CN107287351A - Ring mediated isothermal amplification combination lateral flow ELISA test strip Koi herpesvirus - Google Patents
Ring mediated isothermal amplification combination lateral flow ELISA test strip Koi herpesvirus Download PDFInfo
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- CN107287351A CN107287351A CN201710569444.0A CN201710569444A CN107287351A CN 107287351 A CN107287351 A CN 107287351A CN 201710569444 A CN201710569444 A CN 201710569444A CN 107287351 A CN107287351 A CN 107287351A
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- 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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
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- 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
Abstract
The invention discloses ring mediated isothermal amplification combination lateral flow ELISA test strip Koi herpesvirus.The invention provides ring mediated isothermal amplification primer set, it is made up of upstream outer primer, downstream outer primer, upstream inner primer and downstream inner primer；The nucleotides sequence of the upstream outer primer is classified as sequence 1；The nucleotides sequence of the downstream outer primer is classified as sequence 2；The nucleotides sequence of the upstream inner primer is classified as sequence 3；The nucleotides sequence of the downstream inner primer is classified as sequence 4.The present invention detects that the method for Koi herpesvirus has higher specificity, sensitivity, practicality and convenience than existing conventional art PCR; can immediately it be detected in actual field condition; be conducive to the outburst of prevention and control Koi herpesvirus, it is significant to the aquaculture economic development of protection China.
Description
Technical field
The invention belongs to virus detection techniques field, and in particular to a kind of ring mediated isothermal amplification combination lateral flow test paper
Bar detects Koi herpesvirus.
Background technology
Koi herpesvirus disease (Koi herpes virus disease, KHVD) in May, 1998 in Israel first
It was found that, it is Koi herpesvirus (Koi herpes virus, KHV), also known as carp herpesviral that its cause of disease was just confirmed by 1999
Type III (CyHV-3).The disease then has large area outburst in the area such as the U.S., Indonesia, UK ＆ EURO continent, Japan.
KHV main infections fancy carp, carp and its common mutation.Latent infection KHV fish body does not show clinical symptoms,
Water temperature can be broken out and dead when suitable.The disease have highly infectious and extremely strong fatal rate, not only can level infect, can also hang down
Direct transfer and broadcast.The cultivation to China's fancy carp, mirror carp, common carp causes huge economic loss in recent years.The Ministry of Agriculture of China, state
No. 2013 announcement of joint bulletin of quality supervision and test quarantine general bureau of family《The inward animal quarantine epidemic disease name of the People's Republic of China (PRC)
Record》In be classified as two class animal epidemics, OIE (OIE) is classified as must notifiable disease.Therefore, do
Good KHV detection and the primary work that monitoring work is that control KHV is propagated.Koi herpesvirus early detection is its prevalence of reduction
Risk, reduce its morbidity and cause the effective means of great economic loss, so simple, quick, highly sensitive inspection method is set up,
And exploitation is particularly important suitable for the Koi herpesvirus detection kit of field application.
Ring mediated isothermal amplification (Loop-mediated isothermal amplification, LAMP) technology is
Notomi is equal to a kind of Novel isothermal nucleic acid amplification method (International Patent Publication No. WO00/28082) of report in 2000, for
4 LAMP primers are designed in 6 sites of target gene, and using a kind of archaeal dna polymerase with strand-displacement activity, (Bst DNA polymerize
Enzyme), it is quick under constant temperature, efficiently, it is high it is special, expand target sequence with sensitivity, be suitable for scene and experiment condition be simpler
Fast detected in single laboratory.The improved method for introducing a pair of Loop primers, further shorten the reaction time.
Lateral flow test strips (Lateral flow dipstick, LFD) are a kind of new detection methods, in reaction
The probe of FITC marks and the LAMP amplified production specific hybrids of biotin labeling, it is to avoid agarose gel electrophoresis or fluorescence
Dyeing visually observes the false positive caused by non-specific amplification, further increases specificity and the sensitivity of reaction.
LFD detections are without special equipment, and product and test strips need to only be immersed buffer solution by operator, and be not required to toxic reagent, be grasped
Make easy and safer.The testing result of LAMP-LFD combination technologies can be visually observed directly.There is control in LFD test strips
Band and detection band, two bands, which develop the color, represents that testing result is the positive, and only control band color developing detection band, which does not develop the color, shows to detect
As a result it is feminine gender.LAMP-LFD associated methods safely, quickly, efficiently, high sensitivity, and without equipment and technology restriction, with it
The irreplaceable advantage of his technology institute.
The content of the invention
A purpose of the invention is to provide a kind of ring mediated isothermal amplification primer set for detecting Koi herpesvirus.
The ring mediated isothermal amplification primer set that the present invention is provided, by upstream outer primer, downstream outer primer, upstream inner primer
With downstream inner primer composition；
The nucleotides sequence of the upstream outer primer is classified as sequence 1；
The nucleotides sequence of the downstream outer primer is classified as sequence 2；
The nucleotides sequence of the upstream inner primer is classified as sequence 3；
The nucleotides sequence of the downstream inner primer is classified as sequence 4.
In above-mentioned primer set, the upstream outer primer, the downstream outer primer, the upstream inner primer and the downstream
The mol ratio of inner primer is 1:1:8:8；
Or 5 ' end mark biotins of the upstream inner primer or the downstream inner primer biotin.
Another object of the present invention is to provide a kind of ring mediated isothermal amplification reagent for detecting Koi herpesvirus.
The ring mediated isothermal amplification reagent that the present invention is provided, including above-mentioned primer set, PCR amplification buffers and DNA
Polymerase；
The concentration of the upstream outer primer and the downstream outer primer is 0.2 μm of ol/L；
The concentration of the upstream inner primer and the downstream inner primer is 1.6 μm of ol/L.
3rd purpose of the invention is to provide a kind of reagent for detecting Koi herpesvirus.
The reagent that the present invention is provided, is made up of above-mentioned primer set and probe；
The nucleotides sequence of the probe is classified as sequence 5,
Or the 5' ends end fluorescein isothiocynate FITC marks of the probe.
4th purpose of the invention is to provide a kind of kit for detecting Koi herpesvirus, including above-mentioned primer set
Or above-mentioned ring mediated isothermal amplification reagent or above-mentioned reagent.
Above-mentioned primer set or above-mentioned ring mediated isothermal amplification reagent or above-mentioned reagent or above-mentioned kit exist
Application in detection or auxiliary detection Koi herpesvirus is also the scope of protection of the invention；
Or, above-mentioned primer set or above-mentioned ring mediated isothermal amplification reagent or above-mentioned reagent or above-mentioned kit
Application in detection or auxiliary detection Koi herpesvirus product is prepared is also the scope of protection of the invention；
Or above-mentioned primer set or above-mentioned ring mediated isothermal amplification reagent or above-mentioned reagent or above-mentioned kit
Application in detecting or aiding in detection sample to be tested whether to infect Koi herpesvirus is also the scope of protection of the invention；
Or, above-mentioned primer set or above-mentioned ring mediated isothermal amplification reagent or above-mentioned reagent or above-mentioned kit
Application in whether preparation detection or auxiliary detection sample to be tested infect Koi herpesvirus product is also that the present invention is protected
Scope.
5th purpose of the invention is to provide a kind of detection or whether auxiliary detection sample to be tested infects Koi herpesvirus
Method.
The method that the present invention is provided, comprises the following steps：
1) ring mediated isothermal amplification is carried out to sample to be tested with above-mentioned primer set, obtains ring mediated isothermal amplification production
Thing；
2) hybridize the ring mediated isothermal amplification product with the probe in above-mentioned reagent, obtain hybrid product；
3) hybrid product described in lateral flow ELISA test strip, it is described to treat if the detection band of the reagent strip develops the color 2
This infection of test sample or candidate infection KHV, if the detection band of the reagent strip develops the color 1, the sample to be tested is uninfected by or waited
Choosing is uninfected by KHV.
6th purpose of the invention is to provide a kind of detection or whether auxiliary detection sample to be tested infects Koi herpesvirus
Method.
The method that the present invention is provided, comprises the following steps：
1) ring mediated isothermal amplification is carried out to sample to be tested with above-mentioned primer set, obtains ring mediated isothermal amplification production
Thing；
2) ring mediated isothermal amplification product described in electrophoresis detection, if containing amplified production, sample to be tested infection or
Candidate infects KHV, if not containing the amplified production, the sample to be tested is uninfected by or candidate is uninfected by KHV.
In the above method, the template of the ring mediated isothermal amplification is the genomic DNA or KHV genomes of sample to be tested
DNA or the material containing KHV gene-specific fragments.
In the above method, the condition of the ring mediated isothermal amplification is 63 DEG C of water-bath 45min；
Or 63 DEG C of hybridization 5min of the condition of the hybridization.
KHV provided by the present invention LAMP-LFD detection methods have the following advantages that：
First, high sensitivity, can be to 320pg to KHV detection minimum, detection spirit of its detection sensitivity than normal PCR
Sensitivity is high 10 times；
2nd, strong specificity, specific primer used is designed according to six different zones in KHV TK genes, and is added
Enter distinguished sequence as DNA specific probes；
3rd, detection time is short, can obtain testing result within 50 minutes or so, and 2-4h is saved than Standard PCR detection time；
4th, instrument and equipment requires low, it is not necessary to PCR instrument, electrophoresis apparatus and gel imaging system, only needs a constent temperature heater
Detection can be completed；
5th, substantially, whole detection process is not related to complex and expensive instrument and equipment to easy to operate, result, is slightly given birth to molecule
The personnel on thing basis can complete whole operation, can directly observe by the naked eye and differentiate, testing result is clearly obvious；
6th, to experimenter and more environment-friendly, detection process need not carry out gel electrophoresis, therefore without using poisonous examination
Agent.
The present invention has detects that KHV method has higher specificity, sensitivity, practicality than existing traditional PCR technique
Property and convenience, can immediately be detected in actual field condition, be conducive to the outburst of prevention and control Koi herpesvirus, to protection
China's aquaculture economic development is significant.
Brief description of the drawings
Fig. 1 is the LAMP-LFD primer and probe design diagrams of KHV-TK genes, and primer and probe is marked by horizontal line.
Fig. 2 is the sensitivity that LAMP detects KHV.
M is 200bp DNA marker, and 0 is negative control, and 1 swimming lane is 1 (template concentrations 32ng/ μ L), and 2-5 is followed successively by
10-2、10-3、10-4、10-5Times concentration genomic DNA.
Fig. 3 is that LAMP-LFD detects KHV sensitivity.
M is 200bp DNA marker, and 0 is negative control, and 1 swimming lane is 1 (template concentrations 32ng/ μ L), and 2-5 is followed successively by
10-2、10-3、10-4、10-5Times concentration genomic DNA.
Fig. 4 is the specificity that LAMP detects KHV.
M is 200bp DNA marker, and 0 is negative control, and 1-3 is followed successively by：With KHV, SVCV
(Spring Viraemia of Carp Virus, SVCV), GCRV (Grass carp reovirus, GCRV)
Specific gene recombinant plasmid as template LAMP reaction products.
Fig. 5 is the specificity that LAMP-LFD detects KHV.
M is 200bp DNA marker, and 0 is negative control, and 1-3 is followed successively by：With KHV, SVCV
(Spring Viraemia of Carp Virus, SVCV), GCRV (Grass carp reovirus, GCRV)
Specific gene recombinant plasmid as template LAMP reaction products.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
The technical solution adopted in the present invention includes three below part：
1) provide 2 pairs be used for KHV LAMP primer sequence, i.e., length be respectively 19bp, 18bp, 38bp, 38bp, 17bp and
20bp oligonucleotide sequence, is named as KHV-TK-F3, KHV-TK-B3, KHV-TK-FIP, KHV-TK-BIP successively；
2) LAMP reaction systems are prepared, sample template are expanded by LAMP response procedures, it is determined that optimal reaction
System and reaction condition；
3) design KHV-TK-HP is DNA probe, and LAMP amplifications are analyzed with reference to LFD technologies.
The present invention is described in further detail with reference to embodiments.
KHV is recorded in the following literature：Rahmati‐Holasoo H,Zargar A,Ahmadivand S,et
al.First detection of koi herpesvirus from koi,Cyprinus carpio L.experiencing
mass mortalities in Iran:clinical,histopathological and molecular study[J]
.Journal of fish diseases,2016,39(10):1153-1163。
SVCV is recorded in the following literature：Shivappa R,Kozlowicz S,Rolland J,et al.Spring
viremia of carp in the United States of America:evaluation of current
diagnostics[J].Diseases in Asian Aquaculture VI.Fish Health Section,Asian
Fisheries Society,Manila,Philippines,2008:143-156。
GCRV records Jabbar A, Narankhajid M, Nolan M J, et al.A first in the following literature
insight into the genotypes of Echinococcus granulosus from humans in Mongolia
[J].Molecular and cellular probes,2011,25(1):49-54。
Embodiment 1, LAMP-LFD technology for detection KHV methods are set up
First, the design of LAMP primer
The TK genes (AB375390) of KHV disclosed in NCBI are carried out after homology analysis, and compared approximate with KHV
Kind TK genes sequence, so as to design and filter out following 2 pairs of primers, and a probe, sequence is as follows：
KHV-TK-F3：5'-TGGACGGGGACTTTATGCA-3'(sequences 1)；
KHV-TK-B3：5'-AGACAGGGACGACACACC-3'(sequences 2)；
KHV-TK-FIP：
5'-BIO-TTCATGCACACCGCCGTCAGGCAGCCCTTCAAGCAGGT-3'(sequences 3)；
KHV-TK-BIP：
5'-ACGCACCCTTCACCGTCAGA-AAGACTCGGCGCCTCCAA-3'(sequences 4)；
KHV-TK-HP：5'-FITC-GTGCAAGATGCGCGACGCAC-3'(sequences 5).
Wherein KHV-TK-FIP is 5' ends biotin labeling primer, and KHV-TK-HP is 5' ends fluorescein isothiocynate FITC
Label probe；
KHV TK gene LAMP amplimers sequence location such as Fig. 1 (F1, F2, F3, B1, B2, B3) notes：FIP be F2 with
Be connected F1c (F1c is F1 complementary series) gained, and BIP is obtained by B2 is connected with B1c (B1c is B1 complementary series).It is shown.According to
Above-mentioned primer sequence, in Shanghai, Sheng Gong companies carry out LAMP primer synthesis.
2nd, LAMP amplifications and condition optimizing
The recombinant plasmid dna for extracting Koi herpesvirus sick (Koi herpes virus disease, KHVD) is used as mould
Plate carries out amplified conditions optimization.
Preparing first has optimal amplification efficiency and the specific reaction system of detection.LAMP reaction systems in the present invention
For：Each primer concentration is：Each 0.2 μm of ol/L of FIP-bio and BIP each 1.6 μm of ol/L, F3 and B3；1 × buffer solution is constituted and concentration
For：20mmol/L Tris-HCl(pH8.8)、50mmol/L KCl、2mmol/L MgSO4、10mmol/L(NH4)2SO4, 0.1%
Tween-20,6mmol MgSO4, 1.4mmol/L dNTPs, 8U Bst archaeal dna polymerases and 1-2 μ L sample DNA profilings, plus
Distilled water makes reaction system cumulative volume be 25 μ L.LAMP reaction systems are shown in Table 1.
The LAMP reaction systems of table 1
By above-mentioned LAMP reaction systems, LAMP reacts under the following conditions, obtains LAMP reaction products：
65 DEG C of water bath with thermostatic control 60min carry out amplified reaction, afterwards 80 DEG C of water-bath 5min terminating reactions.During the course of the reaction,
Amplification is too high or too low for temperature to be all unfavorable for LAMP reactions.Using the primer that provides of the present invention and use present invention determine that reaction
System, is respectively compared the influence reacted under the conditions of different temperatures (61 DEG C, 63 DEG C, 65 DEG C) LAMP, passes through Ago-Gel electricity
Swimming method finally determines temperature needed for optimum reacting time and reaction.
By 63 DEG C of analysis of experimental results final choice, the optimum reaction condition that 45min is expanded as follow-up LAMP.
3rd, LFD is detected
After LAMP amplifications terminate, the 2 μ l 10mmol/L HP probes (end for making it in reaction tube is added into reaction tube
Concentration is 0.8mmol/L), 63 DEG C of hybridization 5min obtain hybridization reaction product.
10 μ L hybridization reaction products are taken to be added to LFD test strips (Yousida Biological Technology Co., Ltd., Hangzhou, article No.：
D003-03) in sample pad, and LFD test strips are dipped vertically into 100 μ L Buffer solution (being carried in kit), observation examination
Whether paper slip detection band develops the color.
Developed the color if detection band and represent that the testing result of KHV in the sample is the positive if 2, that is, infect KHV or candidate's sense
KHV is contaminated, represents that sample KHV testing result is feminine gender if colour developing one, that is, is uninfected by KHV or candidate is uninfected by KHV.
Embodiment 2, LAMP-LFD detect the evaluation of KHV sensitivity
By KHV genomic DNA template original concentration (32ng/ μ L) by 10 times of gradient dilutions, respectively as reaction template.
Detection is carried out to KHV with LAMP, LAMP-LFD respectively and compares sensitivity.
LAMP is detected：Using the genomic DNA of above-mentioned gradient dilution as template, with the two LAMP amplifications in embodiment 1
Reaction system and reaction condition carry out LAMP amplifications, obtain amplified production；Electrophoresis detection amplified production.
If electrophoresis detection has amplified production, KHV testing result is the positive in the sample, that is, infects KHV or candidate
Infect KHV, if electrophoresis detection is without amplified production, then it represents that sample KHV testing result for feminine gender, that is, be uninfected by KHV or
Candidate is uninfected by KHV.
As a result as shown in Figure 2, it can be seen that LAMP reaction products amount is with template concentrations (10-2、10-3、10-4、10-5) reduction
And reduce, its minimum template concentrations detected is 10-5Times diluted concentration.
LAMP-LFD is detected：Using the genomic DNA of above-mentioned gradient dilution as template, with the LAMP in two in embodiment 1
The reaction system and reaction condition of amplification carry out LAMP amplifications, obtain amplified production；According still further to the method in three in embodiment 1
HP probes are added into LAMP amplified productions to continue to react, and obtain hybridization reaction product.ELISA test strip hybridization reaction product.
Developed the color if detection band and represent that the testing result of KHV in the sample is the positive if 2, that is, infect KHV or candidate's sense
KHV is contaminated, represents that sample KHV testing result is feminine gender if colour developing one, that is, is uninfected by KHV or candidate is uninfected by KHV.
As a result as shown in Figure 3, it can be seen that LAMP reaction products amount is reduced and reduced with template concentrations, line color is detected
Shoal step by step, the minimum template concentrations being able to detect that in this approach are 10-2Times diluted concentration.
From the above, it can be seen that 10 times of gradient dilutions of KHV genomic DNA templates are it is demonstrated experimentally that the minimum dilutions of LAMP-LFD
Concentration is 10-2, the minimum diluted concentration of LAMP detections is 10-5, template concentrations are 320pg/ μ L.The above results show the present invention
Sensitivity of primer sets when ensure that detection.
Embodiment 3, LAMP-LFD detections evaluation specific to KHV
Respectively with the DNA of the specific gene containing KHV, the DNA of the specific gene containing SVCV, the specific gene containing GCRV
DNA is used as negative control as template by template of distilled water.
The DNA of the specific gene containing KHV is by KHV specific fragment (NCBI accession number：AB375390, sequence 6) insert
Enter the plasmid that the SmaI sites in pUC57 carriers are obtained.
The DNA of the specific gene containing SVCV is by SVCV specific fragment (NCBI accession number：AY527273 sequences 7) insert
Enter the plasmid that the SmaI sites in pUC57 carriers are obtained.
The DNA of the specific gene containing GCRV is by GCRV specific fragment (NCBI accession number：HQ231204 sequences 8) insert
Enter the plasmid that the SmaI sites in pUC57 carriers are obtained.
LAMP amplifications are carried out with the reaction system and reaction condition of the two LAMP amplifications in embodiment 1, LAMP expansions are obtained
Increase production thing；HP probes are added into LAMP amplified productions to continue to react, hybridized according still further to the method in three in embodiment 1
Reaction product.
1.5% agarose gel electrophoresis detects LAMP amplified productions, as a result as shown in Figure 4, it can be seen that using KHV as mould
The reaction of plate is positive, and is negative with SVCV, GCRV for the reaction of template, and the reaction using distilled water as template is also negative.
ELISA test strip hybridization reaction product, develops the color if detection band and represents that the testing result of KHV in the sample is if 2
The positive, that is, infect KHV or candidate infection KHV, represents that sample KHV testing result is feminine gender if colour developing one, i.e., not
Infection KHV or candidate are uninfected by KHV.
As a result as shown in Figure 5, it can be seen that the reaction using KHV as template is positive, detection line shows red stripes, and with
SVCV, GCRV are negative for the reaction of template, and the reaction using distilled water as template is also negative, and detection line is unchanged.
The above results show, illustrate that the LAMP-LFD detection methods for the KHV that the present invention is provided can guarantee that only there is spy to KHV
The opposite sex, does not occur cross reaction with other aquatic products virus.
Sequence table
<110>Agricultural University Of Hebei
<120>Ring mediated isothermal amplification combination lateral flow ELISA test strip Koi herpesvirus
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 19
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<213>Artificial sequence
<220>
<223>
<400> 1
tggacgggga ctttatgca 19
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<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 2
agacagggac gacacacc 18
<210> 3
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<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 3
ttcatgcaca ccgccgtcag gcagcccttc aagcaggt 38
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<213>Artificial sequence
<220>
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<400> 4
acgcaccctt caccgtcaga aagactcggc gcctccaa 38
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<220>
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gtgcaagatg cgcgacgcac 20
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aacgcgggcc agctgaacat ttttgtgaag ttgacaggtt tgacaaagtg ttgagagggc 60
ccgtgatgcc aaccacttaa tcgcgaggtt tcgcgcagct cctagagccg cgctgcgggc 120
cctacgcctc tctttatctc gcagccgcac caccgccgca cactctgaaa aaggatggct 180
atgctggaac tggtgatcgg acccatgttc gcgggcaaga gcacagagag ctgcaggcgg 240
ctggagcgtc tgtcctacag cgggcgacgc tgcatcgccg tcaagcacgc catagaccag 300
cgctacaccg aagagtccaa ggtggccatg cacagcggcg cgacctaccc ggccatctcc 360
gcgggttacc tgtacgaggt gatgcagcgt ctggaggaat acgacgccgt ggccgtcgac 420
gagggacagt tcttccccga cctctacgag ggagtcgtgc agctgctgac cgcgggcaag 480
tacgtgatcg tggcggcgct ggacggggac tttatgcagc agcccttcaa gcaggtgacg 540
gcgttggtgc ccatggcgga caagctggac aagctgacgg cggtgtgcat gaagtgcaag 600
atgcgcgacg cacccttcac cgtcagaatc tctcagggca cggacctggt ccaggttgga 660
ggcgccgagt cttaccaggc ggtgtgtcgt ccctgtctca cggggttcag gatggcccag 720
tacgagctgt acggtccgcc gcctcctcct cctgcgcata atctactggg tgcgcccatc 780
gtgtcagccg ctccacctcg ttcttgtaac atctatcctg tgatggtgtg tgtggaacca 840
ataaaataat gtgcgacttg aatatggttg tacgggtttt tttaacaaaa actaaactac 900
cgaaacacga aacacttgct ctgagcgact ttgcgtccaa tactttaaaa aaaaggagat 960
attaaatata gttcaaacgt ttattgggat acacaca 997
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aacagacatc atgtctatca tcagctacat cgcattcctt ttgctaattg actccacatt 60
tggaatcccc atatttgttc catccgggca gaatatatca tggcaacctg taattcagcc 120
atttgattat caatgtccaa tacacggaaa tctacctaac acaatgggat tgagtgccac 180
caaattgaca ataaaatctc catctgtctt cagtacagat aaagtttctg gatggatctg 240
ccatgcagct gaatggaaaa caacttgtga ttacagatgg tacggacccc aatatataac 300
ccacagtatt catccaatca gtcctaccat agatgaatgc aagagaatca tttcaaggat 360
tgcatcagga actgatgaag atctggggtt tccccctcaa agttgcggat gggcatctgt 420
cacaacagtg tcaaatacta attacaaggt agtaccccat tctgttcatt tggagccgta 480
cggaggacac tggatcgatc atgaattcaa tgggggcgaa tgcagagaaa aagtgtgtga 540
aatgaaaggg aaccactcta tttggatcac agatgagacc gtgcagcatg aatgtgaaaa 600
gcacatagag gaagttgaag gaattatgta cgggaatgct ccgagagggg atgcaatata 660
tattaacaac tttattatag ataaacatca tagagtatac agattcgggg ggtcttgtcg 720
aatgaaattc tgtaataaag atggtataaa attcacaaga ggagactggg tagaaaaaac 780
agctgaaaca ttgacgaata tttatgcaaa tatacctgaa tgtgctgatg gaacgttggt 840
atctggtcac cgacctggat tagacttgat tgacacagtc ttcaatttgg aaaatgtggt 900
agaatatact ttgtgtgaag ggactaaaag aaaaatcaat aaccaagaaa agttgacgtc 960
agtggatttg agttatttgg ccccaagaat tggagggttc ggatcagtat tcagagtgag 1020
aaacggaaca ttagagagag ggagcactac ttatatcaag atagaagtag agggacctat 1080
tgtcgactcg ttgaatggaa cagatccgag aaccaacgcc tcaagagtat tttgggacga 1140
ctgggagtta gatggcaata tatatcaggg ctttaatggt gtatataaag ggaaagatgg 1200
gaagatccat attcccttga atatgataga atcaggaatc atagatgatg aacttcaaca 1260
tgctttccaa gccgatatta tccctcatcc tcattatgac gacgatgaaa tccgagagga 1320
cgatatattc ttcgataata ctggagaaaa tggaaatccc gtggatgcag tggtagaatg 1380
ggtcagtggg tggggaacta gtctaaaatt ctttggcacg actctggtcg ccctgatttt 1440
gatctttctg ctcatcaggt gctgtgttgc ttgcacttat ttgatgaaga agagtaaacg 1500
gcctgcaaca gaatcacacg aaatgcggtc cttcgtttga gagatagcaa attttaagca 1560
aagaccaaga tattatctta ataggtgtat gaaaaaaa 1598
<210> 8
<211> 1560
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 8
gtaatttaga cctttcctat catccgcggt atcaaccagt ctttaacatg tatctggaac 60
tgttcatcgc tgtgatgtgg cacttcatgc tcgctttctg cgcgtatgtg tggtacatac 120
atggtcctac ccttcgacgc ctctaccaac gcctgactgc tactactgct aacgcaatct 180
gccaaacgga ctctacttac gtctgcactg caactgtttc caccatgaca gatgatctga 240
ctaacgcttc ctcttccgcc tccacctcca gtactgcttc ctcggctacg accagttttg 300
tagctctgcc tcgtctgtcg cttgatgatt tgaagaccca ttccaaattc cgatggctgt 360
ggttttctaa taacccatta accattgact tatccactga cgaactcacg actcctttca 420
atagtcctca gcttcatgat gtcctgttca tgcctcaata tactctcgca gcttatccct 480
atatgcatcg ctccctccat ttggcgctct actttcgctt cggctataac atgcgctacc 540
tgctacgcga acatcctggc attccaccgc ttcgcgtcac ctgcccatct atgcgtgtgc 600
tattcatgga caaacagcgc aataaattgg aagagcctgt ctgttctatt tgtcgcctgt 660
ggattgtgga cggccctcag cttcccatcg gtaactgtgg cagaaagtac aaagtccatg 720
agatattcaa cgctttcagt ctcgagcctc accatattgt aaatgctttt gacgtcatcc 780
caatcatctc tctcctcaaa tgcctgcaac tggatatctc tatgctggcc ggcttgtctc 840
tcaccgcatt gacgcagctt tcagaattat caagagatcc agacgttaca cccgctggct 900
ttcttgctgc tattcaattc gatggtcaca cttccaccgt ggattgccgt ggcgatcgtt 960
tatattcaca tcctatgtta ctgatcttgt ttgctttgtt ttgctaccgt cgccgttccc 1020
gtgtttattt ccgtgaatgc tacttgcgcg actaccgccg cttgacttca tccctacccg 1080
atatggttcc ctactcccgc accccaggca aggcaaaggc taagagccca taaatgtaat 1140
ggcaaggtct agtgtaaata tgtacataaa tgttgaatat cattacgctt tgcgatgctt 1200
agagtataac aaaaatgaaa aactacataa aaacttcaaa acaagatatg tgtataacca 1260
agctgtatat gttcgctgaa aaatcaaaaa gatcaagctt cgcaaaacta tattgtttat 1320
cgctgtatat gtacgcataa aaaccaaaaa gaacaaatat attcatcacg ctccatgtta 1380
atacactggc attccatatc ctagcgcggt cactgtaaat atgtataaca cggacgatat 1440
taacgtcctc actgtaaata cgtacagcac agatgcaata gagaggactg tttgatagcg 1500
ggtggcgtgt tttcgacagc ctgcgggtgg caacgcatgt acagagggtc tatttgcatc 1560
Claims (10)
1. it is a kind of detect Koi herpesvirus ring mediated isothermal amplification primer set, by upstream outer primer, downstream outer primer, on
Swim inner primer and downstream inner primer composition；
The nucleotides sequence of the upstream outer primer is classified as sequence 1；
The nucleotides sequence of the downstream outer primer is classified as sequence 2；
The nucleotides sequence of the upstream inner primer is classified as sequence 3；
The nucleotides sequence of the downstream inner primer is classified as sequence 4.
2. primer set according to claim 1, it is characterised in that：The upstream outer primer, the downstream outer primer, institute
The mol ratio for stating upstream inner primer and the downstream inner primer is 1:1:8:8；
Or the upstream inner primer or downstream inner primer mark biotin.
3. a kind of ring mediated isothermal amplification reagent for detecting Koi herpesvirus, including complete described in claim 1 or 2 are drawn
Thing, PCR amplification buffers and archaeal dna polymerase；
The concentration of the upstream outer primer and the downstream outer primer is 0.2 μm of ol/L；
The concentration of the upstream inner primer and the downstream inner primer is 1.6 μm of ol/L.
4. a kind of reagent for detecting Koi herpesvirus, is made up of the primer set and probe described in claim 1 or 2；
The nucleotides sequence of the probe is classified as sequence 5,
Or the 5' ends end fluorescein isothiocynate FITC marks of the probe.
5. a kind of kit for detecting Koi herpesvirus, including primer set or claim 3 described in claim 1 or 2
Reagent described in described ring mediated isothermal amplification reagent or claim 4.
6. ring mediated isothermal amplification reagent described in primer set or claim 3 or claim described in claim 1 or 2
Application of the kit described in reagent or claim 5 in detecting or aiding in detection Koi herpesvirus described in 4；
Or, the ring mediated isothermal amplification reagent or right described in the primer set or claim 3 described in claim 1 or 2 will
The kit described in reagent or the claim 5 described in 4 is sought in detection or auxiliary detection Koi herpesvirus product is prepared
Using；
Or ring mediated isothermal amplification reagent or claim described in the primer set or claim 3 described in claim 1 or 2
The kit described in reagent or claim 5 described in 4 is detecting or aided in whether detection sample to be tested infects fancy carp blister sore
Application in poison；
Or, the ring mediated isothermal amplification reagent or right described in the primer set or claim 3 described in claim 1 or 2 will
The kit described in reagent or claim 5 described in 4 is asked to prepare detection or aiding in whether detection sample to be tested infects fancy carp
Application in herpesviral product.
7. a kind of method for detecting or aiding in detection sample to be tested whether to infect Koi herpesvirus, comprises the following steps：
1) ring mediated isothermal amplification is carried out to sample to be tested with the primer set described in claim 1 or 2, obtains ring mediated isothermal
Amplified production；
2) hybridize the ring mediated isothermal amplification product with the probe in the reagent described in claim 4, obtain hybridization production
Thing；
3) hybrid product described in lateral flow ELISA test strip, it is described to treat test sample if the detection band of the reagent strip develops the color 2
This infection or candidate infection KHV, if the reagent strip detection band develop the color 1, the sample to be tested be uninfected by or candidate not
Infect KHV.
8. a kind of method for detecting or aiding in detection sample to be tested whether to infect Koi herpesvirus, comprises the following steps：
1) ring mediated isothermal amplification is carried out to sample to be tested with the primer set described in claim 1 or 2, obtains ring mediated isothermal
Amplified production；
2) ring mediated isothermal amplification product described in electrophoresis detection, if containing amplified production, the sample to be tested infection or candidate
KHV is infected, if not containing the amplified production, the sample to be tested is uninfected by or candidate is uninfected by KHV.
9. the method according to claim 7 or 8, it is characterised in that：The template of the ring mediated isothermal amplification is to contain KHV
The material of gene-specific fragments.
10. according to any described method in claim 7-9, it is characterised in that：
The condition of the ring mediated isothermal amplification is 63 DEG C of water-bath 45min；
Or 63 DEG C of hybridization 5min of the condition of the hybridization.
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| CN201710569444.0A CN107287351A (en) | 2017-07-13 | 2017-07-13 | Ring mediated isothermal amplification combination lateral flow ELISA test strip Koi herpesvirus |
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| CN201710569444.0A CN107287351A (en) | 2017-07-13 | 2017-07-13 | Ring mediated isothermal amplification combination lateral flow ELISA test strip Koi herpesvirus |
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| CN201710569444.0A Pending CN107287351A (en) | 2017-07-13 | 2017-07-13 | Ring mediated isothermal amplification combination lateral flow ELISA test strip Koi herpesvirus |
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| CN108330216A (en) * | 2018-05-22 | 2018-07-27 | 北京科弘生物技术有限公司 | Ring mediated isothermal amplification combination lateral flow ELISA test strip grass carp reovirus |
| CN109913583A (en) * | 2018-12-30 | 2019-06-21 | 广州动佰生物科技有限公司 | A kind of primer and its method of quick detection 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) |
| CN110964848A (en) * | 2019-10-29 | 2020-04-07 | 北京市水产技术推广站 | RAA amplification primer and probe for rapidly detecting carp herpesvirus II, detection kit and use method |
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| CN110964848A (en) * | 2019-10-29 | 2020-04-07 | 北京市水产技术推广站 | RAA amplification primer and probe for rapidly detecting carp herpesvirus II, detection kit and use method |
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