CN104032037A - Cyprinid herpesvirus detection kit and detection method thereof - Google Patents
Cyprinid herpesvirus detection kit and detection method thereof Download PDFInfo
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Abstract
The invention relates to a Cyprinid herpesvirus detection kit and a detection method thereof, belonging to the technical field of virus genome detection. The detection kit comprises a 10* reaction buffer solution, a 1U/mu L Taq enzyme, a primer set composed of 10 mu M of P1F and 10 mu M of P1R, a primer set composed of 10 mu M of P2F and 10 mu M of P2R, pure water and a 15 mu g/mL positive DNA. The kit can be used for simultaneously detecting genomes of carp pox herpesvirus, haematopoietic necrosis herpesvirus of goldfish and Koi herpesvirus. The method has the advantages of excellent visuality, favorable sensitivity and high specificity, has favorable effects on detecting trace amounts of Cyprinid herpesvirus genome in the long-storage-time specimen, and can be used for Cyprinid-herpesvirus-infected laboratory detection and Cyprinid herpesvirus molecule epidemiological survey.
Description
Technical field
The invention belongs to viral genome detection technique field, be specifically related to a kind of carp simplexvirus (Cyprinid herpesvieus, CYHV) detection kit and detection method thereof.
Background technology
Simplexvirus is linear dsdna virus, can cause Mammals, the multiple mortality disease of bird and hydrocoles, the simplexvirus Zhu that wherein causes fish serious disease is Yaoed You Channel-catfish simplexvirus, cazppox virus (carp pox herpesvirus), goldfish hematopoietic tissue necrosis virus (haematopoietic necrosis herpesvirus of goldfish, and Koi herpesvirus (Koi herpesvirus) HNHV), rear three belongs to carp simplexvirus, cazppox virus belongs to carp simplexvirus 1 type (Cyprinid herpesvirus 1, CYHV-1), goldfish hematopoietic tissue necrosis virus belongs to carp herpes virus type 2 (Cyprinid herpesvirus 2, CYHV-2), Koi herpesvirus belongs to carp simplexvirus 3 types (Cyprinid herpesvirus 3, CYHV-3).CYHV-1 can cause epithelioma papulosum cyprinorum, and main harm carp, crucian and orfe etc., affect the growth of fish, reduces the commodity value of fish, can cause larger financial loss.CYHV-2 can cause the hematopoietic tissue necrosis disease of goldfish and crucian, and this disease found in Aichi, Japan goldfish plant first in 1992, breaks out afterwards and popular in the states such as the U.S., TaiWan, China, Australia and area, and its rate of causing death reaches as high as 100%.2008-2013 causes the crucian cultivation base Yancheng, Jiangsu Province area cultivation crucian of Largest In China to break out carp spleen renal necrosis disease, causes the cultivation crucian death of several ten thousand mu.And CYHV-3 also can cause the blister sore viral disease of carp and bright and beautiful carp, once morbidity, mortality ratio reaches more than 80%.Can be used for preventing because above three kinds of carp blister sore viral diseases also do not have effective vaccine, therefore develop the detection kit of above three kinds of carp blister sore viral diseases, be that current cyprinid fish cultivation needs the subject matter solving.
Because above three kinds of carp simplexviruss all can infect most cyprinid fish, can detect again the detection method of above three kinds of carp simplexviruss due to current shortage simultaneously, this just causes trouble to need to detect above three kinds of cause of diseases simultaneously.The PCR method that can simultaneously detect above three kinds of carp simplexviruss and the sleeve type PCR detection method of the present invention development, made up the defect of this technical field, and for above three kinds of carp simplexviruss time, monitoring provides powerful measure.
Summary of the invention
The problem existing for prior art, the object of the invention is to design provides a kind of carp simplexvirus detection kit and detection method thereof.The method can detect the genome of cazppox virus (carp pox herpesvirus), goldfish hematopoietic tissue necrosis virus (haematopoietic necrosis herpesvirus of goldfish, HNHV) and Koi herpesvirus (Koi herpesvirus) simultaneously.The method intuitively superior, susceptibility good, specificity is high, to detecting the respond well of carp hsv gene group in trace, shelf time sample of a specified duration, the laboratory that can be used for carp herpesvirus infection is detected and the Molecule Epidemiology Investigation of carp simplexvirus.
In order to achieve the above object, the present invention adopts following technical scheme:
A, two pairs of universal primers for carp simplexvirus I type, II type and III type of design
Designed two pairs of carp simplexvirus universal primers in the present invention, from GenBank, obtain whole three of carp herpetoviridae and belong to representative strains whole genome sequence, Cyprinid herpesvirus 1 strain NG-J1, Cyprinid herpesvirus 2 strain ST-J1, Cyprinid herpesvirus 3 KHV-U are totally 3 strain carp hsv gene encoding amino acid sequence and genom sequences.All aminoacid sequences and genom sequence are carried out to program arrangement, and the region that searching aminoacid sequence and corresponding genome sequence are all guarded is as design of primers district (in table 1).There is not yet the research report that can simultaneously detect whole three the type universal primers designs of carp simplexvirus both at home and abroad.
Table 1 carp simplexvirus design of primers
B, a kind of carp simplexvirus detection kit, comprise and the centrifuge tube of 6 1.5mL 10 × reaction buffer be housed respectively, Taq enzyme (1U/ μ L), the each 10 μ M of primer P1F/1R, the each 10 μ M of primer P1F/P1R, pure water, positive DNA (15 μ g/mL).
The formula of 10 described × reaction buffer is: Tris-HCl 100mM, KCl 500mM, MgC
l220mM, dNTPs 2.5mM.
The method that C, carp hsv gene group detect
A, use virus-specific DNA extraction test kit (QIAamp DNA Mini Kit) extract the total DNA of sample;
B, nest-type PRC: described a kind of carp simplexvirus detection method, it is characterized in that comprising the following steps: 1) with carp simplexvirus Auele Specific Primer P1F/P1R, DNA sample to be measured is carried out to first round pcr amplification, its reaction conditions is 95 DEG C of denaturation 3min, 94 DEG C of sex change 30s, 58 DEG C of renaturation 30s, 72 DEG C are extended l.5 min, circulate 35 times, and last 72 DEG C are extended 10min; 2) in first round pcr amplification product, do not observe object nucleic acid belt, further adopt carp simplexvirus Auele Specific Primer P2F/P2R to carry out second to first round pcr amplification product and take turns pcr amplification, its reaction conditions is 95 DEG C of denaturation 3min, 94 DEG C of sex change 30s, 58 DEG C of renaturation 30s, 72 DEG C are extended l min, circulate 35 times, and last 72 DEG C are extended 10min;
C, 1.5% agarose gel electrophoresis are observed viral nucleic acid amplification, and a specific nucleic acid band appears in first round pcr amplification product at 1213bp place, and second takes turns pcr amplification product occurs a specific nucleic acid band at 952bp place.
D, result judge: take turns PCR while there is corresponding amplified band when detecting sample and positive control at first round PCR or second, result is positive; In the time detecting sample without not of uniform size the causing of amplified band of band or stripe size and positive control, result is negative; When positive control is during without band, show related reagent degraded in test kit, test kit lost efficacy; In the time there is band in negative control, in operating process, there is reagent contamination, detected result is invalid.
For the carp herpes virus DNA that ensures various plant types can be detected effectively, the present invention has selected terminal enzyme (terminase) gene coding region that the full genome of carp simplexvirus is guarded the most to design universal primer P1F, P1R, P2F and the P2R for CYHV-1, CYHV-2 and CYHV-3.In order to strengthen the specificity of primer, on the basis of conserved regions screening primer, apply degenerated primer, like this can be with the increase different virus genome of 3 genus of all carp simplexviruss of pair of degenerate primers.Consider in the time carrying out viruses molecule epidemiology survey simultaneously, need to obtain the virus genome sequence of certain length in order to distinguish virus strain gene difference, therefore selected the primer of amplification moderate-length fragment when design of primers, to be applied in the time carrying out viruses molecule epidemiology survey.Wherein cazppox virus (CYHV-1) terminal enzyme (terminase) gene order is as shown in SEQ ID NO:5, crucian hematopoietic tissue necrosis virus (CYHV-2) terminal enzyme (terminase) gene order is as shown in SEQ ID NO:6, and Koi herpesvirus (CYHV-3) terminal enzyme (terminase) gene order is as shown in SEQ ID NO:7.
Detect the susceptibility of carp herpes virus DNA in order further to increase this law, for the recall rate of the low copy sample of virus, primer sets P1F/P1R and P2F/P2R can react for sleeve type PCR, detecting with primer sets P1F/P1R negative in the situation that, recycling primer sets P2F/P2R carries out second to primer sets P1F/P1R amplified production and takes turns PCR, the recall rate that can significantly improve so low copy sample, prevents false negative.
The present invention compared with prior art, has following advantages:
1, in the present invention, two pairs of designed carp simplexvirus universal primers design for carp simplexvirus conserved sequence, can amplify any carp simplexvirus of having found, and positive rate is high, is applicable to the carp simplexvirus sample of clinical detection unknown species.
2, in the present invention, two pairs of designed carp simplexvirus universal primers can be for sleeve type PCR, and sensitivity is equivalent to 10 of regular-PCR
3doubly, reduce carp simplexvirus and detected false-negative possibility.
3, in the present invention, designed carp simplexvirus universal primer group P1F/P1R and P2F/P2R have adopted the method for design of degenerated primer, increase the broad spectrum that detects carp simplexvirus, reduce false-negative possibility, greatly improved the positive rate of carp simplexvirus.
The carp hsv gene group detection method of 4, establishing in the present invention, select the special DNA extraction test kit for minim DNA, low copy Virus Sample, the poor carp herpes virus DNA of preservation condition are also had to good detection effect, to number of freezing and thawing nearly the band poison sample of 20 times still there is 100% recall rate, can be used for clinical, laboratory and detect and epidemiological study.
Brief description of the drawings
Fig. 1 is the specific amplification figure that the present invention detects carp hsv gene group;
Fig. 2 is the susceptibility amplification figure that the present invention detects carp hsv gene group;
Fig. 3 is the amplification figure of the present invention for carp simplexvirus multigelation tissue samples.
M:DL2000 DNA Marker in Fig. 1; L:CYHV-1; 2:CYHV-2; 3:CYHV-3; 4:MRNV; 5:WSSV; 6:GCRV; 7:ddH
2o contrast.
M:DL5000 DNA Marker in Fig. 2; L-10:CYHV-2 DNA starts to increase after 10 times of dilutions from 0.57ug; In 11-20:1-10, first round PCR product carries out sleeve type PCR amplification again.
M:DL2000 DNA Marker in Fig. 3; L-8: the CYHV-2 positive that difference freeze thawing is 8,12,16,20,24,28,32,36 times; 9:ddH
2o contrast.
Embodiment
Below in conjunction with embodiment, principle of the present invention and feature are described, illustrated embodiment, only for explaining the present invention, is not intended to limit scope of the present invention.
EXAMPLE l: PCR method detects the genomic dna of carp simplexvirus positive template
A, synthetic two pairs of carp simplexvirus universal primers, transfer to Nanjing Genscript Biotechnology Co., Ltd. to carry out:
a：CYHV PlF/P1R
Trip primer P1F is 5'-CGCTGAGMATGTCGTCRTTGTC-3', this sequence as shown in SEQ ID NO:1,
Downstream primer P1R is 5'-GGSACCAAYCAGATTCACATCA-3', and this sequence is as shown in SEQ ID NO:2;
b: CYHV P2F/P2R
Trip primer P2F is 5'-CTTSACCRTRTTCATCTGRGCCA-3', this sequence as shown in SEQ ID NO:3,
Downstream primer P2R is 5'-CTACAAGCCCGACCARATAGAGA-3', and this sequence is as shown in SEQ ID NO:4.
B, a kind of carp simplexvirus detection kit, comprise and the centrifuge tube of 6 1.5mL 10 × reaction buffer be housed respectively, Taq enzyme (1U/ μ L), the each 10 μ M of primer P1F/P1R, the each 10 μ M of primer P1F/P1R, pure water, positive DNA (15 μ g/mL).
The formula of 10 described × reaction buffer is: Tris-HCl 100mM, KCl 500mM, MgCl
220mM, dNTPs 2.5mM.
C, carp hsv gene group detection method:
1, the preparation of positive template
Take virus-positive crucian, use total DNA extraction test kit (QIAamp DNA Mini Kit) to extract the total DNA of sample as positive template.Negative control (normal virus-free crucian) is set simultaneously.
2, nest-type PRC reaction
First round PCR: get above-mentioned total DNA 1 μ L, add in the reaction system of cumulative volume 50 μ L, wherein containing 1U Taq archaeal dna polymerase, 5 μ L 10 × reaction buffers and the each l0pmol of Auele Specific Primer (PlF/P1R).After fully mixing, put into the reaction of pcr amplification instrument.Circulating reaction parameter is 95 DEG C of denaturation 3min, 94 DEG C of sex change 30s, and 58 DEG C of renaturation 30s, 72 DEG C are extended l.5 min, circulate 35 times, and last 72 DEG C are extended 10min.
Second takes turns PCR: get above-mentioned first round pcr amplification product 0.5 μ L, add in the reaction system of cumulative volume 50 μ L, wherein containing 1U Taq archaeal dna polymerase, 5 μ L 10 × reaction buffers and the each l0pmol of Auele Specific Primer (P2F/P2R).After fully mixing, put into the reaction of pcr amplification instrument.Circulating reaction parameter is 95 DEG C of denaturation 3min, 94 DEG C of sex change 30s, and 58 DEG C of renaturation 30s, 72 DEG C are extended l min, circulate 35 times, and last 72 DEG C are extended 10min.
3, agarose gel electrophoresis
Get 5 μ L pcr amplification products and containing electrophoresis on 1. 5% sepharoses of 5 smelling of μ g/ml second pyridines (EB), 5V/cm, observations on ultraviolet transmission reflectometer after 30min, first round PCR object amplified fragments is 1213bp; Second to take turns PCR object amplified fragments be 952bp.
4, the specificity of PCR method
Result shows that CYHV-1, CYHV-2 and CYHV-3 nucleic acid amplification product are through agarose gel electrophoresis, occurs a specific nucleic acid band at 1213bp place; After Aeromonas hydrophila (AH), white spot syndrome virus (WSSV), GCRV (GCRV) and negative control amplified production electrophoresis, have no specific nucleic acid band (see figure 1).The designed primer of the present invention is through its specificity of DNA STAR Primer Select software detection, result shows that the genomic dna sequence of 1 strain cazppox virus (CYHV-1), goldfish hematopoietic tissue necrosis virus (CYHV-2) and the 3 strain Koi herpesvirus (CYHV-3) of above-mentioned two pairs of primers (PlF/P1R and P2F/P2R) to known complete sequence all only produces an object fragment product while amplification, without non-specific amplification.
5, the susceptibility of PCR method
Total DNA that 0.1g positive is extracted carries out quantitatively as 0.78ug on foranalysis of nucleic acids instrument, and through 10 times of serial dilutions, the high dilution that this law can detect is 10
-5, be equivalent to the viral DNA in 7.8pg nucleic acid; To extent of dilution is too high and first round PCR does not detect sample carries out two-wheel PCR detection, first round PCR primer adopts CYHV P1F/P1R, and object amplified fragments size is 1213bp, and second takes turns PCR primer adopts CYHV P2F/P2R, object amplified fragments size is 952bp, system and reaction conditions as above 2.PCR is high 1000 times for result sleeve type PCR remolding sensitivity single, and it is 10 that high dilution can reach
-8(as shown in Figure 2).
Embodiment 2:PCR method detects CYHV-2 DNA in multigelation sample
Get the rear 30 minutes multigelations of-80 DEG C of 30 minutes/room temperatures respectively of virus-positive sample packing EP pipe.The sample of different number of freezing and thawing is carried out to DNA extraction, by using CYHV novel primer (PlF/P1R, P2F/P2R) to be in charge of nido amplification to every a nucleic acid sample extracting.
1, DNA extraction
Get multigelation sample 0.1g, with total DNA extraction test kit (QIAamp DNA Mini Kit), extract total DNA by test kit explanation.
2, nest-type PRC reaction
First round PCR: get above-mentioned total DNA 1 μ L, add in the reaction system of cumulative volume 50 μ L, wherein containing 1U Taq archaeal dna polymerase, 5 μ L 10 × reaction buffers and the each l0pmol of Auele Specific Primer (PlF/P1R).After fully mixing, put into the reaction of pcr amplification instrument.Circulating reaction parameter is 95 DEG C of denaturation 3min, 94 DEG C of sex change 30s, and 58 DEG C of renaturation 30s, 72 DEG C are extended l.5 min, circulate 35 times, and last 72 DEG C are extended 10min.
Second takes turns PCR: get above-mentioned first round pcr amplification product 1 μ L, add in the reaction system of cumulative volume 50 μ L, wherein containing 1U Taq archaeal dna polymerase, 5 μ L 10 × reaction buffers and the each l0pmol of Auele Specific Primer (P2F/P2R).After fully mixing, put into the reaction of pcr amplification instrument.Circulating reaction parameter is 95 DEG C of denaturation 3min, 94 DEG C of sex change 30s, and 58 DEG C of renaturation 30s, 72 DEG C are extended l min, circulate 35 times, and last 72 DEG C are extended 10min;
3, agarose gel electrophoresis
Get 5 μ L pcr amplification products containing electrophoresis on 1. 5% sepharoses of 5 smelling of μ g/ml second pyridines (EB), 5V/cm, observations on ultraviolet transmission reflectometer after 30min, second to take turns PCR object amplified fragments be 952bp.Visible through agarose gel electrophoresis, the sample that multigelation is 28 times adopts this law also can detect (as shown in Figure 3).
SEQUENCE LISTING
<110> Zhejiang Institute of Fresh Water Aquatic Products
<120> carp simplexvirus detection kit and detection method thereof
<130> 1
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213> synthetic
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cgctgagmat gtcgtcrttg tc 22
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ggsaccaayc agattcacat ca 22
<210> 3
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<400> 3
cttsaccrtr ttcatctgrg cca 23
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<213> synthetic
<400> 4
ctacaagccc gaccaratag aga 23
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<211> 1213
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<213> cazppox virus
<400> 5
cgctgagtat gtcgtctttg tcaaacgtcc ctgcattgcg cttcttgcca ccggtgtcta 60
acattggctg gttgtttgcg tctgagacca ccttcaccct cttcatctgt gccagaagct 120
tgtttaaatt gatacctgta tagttaaata aaagctcttt gactttcaac gcgtctactt 180
caagttgttg tgctatgctg aatgccagct gctccggatc cctcccgtcg caaacggtct 240
cggagaatgt ctccgcgtcc ttttgcagca actgggacgc aagggatccg gtggcctgcg 300
tagagggttt gaggttcagg ctgtgttcag catgcttgag tcgcctgttg aagctgttca 360
tgaccatctg tcccactgcg aagaccacat cagccctgcc taccttgtag accctgagcg 420
cgtccgcgaa gcccaggaac tgctggagct tgtttctccc cacaatcttt cctggctgtg 480
ggttggcaac atccttggtg ctgtcgatgt aggcaaacac attggtgaag cccagttcca 540
ggcaccagtt cgatatggcc gtccagaggt tgctcacaga ctccgcaaag gtgttggact 600
cgatgacaac agcaatctcc agctcgggga acatgagcct gctcacaagg atgtgagtcc 660
tgagtaccag ctcatgcagc ctgctgctgt atgagacgtg gttgcttatg tcaagctcat 720
cgagaccaaa gatgatcata cggggcatcc ctttgtgggt ggtgtggaca gaggtgcaca 780
ctccgatgtt ggacacggtc cccgtggacc aggttgggtc caccgaaatg acaaccaggg 840
actccagtgg ctgcttgaac acagcgtggt tgacctgtgt ggtgttgagc ataaagttga 900
gcgctcccgt ctcgaagggg tgattgtcag gcttcacagc cagtgtgtgg cctccagtga 960
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<213> crucian hematopoietic tissue necrosis virus
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cgctgaggat gtcgtctttg tcgaacgacc cagagtttct ctttttacca cccgtgtcca 60
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tgtttaaatt gacacctgtg caattgaata aaagttcttt gatcttcatc gggtctatct 180
ctaattgttg tgctatactg aacgccagct ggtctggatt cttgccgtcg cacacggtct 240
ccgagaaact ctcggcgtct ctctggagaa gctgaaacga gagagacccg gtactgtgcg 300
tggtcggttt gagtttgaga gcgtgctcgg cctgcttgag cttgctcacg aaactgttga 360
gcaccatctg tcccacggcg aacacaacgt ccgccctggc gatcttgtaa accctcagag 420
cgtccgcgaa cccgagaaac tgctgcagct tgtttctacc gaccatcttg ccgggttgag 480
gattgagtac gtccttggtc ttgtcgacga acgcaaagac gttttggagt ccaatctcca 540
gacaccagtt tgagatggac gcccacaggt tactcacgga ctctgcgaac gtgttggact 600
cgatgactat ggccacgtcg aggtccggga acatgagcct gctcaccatg atgtgagtcc 660
tgagcacgag ctcgtgcagt ctcgagctgt agctgacgtg attgctgatg tcgatctcgt 720
ccaggccaaa cactatcatc ctgggcatac cgtgtctgat ggtgtgcacc gtggtgcaga 780
cgccgatgtt tgacaccgta ccggtagacc atgtgggatc gatggatatg accaccagag 840
actctgtcgg ttgcttgaag acggcgtggt tgaccggtac agtgttcatc ataaaattga 900
gagcgcccgt ctcgaaggga tgattgtccg gcttgatggc gagagtgtgt ccacccgtca 960
actccatctc gaaggcgccg tcgcacacca ggttcatgag ttctttgacg ccttcctcga 1020
tctctatgtg gtcgggcttg tagatgtcgt tgcagacgca gctgatgcct tcctcctcgg 1080
catgggcctt gcacttgtat ttgtaatcta tgatttgagc caaagtctcc ccattttctt 1140
gtttgacgtc tcccacgtgc gatatccaag agtcggccga tcccggagag ctgatgtgaa 1200
tctgcttggt ccc 1213
<210> 7
<211> 1213
<212> DNA
<213> Koi herpesvirus
<400> 7
cgctgaggat gtcgtccttg tcgacggtac ccgcggcctt cttcttgccc ccggtatcca 60
gcacgggctg attgttggcg tccgagatca ccttcacctt cttcatctgc gccaacagtt 120
tgtttaaatt gacacctgtg tagttgaata aaagctcttt aattttcatc gggtctacgt 180
ctagttgttg tgccacgctg aacgctagct gctcggggtc cttgccgtcg cacaccgtct 240
ctgagaaggt gtcggcgtcc cggtgcagca gctgagaggc cagggagccc gtgaggtggg 300
tctggggttt gatcttgagc gagtgctccg agagcttgag cctcctgatg aagctgttca 360
tgaccatggg tcccacagca aacaccacgt cggcgtggcc gatcttgtag acgcgcatgg 420
cgtcggcaaa ggccagaaac tgctgcagct tctgcctccc gatcatcttg cccggctgcg 480
agttgaccac gtccttggtc ttgtccacga acgcgaacac gttctgcgcc ccgctctcga 540
ggcaccagtt ggccaccgcg gcccagaggt tgctcacgct ctcggcgaag gtgttggact 600
cgatcacgat ggcgacgtcg aggtcgggga acatgaggcg gctcaccatg atgtgcgtgg 660
tgagcaccag ctcgtgcaac cgcgagctgt agctgacgtg gttgctgatg tcgagctcgt 720
ccaggccaaa cactatgatg cgcggcatgc cgttccggac ggtgtgcacc gacgtgcaga 780
cgccgatgtt ggagacggtg cccgtcgacc aggtcgggtc gatggagatg acaaccaggg 840
actctgagga ctgcttgaac acggtgtggt tgacggggat gttgttcatc atgaagttga 900
gggcgcccgt ctcgaagggc ctgttatcgg gcttgacgtt gagcgtgtgg ccaccggtca 960
gctccatctc gaaggcgccg tcgcacacca tgttcatgag ttccttgacg ccctcctcga 1020
tctctatatg gtcgggcttg tagatgtcgt tgcagacgca gctgatgccg tcctcctcgg 1080
cgtgcgcctt gcacttgtac ttgtaatcta tgatttgagc cagggtctcg ccgttttctt 1140
gcttcacgtc tccgacgtgc gaaatccaag agtcagccga gccgggagag ctgatgtgaa 1200
tctgcttggt ccc 1213
Claims (3)
1. a carp simplexvirus detection kit, is characterized in that this detection kit comprises the positive DNA of primer sets P2F/P2R, pure water and the 15 μ g/mL of 10 × reaction buffer, the Taq enzyme of 1U/ μ L, the primer sets P1F/P1R of each 10 μ M, each 10 μ M;
10 described × reaction buffer is: Tris-HCl 100mM, KCl 500mM, MgCl
220mM and dNTPs 2.5mM;
In described primer sets P1F/P1R: upstream primer P1F sequence is as shown in SEQ ID NO:1, and downstream primer P1R sequence is as shown in SEQ ID NO:2;
In described primer sets P2F/P2R: upstream primer P2F sequence is as shown in SEQ ID NO:3, and downstream primer P2R sequence is as shown in SEQ ID NO:4.
2. utilize carp simplexvirus detection kit as claimed in claim 1 to detect a method for carp simplexvirus, it is characterized in that:
To detect the DNA of sample as template, carry out pcr amplification with primer sets P1F/P1R, too low and while having no amplification of nucleic acid band when the contained viral DNA amount of sample, then taking first round pcr amplification product as template, adopt primer sets P2F/P2R to carry out second and take turns PCR; In the time that first round pcr amplification product occurs that at 1213bp place specific nucleic acid band or second is taken turns pcr amplification product at a specific nucleic acid band of 952bp place appearance, be judged as and in sample, contain carp herpes virus DNA.
3. detection method as claimed in claim 2, the first round and second described in it is characterized in that takes turns PCR reaction conditions and is: 95 DEG C of denaturation 3min, 94 DEG C of sex change 30s, 58 DEG C of renaturation 30s, 72 DEG C are extended l min, circulate 33 times, and last 72 DEG C are extended 8min.
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| CN106801049A (en) * | 2017-02-14 | 2017-06-06 | 中国水产科学研究院珠江水产研究所 | The extracting method of the type DNA of carp herpesviral 3 of latent infection, sleeve type PCR detection method and its kit |
| CN108384896A (en) * | 2018-05-08 | 2018-08-10 | 张朝明 | A kind of RT-PCR detection kit and its detection method of Koi herpesvirus |
| CN110592270A (en) * | 2018-06-13 | 2019-12-20 | 杭州众测生物科技有限公司 | RAA constant-temperature fluorescence detection method and reagent for Koi Herpesvirus (KHV) |
| CN113046479A (en) * | 2021-03-19 | 2021-06-29 | 中国水产科学研究院珠江水产研究所 | Koi herpesvirus RPA primer group, probe and detection kit and application thereof |
| CN113046479B (en) * | 2021-03-19 | 2021-11-26 | 中国水产科学研究院珠江水产研究所 | Koi herpesvirus RPA primer group, probe and detection kit and application thereof |
| CN114085931A (en) * | 2022-01-24 | 2022-02-25 | 中国水产科学研究院黄海水产研究所 | Primer group for detecting abalone herpesvirus and application of primer group in construction of virus detection method and kit |
| CN116267799A (en) * | 2022-12-30 | 2023-06-23 | 中国科学院水生生物研究所 | Method for preparing crucian carp resistant to herpesvirus |
| CN116267799B (en) * | 2022-12-30 | 2024-04-09 | 中国科学院水生生物研究所 | Method for preparing crucian carp resistant to herpesvirus |
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Application publication date: 20140910 Assignee: Chengdu Tianbang Biological Products Co., Ltd. Assignor: Zhejiang Institute of Fresh Water Aquatic Products Contract record no.: 2018330000117 Denomination of invention: Cyprinid herpesvirus detection kit and detection method thereof Granted publication date: 20160120 License type: Common License Record date: 20181105 |