KR20170025453A - Primer set for diagnosing honeybee virus-diseases, method for diagnosing honeybee viral diseases by using the same and kit using the same - Google Patents
Primer set for diagnosing honeybee virus-diseases, method for diagnosing honeybee viral diseases by using the same and kit using the same Download PDFInfo
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Abstract
The present invention relates to a method for the treatment of acute bee paralysis virus (ABPV) and acute paralytic virus (SBP), black queen bee venom virus (BQCV), cashmere virus (KBV) The present invention relates to a primer set for diagnosing viral diseases of a bee, a method for simultaneously diagnosing a virus disease of a bee using the same, and a kit for simultaneous diagnosis of a virus disease of a bee including the same. The time and cost of diagnosing the virus of a species can be reduced and it can be usefully used to diagnose a large number of samples.
Description
The present invention relates to a primer set for the detection of bee virus disease, a method and a kit for diagnosing bee virus disease using the same, and more particularly, to a primer set for diagnosing bee virus disease, which comprises SBE virus, black queen bee venom virus (BQCV) A method for simultaneous diagnosis of a viral disease of a bee using the primer set for diagnosing a viral disease of a bee composed of a wing disease virus (DWV), an Israeli acute paralysis virus (IAPV) and an acute bee paralysis virus (ABPV) The present invention relates to a kit for simultaneous diagnosis of viral diseases in a bee containing a virus.
There are more than 18 species of bee diseases caused by viruses. Especially, the major virus causing great damage to Korean beekeeping farmers is Sacbrood virus (SBV), which has been causing great damage to Korea since 2009, The virus has been identified as an infectious virus (BSE), an infectious virus (BSE), a black queen cell virus (BQCV), Kashmir bee virus (KBV), Deformed wing virus (DWV), Israeli acute paralysis virus The detection rate of viral diseases in domestic bees has been increasing steadily (Korean J. Apiculture 23 (2); 153-159 (2008)), and acute bee paralysis virus (ABPV).
Among the major bee viruses, it is a viral infectious disease caused by bee larvae from spring to summer, when the larvae grow from the incubator. The infected larvae become rotten and become swollen due to the fluid filled in the body like water pockets, It is also called chungju money bottle because it is dried.
And the black queen bee viral disease is a viral infectious disease infected only with the queen bee larva, and it is a disease that rot rots down to become a pupa. The wing disease is mainly spread by the bee mite (Varroa Destructor), and the bee pupa that is infected with the virus is born with a shrunken wing and shows paralysis of the legs and trunk.
The virus is caused by the worm disease, which belongs to Picornaviridae as an RNA-type virus. The viruses responsible for the Kashmir virus, the Israeli acute paralysis virus and the acute bee paralysis virus belong to the Dicistroviridae virus type.
They are also known to be infected by bee mite (Varroa Destructor). When they are infected with these viruses, bees can not fly and appear to crawl, wing and body swing abnormally, abdomen to swell, The surface can look extraordinarily smooth.
However, this is largely unobservable, and if the symptoms become noticeably worse, the rod is annihilated, so the infected rod must be incinerated to remove the infectious agent, and measures should be taken to supply the bee with sufficient nutrition.
Until now, no appropriate treatment for viral diseases has been proposed, and only the strong group and maintenance are known to be the only precautions that can lead to natural treatment.
In general, honeybees are mixed with more than one disease. It is very difficult to distinguish viral diseases from symptoms alone. If swift prescriptions are not made, infected swamps will suffer collapsing damages, resulting in a decisive damage to their productivity.
As a conventional bee disease diagnosis method, MLPA (Multiplex Ligation-dependent Probe Amplification) analysis (DeSmet L1, Ravoet J, de Miranda JR, Wenseleers T, Mueller MY, Moritz RF, de Graaf DC., 2012, BeeDoctor, a versatile MLPA-based diagnostic tool for screening bee viruses.Plos ONE 7 (10): e47953. doi: 10.1371 / journal.pone.0047953.), microarray (Glover RH1, Adams IP, Budge G, Wilkins S , Boonham N., 2011. Detection of honey bee (Apis mellifera) viruses with an oligonucleotide microarray.J Invertebr Pathol. 2011 Jul; 107 (3): 216-9.), PCR (Ploymerase chain reaction) Among them, the MLPA analysis and the microarray diagnosis method require very sophisticated technology and high cost.
On the other hand, the PCR method is simple and can be practiced in most laboratories, and has been mainly used to diagnose bee disease. Since the general PCR method uses a single target RT-PCR method that can diagnose only one type of disease using one primer set (Japanese Patent Application Laid-Open No. 10-2010-0024539) To diagnose a species of bee disease, PCR for each disease should be performed several times.
Since the vast majority of these infectious diseases, viral diseases, are required to be continuously monitored because of the time and expense required for a large number of samples to be pathologically evaluated using the above methods, a faster diagnosis method of disease is urgently required .
While researching methods to simultaneously diagnose six major viruses causing diseases in bees (SBV, BQCV, KBV, DWV, IAPV, and ABPV), we set up a primer set specific for six bee viruses By establishing optimal conditions of the multiplex reverse transcription polymerase chain reaction (Multiplex RT-PCR) method which can reduce the interference between the 6 types of primers and enable pathological evaluation even in a small amount of samples, .
Accordingly, it is an object of the present invention to provide a primer set capable of accurately, promptly, and effectively diagnosing six major viruses (SBV, BQCV, KBV, DWV, IAPV, and ABPV) causing diseases in bees.
It is another object of the present invention to provide a method for rapidly and effectively diagnosing bee virus disease using the primer set.
Yet another object of the present invention is to provide a kit that can quickly and effectively diagnose bee virus disease by including the primer set.
In order to achieve the above object, the present invention provides a primer pair comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2; A primer pair consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4; A primer pair consisting of the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6; A primer pair consisting of the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8; A pair of diagnostic primers consisting of the forward primer of SEQ ID NO: 9 and the reverse primer of SEQ ID NO: 10; And a primer pair consisting of a forward primer of SEQ ID NO: 11 and a reverse primer of SEQ ID NO: 12, and a primer set for the detection of bee virus disease.
In addition, the present invention provides a method for diagnosing bee virus disease using the primer set.
Further, the present invention provides a kit for the diagnosis of bee virus disease comprising the primer set.
A kit including simultaneous diagnosis of bees disease using the above primer set, and a kit including the primer set, which are used for simultaneous diagnosis of six major viruses (SBV, BQCV, KBV, DWV, IAPV and ABPV) Can exert an excellent effect of specifically detecting the bee virus and can save time and cost consumed thereon and thus can be usefully used for the pathology of various samples.
Figure 1 shows the results of single target RT-PCR and multiplex RT-PCR of six bee viral diseases.
Fig. 2 shows the sensitivity test results of six kinds of bee viruses.
Figure 3 shows the sensitivity test results for the multiplex bee disease virus Multiplex RT-PCR.
FIGS. 4A and 4B show the nucleotide sequence analysis results of the multiplex PCR products of 6 kinds of honey bee disease viruses.
FIG. 5 shows the result of applying the Multiplex RT-PCR method to six major viral diseases of bees in the domestic sample diagnosis.
A primer pair consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2; A primer pair consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4; A primer pair consisting of the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6; A primer pair consisting of the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8; A pair of diagnostic primers consisting of the forward primer of SEQ ID NO: 9 and the reverse primer of SEQ ID NO: 10; And a primer pair consisting of the forward primer of SEQ ID NO: 11 and the reverse primer of SEQ ID NO: 12, and a primer set for the detection of bee virus disease.
In the primer set for the detection of bee virus disease according to the present invention, the primer pair consisting of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 may be used for diagnosing insect blooding virus (SBV).
In the primer set for the bee virus disease diagnosis of the present invention, the primer pair consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4 may be used for diagnosis of black queen bee virus (BQCV).
In the primer set for diagnosing bee virus disease according to the present invention, the primer pair consisting of the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6 may be used for diagnosis of cashmere virus (KBV).
In the primer set for the bee virus disease diagnosis of the present invention, the primer pair consisting of the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8 is for diagnosis of wing disease virus (DWV) have.
In the primer set for the detection of bee virus disease according to the present invention, the primer pair consisting of the forward primer of SEQ ID NO: 9 and the reverse primer of SEQ ID NO: 10 may be used for the diagnosis of Israel Acute Paralytic Virus (IAPV).
In the primer set for the bee virus disease diagnosis of the present invention, the primer pair consisting of the forward primer of SEQ ID NO: 11 and the reverse primer of SEQ ID NO: 12 may be used for the diagnosis of acute bee paralysis virus (ABPV).
In the primer set for the bee virus disease diagnosis of the present invention, the primer pair consisting of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 can generate the amplification product of SEQ ID NO: 13 (192 bp).
In the primer set for the bee virus disease diagnosis of the present invention, the primer pair consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4 can generate the amplification product of SEQ ID NO: 14 (317 bp).
In the primer set for the bee virus disease diagnosis of the present invention, the primer pair consisting of the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6 can generate the amplification product of SEQ ID NO: 15 (412 bp).
In the primer set for the bee virus disease diagnosis of the present invention, the primer pair consisting of the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8 can generate the amplification product of SEQ ID NO: 16 (479 bp).
In the primer set for the bee virus disease diagnosis of the present invention, the primer pair consisting of the forward primer of SEQ ID NO: 9 and the reverse primer of SEQ ID NO: 10 can generate the amplification product of SEQ ID NO: 17 (725 bp).
In the primer set for the bee virus disease diagnosis of the present invention, the primer pair consisting of the forward primer of SEQ ID NO: 11 and the reverse primer of SEQ ID NO: 12 can generate the amplification product of SEQ ID NO: 18 (900 bp).
The present invention also relates to a method for diagnosing bee virus disease using the above-described primer set.
The method for diagnosing the bee virus disease of the present invention is preferably a multiplex RT-PCR method which is more advanced than the single target RT-PCR method which can diagnose only one disease in a single experiment, This method is a PCR method capable of simultaneously diagnosing various diseases at one time.
However, in the multiple RT-PCR, since the PCR is performed using several kinds of primers in a single tube, interference may occur between the primers. As the number of the primers increases, the possibility increases , It is important to establish optimal conditions by modifying various conditions in performing PCR.
The method for diagnosing the bee virus disease according to the present invention comprises the steps of (a) extracting genomic DNA from a sample, (b) extracting the template DNA extracted in the step (a) with the primer set using Multiplex RT- PCR), and (c) fractionating the amplification product produced by the step (c) to diagnose bee virus disease.
In the method for diagnosing the bee virus disease according to the present invention, the bee virus disease is selected from the group consisting of SBV, black queen beetle virus (BQCV), cashmere virus (KBV), wing disease virus (DWV) Acute paralytic virus (IAPV) and acute bee paralysis virus (ABPV).
In the method for diagnosing the bee virus disease according to the present invention, the reverse transcription polymerase chain reaction is a method for selectively amplifying a specific DNA fragment, which is disclosed in Miller, HI (WO 89/06700) Which is incorporated herein by reference.
In the method for diagnosing the bee virus disease according to the present invention, the reverse transcription polymerase chain reaction can be performed using various DNA polymerase enzymes in the amplification reaction, for example, a "Klenow fragment" of E. coli DNA polymerase I, , Thermostable DNA polymerase and bacteriophage T7 DNA polymerase.
In the method for diagnosing the bee virus disease of the present invention, preferably, the polymerase is a thermostable DNA polymerase obtainable from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu).
In the method for diagnosing the bee virus disease of the present invention, the amplification reaction solution may include a dNTP mixture (dATP, dCTP, dGTP, dTTP), a PCR buffer, and a DNA polymerase joiner.
On the other hand, in the method for diagnosing bee virus disease according to the present invention, it is preferable to provide an excessive amount of components necessary for the reaction in the reaction vessel when the amplification reaction by the polymerase chain reaction is carried out. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component.
In the method of diagnosing the bee disease virus of the present invention, it is required to provide to the reaction mixture enough to achieve a desired degree of amplification is the joinja, dATP, dCTP, dGTP and dTTP, such as Mg + 2. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, it is desirable that the buffer make all enzymes close to optimal reaction conditions.
Therefore, in the method for diagnosing the bee virus disease of the present invention, the amplification process can be carried out in a single reaction without changing the conditions such as addition of reactants.
The present invention also relates to a bee virus disease diagnosis kit comprising the above-described primer set.
In the kit for diagnosing bee virus disease according to the present invention, the bee viral disease is selected from the group consisting of SBV, BQCV, KBV, DWV, The disease virus (IAPV) and the acute bee paralysis virus (ABPV).
When the bee virus disease diagnosis kit of the present invention is applied to a PCR amplification process, a reagent necessary for PCR amplification such as a buffer, a DNA polymerase (for example, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermisflavus , Thermostable DNA polymerase from Thermococcus literalis or Pyrococcus furiosus (Pfu), DNA polymerase joins and dNTPs, and the like.
The bee virus disease diagnostic kit of the present invention can be produced from a number of separate packaging or compartments containing the reagent components described above.
Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following examples, but includes modifications of equivalent technical ideas.
< Example 1> Major Bee disease virus for six species primer set
The major viruses that cause bee diseases are Sacbrood virus (SBV), Black queen cell virus (BQCV), Kashmir bee virus (KBV), Deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), Acute bee paralysis virus ) Was established.
Herein, 5 kinds of primer sets (SBV, BQCV, KBV, DWV, ABPV) were selected by considering the PCR product size and annealing temperature amplified by referring to related papers , And one species (IAPV) was constructed in-house and then established a primer set of Multiplex RT-PCR for simultaneous diagnosis of six bee virus diseases.
After securing positive samples for each virus, Single target RT-PCR was performed using viral primers, and amplification products were electrophoresed to confirm the size of the DNA.
As a result of analysis of the nucleotide sequence of the amplified DNA, it was confirmed that it coincided with each viral genetic information, and it was cloned and used as positive DNA for each bee virus. The primer sequences used in this experiment are shown in Table 1.
The following primer sets were selected so that the amplified major bee virus DNAs were different in size, and electrophoresis was performed on the agarose gel, so that the amplification product of six kinds of bee virus DNA was clearly identified.
< Example 2> Bee's Multiplex RT-PCR for six major viral diseases PCR Establish condition
In order to establish the multiplex PCR RT-PCR conditions using the 6-virus primer of Example 1, gradient PCR was performed to find the annealing temperature at which all six virus DNAs can be amplified.
As shown in FIG. 1, when single-target PCR was performed for each virus, and 6 kinds of viral DNAs were mixed and simultaneously multiplexed and PCR was performed and confirmed by electrophoresis, the SBV was 192 bp, the BQCV was 317 bp, KBV was 412 bp, DWV was 479 bp, IAPV was 725 bp, and ABPV was 900 bp.
In addition, when the nucleotide sequences of the six amplification products obtained by multiplex PCR were analyzed as shown in FIGS. 4A and 4B, it was confirmed that they corresponded to the genetic information of six kinds of bee viruses.
In order to confirm the minimum virus DNA concentration that each virus primer can amplify, the sensitivity of each virus primer was tested using single target PCR.
As shown in FIG. 2, each virus DNA was diluted 10-fold with 10-fold dilution, and PCR was performed for sensitivity test. As a result, the DNA of SBV was 100 gg, the BQCV was 100 fg, the DWV was 100 fg, the KBV was 100 gg, , And ABPV was found to be capable of pathological emotion up to a concentration of 100 fg.
Multiplex PCR was performed to confirm the minimum concentration of DNA that can be simultaneously diagnosed using six virus primers mixed with six viruses.
For the sensitivity test of the six virus primers, six kinds of viral clones were diluted 10-fold (10 -3 / / ~ to 10 -9 / / ㎕) with 6 kinds of viral clones mixed, RT-PCR was performed.
As shown in FIG. 3, when 6 bees virus-positive DNAs mixed at the same concentration were diluted to 10 -6 / /,, that is, 1 pg concentration, 6 simultaneous diagnoses were confirmed.
Table 2 below shows the composition of Multipkex RT PCR for simultaneous diagnosis of bee diseases. Table 3 shows the optimal PCR conditions for multiplex PCR.
< Example 3> Multiplex RT-PCR method for six major viral diseases of bees in domestic samples
In order to confirm whether the Multiplex RT PCR method established using the primer set of the present invention can be applied, RNA was extracted from a bee sample collected in Korea, and then, using single target RT-PCR, The pathogenesis of bee viral diseases (SBV, BQCV, KBV, DWV, IAPV, ABPV) was performed.
After securing the positive bee samples, Multiplex PCR was used to simultaneously detect six bee viral diseases.
Then, we verified whether the results of honey bee pathological emotion using Multiplex PCR for the domestic bee samples were consistent with the results of single target PCR.
As shown in FIG. 5, the pathological empirical results using Multiplex PCR showed a slightly lower amplification rate compared to the pathological empirical results using single target PCR, but the results of most pathological emotions agreed with each other.
It will be understood by those skilled in the art that various modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. And changes may be made without departing from the spirit and scope of the invention.
<110> Republic of Korea <120> Primer set for diagnosing honeybee virus-diseases, method for diagnosing honeybee viral diseases by using the same and kit using the same <130> 10173 <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for SBV <400> 1 cattgcatgg tttaaaacag t 21 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for SBV <400> 2 gcggtaaata agcactcga 19 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for BQCV <400> 3 ggagatgtat gcgctttatc gag 23 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for BQCV <400> 4 caccaaccgc ataatagcga ttg 23 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for KBV <400> 5 gatgaacgtc gacctattga 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for KBV <400> 6 tgtgggtggc tatgagtca 19 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for DWV <400> 7 tcatcttcaa ctcggctttc tacg 24 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for DWV <400> 8 cgaatcattt tcacgggacg 20 <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer for IAPV <400> 9 gatttgagag atgtatttcc ttctgcgg 28 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for IAPV <400> 10 acacttgcgt tggtcctgaa tgttaatgg 29 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for ABPV <400> 11 ttatgtgtcc agagactgta tcca 24 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for ABPV <400> 12 gctcctattg ctcggttttt cggt 24 <210> 13 <211> 192 <212> DNA <213> Artificial Sequence <220> <223> PCR Product of SBV <400> 13 gcggtaaata agcactcgaa tactcattcc aactattaga aacattcgcg ggcaggaatc 60 tatgtggatt atctgattcc cttcttggtc tatttggcgg tttaactgga tcataaccat 120 caggtggaaa actatctatg ggtaccacac ttcttgatgg ccaataagtt gactgtttta 180 aaccatgcaa tg 192 <210> 14 <211> 317 <212> DNA <213> Artificial Sequence <220> <223> PCR Product of BQCV <400> 14 ggagatgtat gcgctttatc gaggaggagt tcgagttaaa gttgtcactg agaagggtgt 60 ggatttcgtc agagctaccg ttagtcctca acagacttac ggcagtgaag tcgctcctac 120 tactcatatc agtactcctt tggcaataga acaaatacct acaaagggag tcgcagagtt 180 ccaaataccg tactatgctc catgtttgtc atcttcgttt agagcgaatt cggaaacatt 240 ttactatagt tcaggtcgga ataatctcga tatagccact tcacctcctt ccatcaatcg 300 ctattatgcg gttggtg 317 <210> 15 <211> 412 <212> DNA <213> Artificial Sequence <220> <223> PCR Product of KBV <400> 15 atgaacgtcg acctattgaa aaagttaatc gattgaaaac acgagtattc tcaaatggac 60 caatggattt ctctatagct tttcgaatgt attatttggg ctttatagct catttgatgg 120 aaaatcgaat tactaatgag gtgtccatcg gaacgaatgt gtattctcaa gactggagta 180 aaactgttcg caagttgacc aaatttggaa ataaagttat tgcaggtgat ttttcaactt 240 ttgatggatc actgaatgta tgtattatgg aaaaatttgc agatttagcg aatgagtttt 300 ataatgatgg aaaagaaaac aatctgatca gacacgtgtt gttgatggat gtgtacaatt 360 ctgtacatat ctgcaacgac tccgtgtata tgatgactca tagccaccca ca 412 <210> 16 <211> 479 <212> DNA <213> Artificial Sequence <220> <223> PCR Product of DWV <400> 16 tcatcttcaa ctcggctttc tacggatgac gtcaagttat ataaaacgat tagcatgttg 60 catcaaaaat atgatacctc agagtgtgcc aaatgtcaac attggtatgc tccgttgact 120 gatatctacg ttgatgacaa gaaattgttt tggtgtcaga aagagaaaaa gacacttatt 180 gatgtccgaa agttgtcgaa agaagatgtg actgttcaat caaaattgat taatttgtct 240 gttccttgtg gtgaagtgtg tgtgttacat tcaaaatatt ttaattatct tttccataaa 300 gcatggttgt gtgagaaccc aacttggcgc ttaatatata atggtaccaa gaaaggtatg 360 cccgagtatt ttatgaattg tgtggatgaa atttcattag attctaaatt tggtaaagtg 420 aaagtgtggt tgcaagcgat cattgataag tatttaactc gtcccgtgaa aatgattcg 479 <210> 17 <211> 725 <212> DNA <213> Artificial Sequence <220> <223> PCR Product of IAPV <400> 17 gatttgagag atgtatttcc ttctgcggtt gatgaaatgg ctatagggta tgtttgcggc 60 aatccagctg tgaaacatgt tcttacctgg aagatgactg acgcaattca gaaaccaata 120 gcaaacggag atgattgggg tggagttata ccagtgggaa tgcctggtta ttctaaatcc 180 attagaacta caagtatttc agaaacggaa aatcgtgaaa ctgaagtcat agatgccgct 240 ccatgtgaat atgttgctaa catgttctcg tattggcgtg caaccatgtg ttataggatt 300 accgtggtga agacagcttt tcatactggc agactcgaga ttttctttga accaggagtg 360 atacccgtca aacccactgt taataacatt gggcccgatc aggatcaact tacaggagcg 420 gtggctcctt ccgataataa ctataagtac attttggacc tgactaatga tacagaagtt 480 acaatacgtg ttccttttgt ttcaaataag atgttcctta agactgctgg agtctatggt 540 gctaatagtg aaaataactg gaactttcat gagtccttta gtggattctt atgtataaga 600 ccagtcacta aattgatggc tcctgatact gtgtctgaca atgtatctat agttgtttgg 660 aagtgggcag aagatgtagt agtagtagaa ccgaaaccat taacattcag gaccaacgca 720 agtgt 725 <210> 18 <211> 900 <212> DNA <213> Artificial Sequence <220> <223> PCR Product of ABPV <400> 18 ttatgtgtcc agagactgta tccataatgt gtcgatagtt gtatggaagt gggctgaaga 60 tgtggtagta gtagaaccta agcctttact ttcaggacca acgcaagtgt ttcaaccacc 120 tgtaacatct gcagattcta tcaataccat agatgcttca atgcaaatta acttagcaaa 180 taaagctgat gaaaatgtag ttacattctt tgattctgat gatgctgaag aaaggaacat 240 ggaagcatta ttgaaaggaa gtggtgaaca aatcatgaat ttgagatcct tactaagaac 300 gtttaggacc atatcagaaa attggaattt accacctaac acaaaaacag caataacaga 360 tttgactgac gttgcggata aagaaggtag ggattatatg tcttatttat catacatcta 420 tagattctat agaggaggga gaagatataa gtttttcaat acaacagctt tgaaacaatc 480 tcaaacttgc tatgttcgta gcttcttaat accgcgatat tatactgctg ataacacaaa 540 caacgatgga ccttcacata taacatatcc agttcttaat ccagttcatg aagtagaagt 600 accatactat tgtcaatata ggaaattacc agtagcatct acaaccgaca aagggtatga 660 tgcatctttg atgattattatt ctaatgttgg taccaatcaa attgttgctc gagctggtaa 720 tgatgatttc acttttggat ggctcatagg aacaccgcaa actcaaggaa taacgagaac 780 ggaaactaaa taaatgaatg aagatgtact ttagaggaca gacataccct cttttgtatg 840 gctatagtct aaatttttca gataatttca atttggaccg aaaaaccgag caataggagc 900 900
Claims (12)
(a) extracting genomic DNA from a sample;
(b) amplifying the template DNA extracted in the above step using the primer set by Multiplex RT-PCR; And
(c) fractionating the amplification product produced by said step to diagnose a bee virus disease.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108318683A (en) * | 2018-03-27 | 2018-07-24 | 中国农业科学院蜜蜂研究所 | A kind of quick diagnosis Israel acute paralysis virus test paper and its application |
| KR20190028274A (en) * | 2017-09-08 | 2019-03-18 | 대한민국(농림축산식품부 농림축산검역본부장) | A primer for the diagnosis of parasitic and bacterial diseases of bees, a method and kit for diagnosing parasitic and bacterial diseases of bees using the same |
| KR20200025622A (en) | 2018-08-31 | 2020-03-10 | 주식회사 제넷바이오 | Composition for diagnosis of Korean sacbrood virus infectious and diagnostic kit containing thereof |
| CN112538548A (en) * | 2020-12-04 | 2021-03-23 | 福州海关技术中心 | Primer group and probe for detecting bee Klishmi virus and detection method |
| KR20230036318A (en) | 2021-09-07 | 2023-03-14 | 경기도 | Diagnostic Kits and Diagnostic Methods for Diagnosis of Bee Diseases such as Acute bee paralysis virus, Chronic bee paralysis virus, Deformed wing virus and Acarapis woodi |
| KR20230036317A (en) | 2021-09-07 | 2023-03-14 | 경기도 | Diagnostic Kits and Diagnostic Methods for Diagnosis of Bee Diseases such as Kashmir bee virus, Black queen cell virus, Israeli acute paralysis virus and Apocephalus borealis |
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2015
- 2015-08-28 KR KR1020150121907A patent/KR20170025453A/en active Search and Examination
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20190028274A (en) * | 2017-09-08 | 2019-03-18 | 대한민국(농림축산식품부 농림축산검역본부장) | A primer for the diagnosis of parasitic and bacterial diseases of bees, a method and kit for diagnosing parasitic and bacterial diseases of bees using the same |
| CN108318683A (en) * | 2018-03-27 | 2018-07-24 | 中国农业科学院蜜蜂研究所 | A kind of quick diagnosis Israel acute paralysis virus test paper and its application |
| KR20200025622A (en) | 2018-08-31 | 2020-03-10 | 주식회사 제넷바이오 | Composition for diagnosis of Korean sacbrood virus infectious and diagnostic kit containing thereof |
| CN112538548A (en) * | 2020-12-04 | 2021-03-23 | 福州海关技术中心 | Primer group and probe for detecting bee Klishmi virus and detection method |
| KR20230036318A (en) | 2021-09-07 | 2023-03-14 | 경기도 | Diagnostic Kits and Diagnostic Methods for Diagnosis of Bee Diseases such as Acute bee paralysis virus, Chronic bee paralysis virus, Deformed wing virus and Acarapis woodi |
| KR20230036317A (en) | 2021-09-07 | 2023-03-14 | 경기도 | Diagnostic Kits and Diagnostic Methods for Diagnosis of Bee Diseases such as Kashmir bee virus, Black queen cell virus, Israeli acute paralysis virus and Apocephalus borealis |
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