KR20210065702A - Method for detecting maritime virus, and primer set, composition and kit for the same - Google Patents

Abstract

The present invention relates to a method for detecting marine virus, and a primer set, composition and kit therefor, and more specifically, to a method for detecting marine virus, which comprises the steps of: a) isolating DNA or RNA from a sample; b) performing a polymerase chain reaction by using as a template, DNA isolated from the sample; or cDNA synthesized by using RNA isolated from the sample as a template, and by using a primer set selected from among a primer set consisting of a forward primer having a sequence of SEQ ID NO: 1 and a reverse primer having a sequence of SEQ ID NO: 2; and a primer set consisting of a forward primer having a sequence of SEQ ID NO: 3 and a reverse primer having a sequence of SEQ ID NO: 4; and c) detecting an amplification product including the forward primer and the reward primer from among the amplification product generated by the polymerase chain reaction.

Description

Marine virus detection method, and primer set, composition and kit therefor {Method for detecting maritime virus, and primer set, composition and kit for the same}

The present invention relates to a method for detecting a marine virus, and a primer set, composition and kit for the same.

Viral hemorrhagic septicemia virus (VHSV) is classified as Novirhabdovirus of Rhabdoviridae. A variety of marine and freshwater fish species are known to be susceptible to this VHSV. The virus can cause systemic hemorrhage in internal organs, skin, muscles and other fish tissues, and can cause serious damage to marine and freshwater fish farms. In recent years, fish farms have become common to house large quantities of fish, and VHSV can be transmitted horizontally through waterways. Severe outbreaks of VHSV have occurred in Europe, the Great Lakes of the United States and elsewhere, causing a worldwide problem.

Red sea bream iridovirus (RSIV) is one of the megalocytiviruses and has a large dsDNA genome that can infect a variety of vertebrates and invertebrates. RSIV infection has been found in freshwater and marine fish species in Korea, and, like VHSV, can be transmitted horizontally through water, resulting in a high infection rate.

Recently, molecular biological methods such as PCR or other amplification tools and serological methods based on antibodies, such as enzyme-linked immunosorbent assay (ELISA), have been used to rapidly detect most pathogens or microorganisms, Many molecular biological methods for detection have been developed. General PCR is a classic and reliable molecular biological method for detecting marine pathogens. However, PCR products have the disadvantage of having to be visualized by gel electrophoresis, which can be time-consuming and technically cumbersome.

Lateral flow assay (LFA) is a technique that uses a paper-based device to detect a target substance in a mixture, and results can be displayed in a short time (5 to 30 minutes). PCR-LFA is a method for rapidly detecting PCR products by combining PCR and LFA. The PCR product is then double-labeled by a gene-specific primer set. Detection is then carried out by recognizing this labeled PCR product on the surface of the sideflow strip carrying the test and control lines. PCR-LFA has several advantages. This method is simpler than the existing PCR-gel electrophoresis method, so it is possible only with a minimum of simple training personnel. In addition, PCR-LFA is capable of on-site diagnosis, enabling fast and easy detection of pathogens in the field environment.

The present inventors developed a method that can detect marine viruses such as VHSV and RSIV, which are serious problems as described above, using PCR, in particular, a method to which lateral flow analysis can be applied. It was intended to make it possible to more easily manage the contamination and spread of these viruses by making it possible to check the contamination.

Al-Hussinee, L, and Lumsden, J (2011): Detection of VHSV IVb within the gonads of Great Lakes fish using in situ hybridization. Diseases of Aquatic Organisms 95, 81-86.

Therefore, the main object of the present invention is to provide a method capable of detecting marine viruses such as VHSV and RSIV simply and quickly by using a method in which PCR, particularly lateral flow analysis, is applied together.

Another object of the present invention is to provide a primer set, a composition and a kit for application to the detection method as described above.

According to one aspect of the present invention, the present invention comprises the steps of: a) isolating DNA or RNA from a sample; b) DNA isolated from the sample; or cDNA synthesized using RNA isolated from a sample as a template; a primer set consisting of a forward primer consisting of the sequence of SEQ ID NO: 1 and a reverse primer consisting of the sequence of SEQ ID NO: 2; And a primer set consisting of a forward primer consisting of the sequence of SEQ ID NO: 3 and a reverse primer consisting of the sequence of SEQ ID NO: 4; performing a polymerase chain reaction using a primer set selected from among; and c) detecting an amplification product including the forward primer and the reverse primer among the amplification products generated by the polymerase chain reaction;

In the detection method of the present invention, the polymerase chain reaction is denaturation at 94 to 96°C for 20 to 40 seconds, annealing at 54 to 56°C for 20 to 40 seconds, and extension at 71 to 73°C for 20 to 40 seconds. It is preferable to carry out under the conditions of repeating 30 to 40 cycles.

In the detection method of the present invention, forward and reverse primers labeled with different labeling materials are used in step b), and both the labeling material of the forward primer and the labeling material of the reverse primer are included in step c). It is preferable to detect the amplification product.

In the detection method of the present invention, it is preferable to perform the detection in step c) using a side flow detection method.

According to another aspect of the present invention, the present invention provides a primer set for detecting marine viruses comprising a forward primer consisting of the sequence of SEQ ID NO: 1 and a reverse primer consisting of the sequence of SEQ ID NO: 2.

According to another aspect of the present invention, the present invention provides a primer set for detecting marine viruses comprising a forward primer consisting of the sequence of SEQ ID NO: 3 and a reverse primer consisting of the sequence of SEQ ID NO: 4.

In the primer set of the present invention, it is preferable that the forward primer and the reverse primer are labeled with different labeling substances.

It is preferable that the primer set of the present invention is for applying the lateral flow detection method.

According to another aspect of the present invention, the present invention provides a primer set consisting of a forward primer consisting of the sequence of SEQ ID NO: 1 and a reverse primer consisting of the sequence of SEQ ID NO: 2; And a primer set consisting of a forward primer consisting of the sequence of SEQ ID NO: 3 and a reverse primer consisting of the sequence of SEQ ID NO: 4; Provided is a composition for detecting marine viruses comprising a primer set selected from among.

In the composition of the present invention, the forward primer and the reverse primer are preferably labeled with different labeling substances.

The composition of the present invention is preferably for applying the lateral flow detection method.

According to another aspect of the present invention, the present invention provides a primer set consisting of a forward primer consisting of the sequence of SEQ ID NO: 1 and a reverse primer consisting of the sequence of SEQ ID NO: 2; And a primer set consisting of a forward primer consisting of the sequence of SEQ ID NO: 3 and a reverse primer consisting of the sequence of SEQ ID NO: 4; Provided is a kit for detecting marine viruses comprising a primer set selected from among.

In the kit of the present invention, the forward primer and the reverse primer are preferably labeled with different labels.

Preferably, the kit of the present invention is for applying the lateral flow detection method.

According to the present invention, marine viruses such as VHSV and RSIV can be detected simply, quickly and with high accuracy using PCR. In particular, the detection method, primer set, composition, and kit of the present invention can be applied to the side flow analysis method, thereby further enhancing the convenience of detection. Accordingly, it is expected that the detection method, primer set, composition and kit of the present invention can greatly contribute to preventing contamination and spread of marine viruses as described above.

1 shows the location of the primers of the present invention on the genome of the target virus. A: RSIV, B: VHSV.
Figure 2 shows the principle of PCR and side flow analysis applied to an embodiment of the present invention. A: PCR, B: side flow analysis.
3 shows the results by agarose gel electrophoresis after PCR according to an embodiment of the present invention. A: RSIV primer used, B: VHSV primer used, NTC: no template control, NNV: nervous necrosis virus, RSIV: red sea bream iridovirus, VHSV: viral hemorrhagic septicemia virus, EHNV: epizootic haematopoietic necrosis virus, IHNV: infectious haematopoietic necrosis virus, ISAV: infectious salmon anemia virus, KHV: koi herpesvirus, SAV: salmon alphavirus, SVC: spring viraemia of carp, IHHNV: infectious hypodermal and haematopoietic necrosis virus, WSSV: white spot syndrome virus, YHV: yellow head virus 1, MrNV: macrobrachium rosenbergii nodavirus, LSNV: laem-singh virus, CCV: channel catfish virus, GIV: grouper iridovirus, AbHV: abalone herpesvirus, OsHV: osteroid herpesvirus 1, BP: baculovirus penaei.
4 shows the results by LFA after PCR according to an embodiment of the present invention. A: RSIV primer used, B: VHSV primer used, NTC: no template control, NNV: nervous necrosis virus, RSIV: red sea bream iridovirus, VHSV: viral hemorrhagic septicemia virus, EHNV: epizootic haematopoietic necrosis virus, IHNV: infectious haematopoietic necrosis virus, ISAV: infectious salmon anemia virus, KHV: koi herpesvirus, SAV: salmon alphavirus, SVC: spring viraemia of carp, IHHNV: infectious hypodermal and haematopoietic necrosis virus, WSSV: white spot syndrome virus, YHV: yellow head virus 1, MrNV: macrobrachium rosenbergii nodavirus, LSNV: laem-singh virus, CCV: channel catfish virus, GIV: grouper iridovirus, AbHV: abalone herpesvirus, OsHV: osteroid herpesvirus 1, BP: baculovirus penaei.
5 shows the results by agarose gel electrophoresis or LFA after PCR by template DNA concentration according to an embodiment of the present invention. A: using RSIV primer, B: using VHSV primer, top: agarose gel electrophoresis result, bottom: LFA result, T: test line, C: control line.
6 shows the results by agarose gel electrophoresis or LFA after PCR according to an embodiment of the present invention targeting fish tissue. A: using RSIV primer, B: using VHSV primer, top: agarose gel electrophoresis result, bottom: LFA result, T: test line, C: control line.

Since the present invention can apply various transformations and can have various embodiments, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

The terms used in the present application are only used to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present application, terms such as “comprise” or “have” are intended to designate that a feature, number, step, operation, component, part, or combination thereof described in the specification exists, but one or more other features It should be understood that this does not preclude the existence or addition of numbers, steps, operations, components, parts, or combinations thereof.

Terms such as first, second, etc. may be used to describe various elements, but the elements should not be limited by the terms. The above terms are used only for the purpose of distinguishing one component from another.

The present invention provides a primer set (first primer set) comprising a forward primer consisting of the sequence of SEQ ID NO: 1 and a reverse primer consisting of the sequence of SEQ ID NO: 2 for detection of marine viruses; And a primer set consisting of a forward primer consisting of the sequence of SEQ ID NO: 3 and a reverse primer consisting of the sequence of SEQ ID NO: 4 (second primer set); It is characterized by using a primer set selected from among.

The primer of the present invention is designed to be suitable for polymerase chain reaction (PCR), and in particular, it can be applied to lateral flow assay (LFA), enabling very simple and rapid detection.

The forward and reverse primers of the present invention can specifically bind to DNA derived from a target marine virus and induce a polymerase to amplify between the binding sites of these primers.

The first primer set of the present invention is a primer set for detecting a marine virus, red sea bream iridovirus (RSIV), and the second primer set is a viral hemorrhagic septicemia virus (VHSV). It is designed as a primer set for

In the present invention, the forward primer and the reverse primer are preferably labeled with different types of labeling substances. If such a labeling material is used, the presence or absence of the target amplification product as described above can be more easily confirmed. In this case, the labeling material may be a conventional labeling material used for labeling oligonucleotides, for example, biotin and fluorescein amidites (FAM).

In the case of using the above labeling material, it can be easily applied to the side flow analysis method. For example, a side flow dipstick containing a detection material capable of detecting the label of the forward primer and a detection material capable of detecting the label of the reverse primer, thereby visually confirming the detection of each label. can be used In this case, the detection material may be, for example, an antibody against a labeling material.

The detection method of the present invention comprises the steps of a) isolating DNA or RNA from a sample; b) DNA isolated from the sample; or cDNA synthesized using RNA isolated from a sample as a template; performing PCR using the primers of the present invention as a template; and c) detecting an amplification product including the forward primer and the reverse primer among the amplification products generated by the PCR.

Step a) is a step for isolating DNA or RNA, which is the genome of the virus, from a sample suspected of being infected with a marine virus, and a conventional method used for isolating DNA or RNA from a sample may be applied.

Since RSIV is a DNA virus, DNA isolation is used to detect RSIV using the first primer set. Since VHSV is an RNA virus, RNA is isolated when VHSV is to be detected using the second primer set. method can be used. In addition, in the case of VHSV, it is necessary to add the step of isolating RNA and then synthesizing it into cDNA.

Step b) is a step for amplifying marine virus-derived DNA from the DNA isolated in step a) or the cDNA. It can be performed using a conventional PCR using a polymerase with the primer of the present invention. can

At this time, for more efficient amplification, PCR conditions were repeated 30 to 40 cycles of denaturation at 94 ~ 96 °C for 20 ~ 40 seconds, annealing at 54 ~ 56 °C for 20 ~ 40 seconds and extension at 71 ~ 73 °C for 20 ~ 40 seconds It is preferable to carry out under the following conditions. More preferably, denaturation at 94.5 to 95.5° C. for 25 to 35 seconds, annealing at 54.5 to 55.5° C. for 25 to 35 seconds, and extension at 71.5 to 72.5° C. for 25 to 35 seconds are repeated 33 to 37 cycles. it's good

Step c) is a step of detecting the target amplification product amplified in step b), and may be performed using a conventional method for detecting the amplification product amplified by a polymerase, for example, agarose gel electrophoresis. Electrophoresis, etc. can be used, but it is preferable to use the lateral flow analysis method for simpler and faster detection. In this case, since the target amplification product is a form including the forward and reverse primers of the present invention, it can be detected using the same.

The present invention provides a primer set, composition, and kit applicable to the method for detecting marine viruses as described above, including the primer.

In addition to the primers of the present invention, the composition for detecting marine viruses of the present invention may further include a buffer for stabilizing them, a reagent used for PCR, and the like, and a primer or probe for amplifying other targets.

The kit for detecting marine viruses of the present invention may further include reagents and instruments used for PCR in addition to the primers of the present invention, and may also include primers or probes for amplification of other targets. In particular, it is preferable to include a device capable of rapidly detecting a target amplification product in a simple manner, such as a side flow dipstick.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and therefore, the scope of the present invention should not be construed as being limited by these examples.

[Example]

1. Method

1-1. Template DNA preparation

The surface protein (CP) gene of red sea bream iridovirus (RSIV) was used as a target gene for RSIV detection using PCR-LFA. CP is one of the important and common proteins in many viruses. Since the protein is produced during viral infection, the gene is an excellent candidate for target detection. The sequence of the complete CP gene was confirmed from a number of RSIV strains, and the conserved region was used as the target gene. This gene was synthesized by requesting Macrogen. After synthesis, the RSIV CP gene was cloned into the pGEM T-easy vector and inserted (see FIG. 1).

In the case of viral hemorrhagic septicemia virus (VHSV), the surface glycoprotein (G) gene was used as a detection target using PCR-LFA. G gene sequences were analyzed from various VHSV strains, and highly conserved regions were used as target genes. This gene was synthesized at the request of Bioneer and cloned into the pGEM T-easy vector (see FIG. 1).

Partial genes for 17 other marine viruses [NNV, Nervous necrosis virus; EHNV, Epizootic haematopoietic necrosis virus (AY187045.1); IHNV, Infectious haematopoietic necrosis virus (X89213.1); ISAV, Infectious salmon anemia virus (NC_006499.1); KHV, Koi herpesvirus (DQ177346.1); SAV, Salmon alphavirus (JQ799139.1); SVC, Spring viraemia of carp (NC_002803.1); IHHNV, Infectious hypodermal and haematopoietic necrosis virus (NC_002190.2); WSSV, White spot syndrome virus (AY126666); YHV, Yellow head virus 1 (EU487200.1); MrNV, Macrobrachium rosenbergii nodavirus (AY222840.1); LSNV, Laem-singh virus (DQ127905.1); CCV, Channel catfish virus (NC_001493.2); GIV, Grouper iridovirus (KX284838); AbHV, Abalone herpesvirus (NC_018874.1); OsHV, Osteroid herpesvirus 1 (NC_005881.2); and BP, Baculovirus penaei (DQ496179.1)] were synthesized in Macrogen and cloned into pGEM T-easy vector and used as a template in the specificity test.

1-2. Plasmid DNA Isolation

E. coli colonies containing RSIV and VHSV target genes were placed in a shaker incubator and cultured in LB medium supplemented with 1% ampicillin at 37° C. After obtaining the cultured E. coli, the plasmid was extracted with AccuPrep® Nano-Plus Plasmid Mini Extraction Kit (Bioneer). The plasmid obtained using the elution buffer included in the kit was used as a template for PCR and PCR-LFA.

1-3. Primers and PCR-LFA

Based on the gene sequence information of each virus, the primers shown in Table 1 were designed.

Primer name primer sequence RSIV F 5'-FAM-aacgtaaccagtgggttca tc-3' RSIV R 5'-Biotin-atgggcaaattaaggtaggcg-3' VHSV F 5'-FAM-aagggcttggtctctgtcc-3' VHSV R 5'-Biotin-aggcagtgatatcactgtgg-3'

The reaction mixture for PCR was prepared by mixing 10 μl of AccuPower® PCR PreMix & Master Mix (Bioneer, Korea), 7 μl of distilled water, 1 μl of template DNA and each primer at a concentration of 10 pmol to make a total of 20 μl.

PCR conditions were as follows: initial denaturation at 95°C for 5 min; denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, extension at 72° C. for 30 seconds, 35 cycles; and final extension at 72° C. for 5 minutes. For the detection of the amplified target gene, the PCR product was electrophoresed on a 1% agarose gel containing EtBr. After agarose gel electrophoresis, PCR products were confirmed by sequencing. Sequencing was commissioned by Macrogen, and the sequencing results were compared with known sequences of RSIV and VHSV genes in NCBI GenBank.

A Genline Hybridetect 1 strip (Milenia Biotec, Giezen, Germany) was used for lateral flow assay (LFA). The PCR product after PCR was used for LFA, and a sample for LFA was prepared by adding 1 μl of the PCR product to 99 μl of Dipstick Assay Buffer. After mixing the PCR product and Dipstick Assay Buffer, the strip was immersed in the mixture and reacted at room temperature for 10 minutes. The results of PCR-LFA were confirmed by visually inspecting the test and control lines (see FIG. 2 ).

1-4. PCR-LFA using fish samples

PCR-LFA was attempted using a tissue sample of halibut ( Paralichthys olivaceus ). Viral genomes were extracted using a viral DNA/RNA extraction kit.

In RSIV detection, PCR-LFA was performed using the extracted DNA directly as a template, and in VHSV detection, the extracted RNA was synthesized into cDNA using a template of SuPrimeScript RT-PCR Premix and used as a template.

The reaction mixture for cDNA synthesis was prepared with 7 μl of distilled water, 1 μl of template, and 10 pmol of each primer concentration to make a final 20 μl, and reacted at 50° C. for 30 minutes. The subsequent procedure was the same as the PCR method. The results were confirmed using both agarose gel electrophoresis and LFA.

2. Results

2-1. Primer set specificity

The amplified PCR product was subjected to 1% agarose gel electrophoresis, and the DNA band was visualized with EtBr and confirmed by comparison with the expected band size. The expected target band sizes were 319 bp for RSIV and 302 bp for VHSV.

As a result of PCR using the plasmid DNA in which each virus gene was cloned as a template, clear and appropriately sized bands were observed for both RSIV and VHSV (see FIG. 3). These PCR results indicate that the primer set is specific for each target viral gene and has no cross-reactivity to 18 other marine virus genes.

2-2. PCR-LFA specificity

To test PCR-LFA, 5'-FAM labeled forward primer and 5'-biotin labeled reverse primer were used. The same PCR method as described above for PCR-LFA was used, and 1 μl of the PCR product was mixed with 99 μl of dipstick assay buffer. The results were confirmed within 10 minutes after the LFA strip was immersed in the mixture (see FIG. 4 ). PCR-LFA results were shown to be as reliable as those of conventional PCR.

2-3. PCR-LFA sensitivity

Serial dilutions of 200 ng to 2 fg of each viral gene-containing plasmid were prepared. These dilutions were used as templates to compare the sensitivity of PCR-LFA with that of conventional PCR.

As a result, conventional PCR was able to detect up to 2 fg of RSIV template and up to 200 fg of VHSV template. PCR-LFA was able to detect up to 2fg of both RSIV and VHSV templates (see Fig. 5). These results indicate that PCR-LFA targeting these two viruses is at least as sensitive as conventional PCR, and may be more sensitive in some circumstances.

2-4. Diagnosis of virus-infected fish

Healthy halibut, RSIV-infected and VHSV-infected halibut were used to determine whether the diagnosis in virus-infected fish tissue also worked well.

Muscle tissue was obtained from each fish, and viral DNA and RNA were extracted. PCR (in case of RSIV) and RT-PCR (in case of VHSV) were performed, and the results were confirmed using agarose gel electrophoresis and LFA, respectively.

As a result, it was shown that the diagnosis using PCR-LFA was reliable not only in viral gene-containing plasmids but also in infected fish samples (see Fig. 6).

Although the present invention has been illustrated and described in relation to specific preferred embodiments, the present invention may be modified and changed in various ways without departing from the technical features or fields of the present invention provided by the following claims. It will be apparent to those of ordinary skill in the art.

<110> Korea Institute of Ocean Science and Technology Research and Business Foundation SUNGKYUNKWAN UNIVERSITY <120> Method for detecting maritime virus, and primer set, composition and kit for the same <130> 04 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 1 aacgtaacca gtgggttcat c 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 2 atgggcaaat taaggtaggc g 21 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 3 aagggcttgg tctctgtcc 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 4 aggcagtgat atcactgtgg 20

Claims (16)

a) isolating DNA or RNA from the sample;
b) DNA isolated from the sample; or cDNA synthesized using RNA isolated from the sample as a template;
A primer set consisting of a forward primer consisting of the sequence of SEQ ID NO: 1 and a reverse primer consisting of the sequence of SEQ ID NO: 2; and
a primer set consisting of a forward primer consisting of the sequence of SEQ ID NO: 3 and a reverse primer consisting of the sequence of SEQ ID NO: 4; performing a polymerase chain reaction using a primer set selected from among; and
c) detecting an amplification product including the forward primer and the reverse primer among the amplification products generated by the polymerase chain reaction; The method of claim 1,
The polymerase chain reaction is
A detection method comprising repeating 30 to 40 cycles of denaturation at 94 to 96° C. for 20 to 40 seconds, annealing at 54 to 56° C. for 20 to 40 seconds, and extension at 71 to 73° C. for 20 to 40 seconds. The method of claim 1,
Using a forward primer and a reverse primer labeled with different labeling substances in step b),
A detection method, characterized in that the amplification product containing both the labeling material of the forward primer and the labeling material of the reverse primer in step c) is detected. 4. The method of claim 3,
A detection method, characterized in that the detection of step c) is performed using a lateral flow detection method. A primer set for detecting marine viruses comprising a forward primer consisting of the sequence of SEQ ID NO: 1 and a reverse primer consisting of the sequence of SEQ ID NO: 2. 6. The method of claim 5,
A primer set, characterized in that the forward primer and the reverse primer are labeled with different labels. 7. The method of claim 6,
The primer set is a primer set, characterized in that for applying the lateral flow detection method. A primer set for detecting marine viruses comprising a forward primer consisting of the sequence of SEQ ID NO: 3 and a reverse primer consisting of the sequence of SEQ ID NO: 4. 9. The method of claim 8,
A primer set, characterized in that the forward primer and the reverse primer are labeled with different labels. 10. The method of claim 9,
The primer set is a primer set, characterized in that for applying the lateral flow detection method. A primer set consisting of a forward primer consisting of the sequence of SEQ ID NO: 1 and a reverse primer consisting of the sequence of SEQ ID NO: 2; and
a primer set consisting of a forward primer consisting of the sequence of SEQ ID NO: 3 and a reverse primer consisting of the sequence of SEQ ID NO: 4; A composition for detecting marine viruses comprising a primer set selected from among. 12. The method of claim 11,
The composition, characterized in that the forward primer and the reverse primer are labeled with different labels. 13. The method of claim 12,
The composition is characterized in that for applying the side flow detection method. A primer set consisting of a forward primer consisting of the sequence of SEQ ID NO: 1 and a reverse primer consisting of the sequence of SEQ ID NO: 2; and
a primer set consisting of a forward primer consisting of the sequence of SEQ ID NO: 3 and a reverse primer consisting of the sequence of SEQ ID NO: 4; A kit for detecting marine viruses comprising a primer set selected from among. 15. The method of claim 14,
The kit, characterized in that the forward primer and the reverse primer are labeled with different labels. 16. The method of claim 15,
The kit is characterized in that for applying the lateral flow detection method.

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