WO2024136540A1 - Composition for detecting spring viraemia of carp, and detection method therefor - Google Patents

Abstract

The present invention relates to a composition for detecting Spring viraemia of carp, and a detection method therefor, and, specifically, to: a primer pair for amplifying genes encoding a matrix protein or a nucleocapsid of Spring viraemia of carp; a composition and a kit for detecting Spring viraemia of carp, each comprising same; and a method for detecting Spring viraemia of carp by using same.

Description

Composition for detecting carp bomb virus and detection method thereof

The present invention relates to a composition for detecting carp bomb virus and a detection method thereof, specifically for amplifying the matrix protein coding gene or nucleocapsid coding gene of carp bomb virus (Spring viraemia of carp). It relates to a primer pair, a composition for detecting carp bomb virus containing the same, a detection kit, and a method for detecting carp bomb virus using the same.

Spring viraemia of carp virus (SVCV) causes lesions such as blackening of the body color, exophthalmos, body surface and gill hemorrhages, anal protrusion, and abdominal distension in freshwater fish such as carp, koi, crucian carp, grass carp, and goldfish. This disease occurs mainly in spring at water temperatures of 10 to 17 C°. Outbreaks have been reported in most European countries and former Soviet Union countries (Belarus, Georgia, Lithuania, Moldova, Russia, and Ukraine), and gradually in Brazil, the United States, Canada, and China. Mortality rates as high as 70% have been reported in fry in Europe, with losses typically ranging from 1-40%. SVCV is designated as a type 1 aquatic infectious disease in Korea and is a disease designated by the World Organization for Animal Health (WOAH).

SVCV, caused by Infection with Spring viraemia of carp, occurs worldwide and infects all freshwater fish (e.g. carp, crucian carp), causing mass mortality. Naturally occurring SVCV infections occur in carp (Cyprinus carpio carpio), koi (Cyprinus carpio koi), crucian carp (Carassius carassius), white lotus (Hypophthalmichthys molitrix), soybean carp (Aristichthys), grass carp (Ctenopharyngodon idella), and goldfish ( Carassius auratus), golden dace (Leuciscus idus), tench (Tinca tinca) and sea bream (Abramis brama), and three species of Indian carp: white carp (Cirrhinus merigala), rohu (Labeo rohita) and South Asian carp (Catla catla). has also been reported to be a host for SVCV.

Various molecular diagnostic methods and kits have been developed to diagnose aquatic biological diseases. In general, diagnostic methods based on electrophoresis using polymerase chain reaction or Real-time Polymerase Chain Reaction (Real-time PCR) using SYBR Green and TaqMan probes are widely used (Korea Aquaculture Society 2004 Fisheries-related Society Joint Academic Conference Presentation Summary 2004 May 13, 2004, pp.387 - 388).

In real-time polymerase chain reaction, a fluorescent reporter material is used and the amplification product increases proportionally as it accumulates in each cycle of amplification. Among the real-time quantitative analysis methods, this is a method using TaqMan. The TaqMan probe uses TaqMan, which has very high specificity, without agarose gel analysis like the existing polymerase chain reaction. The sequence information of the amplified product can be confirmed in a single reaction of about 2 hours, and the amount of nucleic acid in the sample can be accurately measured using a standard sample.

To date, the conventional RT-PCR (reverse-transcription PCR) method, a general genetic diagnosis method, has been designated as the standard diagnostic method for statutory aquatic life infectious diseases. However, in the case of Aphanomyces invadans (A. invadans), the cause of spring viraemia of carp (SVC), polymerase chain reaction and FISH (Fluorescent peptide nucleic acid in-situ hybridisation) methods were developed and OIE However, since real-time PCR-based diagnostic methods and kits have not been developed, the existing conventional PCR method is performed and RT-PCR amplification products are confirmed by electrophoresis, and then the World Organization for Animal Health (World Organization for Animal Health) It is stipulated that diagnosis be performed according to the standards of the Aquatic Animal Health Code of Health, WOAH. Diagnosis of SVCV has the disadvantage of having to isolate RNA from infected tissue and perform RT-PCR, so no diagnostic method using real-time PCR has been reported to date.

The real-time PCR diagnostic method for SVCV registered in WOAH includes Protocol 1 (SVCV n2 F, SVCV n2 R, SVCV n2 P). To diagnose and identify SVCV, RT-PCR is performed according to WOAH standards and a sequencing step is required. It is finally determined through .

Against this technical background, the inventors of the present application identified the causative agent of the shrimp disease through a PCR product for detecting Spring viraemia of carp virus, the causative agent of Spring viraemia of carp (SVC), and identified the cause of the disease. As a result of diligent efforts to develop a method for detecting spring viraemia of carp virus from organisms infected with the causative agent (e.g., shrimp), the base sequences of two genes of spring viraemia of carp virus were obtained from carp. By selecting a genetic marker for identification and/or detection of spring virus disease and amplifying fluorescence using the selected marker, the causative agent of shrimp disease can be identified more quickly and accurately than the existing real-time PCR method. was confirmed, and the outbreak was completed.

The information described in this background section is only for improving the understanding of the background of the present invention, and therefore does not include information about prior art already known to those skilled in the art to which the present invention pertains. You can.

Summary of the Invention

The purpose of the present invention is to provide a composition for detecting spring viraemia of carp (SVC) caused by carp spring virus infection.

The purpose of the present invention is to provide a kit for detecting carp bomb virus disease caused by carp bomb virus infection.

The purpose of the present invention is to provide a method for detecting carp bomb virus disease caused by carp bomb virus infection.

In order to achieve the above object, the present invention includes a primer pair for amplifying the Matrix protein coding gene and/or the Nucleocapsid coding gene of Spring viraemia of carp (SVC), It relates to a composition for detecting carp spring virus disease caused by carp spring virus infection.

The present invention also provides a primer pair for amplifying a matrix protein coding gene or a nucleocapsid coding gene of spring viraemia of carp (SVC). This is about a kit for detecting carp spring virus disease.

The present invention further provides the step of amplifying the nucleic acid extracted from the sample with a pair of primers to amplify the matrix protein coding gene or the nucleocapsid coding gene of spring viraemia of carp (SVC). It relates to a method of detecting carp spring virus disease caused by carp spring virus infection, including.

1A and 1B are results showing an example of the base sequence of a primer pair and a TaqMan probe used in the present invention, respectively (A: primer pair and TaqMan probe targeting a substrate protein coding gene, B: nucleocapsid (Nucleocapsid) gene-targeting primer pair and TaqMan probe).

Figures 2a and 2b show the results of PCR using a primer pair with improved sensitivity and a TaqMan probe for nucleic acids of viruses similar to the causative agent of spring viraemia of carp (SVC), respectively.

Detailed Description and Preferred Embodiments of the Invention

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention provides a carp spring virus ( Spring viraemia of carp : SVC) comprising a primer pair for amplifying a matrix protein coding gene and/or a nucleocapsid coding gene. It relates to a composition for detecting carp spring virus disease caused by viral infection.

The present invention also amplifies the nucleic acid extracted from the specimen with a pair of primers for amplifying the matrix protein coding gene and/or the nucleocapsid coding gene of Spring viraemia of carp (SVC). It relates to a method for detecting carp spring virus disease caused by carp bomb virus infection, including the step of prescribing.

The present invention aims to develop a method for detecting Spring viraemia of carp virus, the causative agent of spring viraemia of carp (SVC), and detecting individuals (e.g., carp) infected with the causative pathogen, It was possible to detect the causative pathogen of aquatic infectious diseases using detection primer pairs and probes with specific base sequences.

As used herein, 'primer' is a single-stranded oligonucleotide that can act as an initiation point for template-directed DNA synthesis under suitable conditions (i.e., four different nucleoside triphosphates and polymerization enzymes) at a suitable temperature and buffer. means.

When performing a real-time polymerase chain reaction, the primer according to the present invention improves the sensitivity by about 3 times compared to using a single primer as a primer pair, enabling sensitive detection and spring viraemia of carp virus. ) can be detected, which has the effect of early detection of carp spring virus disease occurring in freshwater fish, reducing damage, and preparing quarantine measures against the disease.

In this specification, ‘complementary bond’ means that a primer hybridizes with the corresponding nucleic acid strand under conditions for performing a polymerization reaction, and may form a duplex structure. Complementary bonds can be formed even if the complementarity between paired nucleotide sequences forms a Watson-Crick pair or some non-Watson-Crick bonds (Non-Watson-Crick base pair) exist.

If it contains a sequence that can amplify the target, it can be used as a primer if it can amplify the target not only with an exact matching sequence, but also even when additional bp is included or deleted.

The primer pair includes a forward and reverse primer, and the forward primer specifically binds to and amplifies the Matrix protein coding gene or the Nucleocapsid coding gene of Spring viraemia of carp. You can. The reverse primer can be amplified by binding to the matrix protein coding gene or nucleocapsid coding gene and the complementary strand.

For example, the primer pair may include the sequences of SEQ ID NOs: 1 and 2 and/or the sequences of SEQ ID NOs: 4 and 5.

The primer pair may include a marker. A marker may additionally be included in the reverse primer.

In one embodiment, the marker included in the primer is biotin, Cy5, Cy3, FITC, EDANS (5-(2'-aminoethyl)amino-1-naphthalene sulfate), tetramethylrhodamine (TMR), tetramethyl It may be rhodamine isocyanate (TMRITC), x-rhodamine, DIG or an antibody or nanoparticles bound thereto, but is not limited thereto.

Here, by reacting the marker with a binding agent that induces color development, color development or fluorescence expression can be checked and amplification of the target nucleic acid can be detected. At this time, the binder may be streptavidin, but is not limited thereto.

The present invention may also further include a probe that binds to the product amplified by the primer pair. The probe can hybridize complementary to a gene specifically amplified by a primer pair.

In hybridization reactions, the conditions used to achieve a particular level of stringency vary depending on the nature of the nucleic acid being hybridized. For example, the length of the nucleic acid region to be hybridized, the degree of homology, the nucleotide sequence composition (e.g., GC/AT composition ratio), and the nucleic acid type (e.g., RNA, DNA) are considered in selecting hybridization conditions. A further consideration is whether the nucleic acid is immobilized, for example on a filter.

Examples of very stringent conditions include: 2X SSC/0.1% SDS at room temperature (hybridization conditions); 0.2X SSC/0.1% SDS at room temperature (low stringency conditions); 0.2X SSC/0.1% SDS at 42°C (moderate stringency conditions); 0.1X SSC at 68°C (high stringency conditions). The washing process can be performed using one of these conditions, for example, high stringency conditions, or each of the above conditions, in the order described above, for 10 to 15 minutes each, all or all of the conditions described above. It can be performed partially repeatedly. However, as described above, optimal conditions vary depending on the particular hybridization reaction involved and can be determined through experimentation. Generally, high stringency conditions are used for hybridization of probes of interest.

The probe may include, for example, the sequence of SEQ ID NO: 3 or 6. A probe that binds to a product amplified by a primer pair containing the sequences of SEQ ID NO: 1 and 2 may include the sequence of SEQ ID NO: 3. A probe that binds to a product amplified by a primer pair containing the sequences of SEQ ID NO: 4 and 5 may include the sequence of SEQ ID NO: 6. The probe that binds to the product amplified by the primer pair containing the sequences of SEQ ID NO: 1 and 2 is the probe that binds to the product amplified by the primer pair containing the sequence of SEQ ID NO: 3 and the sequence of SEQ ID NO: 4 and 5. It may include the sequence of SEQ ID NO: 6.

In some cases, the probe is detectably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelate, or an enzyme. Appropriately labeling the above probe is a widely known technique in the field, and can be performed through conventional methods.

The amount of the amplification product can be detected by a fluorescence signal. The intercalating method uses an intercalator that binds to the double-stranded DNA of the amplification product to which the probe is bound and displays fluorescence. The 5' end is labeled with a fluorescent substance and the 3' end is labeled with a quencher. There is a method using oligonucleotides, etc.

Specifically, a reporter and a fluorescent quencher capable of quenching the reporter fluorescence may be bound to both ends of the probe. The reporter can be selected from 6-carboxyfluorescein (FAM), and the quencher can be selected from phosphate.

Amplification according to the present invention can be performed through real-time quantitative amplification, for example, real-time polymerase chain reaction (Real-Time PCR). In real-time polymerase chain reaction, the amount of PCR amplification product can be detected by a fluorescence signal. You can. As the real-time polymerase chain reaction progresses, the intensity of the fluorescence signal increases with the increasing amount of polynucleotide, and an amplification profile curve representing the intensity of the fluorescence signal according to the number of amplification cycles is obtained.

In the Real-Time PCR (Real-Time Polymerase Chain Reaction) method of the present invention, the amount of fluorescence begins to be detected by the device as the fluorescent substance is intercalated into the double strand DNA chain during the PCR process. In this way, the threshold cycle (Ct), which is an amplification cycle, occurs at the point when the fluorescence significantly increases beyond the lowest detectable level (background level). There is a linearly proportional relationship between the log value of the initial amount of template and the corresponding limit cycle when amplifying the template through real-time polymerase chain reaction. Therefore, a real-time measurement method of nucleic acid amplification products is provided by obtaining a standard calibration curve by measuring the logarithmic value and limit cycle of the initial amount using a sample whose initial amount of nucleic acid is already known.

The present invention also relates to carp caused by Spring viraemia of carp infection, comprising a primer pair for amplifying the Matrix protein coding gene or the Nucleocapsid coding gene of Spring viraemia of carp. This relates to a kit for detecting spring virus disease (Spring viraemia of carp; SVC).

Depending on the case, the kit may optionally include reagents necessary for performing a nucleic acid amplification PCR reaction, such as polymerase and buffer. The kit according to the present invention may also further include various polynucleotide molecules, various buffers and reagents.

The optimal amount of reagents, buffers or reactants used for a particular reaction in the kit can be determined by a person skilled in the art, and is produced in separate packaging or compartments containing each of the aforementioned primer pairs and/or probes. It can be.

The description of the kit used in carrying out the method according to the present invention and its related configuration can be equally applied to the method.

In one embodiment, the sample includes various samples, and preferably, a biological sample (biosample) is analyzed using the method of the present invention. More preferably, it may be a sample mixed with the virus species described in the present invention or a sample of an individual infected with the virus (e.g., carp, crucian carp, etc.), and may be of plant, animal, human, fungal, bacterial or viral origin. of biological samples can be analyzed. When analyzing samples of mammalian or human origin, the sample may be derived from a specific tissue or organ. Representative examples of tissues include connective, skin, muscle, or nervous tissue. Representative examples of organs include the eyes, brain, lungs, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidneys, gallbladder, stomach, small intestine, testes, ovaries, uterus, rectum, nervous system, Includes glands and internal blood vessels. The biological sample to be analyzed includes any cell, tissue, fluid, or other medium that can be analyzed by the present invention from a biological source, including humans, animals, or consumption by humans or animals. Samples obtained from food prepared for this purpose are included. Additionally, biological samples analyzed include body fluid samples, including blood, serum, plasma, lymph, breast milk, urine, feces, ocular fluid, saliva, semen, brain extracts (e.g., brain pulverized material), spinal fluid, appendix, and spleen. and tonsil tissue extract, but are not limited thereto.

A step of extracting nucleic acids from the specimen may be further included, and extraction of nucleic acids may be performed using, for example, various commercially available kits or extraction reagents.

In the present invention, when extracting target nucleic acid from a specimen, fragments of nucleic acid are generated by reagents (e.g., buffer) or various factors (e.g., vortexing) used for nucleic acid extraction, thereby reducing the sensitivity during PCR, thereby eliminating pathogens. There is a possibility that it may be difficult to detect. In order to improve sensitivity due to nucleic acid fragments, an experiment was performed to compare base sequences of SEQ ID NOs: 1 to 3 and SEQ ID NOs: 1 to 6, respectively. When the fluorescence of the PCR product was measured, the method using SEQ ID NOs. 1 to 6 in two methods for detecting Spring viraemia of carp virus, the cause of spring viraemia of carp (SVC), was SEQ ID NO. 1. It was confirmed that the sensitivity increased about 3 times compared to the method using ~3.

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

[Example 1] Construction of a primer set targeting the matrix protein coding gene and the nucleocapsid coding gene of Spring viraemia of carp, the causative agent of carp spring virus disease (Spring viraemia of carp; SVC)

To produce a set of primers that can specifically bind to the matrix protein coding gene and nucleocapsid coding gene of Spring viraemia of carp, the causative agent of spring viraemia of carp (SVC). For this purpose, the base sequence registered in the nucleotide database of the National Center for Biotechnology Information (NCBI) and the entire gene base sequence were referred to. From this, a set of primers capable of specifically binding to the matrix protein coding gene and the nucleocapsid coding gene of Spring viraemia of carp were created, and these are shown in Table 1 below.

Figure 1a is a nucleotide sequence diagram showing an example of a primer pair and a TaqMan probe that bind to the matrix protein coding gene of Spring viraemia of carp, and Figure 1b is a nucleocapsid coding gene.

[Example 2] Comparison of Sensitivity of Real-Time PCR for Detection of Spring Viraemia of Carp, the Cause of Spring Viraemia of Carp (SVC)

Using the TaqMan probe and primer produced in Example 1, an amplification curve was derived for an RNA sample of Spring viraemia of carp, the cause of carp spring virus disease (Spring viraemia of carp; SVC), and analyzed to determine the causative virus. Detection was performed. PCR was performed using Exicycler96™ (Bioneer, Korea). The composition of the reactants for PCR targeting the matrix protein coding gene is shown in Table 2, and the composition of the reactant for PCR targeting the matrix protein coding gene and the nucleocapsid coding gene is shown in Table 3. As shown in , real-time PCR was performed by creating a PCR master mix and adding 5 μl viral RNA.

Figure 2 shows the results showing the amount of fluorescence when a composition targeting all of the matrix protein coding gene, matrix protein coding gene, and nucleocapsid coding gene was used.

Table 4 shows the amplification conditions and hybridization reaction conditions of the reactants, and shows the process of amplifying and hybridizing the viral RNA sample and then increasing the temperature of the hybridized product. The above process was performed by real-time PCR.

[Example 3] Virus detection method according to the amplification curve of real-time PCR

When attempting to detect a sample by performing real-time PCR using the detection method according to the present invention, a fluorescent reporter can be used by binding to a TaqMan probe as shown in Table 5. For example, as shown in Table 5, check the fluorescence amplification curve so that the virus can be detected with a fluorescent reporter (Detection of Spring viraemia of carp with FAM fluorescence).

[Example 4] Comparison of specificity of Real-Time PCR for detection of Spring viraemia of carp, the cause of carp spring virus disease (Spring viraemia of carp; SVC)

In the present invention, the causative agent of spring viraemia of carp (SVC) and Real-time PCR was performed by adding 5 μL of nucleic acid of similar viruses.

Figures 2a and 2b show the results of PCR using a primer pair with improved sensitivity and a TaqMan probe for the nucleic acid of a virus similar to the causative agent of spring viraemia of carp (SVC).

In the single case, the results were confirmed as shown in Figure 2a and as follows.

In the case of dual, the results were confirmed as shown in Figure 2b and as follows.

[Example 5] Comparison of detection limits and cut-off values of real-time PCR for detection of SVCV, the cause of spring viraemia of carp (SVC)

In the present invention, plasmid DNA containing the sequences of the matrix protein and nucleocapsid region of SVCV, the cause of Epizootic ulcerative syndrome (EUS), was produced, and a PCR reaction targeting the nucleocapsid protein and matrix protein with improved sensitivity was produced. The composition of the PCR reaction targeting only the composition and matrix protein was used (see Tables 2 and 3). For the detection limit and cut-off value analysis, plasmid DNA 3 fold-diluted from 1000 to 1.4 copies/rxn was used, and the detection limit and cut-off value were compared and analyzed using 95% probit regression analysis.

Figure 2a shows the results of analyzing the detection limit and cut-off value for the PCR composition targeting only the matrix protein, and Figure 2b shows the analysis of the detection limit and cut-off value for the PCR composition targeting the matrix protein and nucleocapsid protein. This is one result.

The attached Figure 2b is the result of using the primer mixture of [Table 3], and it was confirmed that the detection limit was about 3 times different than when using the primer mixture of [Table 2]. It was confirmed that the sensitivity of SVCV, the causative agent of carp spring virus disease, increased with the primer mixture according to the present invention [Table 3].

By providing a composition, kit, and detection method that can sensitively molecularly diagnose spring viraemia of carp, the causative agent of carp spring virus disease, through the primer pair according to the present invention, it is simple to determine whether the pathogen is infected. ·It is effective in detecting quickly and accurately.

As above, specific parts of the content of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. It will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Electronic file attached.

Claims (12)

1. Carp spring virus caused by carp spring virus infection, comprising a primer pair for amplifying the matrix protein coding gene and/or the nucleocapsid coding gene of spring viraemia of carp (SVC). Composition for disease detection.
2. The composition of claim 1, wherein the primer pair comprises the sequences of SEQ ID NOs: 1 and 2 and/or the sequences of SEQ ID NOs: 4 and 5.
3. The composition of claim 1, further comprising a probe that binds to the product amplified by the primer pair.
4. The composition according to claim 3, wherein the probe comprises the sequence of SEQ ID NO: 3 or 6.
5. Carp spring virus caused by carp spring virus infection, comprising a primer pair for amplifying the matrix protein coding gene and/or the nucleocapsid coding gene of spring viraemia of carp (SVC). Bottle detection kit.
6. The kit according to claim 5, wherein the primer pair comprises the sequences of SEQ ID NOs: 1 and 2 and/or the sequences of SEQ ID NOs: 4 and 5.
7. The kit according to claim 5, further comprising a probe that binds to the product amplified by the primer pair.
8. The kit according to claim 7, wherein the probe comprises the sequence of SEQ ID NO: 3 or 6.
9. Carp comprising the step of amplifying the nucleic acid extracted from the sample with a pair of primers for amplifying the matrix protein coding gene or nucleocapsid coding gene of spring viraemia of carp (SVC). Method for detecting carp spring virus disease caused by spring virus infection.
10. The detection method according to claim 9, wherein the primer pair comprises the sequences of SEQ ID NOs: 1 and 2 and/or the sequences of SEQ ID NOs: 4 and 5.
11. The detection method according to claim 9, comprising adding a probe that binds to the product amplified by the primer pair.
12. The detection method according to claim 11, wherein the probe comprises the sequence of SEQ ID NO: 3 or 6.

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