WO2017122897A1 - Marqueur génétique servant à détecter le virus responsable de l'iridovirose de la daurade japonaise, et procédé de détection du virus causal utilisant le marqueur - Google Patents

Marqueur génétique servant à détecter le virus responsable de l'iridovirose de la daurade japonaise, et procédé de détection du virus causal utilisant le marqueur Download PDF

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WO2017122897A1
WO2017122897A1 PCT/KR2016/009374 KR2016009374W WO2017122897A1 WO 2017122897 A1 WO2017122897 A1 WO 2017122897A1 KR 2016009374 W KR2016009374 W KR 2016009374W WO 2017122897 A1 WO2017122897 A1 WO 2017122897A1
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seq
virus
rsiv
sea bream
nucleotide sequence
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Korean (ko)
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조미영
박명애
지보영
황성돈
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대한민국(관리부서:국립수산과학원)
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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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B23—MACHINE TOOLS; METAL-WORKING NOT OTHERWISE PROVIDED FOR
    • B23K—SOLDERING OR UNSOLDERING; WELDING; CLADDING OR PLATING BY SOLDERING OR WELDING; CUTTING BY APPLYING HEAT LOCALLY, e.g. FLAME CUTTING; WORKING BY LASER BEAM
    • B23K37/00—Auxiliary devices or processes, not specially adapted to a procedure covered by only one of the preceding main groups
    • B23K37/04—Auxiliary devices or processes, not specially adapted to a procedure covered by only one of the preceding main groups for holding or positioning work
    • B23K37/047—Auxiliary devices or processes, not specially adapted to a procedure covered by only one of the preceding main groups for holding or positioning work moving work to adjust its position between soldering, welding or cutting steps
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B23—MACHINE TOOLS; METAL-WORKING NOT OTHERWISE PROVIDED FOR
    • B23K—SOLDERING OR UNSOLDERING; WELDING; CLADDING OR PLATING BY SOLDERING OR WELDING; CUTTING BY APPLYING HEAT LOCALLY, e.g. FLAME CUTTING; WORKING BY LASER BEAM
    • B23K31/00—Processes relevant to this subclass, specially adapted for particular articles or purposes, but not covered by only one of the preceding main groups
    • B23K31/02—Processes relevant to this subclass, specially adapted for particular articles or purposes, but not covered by only one of the preceding main groups relating to soldering or welding
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B23—MACHINE TOOLS; METAL-WORKING NOT OTHERWISE PROVIDED FOR
    • B23K—SOLDERING OR UNSOLDERING; WELDING; CLADDING OR PLATING BY SOLDERING OR WELDING; CUTTING BY APPLYING HEAT LOCALLY, e.g. FLAME CUTTING; WORKING BY LASER BEAM
    • B23K37/00—Auxiliary devices or processes, not specially adapted to a procedure covered by only one of the preceding main groups
    • B23K37/04—Auxiliary devices or processes, not specially adapted to a procedure covered by only one of the preceding main groups for holding or positioning work
    • B23K37/0461—Welding tables
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B25—HAND TOOLS; PORTABLE POWER-DRIVEN TOOLS; MANIPULATORS
    • B25H—WORKSHOP EQUIPMENT, e.g. FOR MARKING-OUT WORK; STORAGE MEANS FOR WORKSHOPS
    • B25H1/00—Work benches; Portable stands or supports for positioning portable tools or work to be operated on thereby
    • B25H1/10—Work benches; Portable stands or supports for positioning portable tools or work to be operated on thereby with provision for adjusting holders for tool or work
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B25—HAND TOOLS; PORTABLE POWER-DRIVEN TOOLS; MANIPULATORS
    • B25H—WORKSHOP EQUIPMENT, e.g. FOR MARKING-OUT WORK; STORAGE MEANS FOR WORKSHOPS
    • B25H1/00—Work benches; Portable stands or supports for positioning portable tools or work to be operated on thereby
    • B25H1/14—Work benches; Portable stands or supports for positioning portable tools or work to be operated on thereby with provision for adjusting the bench top
    • B25H1/18—Work benches; Portable stands or supports for positioning portable tools or work to be operated on thereby with provision for adjusting the bench top in inclination
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B63—SHIPS OR OTHER WATERBORNE VESSELS; RELATED EQUIPMENT
    • B63B—SHIPS OR OTHER WATERBORNE VESSELS; EQUIPMENT FOR SHIPPING 
    • B63B25/00—Load-accommodating arrangements, e.g. stowing, trimming; Vessels characterised thereby
    • B63B25/02—Load-accommodating arrangements, e.g. stowing, trimming; Vessels characterised thereby for bulk goods
    • B63B25/08—Load-accommodating arrangements, e.g. stowing, trimming; Vessels characterised thereby for bulk goods fluid
    • B63B25/12—Load-accommodating arrangements, e.g. stowing, trimming; Vessels characterised thereby for bulk goods fluid closed
    • B63B25/16—Load-accommodating arrangements, e.g. stowing, trimming; Vessels characterised thereby for bulk goods fluid closed heat-insulated
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B63—SHIPS OR OTHER WATERBORNE VESSELS; RELATED EQUIPMENT
    • B63B—SHIPS OR OTHER WATERBORNE VESSELS; EQUIPMENT FOR SHIPPING 
    • B63B2221/00—Methods and means for joining members or elements
    • B63B2221/02—Methods and means for joining members or elements by welding

Definitions

  • the present invention relates to a gene marker for the identification and detection of the causative virus of sea bream iridovirus disease, which is the causative virus of aquatic organisms, and to a method for discriminating and detecting the causal virus using the same.
  • Iridovirus (RSIV) / specific genes of Infectious Spleen and Kidney Necrosis Virus (ISKNV); Or (ii) selecting and amplifying a DNA sequence encoding a specific gene that distinguishes RSIV from RSIV / RSKNV, and then hybridizing and hybridizing a peptide nucleic acid (PNA) that specifically recognizes the amplification product.
  • ISKNV Infectious Spleen and Kidney Necrosis Virus
  • the present invention relates to a method of obtaining a melting curve for each temperature by controlling a temperature of a product, and determining a virus type from a melting temperature or analyzing whether a fish is infected with the virus type by analyzing the obtained melting curve.
  • Red sea bream iridoviral disease is a viral disease that causes serious damage in major marine fish farming in Korea.
  • Various molecular diagnostic methods and kits have been developed as a method for diagnosing fish viral diseases, and after performing a general polymerase chain reaction (PCR) method, an amplification product of the reaction is confirmed by electrophoresis or a TaqMan probe. Or, the method of confirming with real-time PCR using SYBR Green is mainly used.
  • PCR polymerase chain reaction
  • Red sea bream iridovirus disease not only occurs mainly in cultured red snapper, but is also an important cause of death in more than 30 marine cultured fishes, and is known to occur mainly in perch and flounder.
  • the first occurrence of RSIVD was recorded in 1990 from aquaculture red snapper on Shikoku Island, Japan, and RSIVD has become a major cause of mass mortality in red snapper.
  • RSIV and ISKNV should be simultaneously analyzed for accurate diagnosis of RSIVD.
  • serological methods such as staining using tissue smear samples and IFAT using MAbs are used.
  • molecular diagnosis conventional PCR using two types of primers is performed according to OIE standard. do.
  • OIE protocol 1 OIE 1
  • OIE protocol 2 OIE 4
  • RSIV / ISKNV is used when OIE 1 is used. While all can be detected, only RSIV can be detected when using OIE 4. Therefore, for the diagnosis and determination of RSIV / ISKNV, two PCR steps should be performed according to the OIE standard.
  • the present inventors discriminate fish disease virus without sequencing step or before sequencing step whether RSIV / ISKNV or PCR product for identifying or detecting RSIV / ISKNV or RSIV, which is the causative virus of aquatic biological infection.
  • RSIV Red Sea Bream Iridovirus
  • ISKNV ISKNV
  • a nucleotide sequence of a specific gene that distinguishes RSIV from RSIV / RSKNV is selected as a gene marker for virus type discrimination and / or detection, using peptide nucleic acid and primer pairs specific for the gene marker.
  • Different virus types show different amplification and melting curves of fluorescence By ensuring that the effect of a simple, rapid, accurate determination of the type of fish diseases causing virus, and have completed the present invention.
  • An object of the present invention is to provide a genetic marker, primer pair, and PNA probe for the identification or detection of the causative virus of sea bream iridovirus disease which is a marine organism infectious disease causative virus.
  • Another object of the present invention is to provide a composition and kit for discriminating or detecting a causative virus of red seabream iridovirus disease comprising the primer pair and the PNA probe.
  • Another object of the present invention is to determine the type of the causal virus or cause the individual by obtaining the Tm value according to hybridization of the PNA probe to the region of the virus marker gene specific virus markers amplified using the primer pair
  • the present invention provides a method for detecting a virus infection.
  • the present invention is to determine or detect the Red Sea Bream Iridovirus (RSIV) or infectious spleen and kidney necrosis virus (ISKNV), a causative agent of aquatic organisms infectious disease represented by the nucleotide sequence of SEQ ID NO: 7 Provide a genetic marker for RSIV or infectious spleen and kidney necrosis virus (ISKNV), a causative agent of aquatic organisms infectious disease represented by the nucleotide sequence of SEQ ID NO: 7
  • RSIV Red Sea Bream Iridovirus
  • ISKNV infectious spleen and kidney necrosis virus
  • the present invention also provides a genetic marker for the identification or detection of Red Sea Bream Iridovirus (RSIV), which is a causative agent of aquatic organisms infectious disease represented by the nucleotide sequence of SEQ ID NO: 8.
  • RSIV Red Sea Bream Iridovirus
  • the present invention is also used for the identification or detection of Red Sea Bream Iridovirus (RSIV) or infectious spleen and kidney necrosis virus (ISKNV), a causative agent of aquatic organisms infectious diseases represented by the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 Provide primer pairs.
  • RSIV Red Sea Bream Iridovirus
  • ISKNV infectious spleen and kidney necrosis virus
  • the present invention also provides a primer pair for the identification or detection of Red Sea Bream Iridovirus (RSIV), which is a causative agent of aquatic organisms infectious diseases represented by the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6.
  • RSIV Red Sea Bream Iridovirus
  • the present invention also provides a PNA probe for the identification or detection of Red Sea Bream Iridovirus (RSIV) or infectious spleen and kidney necrosis virus (ISKNV), a causative agent of aquatic organisms infectious disease represented by the nucleotide sequence of SEQ ID NO: 1 do.
  • RSIV Red Sea Bream Iridovirus
  • ISKNV infectious spleen and kidney necrosis virus
  • the present invention also provides a PNA probe for discriminating or detecting red sea bream iridovirus (RSIV), which is a causative agent of aquatic organisms infectious disease represented by the nucleotide sequence of SEQ ID NO: 2.
  • RSIV red sea bream iridovirus
  • the present invention also provides a composition and kit for discriminating or detecting a causative virus of aquatic infectious diseases comprising the primer pair and the PNA probe.
  • the present invention also provides
  • step (c) obtaining a melting curve for each temperature while increasing the temperature of the product hybridized with the PNA probe in step (b);
  • step (d) determining the virus type of the causative virus of aquatic organisms infectious diseases from the melting temperature or detecting whether the fish is infected with the virus type by analyzing the melting curve obtained in step (c);
  • It provides a method for determining or detecting aquatic infectious diseases causal virus comprising a.
  • 1 is a conceptual diagram showing the technical characteristics of the step of obtaining the amplification curve for determining the virus type or virus infection of the individual.
  • FIG. 2 is a schematic diagram showing a step of obtaining a melting curve according to hybridization of peptide nucleic acids in a virus type determination method and an individual virus infection detection method.
  • Figure 3 is a gene position diagram for explaining the nucleotide sequence region included in the primer and peptide nucleic acid in the amplification product derived by the detection "PCR protocol 1 (OIE 1)" method according to the OIE standard of RSIV / ISKNV.
  • OIE 1 PCR protocol 1
  • Figure 4 is a gene position diagram for explaining the nucleotide sequence region contained in the primer and peptide nucleic acid in the amplification product derived by the detection "PCR protocol 2 (OIE 4)" method according to the OIE standard of RSIV
  • FIGS. 5 and 6 show amplification curves and melting curves according to the RSIV / ISKNV detection method using primers and peptide nucleic acids for each virus type described in FIGS. 1 to 4.
  • the present invention provides a marker for identifying RSIV / ISKNV, which is a causative agent of red snapper virus, and developing a method for detecting an individual (eg, a fish) infected with the causative virus. And a causal virus of aquatic organism infectious disease using a primer pair and a peptide nucleic acid probe (PNA probe) for determining the virus type corresponding to the marker, and the type of each causal virus could be determined.
  • PNA probe peptide nucleic acid probe
  • the cause virus can be identified / detected by type
  • an oligomer mixture comprising a PNA probe for RSIV / ISKNV detection (SEQ ID NO: 1) and a primer pair (SEQ ID NO: 3 and SEQ ID NO: 4) capable of simultaneously detecting RSIV and ISKNV;
  • oligomer mixtures comprising a detection PNA probe (SEQ ID NO: 2) and a primer pair (SEQ ID NO: 5 and SEQ ID NO: 6) specific for RSIV;
  • composition or kit comprising a red sea bream iridovirus causative virus was able to detect and determine the type of each causal virus.
  • the present invention in one aspect, for the identification or detection of Red Sea Bream Iridovirus (RSIV) or infectious spleen and kidney necrosis virus (ISKNV), which is the causative agent of aquatic organisms infectious disease represented by the nucleotide sequence of SEQ ID NO: 7 It relates to genetic markers.
  • RSIV Red Sea Bream Iridovirus
  • ISKNV infectious spleen and kidney necrosis virus
  • the present invention relates to a genetic marker for identifying or detecting red sea bream iridovirus (RSIV), which is a causative agent of aquatic organisms infectious disease represented by the nucleotide sequence of SEQ ID NO: 8.
  • RSIV red sea bream iridovirus
  • the present invention in another aspect, the determination of the Red Sea Bream Iridovirus (RSIV) or infectious spleen and kidney necrosis virus (ISKNV), a causative agent of aquatic organisms infectious diseases represented by the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 Or to a primer pair for detection.
  • RSIV Red Sea Bream Iridovirus
  • ISKNV infectious spleen and kidney necrosis virus
  • the present invention relates to a pair of primers for the identification or detection of Red Sea Bream Iridovirus (RSIV), a causative agent of aquatic organisms infectious diseases represented by the nucleotide sequences of SEQ ID NOs: 5 and 6.
  • RSIV Red Sea Bream Iridovirus
  • the present invention provides a PNA for the determination or detection of Red Sea Bream Iridovirus (RSIV) or infectious spleen and kidney necrosis virus (ISKNV), which is a causative agent of aquatic organisms infectious disease represented by the nucleotide sequence of SEQ ID NO: 1 Relates to a probe.
  • RSIV Red Sea Bream Iridovirus
  • ISKNV infectious spleen and kidney necrosis virus
  • the present invention relates to a PNA probe for discriminating or detecting red sea bream iridovirus (RSIV), which is a causative agent of aquatic biological infectious disease represented by the nucleotide sequence of SEQ ID NO: 2.
  • RSIV red sea bream iridovirus
  • a reporter and a fluorescent material of a quencher capable of quenching the reporter fluorescence may bind to both ends.
  • the reporter is reported as FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, Cy3 and Cy5.
  • the quencher may be one or more selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, but is not limited thereto, preferably Dabcyl You can use (FAM-labeled).
  • TAMRA 6-carboxytetramethyl-rhodamine
  • BHQ1, BHQ2 and Dabcyl but is not limited thereto, preferably Dabcyl You can use (FAM-labeled).
  • PNA Peptide nucleic acid
  • Peptide nucleic acid is artificially synthesized as one of the gene recognition materials, such as LNA (Locked nucleic acid) or MNA (Mopholino nucleic acid), the basic skeleton is composed of polyamide (polyamide).
  • PNA has very high affinity and selectivity, and has high stability against nucleases, so that it is not degraded by existing restriction enzymes.
  • thermal properties and chemical properties are high and easy to store and not easily decomposed.
  • PNAs form double strands through hybridization with native nucleic acids of complementary base sequences.
  • PNA / DNA double strands are more stable than DNA / DNA double strands and PNA / RNA double strands are more stable than DNA / RNA double strands.
  • PNA has a greater ability to detect single nucleotide polymorphism (SNP) than natural nucleic acid because of its large degree of double strand instability due to single base mismatch.
  • SNP single nucleotide polymorphism
  • the PNA-DNA binding ability is much better than the DNA-DNA binding force, so that there is a difference of about 10 to 15 ° C even in one nucleotide miss match.
  • the difference in binding force it is possible to detect SNP (Single-nucleotide polymorphism) and change in nucleotides of In / Del.
  • the length of the PNA sequence according to the present invention is not particularly limited, but may be produced in a length of 12 to 18mer so as to include a specific sequence (eg, nucleotide variation or single nucleotide polymorphism (SNP)) according to the virus type. have.
  • the PNA probe may be designed to have a desired Tm value by adjusting the length of the PNA probe, or even a PNA probe of the same length may be adjusted by changing the base sequence.
  • PNA probes have a higher binding force than DNA and have a higher basic Tm value, so that the PNA probe can be designed with a shorter length than DNA, so that even neighboring base mutations or SNPs can be detected.
  • the difference in Tm value is very small, about 0.5 ° C, which requires additional analysis program or detailed temperature change or correction. Therefore, when two or more base mutations or SNPs appear, the analysis is performed. Although it was difficult, the PNA probe according to the present invention is not affected by the sequence and SNP of the PNA probe, and thus can be easily analyzed.
  • HRM High Resolution Melt
  • the PNA probe when the PNA probe includes 14 base sequences, it is preferable to have a sequence corresponding to the base mutation or SNP region of the virus at one or more positions of the center sequences.
  • the PNA probe may have a structural modification including a sequence corresponding to a nucleotide variation or SNP site of the virus in the center of the base sequence, through which the melting temperature with the perfect nucleic acid (perfect match) Tm) can make the difference even larger.
  • the present invention relates to a composition and kit for determining or detecting aquatic infectious agent causative virus comprising the primer pair and the PNA probe.
  • the present invention provides a composition for detecting a red seabream iridovirus causative virus comprising a primer pair represented by a nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4, and a PNA probe represented by a nucleotide sequence of SEQ ID NO: 1 Can provide.
  • Red Sea Bream Iridovirus comprising a primer pair represented by the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6, and a PNA probe represented by the nucleotide sequence of SEQ ID NO: 2 It is possible to provide a kit for discrimination or detection.
  • kits for detecting a red snapper virus causing virus comprising a primer pair represented by a nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4, and a PNA probe represented by a nucleotide sequence of SEQ ID NO: 1.
  • Red Sea Bream Iridovirus comprising a primer pair represented by the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6, and a PNA probe represented by the nucleotide sequence of SEQ ID NO: 2
  • RSIV Red Sea Bream Iridovirus
  • kits of the present invention may optionally include reagents necessary to conduct target nucleic acid amplification reactions (eg, PCR reactions) such as buffers, DNA polymerase cofactors and deoxyribonucleotide-5-triphosphates.
  • reagents necessary to conduct target nucleic acid amplification reactions eg, PCR reactions
  • the kits of the present invention may also include various polynucleotide molecules, reverse transcriptases, various buffers and reagents, and antibodies that inhibit DNA polymerase activity.
  • the optimum amount of reagent used in a particular reaction of the kit can be easily determined by those skilled in the art having learned the disclosure herein.
  • the equipment of the present invention can be manufactured in a separate package or compartment containing the aforementioned components.
  • the kit may consist of a PNA-based multiple assay kit capable of detecting and / or discriminating gene sequences for distinguishing RSIV / ISKNV, comprising one 2x qPCR premix tube and one oligomer mix tube. tube), wherein the oligomer mixture may be characterized by comprising one or more TaqMan probes, PNA probes of SEQ ID NO: 1 or SEQ ID NO: 2 and primers selected from two or more primers of SEQ ID NO: 3 to SEQ ID NO: 6 (See Table 1).
  • a single base mutation of a target nucleic acid and a mutation due to a deletion or insertion of a base can be effectively detected through a lysis curve analysis by a PNA probe, and thus the species of a virus can be determined.
  • the present invention (i) specific genes of Red Sea Bream Iridovirus (RSIV) / Infectious Spleen and Kidney Necrosis Virus (ISKNV); Or (ii) performing detection PCR according to OIE criteria for RSIV and / or RSKNV for identification or detection according to a specific gene that distinguishes RSIV from RSIV / RSKNV, and then sequenced the gene sequence corresponding to the PCR product.
  • RSIV Red Sea Bream Iridovirus
  • ISKNV Infectious Spleen and Kidney Necrosis Virus
  • step (c) obtaining a melting curve for each temperature while increasing the temperature of the product hybridized with the PNA probe in step (b);
  • step (d) determining the virus type of the causative virus of aquatic organisms infectious diseases from the melting temperature or detecting whether the fish is infected with the virus type by analyzing the melting curve obtained in step (c);
  • It relates to a method for determining or detecting aquatic infectious agent virus comprising a.
  • step (c) obtaining a melting curve for each temperature while increasing the temperature of the product hybridized with the PNA probe in step (b);
  • step (D) through the analysis of the melting curve obtained in the step (c) it can provide a method for detecting a red seabream iridovirus causal virus comprising the step of detecting whether or not the sea urchin seaweed virus causing virus from the melting temperature.
  • step (c) obtaining a melting curve for each temperature while increasing the temperature of the product hybridized with the PNA probe in step (b);
  • step (d) detecting red sea bream iridovirus (RSIV) from the melting temperature by analyzing the melting curve obtained in step (c) or determining whether the virus is infected with red sea bream iridovirus (red) Sea Bream Iridovirus (RSIV) can be provided for the identification or detection method.
  • RSIV red sea bream iridovirus
  • the amplification curve may be obtained by additionally including a TaqMan probe when amplifying the gene marker sequence using the primer pair.
  • two or more target nucleic acids are used, and the reporter labeled on the PNA probe is different for each target nucleic acid, thereby determining or detecting the virus type of at least one aquatic infectious agent causative virus through detection of the two or more target nucleic acids.
  • the amplification may be performed by a real-time polymerase chain reaction (PCR) method.
  • PCR polymerase chain reaction
  • a sample sample includes various samples, and preferably, a biosample is analyzed using the method of the present invention. More preferably, the sample may be a sample mixed with the virus species described in the present invention or a sample of an individual infected with the virus (for example, fish, etc.), and may be a plant, animal, human, fungus, bacteria, or organism of viral origin. Samples can be analyzed. When analyzing a sample 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 nerve tissue.
  • organs include eyes, brain, lungs, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, small intestine, testes, ovaries, uterus, rectum, nervous system, Glands and internal vessels are included.
  • the biosample to be analyzed includes any cell, tissue, fluid from a biological source, or any other medium that can be well analyzed by the present invention, which is the consumption of humans, animals, humans or animals. Samples obtained from food prepared for use are included.
  • the biological sample to be analyzed includes a bodily fluid sample, which includes blood, serum, plasma, lymph, breast milk, urine, feces, ocular fluid, saliva, semen, brain extracts (e.g., brain grinds), spinal fluid, appendix, spleen And tonsil tissue extracts, but is not limited thereto.
  • a bodily fluid sample which includes blood, serum, plasma, lymph, breast milk, urine, feces, ocular fluid, saliva, semen, brain extracts (e.g., brain grinds), spinal fluid, appendix, spleen And tonsil tissue extracts, but is not limited thereto.
  • 'Target nucleic acid', 'synthetic DNA' or 'synthetic oligo' of the present invention means a nucleic acid sequence (including base mutation or SNP) to be detected or not, and encodes a protein having a physiological and biochemical function.
  • a specific site of the nucleic acid sequence of the target gene 'and is annealed or hybridized with a primer or probe under hybridization, annealing or amplification conditions.
  • Hybridization' of the present invention is meant that complementary single stranded nucleic acids form a double-stranded nucleic acid.
  • Hybridization can occur when the complementarity between two nucleic acid strands is perfect or even when some mismatch base is present.
  • the degree of complementarity required for hybridization may vary depending on the hybridization conditions, and may be particularly controlled by temperature.
  • the melting curve analysis may be performed by a method of Fluorescence Melting Curve Analysis (FMCA).
  • FMCA Fluorescence Melting Curve Analysis
  • the PNA probe including the reporter and the quencher of the present invention hybridizes with the target nucleic acid and generates a fluorescence signal, and as the temperature increases, the PNA probe rapidly melts with the target nucleic acid at an appropriate melting temperature of the probe, thereby extinguishing the fluorescent signal.
  • the PNA probe shows the expected melting temperature (Tm) value when it is in perfect hybridization with the target nucleic acid sequence, but is incompletely mismatched with the target nucleic acid in which the base mutation is present. It is characterized by showing (Tm) value.
  • the 'base mutation' of the present invention is a mutation in the nucleotide sequence of the target nucleic acid, characterized in that it comprises a single nucleotide polymorphism (SNP), as well as a mutation occurs by substitution, deletion or insertion of the base
  • the PNA probe of the present invention may be analyzed through a melting curve analysis that a mutation occurs due to substitution, deletion or insertion of the SNP of the target nucleic acid or the base of the target nucleic acid.
  • a reporter and a fluorescent material of a quencher capable of quenching the reporter fluorescence may bind to both ends.
  • the reporter is reported as FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, Cy3 and Cy5.
  • the quencher may be one or more selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, but is not limited thereto, preferably Dabcyl You can use (FAM-labeled).
  • TAMRA 6-carboxytetramethyl-rhodamine
  • BHQ1, BHQ2 and Dabcyl but is not limited thereto, preferably Dabcyl You can use (FAM-labeled).
  • the Tm value is also changed according to the difference between the nucleotides of the PNA probe and the nucleotides of the DNA complementarily binding thereto, thereby facilitating the development of an application using the same.
  • PNA probes are analyzed using a hybridization method that is different from the hydrolysis method of TaqMan probes. Probes that play similar roles include molecular beacon probes and scorpion probes. There is.
  • a probe containing a base (s) that recognizes a particular sequence and a set of forward / reverse primers for PCR Do.
  • the PCR conditions can be used in the conventional method, after the completion of the PCR (melting) process is required and each time the increase in 0.5 °C to obtain the Tm value by measuring the intensity of fluorescence.
  • the general real-time PCR (real-time PCR) device is widely spread and has the advantage that does not require additional program purchase or minute temperature changes, such as high resolution melting (HRM).
  • HRM high resolution melting
  • the melting curve analysis of the present invention is a method for analyzing the melting temperature of a double-stranded nucleic acid formed of a target nucleic acid DNA or RNA and a probe. Such a method is called a melting curve analysis because it is performed by Tm analysis or analysis of the said double chain melting curve, for example.
  • a probe complementary to a particular nucleotide sequence (including base mutation or SNP) of the detection target (target) a hybrid (double chain DNA) of the target single-stranded DNA of the detection sample with the probe is formed.
  • heat processing is performed to this hybrid formation body, and the dissociation (fusion) of a hybrid with temperature rise is detected by the change of signals, such as absorbance.
  • the Tm value is determined based on the detection result to determine the presence or absence of a specific nucleotide sequence.
  • the Tm value is higher as the homology of the hybrid body is higher, and lower as the homology is lower. For this reason, the Tm value (evaluation reference value) is calculated
  • the measured value is about the same as the evaluation reference value, it can be determined that there is a match, that is, a specific sequence exists in the target DNA, and if the measured value is lower than the evaluation reference value, there is a mismatch, that is, a mutation in the target DNA. You can judge that you do not.
  • Fluorescence melting curve analysis of the present invention is a method of analyzing the melting curve using a fluorescent material, more specifically, the melting curve can be analyzed using a probe containing a fluorescent material.
  • the fluorescent material may be a reporter and a quencher, and may be an intercalating fluorescent material.
  • the Real-Time Polymerase Chain Reaction (PCR) method of the present invention allows the fluorescent material to be interchelated with a double strand DNA chain during the PCR process and increases the temperature with the amplification of the PCR product.
  • the pattern of the melting curve that reduces the amount of fluorescent material present between the double strands of DNA, in particular the temperature (Tm) at which the DNA is fused (denatured) is analyzed to determine the specific sequence (base mutation, including SNP). With or without virus types can be detected and / or determined.
  • the PNA probe may generate a fluorescent signal after hybridization with a target nucleic acid. As the temperature increases, it rapidly melts from the target nucleic acid at the proper melting temperature of the probe, thereby extinguishing the fluorescent signal.
  • the present invention is capable of detecting the presence or absence of base mutation of the target nucleic acid through high resolution fluorescence melting curve analysis (FMCA) obtained from the fluorescence signal according to the temperature change.
  • FMCA fluorescence melting curve analysis
  • the PNA probe according to the present invention shows the expected melting temperature (Tm) value when it is in perfect hybridization with the target nucleic acid sequence, but is incompletely mismatched with the target nucleic acid in which the base mutation is present. It may be characterized by showing a low melting temperature (Tm) value.
  • Example 1 Gene marker for discrimination or detection of causative virus of sea bream iridovirus, and primer and PNA probe specific for the virus
  • RSIV Red Sea Bream Iridovirus
  • ISKNV Infectious Spleen and Kidney Necrosis Virus
  • the sequence of the gene fragment synthesized by the "PCR protocol 1 (OIE 1)" method for detecting the OIE standard of RSIV / ISKNV is transferred to the nucleotide database (nucleotide database) of the National Center for Biotechnology Information (NCBI). By comparing with the registered nucleotide sequence, we tried to secure the nucleotide sequence of the gene for each virus type.
  • OIE 1 PCR protocol 1
  • RSIV / ISKNV represented by 5'-CCATGTACAACATGCTC-3 '(SEQ ID NO: 7) was secured, and the nucleotide sequence was selected as a gene marker for identifying or detecting RSIV / ISKNV.
  • a primer pair (Forward primer 1, Reverse primer 1) represented by SEQ ID NO: 3 and SEQ ID NO: 4 as a primer for DNA amplification of the genetic marker and PNA 1 represented by SEQ ID NO: 1 as a hybridization probe of the genetic marker Produced.
  • FIG. 3 is a nucleotide sequence diagram showing a part of the gene of RSIV / ISKNV, a base sequence for discrimination / detection, and a base sequence of PNA derived therefrom, and the base sequence of the PNA probe is indicated in blue.
  • the sequence of the gene fragment synthesized by the "PCR protocol 2 (OIE 4)" method for detecting the OIE standard of RSIV / ISKNV is transferred to the nucleotide database (nucleotide database) of the National Center for Biotechnology Information (NCBI). By comparing with the registered nucleotide sequence, we tried to secure the nucleotide sequence of the gene for each virus type.
  • a base sequence having a single nucleotide polymorphism (SNP) of RSIV capable of discriminating between RSIV and ISKNV represented by 5'-ACCAAGTTCATCATCT-3 '(SEQ ID NO: 8) is obtained, and the base sequence is RSIV.
  • SNP single nucleotide polymorphism
  • a primer pair (Forward primer 2, Reverse primer 2) represented by SEQ ID NO: 5 and SEQ ID NO: 6 as a primer for DNA amplification of the genetic marker and PNA 2 represented by SEQ ID NO: 2 as a hybridization probe of the genetic marker Produced.
  • FIG. 4 is a nucleotide sequence diagram showing an example of a part of the gene of RSIV / ISKNV and a base sequence for discrimination / detection and a PNA derived therefrom, and the base sequence of the PNA probe is indicated in blue.
  • PNA probe and primer sequences according to the present invention were determined and shown in Table 1.
  • TaqMan and PNA probes were prepared by combining FAM, TexasRed and Cy5, respectively, so that the same fluorescence was not included. Then, PNA probes were prepared using the nucleotide sequences, reporters, and quenchers as shown in Table 1.
  • the PNA probe used in the present invention was designed using PNA probe designer (Applied biosystems, USA), PNA probe was synthesized by HPLC purification method in Panazine (Panagene, Korea), the purity of all synthesized probes using mass spectrometry (Needed to avoid unnecessary secondary structures of the probe for more effective binding with the target nucleic acid).
  • amplification curves and lysis curves were derived for DNA samples of red snapper Iridovirus disease causative virus, and analyzed for the identification or detection of causal viruses.
  • a TaqMan probe (and corresponding primer set) for identifying or detecting the causative virus according to the OIE (Office of International Epizootics) standard may be used.
  • PCR was performed using the CFX96 TM Real-Time System (BIO-RAD, USA), and all PCR conditions used asymmetric PCR to generate single stranded target nucleic acids.
  • the composition of the reactants for asymmetric PCR is shown in Table 2.
  • Table 2 The composition of the reactants for asymmetric PCR is shown in Table 2.
  • real-time PCR was performed by adding 1 ⁇ l viral DNA.
  • 1 ⁇ l of the template DNA of each virus was added, followed by PCR.
  • Table 3 shows the amplification conditions and hybridization conditions of the reactants, and shows the process of amplifying and hybridizing the viral DNA sample and then raising the temperature of the hybridized product. The procedure was performed by real-time PCR.
  • various fluorescent reporters may be combined with PNA probes as shown in Table 1 below.
  • the virus-specific fluorescence amplification curve is confirmed so that the virus type can be determined by the fluorescent reporter (RSIV / ISKNV detection by TexasRed fluorescence).
  • the melting curve analysis was performed as in Example 2, and then the resulting fluorescent signal and Tm value were digitized to the perfect match temperature. That is, a range of ⁇ 2 ° C. of the perfect match temperature is made, and if the Tm value for an unknown viral DNA sample is within the above range, the virus type can be determined and specified.
  • 5 and 6 show amplification curves and fusion curves for each fluorescence obtained by applying peptide nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 2 to a viral DNA sample according to the present invention. Or it was shown that it can be detected.
  • the present invention selects genetic markers for the identification and / or detection of causative viruses of red seabream iridovirus disease, and (i) Red Sea Bream Iridovirus (RSIV) / Infectious Spleen and Kidney Necrosis Virus. , ISKNV) specific genes; Or (ii) using a peptide nucleic acid and primer pair specific for a specific gene marker that distinguishes RSIV from RSIV / RSKNV, resulting in amplification and melting curves of different fluorescence for each virus type. There is an effect capable of accurately determining and detecting whether the fish is infected with the virus.
  • RSIV Red Sea Bream Iridovirus
  • ISKNV Red Sea Bream Iridovirus

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Abstract

La présente invention concerne un marqueur génétique permettant de distinguer et de détecter un virus responsable d'une maladie infectieuse touchant les poissons marins, autrement dit le virus responsable de l'iridovirose de la daurade japonaise (RSIV), et un procédé de distinction et de détection d'un virus causal utilisant le marqueur et, plus précisément, un procédé consistant à : sélectionner et amplifier une séquence de base d'ADN codant (i) un gène particulier du virus responsable de l'iridovirose de la daurade japonaise (RSIV)/virus de la nécrose infectieuse de la rate et du rein (ISKNV) ou (ii) un gène particulier qui distingue la RSIV de RSIV/RSKNV; puis à hybrider un acide nucléique peptidique (PNA) qui reconnaît spécifiquement le produit amplifié; obtenir une courbe de fusion basée sur la température en régulant la température du produit hybridé; et déterminer un type de virus à partir d'une température de fusion par analyse de la courbe de fusion obtenue, ou détecter le statut d'infection des poissons par le type viral.
PCT/KR2016/009374 2016-01-15 2016-08-24 Marqueur génétique servant à détecter le virus responsable de l'iridovirose de la daurade japonaise, et procédé de détection du virus causal utilisant le marqueur WO2017122897A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113215167A (zh) * 2021-05-24 2021-08-06 广西科学院 核酸适配体及其在检测大口黑鲈虹彩病毒感染的细胞中的应用
CN117385053A (zh) * 2023-10-08 2024-01-12 山东省渔业发展和资源养护总站 一种与海水鲈鱼生长性状相连锁的分子标记位点

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101642784B1 (ko) * 2016-01-15 2016-07-27 대한민국 바이러스성 출혈성 패혈증 바이러스 판별용 유전자 마커, 및 이를 이용한 원인바이러스의 판별방법
CN110042174A (zh) * 2019-04-09 2019-07-23 广东省农业科学院动物卫生研究所 海鲈虹彩病毒的检测方法与试剂盒
CN114807149B (zh) * 2022-03-21 2023-11-17 华南农业大学 一种特异性识别大口黑鲈虹彩病毒的核酸适配体及其应用
CN116555433B (zh) * 2022-12-28 2024-03-19 中国水产科学研究院南海水产研究所 一种用于鉴别黄鳍棘鲷和平鲷的引物、试剂盒及应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102559928A (zh) * 2011-12-28 2012-07-11 中国海洋大学 特异性引物组、包含其的试剂盒、使用方法及检测方法
KR20150028063A (ko) * 2013-09-05 2015-03-13 주식회사 시선바이오머티리얼스 리포터 및 소광자가 결합된 프로브를 이용한 액상형 어레이 및 이를 이용한 표적핵산 또는 돌연변이 검출방법
KR101557844B1 (ko) * 2013-11-29 2015-10-06 한국해양과학기술원 이리도바이러스를 검출하기 위한 등온증폭 반응용 프라이머 세트, 이를 포함하는 프라이머 조성물, 및 이를 이용한 이리도바이러스 검출방법

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102559928A (zh) * 2011-12-28 2012-07-11 中国海洋大学 特异性引物组、包含其的试剂盒、使用方法及检测方法
KR20150028063A (ko) * 2013-09-05 2015-03-13 주식회사 시선바이오머티리얼스 리포터 및 소광자가 결합된 프로브를 이용한 액상형 어레이 및 이를 이용한 표적핵산 또는 돌연변이 검출방법
KR101557844B1 (ko) * 2013-11-29 2015-10-06 한국해양과학기술원 이리도바이러스를 검출하기 위한 등온증폭 반응용 프라이머 세트, 이를 포함하는 프라이머 조성물, 및 이를 이용한 이리도바이러스 검출방법

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KIM, JIN WOO ET AL.: "Diagnosis Case of Viral Hemorrhagic Septicemia (VHS) for Adult Paralichthys Olivaceus", KOREAN JOURNAL OF FISHERIES AND AQUATIC SCIENCES, vol. 45, no. 6, 2012, pages 666 - 674, XP055401235 *
MANUALS OF DIAGNOSTIC TESTS FOR AQUATIC ANIMALS, 2009, XP055400683 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113215167A (zh) * 2021-05-24 2021-08-06 广西科学院 核酸适配体及其在检测大口黑鲈虹彩病毒感染的细胞中的应用
CN113215167B (zh) * 2021-05-24 2023-11-17 广西科学院 核酸适配体及其在检测大口黑鲈虹彩病毒感染的细胞中的应用
CN117385053A (zh) * 2023-10-08 2024-01-12 山东省渔业发展和资源养护总站 一种与海水鲈鱼生长性状相连锁的分子标记位点
CN117385053B (zh) * 2023-10-08 2024-04-05 山东省渔业发展和资源养护总站 一种与海水鲈鱼生长性状相连锁的分子标记位点

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