L, N:7.13 × 107 copies /μL。
Specific embodiment five
Sample pre-treatments are carried out according to related national standard method by doubtful foodstuff samples 2 to be measured, prepare DNA profiling, sample is through upper
State three method LAMP of specific embodiment reaction (centrifugal type microfludic biochip) detection after, A sample using hydroxynaphthol blue into
Row display, B pass through SYBR-Green I dyeing.As a result as shown in figure 3, it is bad containing pancreas in Sample A Sample as the result is shown
In B sample, it is sick to contain infectious spleen renal necrosis for dead virus, spring viremia of carp virus disease, yellow head virus, microsporidians
Poison, penaeus monodon baculovirus.It is verified through physiological and biochemical test, the virus containing detection in two above sample.
Above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art it is common
Within the essential scope of the present invention, the variations, modifications, additions or substitutions made also should belong to protection of the invention to technical staff
Range.
Sequence table
<110>University Of Ningbo
<120>a kind of high-throughput quantification detection kit of aquiculture animal virus
<130>
<160> 56
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>pancreas necrosis virus VP3-F3
<400> 1
ACAGTGTGTACGGCCTCC 18
<210> 2
<211> 18
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<213>artificial sequence
<220>
<223>pancreas necrosis virus VP3-B3
<400> 2
GTTCTCCGTGGTGCATCG 18
<210> 3
<211> 41
<212> DNA
<213>artificial sequence
<220>
<223>pancreas necrosis virus VP3-FIP
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CTGCCTCCGTTCTGTGCGAATAGAACCAGCACCAGAGGAGT 41
<210> 4
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<212> DNA
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AGGTCCCGACCAGGACCAAAGGGTCGTCGTTTCATCTGTC 40
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<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>giant salamander irido virus ICP46-F3
<400> 5
TGATGGAGATGGGTCCAGAG 20
<210> 6
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>giant salamander irido virus ICP46-B3
<400> 6
CTCGGTTCCGGGTATGCA 18
<210> 7
<211> 40
<212> DNA
<213>artificial sequence
<220>
<223>giant salamander irido virus ICP46-FIP
<400> 7
ATCCTCTCCCTGACGTCCCACCCCACATCGAAGTCCTGTG 40
<210> 8
<211> 42
<212> DNA
<213>artificial sequence
<220>
<223>giant salamander irido virus ICP46-BIP
<400> 8
TGCTACCTCGATCTGACCGAGCCATCAGCCTCCTTGTCATCC 42
<210> 9
<211> 21
<212> DNA
<213>artificial sequence
<220>
<223>lymphocystis disease virus orf46-F3
<400> 9
GACCCAATACAGAAAGTATGG 21
<210> 10
<211> 24
<212> DNA
<213>artificial sequence
<220>
<223>lymphocystis disease virus orf46-B3
<400> 10
GTTTATGGATCTTCTCAATGATTG 24
<210> 11
<211> 45
<212> DNA
<213>artificial sequence
<220>
<223>lymphocystis disease virus orf46-FIP
<400> 11
AATGACGGATTTTCCAGAACCAGACGGATATTGGTGGTATGAAAC 45
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<211> 48
<212> DNA
<213>artificial sequence
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<223>lymphocystis disease virus orf46-BIP
<400> 12
AATACCTGCGGCAATTGTAATTTCGATAAAACAGGAAGGAAATATGGT 48
<210> 13
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>bright and beautiful lithium herpesviral ribonucleotide reductase small subunit-F3
<400> 13
TTCTGGGTCATTTGCGGC 18
<210> 14
<211> 20
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<213>artificial sequence
<220>
<223>bright and beautiful lithium herpesviral ribonucleotide reductase small subunit-B3
<400> 14
GGGGTGTCTTTAGTGCCTCT 20
<210> 15
<211> 41
<212> DNA
<213>artificial sequence
<220>
<223>bright and beautiful lithium herpesviral ribonucleotide reductase small subunit-FIP
<400> 15
AGTGTAGACCTTCGTCACGGCTTGGCTGAAGAAACGTTCGC 41
<210> 16
<211> 42
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AGGCAAATGGCAAACAAACCGTTCAATGTCAACGGCATCAGA 42
<210> 17
<211> 19
<212> DNA
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<400> 17
CACGGACAAGGATGCCATT 19
<210> 18
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>infectious spleen and kidney necrosis virus ORF102R-B3
<400> 18
TAGCTCCCGCAGAATTGC 18
<210> 19
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<223>infectious spleen and kidney necrosis virus ORF102R-FIP
<400> 19
TCGGCATGTCTTGTGCGTAGCTGACTGAGATGTGCCGCTA 40
<210> 20
<211> 41
<212> DNA
<213>artificial sequence
<220>
<223>infectious spleen and kidney necrosis virus ORF102R-BIP
<400> 20
ATTGCCTGCCTGTCAATGACGTCATTGGGCCTTCTCGTAGG 41
<210> 21
<211> 25
<212> DNA
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<400> 21
ATACTAATTATAGAGTAGTACCCCA 25
<210> 22
<211> 21
<212> DNA
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<223>spring viremia of carp virus disease G-protein-B3
<400> 22
TGATTCCTTCAACTTCCTCTA 21
<210> 23
<211> 42
<212> DNA
<213>artificial sequence
<220>
<223>spring viremia of carp virus disease G-protein-FIP
<400> 23
TCGCCCCCATTAAATTCGTGTTCTGTTCATTTAGAGCCATAC 42
<210> 24
<211> 43
<212> DNA
<213>artificial sequence
<220>
<223>spring viremia of carp virus disease G-protein-BIP
<400> 24
ATGCAGAGAAAAAGTGTGCGAAATGATGTTTTGCACACTCGTG 43
<210> 25
<211> 25
<212> DNA
<213>artificial sequence
<220>
<223>penaeus monodon baculovirus ring superfamily proteins-F3
<400> 25
AGATATGAAAGCTAGAATTCAGAAG 25
<210> 26
<211> 23
<212> DNA
<213>artificial sequence
<220>
<223>penaeus monodon baculovirus ring superfamily proteins-B3
<400> 26
TTTTTGCACTAGAATATCCTTCT 23
<210> 27
<211> 48
<212> DNA
<213>artificial sequence
<220>
<223>penaeus monodon baculovirus ring superfamily proteins-FIP
<400> 27
TCTCCTTAACTTCCTGCCAATGTAATATCCAAATCGATAAAGGAGGTT 48
<210> 28
<211> 49
<212> DNA
<213>artificial sequence
<220>
<223>penaeus monodon baculovirus ring superfamily proteins-BIP
<400> 28
TAGAGAGGCGATCTATAATGTCCACGCCAATAGTTTTCTTAATTTCAAG 49
<210> 29
<211> 24
<212> DNA
<213>artificial sequence
<220>
<223>hepatopancreatic parvovirus shape virus nonstructural protein 1-F3
<400> 29
TTTATGATCATAGAGAACATGAGG 24
<210> 30
<211> 21
<212> DNA
<213>artificial sequence
<220>
<223>hepatopancreatic parvovirus shape virus nonstructural protein 1-B3
<400> 30
ATTTGTCATTACAGCTGTGTT 21
<210> 31
<211> 45
<212> DNA
<213>artificial sequence
<220>
<223>hepatopancreatic parvovirus shape virus nonstructural protein 1-FIP
<400> 31
CCATGCATGATATCCATAAAGTCCTTAGAGTCAAGACCAAAGACG 45
<210> 32
<211> 44
<212> DNA
<213>artificial sequence
<220>
<223>hepatopancreatic parvovirus shape virus nonstructural protein 1-BIP
<400> 32
TGCCCAAGATTAACTGTATGATGTCTAGACCAGTTAAAGCCTCA 44
<210> 33
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>yellow head virus helicase-F3
<400> 33
CGCAGATGCAAGAGACTT 18
<210> 34
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>yellow head virus helicase-B3
<400> 34
GGATTGAGATGACAGATGGC 20
<210> 35
<211> 42
<212> DNA
<213>artificial sequence
<220>
<223>yellow head virus helicase-FIP
<400> 35
TAGCGAAGATGATGCCACCGCCTCAAGAATCAGAAGACAAAG 42
<210> 36
<211> 45
<212> DNA
<213>artificial sequence
<220>
<223>yellow head virus helicase-BIP
<400> 36
TCAACCCGTGGTATTTTATGTCCGAGTAGATTTGAATGTCAGTGG 45
<210> 37
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>37 kDa coat protein-F3 of infectious subcutaneous and haematopoietic necrosis virus
<400> 37
TCTATGGTCTAAAGAGCAGC 20
<210> 38
<211> 19
<212> DNA
<213>artificial sequence
<220>
<223>37 kDa coat protein-B3 of infectious subcutaneous and haematopoietic necrosis virus
<400> 38
GGGGTGTCTGTAAATGTGA 19
<210> 39
<211> 45
<212> DNA
<213>artificial sequence
<220>
<223>37 kDa coat protein-FIP of infectious subcutaneous and haematopoietic necrosis virus
<400> 39
CATCCGTAGGTCTTCATCATTGATTGACAGTTCAGCAACAGAAAC 45
<210> 40
<211> 40
<212> DNA
<213>artificial sequence
<220>
<223>37 kDa coat protein-BIP of infectious subcutaneous and haematopoietic necrosis virus
<400> 40
TAAAGCAGGCGTAGTGATGCAGAGTCTCAAATGATGTGCC 40
<210> 41
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>dead nodavirus RdRp-F3 is stolen
<400> 41
TGCGATCGAGTTGAAGGC 18
<210> 42
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>dead nodavirus RdRp-B3 is stolen
<400> 42
CTTTATCGGCGGCATTTTGG 20
<210> 43
<211> 42
<212> DNA
<213>artificial sequence
<220>
<223>dead nodavirus RdRp-FIP is stolen
<400> 43
CGCAGCTCCACCATACAATCGAATCTCGTGACAGATGCCCTT 42
<210> 44
<211> 42
<212> DNA
<213>artificial sequence
<220>
<223>dead nodavirus RdRp-BIP is stolen
<400> 44
AAGACTGAAGCGCAAAACCAGCCCAGGAACCATCATTCGTCA 42
<210> 45
<211> 19
<212> DNA
<213>artificial sequence
<220>
<223>Leucodermia virus non-structural protein 1-F3
<400> 45
GGCATTACCTTCACGGATA 19
<210> 46
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>Leucodermia virus non-structural protein 1-B3
<400> 46
TCACCCAAAGAGTCCTCC 18
<210> 47
<211> 49
<212> DNA
<213>artificial sequence
<220>
<223>Leucodermia virus non-structural protein 1-FIP
<400> 47
GGCTGAGAGAACTGGAAACTATTTTCTGTAGATGATTGTGTTATACCTG 49
<210> 48
<211> 41
<212> DNA
<213>artificial sequence
<220>
<223>Leucodermia virus non-structural protein 1-BIP
<400> 48
CTCCGGTTGGTGATGAAGCACCAGAACTGGATAACATGGAA 41
<210> 49
<211> 21
<212> DNA
<213>artificial sequence
<220>
<223>minimum viral capsid proteins gene-F3
<400> 49
CGTCATTAGTAATCCTCGGAA 21
<210> 50
<211> 25
<212> DNA
<213>artificial sequence
<220>
<223>minimum viral capsid proteins gene-B3
<400> 50
CAGTTTTAAAGTCATTGAGAACAAC 25
<210> 51
<211> 43
<212> DNA
<213>artificial sequence
<220>
<223>minimum viral capsid proteins gene-FIP
<400> 51
TGACCCAACCGTCAATGTGTATTGGATTGACATTGATCTTTCA 43
<210> 52
<211> 44
<212> DNA
<213>artificial sequence
<220>
<223>minimum viral capsid proteins gene-BIP
<400> 52
ATAGGATCACTAAACTTGGTCCATCCTTGTAATCTCCTGGTGCA 44
<210> 53
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>viral nervous necrosis RNA Dependent RNA pol gene-F3
<400> 53
GCATAAACCTGGCGATCTCG 20
<210> 54
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>viral nervous necrosis RNA Dependent RNA pol gene-B3
<400> 54
TGCGGACCAGCAATACGA 18
<210> 55
<211> 40
<212> DNA
<213>artificial sequence
<220>
<223>viral nervous necrosis RNA Dependent RNA pol gene-FIP
<400> 55
GGCGTAACACTTGCCCTGCTTACCAAGCAGTTGGTAGTGC 40
<210> 56
<211> 41
<212> DNA
<213>artificial sequence
<220>
<223>viral nervous necrosis RNA Dependent RNA pol gene-BIP
<400> 56
TGGACCGTTGACTCAAACTGCTCGATTGTGGCAAGCTCGTT 41
Claims (3)
1. a kind of high-throughput quantification detection kit of aquiculture animal virus, it is characterised in that: including 14 kinds of aquacultures
The LAMP detection primer group of animal virus, gene order are as shown in the table:
。
2. a kind of high-throughput quantification detection kit of aquiculture animal virus according to claim 1, feature exist
In: the LAMP reaction system of the detection kit and the final concentration of each ingredient are as follows: sample template DNA 1 μ L, 2.5 μ L
10 × reaction buffer, 1.5 μ L concentration are the MgSO of 100mM4, 3.5 μ L concentration are the dNTP of 10mM, and 1 μ L concentration is 8 U/
μl Bst DNA polymerase, 1 μ L FIP/BIP primer sets solution, 1 μ L F3/B3 primer sets solution and 4 μ L glycine betaines, are then used
Distilled water supplies 25 μ L；Wherein the FIP/BIP primer sets solution be respectively pancreas necrosis virus described in claim 1,
Giant salamander irido virus, lymphocystis disease virus, bright and beautiful lithium herpesviral, infectious spleen and kidney necrosis virus, spring viremia of carp virus disease, Penaeus monodon
Baculoviral, hepatopancreatic parvovirus shape virus, yellow head virus, infectious subcutaneous and haematopoietic necrosis virus steal dead wild field
Village's virus, Leucodermia virus, minimum viral and viral nervous necrosis FIP/BIP primer sets solution, each primer final concentration
It is 40 μM, the F3/B3 primer sets solution is respectively pancreas necrosis virus described in claim 1, giant salamander iris disease
Poison, lymphocystis disease virus, bright and beautiful lithium herpesviral, infectious spleen and kidney necrosis virus, spring viremia of carp virus disease, penaeus monodon baculovirus,
Hepatopancreatic parvovirus shape virus, yellow head virus, infectious subcutaneous and haematopoietic necrosis virus steal dead nodavirus, are white
The F3/B3 primer sets solution of spot syndrome virus, minimum virus and viral nervous necrosis, each primer final concentration is 5 μM.
3. a kind of high-throughput quantification detection kit of aquiculture animal virus according to claim 2, feature exist
In: the detection kit further includes color developing agent, and the color developing agent is calcein/manganese chloride solution or hydroxyl naphthols
Blue solution or SYBR-Green I, wherein calcein concentration is 75 μM in calcein/manganese chloride solution, manganese chloride concentration
It is 500 μM；The hydroxynaphthol blue solution concentration is 150 μM, the calcein/manganese chloride solution or described
The additive amount of hydroxynaphthol blue solution is 1 hole μ L/, and the additive amount of the SYBR-Green I color developing agent is 5 holes μ L/.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112048572A (en) * | 2020-09-17 | 2020-12-08 | 福建省水产研究所（福建水产病害防治中心） | LAMP technology-based visual rapid detection kit for shrimp health system |
| CN112981006A (en) * | 2021-01-21 | 2021-06-18 | 宁波爱基因科技有限公司 | Efficient quintuplet shrimp disease detection primer and kit |
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| CN112981006A (en) * | 2021-01-21 | 2021-06-18 | 宁波爱基因科技有限公司 | Efficient quintuplet shrimp disease detection primer and kit |
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| CN110106283B (en) | 2022-12-09 |
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