WO2023121337A1 - Composition for detecting white spot syndrome virus and method for detecting same - Google Patents

Composition for detecting white spot syndrome virus and method for detecting same Download PDF

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WO2023121337A1
WO2023121337A1 PCT/KR2022/021054 KR2022021054W WO2023121337A1 WO 2023121337 A1 WO2023121337 A1 WO 2023121337A1 KR 2022021054 W KR2022021054 W KR 2022021054W WO 2023121337 A1 WO2023121337 A1 WO 2023121337A1
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wssv
white spot
syndrome virus
seq
spot syndrome
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French (fr)
Korean (ko)
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김소민
윤동빈
김민정
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(주)바이오니아
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the present invention relates to a composition for detecting white spot syndrome virus and a method for detecting the same, and specifically, a pair of primers for amplifying VP664 and/or VP28, which are genes expressing envelope proteins of white spot syndrome virus, and a white spot syndrome virus comprising the same. It relates to a composition for detection, a detection kit, and a method for detecting white spot syndrome virus using the same.
  • WSD White spot disease
  • Cephalothorax exoskeleton
  • WSD was first reported in lobster and prawn farms around the west coast in 1993, and since then, economic losses in the shrimp farming industry have been increasing worldwide every year.
  • WSD is highly contagious, and more than 90% of the objects are infected and killed within 3 to 10 days after onset.
  • White spot syndrome virus is a virus that causes WSD, and phylogenetically belongs to the Whispovirus of the Nimaviridae family.
  • WSSV is a double-stranded DNA virus with a morphological length of about 250-380 nm, a width of 80-120 nm, and a genome length of 290-305 kb, which is known to reach a large size among viruses.
  • the OIE defines a wide range of aquatic crustaceans, including seawater, brackish water, and freshwater shrimp, crabs, and crayfish, as WSSV-infected hosts, and also designates wild crustaceans and shellfish as disease vectors. .
  • WSSV can be finally diagnosed by detecting WSSV-specific genes and antigens detected in infected shrimp, crustaceans and shellfish near farms.
  • Most of the methods for diagnosing WSSV are known to use polymerase chain reaction (PCR), in situ hybridization using a gene probe, and immunological antigen diagnostic kits.
  • PCR polymerase chain reaction
  • in situ hybridization using a gene probe and immunological antigen diagnostic kits.
  • a genetic diagnosis method using polymerase chain reaction due to the absence of a standard culturable cell line for WSSV, it is most common to use a genetic diagnosis method using polymerase chain reaction.
  • the nested PCR method and the real-time PCR method, as well as the IQ2000 and IQplus products sold by Taiwan's GeneReach Biotechnology Corporation, are currently used for the diagnosis of WSSV worldwide as genetic diagnosis methods recommended by the OIE.
  • these existing genetic diagnostic methods are designed to detect only one specific gene, and thus have a problem in that the sensitivity and specificity of diagnosis are significantly lowered, making accurate diagnosis difficult in vector organisms and false positives for some negative samples.
  • There is a problem that the reliability of diagnosis is reduced because it can cause a reaction or cause a false negative reaction for a positive sample.
  • WSD is highly contagious and can have a significant impact on imports, exports and aquaculture due to false negatives (false negatives) rather than false positives (false positives).
  • a test method with low sensitivity can also affect the problem that a false negative reaction (false negative) can cause.
  • false negative false negative results may result if fragmentation of the gene of the region to be detected occurs.
  • the inventors of the present application while studying an excellent gene diagnosis method in terms of sensitivity improvement and specificity, used a single primer pair binding to genes VP664 and/or VP28 expressing WSSV outer membrane proteins and each gene.
  • sensitivity to WSSV is improved, enabling detection even in samples with a low degree of infection, and false negative (false negative) rate due to nucleic acid fragmentation during the nucleic acid extraction process It was confirmed that can be significantly lowered, and the present invention was completed.
  • An object of the present invention is to provide a composition for detecting white spot syndrome virus (WSSV).
  • WSSV white spot syndrome virus
  • An object of the present invention is to provide a kit for detecting white spot syndrome virus.
  • An object of the present invention is to provide a method for detecting white spot syndrome virus.
  • the present invention relates to a composition for detecting white spot syndrome virus (WSSV) comprising a pair of primers for amplifying VP664 and/or VP28 genes.
  • WSSV white spot syndrome virus
  • the present invention also relates to a kit for detecting white spot syndrome virus (WSSV), comprising a pair of primers for amplifying VP664 and/or VP28 genes.
  • WSSV white spot syndrome virus
  • the present invention further provides a step of amplifying the nucleic acid extracted from the sample with a primer pair to amplify the VP664 and/or VP28 genes of the white spot syndrome virus (WSSV), spot syndrome virus (WSSV) detection method.
  • WSSV white spot syndrome virus
  • WSSV spot syndrome virus
  • 1 is a schematic diagram of negative detection according to the position of primers for each gene and the presence or absence of gene fragments in the single-tube dual-target real-time chain polymerization of the WSSV diagnostic method according to the present invention.
  • Figure 2 is a schematic diagram showing the detection limit and cut-off value when VP28 and VP664 are simultaneously detected and VP664 is singlely detected in the WSSV diagnostic method according to the present invention.
  • Figure 3 shows the dual target detection method according to the present invention, the real-time PCR method specified in the OIE, a conventional genetic diagnosis method, and the IQ2000 WSSV detection kit developed by Taiwan's GeneReach Biotechnology Corporation (in accordance with the test method in the OIE manual).
  • This is a diagram showing the results of comparative evaluation of WSSV detection rate using shrimp samples obtained through random sampling in Hawaii (USA) and domestic aquaculture.
  • the present invention relates to a composition for detecting white spot syndrome virus (WSSV) comprising a primer pair for amplifying VP664 and/or VP28 genes.
  • WSSV white spot syndrome virus
  • the present invention relates to a composition for detecting white spot syndrome virus (WSSV), comprising a pair of primers for amplifying VP664 and VP28 genes.
  • WSSV white spot syndrome virus
  • the present invention also provides a white spot syndrome virus detection method comprising the step of amplifying a nucleic acid extracted from a specimen with a primer pair for amplifying the VP664 and/or VP28 genes of white spot syndrome virus (WSSV). It is about.
  • the present invention relates to a white spot syndrome virus detection method comprising the step of amplifying nucleic acids extracted from a specimen with a primer pair for amplifying the VP664 and VP28 genes of white spot syndrome virus (WSSV). it's about
  • WSSV can be detected not only in WSSV-infected individuals, but also in vectors and possessing organisms, making it useful for molecular diagnosis for aquatic organisms quarantine, surveillance, and early detection and control of infectious diseases.
  • a 'primer' is a single-stranded oligonucleotide capable of acting as a starting point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) at a suitable temperature and buffer.
  • oligonucleotide that can act as a starting point for template-directed DNA synthesis under appropriate conditions and appropriate temperatures in a buffered solution.
  • the appropriate length of the primer may vary depending on the purpose of use, but is usually 15 to 30 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template.
  • the primer sequence need not be perfectly complementary to the template, but must be sufficiently complementary to hybridize with the template.
  • 'complementary binding' means that a primer hybridizes with a corresponding nucleic acid strand under the conditions of performing a polymerization reaction, and may form a duplex structure. Complementary bonds may be formed even when the complementarity between paired nucleotide sequences forms a Watson-Crick pair or some non-Watson-Crick base pairs exist.
  • the primer pair includes forward and reverse primers, and refers to a primer that binds to the 5'-end and 3'-end of a specific region of a gene to be amplified by a gene amplification reaction, respectively, and acts as an initiation point for DNA synthesis.
  • the primer pair may include sequences of SEQ ID NOs: 1 and 2 or sequences of SEQ ID NOs: 4 and 5.
  • the primer pair including the sequences of SEQ ID NOs: 1 and 2 can bind to the VP28 gene constituting the white spot syndrome virus envelope protein.
  • the primer pair including the sequences of SEQ ID NOs: 4 and 5 can bind to the VP664 gene constituting the white spot syndrome virus envelope protein.
  • the present invention may further include a probe that binds to a product amplified by the primer pair.
  • the probe may complementarily hybridize to a gene specifically amplified by the primer pair.
  • the conditions used to achieve a particular level of stringency vary depending on the nature of the nucleic acids being hybridized. For example, the length of the nucleic acid region to be hybridized, degree of homology, nucleotide sequence composition (eg, GC/AT composition ratio), and nucleic acid type (eg, RNA, DNA) are considered in selecting hybridization conditions. A further consideration is whether the nucleic acid is immobilized, for example on a filter or the like.
  • very stringent conditions are: 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 carried out using one of these conditions, for example, a condition having high stringency, or each of the above conditions can be used, each 10 to 15 minutes in the order described above, all or all of the conditions described above Some iterations can be performed. However, as described above, optimal conditions will vary depending on the particular hybridization reaction involved and can be determined experimentally. In general, conditions with high stringency are used for hybridization of the probes of interest.
  • the probe may include, for example, a sequence of SEQ ID NO: 3 or 6.
  • a probe that binds to a product amplified by the primer pair including the sequences of SEQ ID NOs: 1 and 2 may include the sequence of SEQ ID NO: 3.
  • a probe that binds to the product amplified by the primer pair including the sequences of SEQ ID NOs: 4 and 5 may include the sequence of SEQ ID NO: 6.
  • nucleic acid when nucleic acid is amplified in a single tube together with the primers of SEQ ID NOs: 1 and 2, probe set 1 of SEQ ID NO: 3, primers of SEQ ID NOs: 4 and 5, and SEQ ID NO: 6 set 2, the sensitivity is greatly improved.
  • Real-time polymerase chain reaction was performed using the primer sets and probes binding to the genes VP28 and VP664 expressing the WSSV outer membrane protein according to the present invention, using both genes together with each probe loaded with the same fluorescent material in a single tube.
  • the sensitivity to WSSV can be improved and even if one gene fragment is generated during the nucleic acid extraction process, the probability of false negatives can be significantly reduced due to simultaneous detection of other genes.
  • the probe is detectably labeled, for example, a radioactive isotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelate, or an enzyme.
  • a radioactive isotope for example, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelate, or an enzyme.
  • the amount of the amplification product can be detected by fluorescence signal.
  • a reagent intercalator
  • a reporter and a fluorescent material of a quencher capable of quenching reporter fluorescence may be bound to both ends of the probe.
  • the reporter may be 6-carboxyfluorescein (FAM) or black hole quencher (BHQ), and the quencher may be selected from phosphate.
  • each probe is preferably loaded with the same fluorescent substance.
  • the amplification according to the present invention is performed using methods such as real-time quantitative amplification, for example, Real-Time PCR or Real-time Quantitative Polymerase chain reaction (RQ-PCR). can be done through
  • PCR Real-time polymerase chain reaction
  • NAT nucleic acid amplification technologies
  • the fluorescent substance is intercalated into a double-stranded DNA chain during the PCR process, so that the amount of fluorescence is finally detected by the device.
  • a threshold cycle which is an amplification cycle at a point in time when the amount of fluorescence significantly increases beyond the lowest detectable level (background level)
  • a standard calibration curve is obtained by measuring the log value of the initial amount and the critical period using a sample of which the initial amount of nucleic acid is already known, thereby providing a method for real-time measurement of nucleic acid amplification products.
  • Real-time chain reaction including antigen-antibody hot-start nucleic acid amplification method in real-time polymerization chain reaction, hot-start nucleic acid amplification method using pyrophosphate and pyrophorpophatase, hot-start nucleic acid amplification method using DNA aptamer, etc. Preference is given to using reagents.
  • the DNA of the sample was added to a tube together with the real-time chain amplification reagent and the primer set for WSSV diagnosis, that is, the primers of SEQ ID NOs: 1 and 2, the probe set 1 of SEQ ID NO: 3, the primers of SEQ ID NOs: 4 and 5, and the set 2 of SEQ ID NO: 6 , (a) a preliminary DNA denaturation step of denaturing at a temperature of 91 to 97 ° C. for 1 to 5 minutes; (b) The first PCR reaction was performed at a temperature of 50 to 65° C.
  • conditions of temperature and time in the polymerase chain reaction are values set to produce the polymerase chain reaction product of the present invention, and are appropriately selected by those skilled in the art according to changes in experimental methods and equipment Change to reasonable values It can be.
  • the amplified target sequence may be labeled with a detectable labeling material.
  • the probe labeling material is a fluorescent material, but is not limited to the type of fluorescent material.
  • the labeling substance may be Cy-5 or Cy-3.
  • the present invention also relates to a kit for detecting white spot syndrome virus (WSSV), comprising a pair of primers for amplifying VP664 and/or VP28 genes.
  • WSSV white spot syndrome virus
  • the kit may optionally include reagents necessary for carrying out a nucleic acid amplification PCR reaction, such as a polymerase and a buffer, if necessary.
  • a kit according to the present invention may also further comprise various polynucleotide molecules, various buffers and reagents.
  • Optimal amounts of reagents, buffers, or reactants used for a specific reaction in the kit can be determined by a person skilled in the art, and are prepared in separate packages or compartments containing each of the aforementioned primer pairs and/or probes. It can be.
  • the sample includes shrimp, crab and shellfish, but is not limited thereto.
  • the target sample includes, but is not limited to, white spot syndrome virus-infected individuals, mediators, and organisms.
  • a step of extracting nucleic acids from the sample may be further included, and the extraction of nucleic acids may be performed using, for example, various commercially available kits or extraction reagents.
  • Methods for extracting genomic DNA from samples include a phenol/chloroform extraction method commonly used in the art, an SDS extraction method (Tai et al., Plant Mol. Biol. Reporter, 8: 297-303, 1990), and a CTAB separation method (Cetyl Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980) or a commercially available DNA extraction kit.
  • white spot syndrome virus can be diagnosed even with low-infected specimens, so the sensitivity is very good, and the false negative rate due to gene fragments that can occur during the nucleic acid extraction process can be significantly reduced compared to existing detection methods. has an effect.
  • WSSV white spot syndrome virus
  • WSSV-related genetic information The entire gene sequence was referred to, and GenBank Accession No. See AF332093.3.
  • the entire gene sequences of the Chinese strain (Registration No. AF332093), Thailand strain (Registration No. AF369029) and Taiwanese strain (Registration No. AF440570) were referenced.
  • primer sets targeting the WSSV envelope protein that is, specific primers and probes targeting the VP28 gene and specific primers and probes targeting VP664 are the primers and probes listed in the OIE WSSV test protocol. was used. This is shown in Table 1 below.
  • Plasmid DNA containing WSSV VP664 and VP28 was prepared to analyze the detection limit and cut-off value of the WSSV gene diagnosis method of the present invention and the VP664 gene (International Bureau of Animal Services method) diagnosis method, and to analyze the detection limit and cut-off value 3 fold-diluted plasmid DNA with 1000 ⁇ 1.4 copies/rxn was used. After adding primer sets of SEQ ID NOs: 1 to 6 and SEQ ID NOs: 4 to 6 described in Table 1 to a tube of AccuPower Plus Dualstar qPCR Mastermix (Bioneer), the DNA was denatured at 95°C for 5 minutes.
  • FIG. 2A shows primer sets of SEQ ID NOs: 1 to 3 targeting only VP664, and FIG. 2B shows the detection limit and
  • the detection limit of the primer set targeting VP664 and VP28 together was about 6 times higher than that of the primer set targeting only VP664, and the cut-off value was 0.82. It was confirmed that the primer set targeting VP664 and VP28 of the present invention can increase the sensitivity of the white spot virus to low-concentration samples.
  • nucleic acid was extracted from WSSV-infected shrimp gypsy according to a nucleic acid extraction protocol, and DNA extracted from 10 ⁇ -1 dilution to 10 ⁇ -7 dilution After dilution
  • Example 1 AccuPower Plus Dualstar qPCR with a primer set in which the primer sets of SEQ ID NOs: 1 to 3 and the primer sets of SEQ ID NOs: 4 to 6 described in Table 1 were used, respectively, and the primer sets of SEQ ID NOs: 1 to 6 were mixed.
  • DNA was denatured for 5 minutes at a temperature of 95 ° C. Thereafter, a scanning step (fluorescence measurement) at 95°C for 20 seconds and at 55°C for 30 seconds was performed using a real-time PCR amplifier (Exicycler96 thermal block, BIONEER).
  • the WSSV dual target gene diagnosis method of the present invention is sensitive to the conventional gene diagnosis method, the VP664 gene detection diagnosis method according to the International Bureau of Animal Services manual, the VP 28 gene detection diagnosis method designed through the present invention, and the mixed diagnosis method of VP664 and VP28 gene detection primers. As confirmed, detection of up to 10 times higher dilution in the detection diagnosis method in which VP28 and VP664 detection primers were mixed compared to the conventional genetic diagnosis method, the VP664 gene detection diagnosis method according to the International Animal Services Manual and the VP 28 gene detection diagnosis method designed through the present invention. , and very good sensitivity was confirmed. The results are shown in Table 2 below.
  • the detection rate of the WSSV gene diagnosis method of the present invention and the detection rate of the conventional genetic diagnosis method were randomly sampled in Hawaii (USA) and a domestic aquaculture farm, and the detection rate was compared and evaluated. was performed.
  • nucleic acids were extracted from random samples using a viral DNA/RNA extraction kit according to the manufacturer's manual.
  • the conventional gene diagnosis method detection of the VP664 gene according to the manual of the International Animal Health Service
  • the IQ2000 WSSV detection kit developed by Taiwan's GeneReach Biotechnology Corporation listed as a test method in the manual of the International Animal Health Service
  • Taiwan's GeneReach Biotechnology Corporation listed as a test method in the manual of the International Animal Health Service
  • the conventional gene diagnosis method and the dual target gene diagnosis method of the present invention were performed as in Example 2, and the IQ2000 WSSV detection kit was performed according to the product manual.
  • a composition, kit, and detection method capable of preventing false negatives and false positives that may occur in detecting white spot syndrome virus (WSSV).
  • WSSV white spot syndrome virus
  • real-time polymerase chain reaction is performed using a pair of primers to amplify the genes VP28 and VP664 that express WSSV outer membrane proteins, and using both genes together with each probe loaded with the same fluorescent material in a single tube , it improves the sensitivity to WSSV, and even if one gene fragment is generated during the nucleic acid extraction process, it can significantly reduce the probability of false negatives by simultaneously detecting other genes.

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Abstract

The present invention relates to a composition for detecting a white spot syndrome virus and a method for detecting same and, particularly, to a pair of primers for amplifying VP664 and/or VP28, which are genes expressing envelope proteins of a white spot syndrome virus, a composition for detecting a white spot syndrome virus comprising same, a kit for detection, and a method for detecting a white spot syndrome virus using same.

Description

흰반점 증후군 바이러스 검출용 조성물 및 이의 검출방법Composition for detecting white spot syndrome virus and method for detecting the same
본 발명은 흰반점 증후군 바이러스 검출용 조성물 및 이의 검출방법에 관한 것으로, 구체적으로 흰반점 증후군 바이러스의 외피막 단백질 발현 유전자인 VP664 및/또는 VP28을 증폭하기 위한 프라이머 쌍, 이를 포함하는 흰반점 증후군 바이러스 검출용 조성물, 검출용 키트 및 이를 이용한 흰반점 증후군 바이러스 검출방법에 관한 것이다.The present invention relates to a composition for detecting white spot syndrome virus and a method for detecting the same, and specifically, a pair of primers for amplifying VP664 and/or VP28, which are genes expressing envelope proteins of white spot syndrome virus, and a white spot syndrome virus comprising the same. It relates to a composition for detection, a detection kit, and a method for detecting white spot syndrome virus using the same.
흰반점병(white spot disease; WSD)은 새우의 외골격(두흉갑)에 흰색 반점이 나타나는 질환으로, 수입 냉동 새우 검역, 양식 새우 등의 대량폐사의 원인으로 심각한 경제적 손실을 입히고 있다. 아시아에서 최초로 보고 된 이래로, 현재 대부분의 아시아 국가 및 북아메리카 등의 지역에서 양식 새우의 WSD가 꾸준히 발병하고 있으며, WSD는 세계적으로 새우 양식산업에 심각한 경제적 손실을 입히고 있다. 국제수역사무국 OIE (World Organization for animal health) 및 국내 해양수산부에서도 10 대 전염성 병원체로 지정하여 관리되고 있다.White spot disease (WSD) is a disease in which white spots appear on the exoskeleton (cephalothorax) of shrimp, causing serious economic losses due to quarantine of imported frozen shrimp and mass mortality of farmed shrimp. Since it was first reported in Asia, WSD in farmed shrimp has been steadily occurring in most Asian countries and regions such as North America, and WSD is causing serious economic losses to the shrimp farming industry worldwide. The World Organization for Animal Health (OIE) and the Ministry of Maritime Affairs and Fisheries in Korea also designate and manage them as one of the top 10 infectious pathogens.
우리나라의 경우, 1993년 서해안 일대의 대하 및 보리새우 양식장에서 최초로 WSD가 보고되었으며, 그 후 현재까지 전세계적으로 매년 새우 양식산업의 경제적 손실이 늘고 있다. 특히, 대하 및 보리새우의 경우, WSD가 강한 전염성을 보여 발병된 후 3-10일 이내에 개체의 90% 이상이 감염 및 폐사되고 있다.In the case of Korea, WSD was first reported in lobster and prawn farms around the west coast in 1993, and since then, economic losses in the shrimp farming industry have been increasing worldwide every year. In particular, in the case of prawns and shrimp, WSD is highly contagious, and more than 90% of the objects are infected and killed within 3 to 10 days after onset.
흰반점 증후군 바이러스(white spot syndrom virus; WSSV)는 WSD의 원인 바이러스로, 계통발생학적으로는 니마비리데과(Nimaviridae) 휘스포바이러스속(Whispovirus)에 속한다. 또한, WSSV는 이중나선형 DNA 바이러스로 형태학적 길이는 약 250-380 nm, 넓이는 80-120 nm이고, 유전체의 길이는 290-305 kb로 바이러스 중에서도 큰 사이즈에 이르는 것으로 알려져 있다. 국제수역사무국(OIE)에서는 해수, 기수 및 담수 서식 새우류, 게, 가재 등을 포함하는 넓은 범위의 수생 갑각류를 WSSV 감염 숙주로 정의하고 있으며, 이와 더불어 야생 갑각류 및 조개류 등을 질병 매개체로 지정하고 있다.White spot syndrome virus (WSSV) is a virus that causes WSD, and phylogenetically belongs to the Whispovirus of the Nimaviridae family. In addition, WSSV is a double-stranded DNA virus with a morphological length of about 250-380 nm, a width of 80-120 nm, and a genome length of 290-305 kb, which is known to reach a large size among viruses. The OIE defines a wide range of aquatic crustaceans, including seawater, brackish water, and freshwater shrimp, crabs, and crayfish, as WSSV-infected hosts, and also designates wild crustaceans and shellfish as disease vectors. .
WSSV는 감염 새우, 양식장 인근 갑각류 및 패류에서 검출되는 WSSV 특이적 유전자 및 항원을 검출함으로써 최종 진단될 수 있다. WSSV 진단법은 대부분 중합효소 연쇄반응(polymerase chain reaction; PCR), 유전자 탐침을 이용한 현장혼성화(in situ hybridization) 및 면역학적 항원 진단키트를 이용하는 것으로 알려져 있다. 특히, WSSV는 배양 가능한 표준 세포주의 부재로 인해 중합효소 연쇄반응을 이용한 유전자 진단법을 사용하는 것이 가장 일반화되어 있다.WSSV can be finally diagnosed by detecting WSSV-specific genes and antigens detected in infected shrimp, crustaceans and shellfish near farms. Most of the methods for diagnosing WSSV are known to use polymerase chain reaction (PCR), in situ hybridization using a gene probe, and immunological antigen diagnostic kits. In particular, due to the absence of a standard culturable cell line for WSSV, it is most common to use a genetic diagnosis method using polymerase chain reaction.
현재 국제수역사무국(OIE)에서 권장하고 있는 유전자 진단법으로 Nested PCR법을 이용한 방법과 Real time PCR법 및 대만 GeneReach Biotechnology Corporation에서 판매하고 있는 IQ2000, IQplus 제품이 전세계적으로 WSSV의 진단을 위해 사용되고 있다. 그러나, 이러한 기존의 유전자 진단법은 한가지 특이 유전자만을 검출할 수 있도록 고안되어 있어 진단의 민감도 및 특이도가 현저히 떨어진다는 문제점이 있어 매개체 생물에서의 정확한 진단이 어려울 뿐만 아니라, 일부 음성시료에 대해서 거짓 양성반응을 유발하거나, 양성시료에 대해서 거짓 음성반응을 유발할 수 있어 진단의 신뢰성이 떨어진다는 문제점이 있다. The nested PCR method and the real-time PCR method, as well as the IQ2000 and IQplus products sold by Taiwan's GeneReach Biotechnology Corporation, are currently used for the diagnosis of WSSV worldwide as genetic diagnosis methods recommended by the OIE. However, these existing genetic diagnostic methods are designed to detect only one specific gene, and thus have a problem in that the sensitivity and specificity of diagnosis are significantly lowered, making accurate diagnosis difficult in vector organisms and false positives for some negative samples. There is a problem that the reliability of diagnosis is reduced because it can cause a reaction or cause a false negative reaction for a positive sample.
WSD는 전염성이 매우 높아 거짓 양성반응(위양성)보다는 거짓 음성반응(위음성)으로 인해 수입 및 수출, 양식에 지대한 영향을 줄 수 있다.WSD is highly contagious and can have a significant impact on imports, exports and aquaculture due to false negatives (false negatives) rather than false positives (false positives).
거짓 음성반응(위음성)이 유발할 수 있는 문제로는 낮은 민감도를 가진 검사법도 영향을 줄 수 있지만, 분자진단을 위한 핵산 추출 과정에서 300kb에 달하는 거대한 핵산으로 구성되어 있는 WSD의 경우 핵산 추출과정에서 나타나는 핵산 파편 현상으로 인해 바이러스가 존재를 하지만, 검출하려고 하는 부위 유전자의 파편현상이 발생하게 되면 거짓 음성(위음성)결과를 초래할 수 있다.A test method with low sensitivity can also affect the problem that a false negative reaction (false negative) can cause. Although viruses exist due to nucleic acid fragmentation, false negative (false negative) results may result if fragmentation of the gene of the region to be detected occurs.
이러한 기술적 배경에서, 본 출원의 발명자들은 민감도 향상 및 특이도 측면에서 우수한 유전자 진단법에 관해 연구하던 중, WSSV 외피막 단백질을 발현하는 유전자 VP664 및/또는 VP28과 각 유전자에 결합하는 프라이머 쌍을 통해 단일 튜브에서 두 개 타겟을 동시에 중합효소연쇄반응을 수행할 경우, WSSV에 대한 민감도를 개선시켜 감염정도가 낮은 검체에서도 검출이 가능하고, 핵산 추출과정에서 나타나는 핵산 파편 현상으로 인한 거짓음성(위음성)률을 현저히 낮출 수 있음을 확인하고, 본 발명을 완성하게 되었다.Against this technical background, the inventors of the present application, while studying an excellent gene diagnosis method in terms of sensitivity improvement and specificity, used a single primer pair binding to genes VP664 and/or VP28 expressing WSSV outer membrane proteins and each gene. When polymerase chain reaction is performed on two targets simultaneously in a tube, sensitivity to WSSV is improved, enabling detection even in samples with a low degree of infection, and false negative (false negative) rate due to nucleic acid fragmentation during the nucleic acid extraction process It was confirmed that can be significantly lowered, and the present invention was completed.
본 배경기술 부분에 기재된 상기 정보는 오직 본 발명의 배경에 대한 이해를 향상시키기 위한 것이며, 이에 본 발명이 속하는 기술분야에서 통상의 지식을 가지는 자에게 있어 이미 알려진 선행기술에 대한 정보는 포함하지 않을 수 있다.The above information described in the Background Art section is only for improving the understanding of the background of the present invention, and therefore, information on prior art already known to those skilled in the art to which the present invention belongs will not be included. can
발명의 요약Summary of Invention
본 발명의 목적은 흰반점 증후군 바이러스 (white spot syndrome virus; WSSV) 검출용 조성물을 제공하는 것이다.An object of the present invention is to provide a composition for detecting white spot syndrome virus (WSSV).
본 발명의 목적은 흰반점 증후군 바이러스 검출용 키트를 제공하는 것이다.An object of the present invention is to provide a kit for detecting white spot syndrome virus.
본 발명의 목적은 흰반점 증후군 바이러스 검출방법을 제공하는 것이다.An object of the present invention is to provide a method for detecting white spot syndrome virus.
상기 목적을 달성하기 위하여 본 발명은 흰반점 증후군 바이러스 (white spot syndrome virus; WSSV)의 VP664 및/또는 VP28 유전자를 증폭하기 위한 프라이머 쌍을 포함하는, 흰반점 증후군 바이러스 검출용 조성물에 관한 것이다. In order to achieve the above object, the present invention relates to a composition for detecting white spot syndrome virus (WSSV) comprising a pair of primers for amplifying VP664 and/or VP28 genes.
본 발명은 또한, 흰반점 증후군 바이러스 (white spot syndrome virus; WSSV)의 VP664 및/또는 VP28 유전자를 증폭하기 위한 프라이머 쌍을 포함하는, 흰반점 증후군 바이러스 검출용 키트에 관한 것이다.The present invention also relates to a kit for detecting white spot syndrome virus (WSSV), comprising a pair of primers for amplifying VP664 and/or VP28 genes.
본 발명은 더욱이, 검체에서 추출된 핵산을 흰반점 증후군 바이러스 (white spot syndrome virus; WSSV)의 VP664 및/또는 VP28 유전자를 증폭하기 위한 프라이머 쌍으로 증폭시키는 단계를 포함하는, 흰반점 증후군 바이러스 (white spot syndrome virus; WSSV) 검출방법에 관한 것이다. The present invention further provides a step of amplifying the nucleic acid extracted from the sample with a primer pair to amplify the VP664 and/or VP28 genes of the white spot syndrome virus (WSSV), spot syndrome virus (WSSV) detection method.
도 1은 본 발명에 따른 WSSV 진단법의 단일 튜브 듀얼타겟 실시간연쇄중합반응에서 각 유전자에 대한 프라이머 위치와 유전자 파편 발생 유무에 따른 음양성 검출 모식도이다. 1 is a schematic diagram of negative detection according to the position of primers for each gene and the presence or absence of gene fragments in the single-tube dual-target real-time chain polymerization of the WSSV diagnostic method according to the present invention.
도 2는 본 발명에 따른 WSSV 진단법의 VP28 및 VP664 동시 검출 과 VP664 단일 검출했을 때의 검출 한계 및 cut-off value를 나타내는 모식도이다. Figure 2 is a schematic diagram showing the detection limit and cut-off value when VP28 and VP664 are simultaneously detected and VP664 is singlely detected in the WSSV diagnostic method according to the present invention.
도 3는 본 발명에 따른 듀얼타겟검출법과 종래의 유전자 진단법인 국제수역사무국(OIE)에 명시되어 있는 Real time PCR 법과 대만 GeneReach Biotechnology Corporation 사에서 개발한 IQ2000 WSSV detection kit(국제수역사무국 매뉴얼에 검사법으로 등재) 진단법과 하와이(미국), 국내양식업에서 무작위 샘플링을 통해 확보한 새우 샘플을 이용하여 WSSV 검출률을 비교 평가한 결과를 나타낸 도면이다.Figure 3 shows the dual target detection method according to the present invention, the real-time PCR method specified in the OIE, a conventional genetic diagnosis method, and the IQ2000 WSSV detection kit developed by Taiwan's GeneReach Biotechnology Corporation (in accordance with the test method in the OIE manual). This is a diagram showing the results of comparative evaluation of WSSV detection rate using shrimp samples obtained through random sampling in Hawaii (USA) and domestic aquaculture.
발명의 상세한 설명 및 바람직한 구현예DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.
본 발명은 일 관점에서, 흰반점 증후군 바이러스 (white spot syndrome virus; WSSV)의 VP664 및/또는 VP28 유전자를 증폭하기 위한 프라이머 쌍을 포함하는, 흰반점 증후군 바이러스 검출용 조성물에 관한 것이다. In one aspect, the present invention relates to a composition for detecting white spot syndrome virus (WSSV) comprising a primer pair for amplifying VP664 and/or VP28 genes.
구체적으로, 본 발명은 흰반점 증후군 바이러스 (white spot syndrome virus; WSSV)의 VP664 및 VP28 유전자를 증폭하기 위한 프라이머 쌍을 포함하는, 흰반점 증후군 바이러스 검출용 조성물에 관한 것이다.Specifically, the present invention relates to a composition for detecting white spot syndrome virus (WSSV), comprising a pair of primers for amplifying VP664 and VP28 genes.
본 발명은 또한, 검체에서 추출된 핵산을 흰반점 증후군 바이러스 (white spot syndrome virus; WSSV)의 VP664 및/또는 VP28 유전자를 증폭하기 위한 프라이머 쌍으로 증폭시키는 단계를 포함하는, 흰반점 증후군 바이러스 검출방법에 관한 것이다. The present invention also provides a white spot syndrome virus detection method comprising the step of amplifying a nucleic acid extracted from a specimen with a primer pair for amplifying the VP664 and/or VP28 genes of white spot syndrome virus (WSSV). It is about.
구체적으로, 본 발명은 검체에서 추출된 핵산을 흰반점 증후군 바이러스 (white spot syndrome virus; WSSV)의 VP664 및 VP28 유전자를 증폭하기 위한 프라이머 쌍으로 증폭시키는 단계를 포함하는, 흰반점 증후군 바이러스 검출방법에 관한 것이다.Specifically, the present invention relates to a white spot syndrome virus detection method comprising the step of amplifying nucleic acids extracted from a specimen with a primer pair for amplifying the VP664 and VP28 genes of white spot syndrome virus (WSSV). it's about
흰반점 증후군 바이러스 내에 유전자 두 곳을 동시에 검출함으로써 핵산 추출과정에서 나타나는 핵산 파편(Fragment)에 의해 한 개의 타겟 유전자가 상실되어도 검출이 가능하여, 위음성 발생율을 현저히 낮춰주고, 한 튜브에서 두 개의 유전자를 같은 형광 염료를 이용하여 검출함으로써 낮은 카피수까지 검출이 가능하여, WSSV 감염된 개체뿐만아니라, 매개체 및 보유 생물에서도 WSSV 검출이 가능하여 수산생물 방역, 감시, 전염병 조기 발견 및 통제를 위한 분자진단에 유용하게 사용될 수 있다.By simultaneously detecting two genes in the white spot syndrome virus, it is possible to detect even if one target gene is lost due to a nucleic acid fragment that appears during the nucleic acid extraction process, significantly reducing the false negative rate and detecting the same two genes in one tube. It is possible to detect even low copy numbers by detecting it using a fluorescent dye, so WSSV can be detected not only in WSSV-infected individuals, but also in vectors and possessing organisms, making it useful for molecular diagnosis for aquatic organisms quarantine, surveillance, and early detection and control of infectious diseases. can be used
본 명세서에서 ‘프라이머’는 적합한 온도 및 완충액 내에서 적합한 조건(즉, 4종의 다른 뉴클레오사이드 트리포스페이트 및 중합반응 효소) 하에서 주형-지시 DNA 합성의 개시점으로 작용할 수 있는 단일-가닥 올리고뉴클레오티드를 의미한다. As used herein, a 'primer' is a single-stranded oligonucleotide capable of acting as a starting point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) at a suitable temperature and buffer. means
절한 완충용액 중의 적절한 조건 및 적당한 온도 하에서 주형-지시 DNA 합성의 시작점으로서 작용할 수 있는 단일가닥 올리고뉴클레오티드를 말한다. 상기 프라이머의 적절한 길이는 사용 목적에 따라 달라질 수 있으나, 통상 15 내지 30 뉴클레오티드이다. 짧은 프라이머 분자는 일반적으로 주형과 안정한 혼성체를 형성하기 위해서는 더 낮은 온도를 필요로 한다. 프라이머 서열은 주형과 완전하게 상보적일 필요는 없으나, 주형과 혼성화할 정도로 충분히 상보적이어야 한다. It refers to a single-stranded oligonucleotide that can act as a starting point for template-directed DNA synthesis under appropriate conditions and appropriate temperatures in a buffered solution. The appropriate length of the primer may vary depending on the purpose of use, but is usually 15 to 30 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template. The primer sequence need not be perfectly complementary to the template, but must be sufficiently complementary to hybridize with the template.
본 명세서에서 ‘상보적 결합’은 중합반응을 수행하는 조건에서 프라이머가 대응하는 핵산 가닥과 하이브리다이제이션되는 것을 의미하고, 이합체(duplex) 구조를 형성할 수 있다. 상보적 결합은 쌍을 이루는 뉴클레오티드 서열 간의 상보성이 왓슨-크릭 결합(Watson-Crick pair)을 이루거나, 일부 비 왓슨-크릭 결합(Non-Watson-Crick base pair)이 존재하여도 형성될 수 있다.In the present specification, 'complementary binding' means that a primer hybridizes with a corresponding nucleic acid strand under the conditions of performing a polymerization reaction, and may form a duplex structure. Complementary bonds may be formed even when the complementarity between paired nucleotide sequences forms a Watson-Crick pair or some non-Watson-Crick base pairs exist.
상기 프라이머 쌍은 정방향 및 역방향 프라이머를 포함하고, 유전자 증폭 반응에 의해 증폭되는 유전자의 특정 부위의 5‘-말단 및 3’-말단에 각각 결합하여 DNA 합성의 개시점으로 작용하는 프라이머를 의미한다.The primer pair includes forward and reverse primers, and refers to a primer that binds to the 5'-end and 3'-end of a specific region of a gene to be amplified by a gene amplification reaction, respectively, and acts as an initiation point for DNA synthesis.
예를 들어, 상기 프라이머 쌍은 서열번호 1 및 2의 서열 또는 서열번호 4 및 5의 서열을 포함할 수 있다. For example, the primer pair may include sequences of SEQ ID NOs: 1 and 2 or sequences of SEQ ID NOs: 4 and 5.
상기 프라이머 쌍 중 서열번호 1 및 2의 서열을 포함하는 프라이머 쌍은 흰반점 증후군 바이러스 외피막 단백질을 구성하는 VP28 유전자에 결합할 수 있다. Among the primer pairs, the primer pair including the sequences of SEQ ID NOs: 1 and 2 can bind to the VP28 gene constituting the white spot syndrome virus envelope protein.
상기 프라이머 쌍 중 서열번호 4 및 5의 서열을 포함하는 프라이머 쌍은 흰반점 증후군 바이러스 외피막 단백질을 구성하는 VP664 유전자에 결합할 수 있다. Among the primer pairs, the primer pair including the sequences of SEQ ID NOs: 4 and 5 can bind to the VP664 gene constituting the white spot syndrome virus envelope protein.
본 발명은 또한, 상기 프라이머 쌍에 의해 증폭된 산물에 결합하는 프로브를 추가로 포함할 수 있다. 상기 프로브는 프라이머 쌍에 의해 특이적으로 증폭된 유전자에 상보적으로 혼성화 할 수 있다.The present invention may further include a probe that binds to a product amplified by the primer pair. The probe may complementarily hybridize to a gene specifically amplified by the primer pair.
혼성화 반응에서, 엄격한 특정 수준을 달성하기 위하여 사용되는 조건은 혼성화 되는 핵산의 성질에 따라 다양하다. 예를 들면, 혼성화 되는 핵산 부위의 길이, 상동성 정도, 뉴클레오티드 서열 조성(예를 들면, GC/AT 조성비) 및 핵산 타입(예를 들면, RNA, DNA)등이 혼성화 조건을 선택하는데 고려된다. 추가적인 고려 조건은 핵산이 예를 들면, 필터 등에 고정화되어 있는지의 여부이다.In a hybridization reaction, the conditions used to achieve a particular level of stringency vary depending on the nature of the nucleic acids being hybridized. For example, the length of the nucleic acid region to be hybridized, degree of homology, nucleotide sequence composition (eg, GC/AT composition ratio), and nucleic acid type (eg, RNA, DNA) are considered in selecting hybridization conditions. A further consideration is whether the nucleic acid is immobilized, for example on a filter or the like.
매우 엄격하게 진행되는 조건의 예를 들면 다음과 같다: 실온의 2X SSC/0.1% SDS(혼성화 조건); 실온의 0.2X SSC/0.1% SDS(엄격성이 낮은 조건); 42℃에서의 0.2X SSC/0.1% SDS(보통의 엄격성을 가지는 조건); 68℃에서 0.1X SSC(높은 엄격성을 가지는 조건). 세척 과정은 이들 중 한가지 조건을 사용하여 수행할 수 있고, 예를 들면 높은 엄격성을 가지는 조건, 또는 상기 조건을 각각 사용할 수 있으며, 상기 기재된 순서대로 각각 10~15분씩, 상기 기재된 조건을 전부 또는 일부 반복하여 수행할 수 있다. 그러나 상기에 기술한 바와 같이, 최적 조건은 포함된 특별한 혼성화 반응에 따라 다양하며, 실험을 통하여 결정할 수 있다. 일반적으로, 중요한 프로브의 혼성화에는 높은 엄격성을 가지는 조건이 사용된다.Examples of very stringent conditions are: 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 carried out using one of these conditions, for example, a condition having high stringency, or each of the above conditions can be used, each 10 to 15 minutes in the order described above, all or all of the conditions described above Some iterations can be performed. However, as described above, optimal conditions will vary depending on the particular hybridization reaction involved and can be determined experimentally. In general, conditions with high stringency are used for hybridization of the probes of interest.
상기 프로브는 예를 들어, 서열번호 3 또는 6의 서열을 포함할 수 있다. 서열번호 1 및 2의 서열을 포함하는 프라이머 쌍에 의해 증폭된 산물에 결합하는 프로브는 서열번호 3의 서열을 포함할 수 있다. 서열번호 4 및 5의 서열을 포함하는 프라이머 쌍에 의해 증폭된 산물에 결합하는 프로브는 서열번호 6의 서열을 포함할 수 있다.The probe may include, for example, a sequence of SEQ ID NO: 3 or 6. A probe that binds to a product amplified by the primer pair including the sequences of SEQ ID NOs: 1 and 2 may include the sequence of SEQ ID NO: 3. A probe that binds to the product amplified by the primer pair including the sequences of SEQ ID NOs: 4 and 5 may include the sequence of SEQ ID NO: 6.
구체적으로, 본 발명에 따르면 서열번호 1 및 2의 프라이머와 서열번호 3 프로브 세트 1, 서열번호 4 및 5의 프라이머와 서열번호 6 세트 2와 함께 단일 튜브에서 핵산을 증폭하는 경우 민감도가 매우 향상될 수 있다. Specifically, according to the present invention, when nucleic acid is amplified in a single tube together with the primers of SEQ ID NOs: 1 and 2, probe set 1 of SEQ ID NO: 3, primers of SEQ ID NOs: 4 and 5, and SEQ ID NO: 6 set 2, the sensitivity is greatly improved. can
본 발명에 따른 WSSV 외피막 단백질을 발현하는 유전자 VP28과 VP664와 결합하는 프라이머 세트 및 프로브를 이용하여 단일 튜브에서 두 유전자를 같은 형광물질이 탑재된 각 프로브와 같이 사용하여 실시간중합효소연쇄반응을 수행할 경우, WSSV에 대한 민감도를 개선시켜주고 핵산 추출과정에서 하나의 유전자 파편이 발생이 되더라도 다른 유전자를 동시 검출하기에 위음성이 나타날 확률을 현저히 감소시킬 수 있다.Real-time polymerase chain reaction was performed using the primer sets and probes binding to the genes VP28 and VP664 expressing the WSSV outer membrane protein according to the present invention, using both genes together with each probe loaded with the same fluorescent material in a single tube. In this case, the sensitivity to WSSV can be improved and even if one gene fragment is generated during the nucleic acid extraction process, the probability of false negatives can be significantly reduced due to simultaneous detection of other genes.
경우에 따라서, 프로브는 검출할 수 있도록 표지되며, 예를 들면 방사선 동위원소, 형광 화합물, 바이오 발광 화합물, 화학 발광 화합물, 금속 킬레이트 또는 효소로 표지될 수 있다. 상기와 같은 프로브를 적당하게 표지하는 것은 당해 분야에서 널리 알려진 기술이며, 통상적인 방법을 통하여 수행할 수 있다.Optionally, the probe is detectably labeled, for example, a radioactive isotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelate, or an enzyme. Properly labeling the probe as described above is a technique widely known in the art and can be performed through a conventional method.
상기 증폭 산물의 양은 형광신호에 의해 검출할 수 있다. 프로브가 결합된 증폭산물의 이중 나선 DNA에 결합하여 형광을 나타내는 시약(intercalator)을 사용하는 인터컬레이팅(Intercalating)법, 5' 말단은 형광물질, 3' 말단은 소광자(quencher)로 표지된 올리고뉴클레오티드를 사용하는 방법 등이 있다. The amount of the amplification product can be detected by fluorescence signal. Intercalating method using a reagent (intercalator) that shows fluorescence by binding to the double-stranded DNA of the amplification product to which the probe is bound, the 5' end is labeled with a fluorescent material and the 3' end is labeled with a quencher. There are methods using oligonucleotides and the like.
구체적으로, 상기 프로브 양 말단에 리포터(reporter)와 리포터 형광을 소광(quenching)할 수 있는 소광자 (quencher)의 형광 물질이 결합할 수 있다. 상기 리포터(reporter)는 FAM(6-carboxyfluorescein) 또는 BHQ (Black Hole Quencher), 상기 소광자는 포스페이트 (Phosphate)에서 선택되어 사용할 수 있다.Specifically, a reporter and a fluorescent material of a quencher capable of quenching reporter fluorescence may be bound to both ends of the probe. The reporter may be 6-carboxyfluorescein (FAM) or black hole quencher (BHQ), and the quencher may be selected from phosphate.
구체적으로, 각 프로브는 동일 형광물질이 탑재된 것이 바람직하다.Specifically, each probe is preferably loaded with the same fluorescent substance.
본원발명에 따른 증폭은 실-시간 정량 증폭 예를 들어 실시간 중합효소연쇄반응 (Real-Time PCR) 또는 실시간 중합효소연쇄반응 정량검사(Real-time Quantitative Polymerase chain reaction; RQ-PCR)등의 방법을 통해 수행될 수 있다.The amplification according to the present invention is performed using methods such as real-time quantitative amplification, for example, Real-Time PCR or Real-time Quantitative Polymerase chain reaction (RQ-PCR). can be done through
"실시간연쇄중합효소연쇄반응(Real-time polymerase chain reaction: PCR)" 은 중합효소를 이용하여 표적 핵산에 특이적으로 결합하는 프라이머와 형광물질이 탑재된 프로브 세트로부터 표적 핵산을 실시간으로 증폭하여 검출하는 방법으로, 대표적인 핵산증폭기술(NAT) 중의 하나이다. 실시간 중합효소연쇄반응에 있어 PCR 증폭 산물의 양은 형광 신호에 의해 검출할 수 있다. 실시간 중합효소연쇄반응이 진행되면서 증가하는 폴리뉴클레오티드 양에 따라 형광 신호의 세기가 증가하게 되고, 증폭 사이클 횟수에 따른 형광 신호 세기를 나타내는 증폭 프로파일(amplification profile) 곡선을 얻게 된다."Real-time polymerase chain reaction (PCR)" uses a polymerase to amplify and detect target nucleic acids in real time from a probe set loaded with primers and fluorescent substances that specifically bind to target nucleic acids As a method, it is one of the representative nucleic acid amplification technologies (NAT). In real-time polymerase chain reaction, the amount of PCR amplification product can be detected by fluorescence signal. As the real-time polymerase chain reaction proceeds, the intensity of the fluorescence signal increases according to the amount of polynucleotide, and an amplification profile curve representing the intensity of the fluorescence signal according to the number of amplification cycles is obtained.
본 발명의 Real-time PCR(Real-Time Polymerase Chain Reaction) 방법은, 형광물질이 PCR 과정에서 이중가닥 (double strand) DNA 사슬에 인터컬레이션(intercalation)됨으로써 형광량이 비로소 기기에 감지되기 시작한다. 이렇게 형광량이 최저 감지가능 수준(background level)을 넘어 두드러지게 증가하는 시점의 증폭주기인 한계 주기(threshold cycle, Ct)가 나타난다. 주형 초기량의 로그(log)값과 주형을 실시간 중합효소 연쇄반응을 통하여 증폭할 때 해당하는 한계 주기 사이에는 직선적으로 비례하는 관계를 가지고 있다. 따라서, 핵산의 초기량을 이미 알고 있는 시료를 이용하여 초기량의 로그값과 한계 주기를 측정하여 표준검량곡선을 얻음으로써 핵산 증폭산물의 실시간 측정방법을 제공한다.In the real-time PCR (Real-Time Polymerase Chain Reaction) method of the present invention, the fluorescent substance is intercalated into a double-stranded DNA chain during the PCR process, so that the amount of fluorescence is finally detected by the device. In this way, a threshold cycle (Ct), which is an amplification cycle at a point in time when the amount of fluorescence significantly increases beyond the lowest detectable level (background level), appears. There is a linearly proportional relationship between the log value of the initial amount of the template and the corresponding limit cycle when the template is amplified through real-time polymerase chain reaction. Therefore, a standard calibration curve is obtained by measuring the log value of the initial amount and the critical period using a sample of which the initial amount of nucleic acid is already known, thereby providing a method for real-time measurement of nucleic acid amplification products.
실시간중합연쇄반응에서 항원-항체 핫스타트 핵산 증폭 방법 또는 피로포스페이트와 피로포트파타아제를 이용하는 핫스타트 핵산 증폭 방법, DNA 압타머 (aptamer)를 이용한 핫스타트 핵산 증폭 방법 등이 포함된 실시간연쇄중합반응 시약을 사용하는 것이 바람직하다.Real-time chain reaction including antigen-antibody hot-start nucleic acid amplification method in real-time polymerization chain reaction, hot-start nucleic acid amplification method using pyrophosphate and pyrophorpophatase, hot-start nucleic acid amplification method using DNA aptamer, etc. Preference is given to using reagents.
시료의 DNA를 실시간연쇄증폭반응 시약과 WSSV 진단용 프라이머 세트, 즉 서열번호 1 및 2의 프라이머와 서열번호 3 프로브 세트 1, 서열번호 4 및 5의 프라이머와 서열번호 6 세트 2와 함께 튜브에 첨가하여, (a) 91 내지 97℃의 온도에서 1분 내지 5분간 변성시키는 사전 DNA 변성단계; (b) 1차 PCR 반응을 서열번호 1 및 2의 프라이머와 서열번호 3 세트 1, 서열번호 4 및 5의 프라이머와 서열번호 6 세트 2을 이용하여, 50 내지 65℃의 온도에서 5 내지 25초간; 형광스캔; 처리하는 과정을 30 내지 45회 사이클로 수행하였으며, 이에 한정되는 것은 아니다.The DNA of the sample was added to a tube together with the real-time chain amplification reagent and the primer set for WSSV diagnosis, that is, the primers of SEQ ID NOs: 1 and 2, the probe set 1 of SEQ ID NO: 3, the primers of SEQ ID NOs: 4 and 5, and the set 2 of SEQ ID NO: 6 , (a) a preliminary DNA denaturation step of denaturing at a temperature of 91 to 97 ° C. for 1 to 5 minutes; (b) The first PCR reaction was performed at a temperature of 50 to 65° C. for 5 to 25 seconds using primers of SEQ ID NOs: 1 and 2, SEQ ID NO: 3 set 1, SEQ ID NOs: 4 and 5, and SEQ ID NO: 6 set 2 ; fluorescence scan; The treatment process was performed in 30 to 45 cycles, but is not limited thereto.
더욱이, 상기 중합효소연쇄반응에서 온도 및 시간의 조건은 본 발명의 중합효소연쇄반응 산물을 생성하기 위해 설정된 수치로, 실험방법 및 기기의 변경 등에 따라 통상의 당업자의 적절한 선택에 의해 타당한 수치로 변경될 수 있다.Moreover, the conditions of temperature and time in the polymerase chain reaction are values set to produce the polymerase chain reaction product of the present invention, and are appropriately selected by those skilled in the art according to changes in experimental methods and equipment Change to reasonable values It can be.
또한, 상기 증폭된 표적 서열은 검출 가능한 표지 물질로 표지될 수 있다. 상기 프로브 표지 물질은 형광 물질이나, 형광 물질 종류에 한정되지 않는다. 예컨대, 상기 표지 물질은 Cy-5 또는 Cy-3일 수 있다. In addition, the amplified target sequence may be labeled with a detectable labeling material. The probe labeling material is a fluorescent material, but is not limited to the type of fluorescent material. For example, the labeling substance may be Cy-5 or Cy-3.
본 발명은 또한, 흰반점 증후군 바이러스 (white spot syndrome virus; WSSV)의 VP664 및/또는 VP28 유전자를 증폭하기 위한 프라이머 쌍을 포함하는, 흰반점 증후군 바이러스 검출용 키트에 관한 것이다. The present invention also relates to a kit for detecting white spot syndrome virus (WSSV), comprising a pair of primers for amplifying VP664 and/or VP28 genes.
상기 키트는 경우에 따라, 중합효소, 버퍼와 같은 핵산 증폭 PCR 반응을 실시하는데 필요한 시약을 선택적으로 포함할 수 있다. 본 발명에 따른 키트는 또한 다양한 폴리뉴클레오타이드 분자, 다양한 버퍼 및 시약을 추가로 포함할 수 있다.The kit may optionally include reagents necessary for carrying out a nucleic acid amplification PCR reaction, such as a polymerase and a buffer, if necessary. A kit according to the present invention may also further comprise various polynucleotide molecules, various buffers and reagents.
상기 키트에서 특정 반응을 위해 사용되는 시약, 버퍼 또는 반응물의 최적량은 당업자에 의해 결정될 수 있으며, 앞서 언급된 프라이머 쌍 및/또는 프로브 각각을 포함하는 별도의 포장 또는 컴파트먼트(compartment)로 제작될 수 있다.Optimal amounts of reagents, buffers, or reactants used for a specific reaction in the kit can be determined by a person skilled in the art, and are prepared in separate packages or compartments containing each of the aforementioned primer pairs and/or probes. It can be.
본 발명에 따른 방법을 수행함에 있어 사용되는 키트와 관련된 구성에 대한 설명은 방법에도 동일하게 적용될 수 있다. The description of the configuration related to the kit used in performing the method according to the present invention can be equally applied to the method.
하나의 실시예에서, 상기 검체는 새우류, 게류 및 패류를 포함하나, 이에 한정되는 것은 아니다. 또한, 상기 대상 시료는 흰반점 증후군 바이러스 감염개체, 매개체 및 보유 생물을 포함하나, 이에 한정되는 것은 아니다. In one embodiment, the sample includes shrimp, crab and shellfish, but is not limited thereto. In addition, the target sample includes, but is not limited to, white spot syndrome virus-infected individuals, mediators, and organisms.
상기 검체로부터 핵산을 추출하는 단계를 추가로 포함할 수 있으며, 핵산의 추출은 예를 들어 상업화 되어 공급되고 있는 다양한 키트 또는 추출 시약을 사용하여 수행될 수 있다. 시료로부터 게놈 DNA를 추출하는 방법은 당업계에서 통상적으로 사용되는 페놀/클로로포름 추출법, SDS 추출법 (Tai et al., Plant Mol. Biol. Reporter, 8: 297-303, 1990), CTAB 분리법 (Cetyl Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980) 또는 상업적으로 판매되는 DNA 추출 키트를 이용하여 수행할 수 있다.A step of extracting nucleic acids from the sample may be further included, and the extraction of nucleic acids may be performed using, for example, various commercially available kits or extraction reagents. Methods for extracting genomic DNA from samples include a phenol/chloroform extraction method commonly used in the art, an SDS extraction method (Tai et al., Plant Mol. Biol. Reporter, 8: 297-303, 1990), and a CTAB separation method (Cetyl Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980) or a commercially available DNA extraction kit.
본 발명을 통해 흰반점 증후군 바이러스를 낮은 감염 검체로도 진단가능하여 민감도가 매우 우수하고, 특이도 및 핵산 추출과정에서 발생될 수 있는 유전자 파편에 의한 위음성률을 기존 검출 방법에 비해 현저하게 낮출 수 있는 효과를 가지고 있다.Through the present invention, white spot syndrome virus can be diagnosed even with low-infected specimens, so the sensitivity is very good, and the false negative rate due to gene fragments that can occur during the nucleic acid extraction process can be significantly reduced compared to existing detection methods. has an effect.
실시예Example
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.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 being limited by these examples.
[실시예 1] WSSV VP28 및 VP664 유전자를 표적으로 하는 프라이머 세트 제작[Example 1] Construction of primer sets targeting WSSV VP28 and VP664 genes
흰반점 증후군 바이러스(white spot syndrome virus; WSSV)의 외피막 단백질를 구성하는 유전자, 즉 VP1664 또는 VP28에 특이적으로 결합할 수 있는 프라이머 세트를 제작하기 위해서, 국제유전자은행(NCBI, GenBank)에 등록된 WSSV 관련 유전정보 전체 유전자 염기서열을 참조하였으며, 대표적으로 국제유전자은행(NCBI, GenBank)에 등록되어있는 GenBank Accession No. AF332093.3을 참조하였다. 중국 균주(등록번호 제 AF332093호), 태국 균주(등록번호 제AF369029) 및 대만 균주(등록번호 제 AF440570)의 전체 유전자 염기서열을 참조하였다.In order to prepare a set of primers that can specifically bind to the gene constituting the envelope protein of white spot syndrome virus (WSSV), that is, VP1664 or VP28, WSSV-related genetic information The entire gene sequence was referred to, and GenBank Accession No. See AF332093.3. The entire gene sequences of the Chinese strain (Registration No. AF332093), Thailand strain (Registration No. AF369029) and Taiwanese strain (Registration No. AF440570) were referenced.
이로부터 WSSV 외피막 단백질을 표적으로하는 프라이머 세트, 즉 VP28 유전자 표적으로 하는 특이 프라이머 및 프로브 및 VP664를 표적으로 하는 특이 프라이머 및 프로브는 국제수역사무국(OIE) WSSV 검사 프로토콜에 기입되어 있는 프라이머 및 프로브를 사용하였다. 이를 하기 표 1에 나타내었다.From this, primer sets targeting the WSSV envelope protein, that is, specific primers and probes targeting the VP28 gene and specific primers and probes targeting VP664 are the primers and probes listed in the OIE WSSV test protocol. was used. This is shown in Table 1 below.
Figure PCTKR2022021054-appb-img-000001
Figure PCTKR2022021054-appb-img-000001
[실시예 2] 본 발명의 WSSV 진단법과 VP664 유전자만 검출하는 방법 간의 검출 한계 및 cut-off value 비교[Example 2] Comparison of detection limit and cut-off value between the WSSV diagnosis method of the present invention and the method for detecting only the VP664 gene
본 발명의 WSSV 유전자 진단법과 VP664 유전자(국제수역사무국 방법)진단법의 검출 한계 및 cut-off value 분석을 위해 WSSV VP664와 VP28이 포함 되어있는 Plasmid DNA를 제조하여 검출 한계 및 cut-off value 분석을 위해 1000~1.4 copies/rxn으로 3 fold-dilution된 Plasmid DNA를 이용하였다. 표 1에 기재된 서열번호 1 내지 6의 프라이머 세트와 서열번호 4 내지 6 프라이머 세트와 함께 AccuPower Plus Dualstar qPCR Mastermix(Bioneer)의 튜브에 첨가한 후, 95℃의 온도에서 5분간 DNA를 변성시켰다. 이후, 95℃의 온도에서 20초간, 55℃의 온도에서 30초간, 스캔 단계(형광 측정)처리하는 과정을 실시간 PCR 증폭기기(Exicycler96 thermal block, BIONEER)를 이용하여 실행하였다. VP664 유전자만 검출하는 방법의 결과는 도 2a, 본 발명의 WSSV 진단법의 결과는 도 2b에 나타내었다.Plasmid DNA containing WSSV VP664 and VP28 was prepared to analyze the detection limit and cut-off value of the WSSV gene diagnosis method of the present invention and the VP664 gene (International Bureau of Animal Services method) diagnosis method, and to analyze the detection limit and cut-off value 3 fold-diluted plasmid DNA with 1000~1.4 copies/rxn was used. After adding primer sets of SEQ ID NOs: 1 to 6 and SEQ ID NOs: 4 to 6 described in Table 1 to a tube of AccuPower Plus Dualstar qPCR Mastermix (Bioneer), the DNA was denatured at 95°C for 5 minutes. Thereafter, a scanning step (fluorescence measurement) at 95°C for 20 seconds and at 55°C for 30 seconds was performed using a real-time PCR amplifier (Exicycler96 thermal block, BIONEER). The results of the method for detecting only the VP664 gene are shown in FIG. 2a, and the results of the WSSV diagnosis method of the present invention are shown in FIG. 2b.
도 2a는 VP664만 표적하는 서열번호 1 내지 3 프라이머 세트, 도 2b는 VP664와 VP28을 함께 표적하는 서열번호 1 내지 3 프라이머 세트와 서열번호 4 내지 6 프라이머 세트가 함께 있는 혼합액을 이용하여 검출한계와 cut-off value를 분석한 결과로 VP664와 VP28을 함께 표적하는 프라이머 세트가 VP664만 표적하는 프라이머 세트보다 검출한계는 약 6배 그리고 cut-off value 0.82로 나타났다. 본 발명의 VP664와 VP28을 함께 표적하는 프라이머 세트가 저농도 검체에 대한 흰반점 바이러스의 민감도를 높일 수 있음을 확인하였다.FIG. 2A shows primer sets of SEQ ID NOs: 1 to 3 targeting only VP664, and FIG. 2B shows the detection limit and As a result of analyzing the cut-off value, the detection limit of the primer set targeting VP664 and VP28 together was about 6 times higher than that of the primer set targeting only VP664, and the cut-off value was 0.82. It was confirmed that the primer set targeting VP664 and VP28 of the present invention can increase the sensitivity of the white spot virus to low-concentration samples.
[실시예 3] WSSV 진단법의 VP28 및 VP664 유전자에 대한 각 검출 민감도와 VP28 및 VP664 유전자를 동시 검출했을 때의 민감도 평가[Example 3] Evaluation of each detection sensitivity for the VP28 and VP664 genes of the WSSV diagnosis method and the sensitivity when the VP28 and VP664 genes are simultaneously detected
본 발명의 WSSV 유전자 진단법의 WSSV 검출에 대한 민감도를 확인하기 위하여, WSSV에 감염된 새우 유영각에서 핵산추출 프로토콜에 따라 핵산 추출하여, 10^-1 희석배수부터 10^-7 희석배수까지 추출된 DNA을 희석한 후 [실시예1] 표 1에 기재된 서열번호 1 내지 3의 프라이머 세트와 서열번호 4 내지 6 프라이머 세트를 각 사용한 것과 서열번호 1내지 6 프라이머 세트가 혼합된 프라이머 세트와 함께 AccuPower Plus Dualstar qPCR Mastermix(Bioneer)의 튜브에 첨가한 후, 95℃의 온도에서 5분간 DNA를 변성시켰다. 이후, 95℃의 온도에서 20초간, 55℃의 온도에서 30초간, 스캔 단계(형광 측정)처리하는 과정을 실시간 PCR 증폭기기(Exicycler96 thermal block, BIONEER)를 이용하여 실행하였다.In order to confirm the sensitivity of the WSSV gene diagnosis method of the present invention for WSSV detection, nucleic acid was extracted from WSSV-infected shrimp gypsy according to a nucleic acid extraction protocol, and DNA extracted from 10^-1 dilution to 10^-7 dilution After dilution [Example 1] AccuPower Plus Dualstar qPCR with a primer set in which the primer sets of SEQ ID NOs: 1 to 3 and the primer sets of SEQ ID NOs: 4 to 6 described in Table 1 were used, respectively, and the primer sets of SEQ ID NOs: 1 to 6 were mixed. After adding to the tube of Mastermix (Bioneer), DNA was denatured for 5 minutes at a temperature of 95 ° C. Thereafter, a scanning step (fluorescence measurement) at 95°C for 20 seconds and at 55°C for 30 seconds was performed using a real-time PCR amplifier (Exicycler96 thermal block, BIONEER).
본 발명의 WSSV 듀얼 타겟 유전자 진단법은 종래의 유전자 진단법인 국제수역사무국 매뉴얼에 따른 VP664 유전자 검출 진단법 및 본 발명을 통해 디자인된 VP 28 유전자 검출 진단법과 VP664와 VP28 유전자 검출 프라이머가 혼합된 진단법에 대한 민감도를 확인한 바 종래의 유전자 진단법인 국제수역사무국 매뉴얼에 따른 VP664 유전자 검출 진단법 및 본 발명을 통해 디자인된 VP 28 유전자 검출 진단법에 비해 VP28, VP664 검출 프라이머가 혼합된 검출 진단법에서 10배 높은 희석배수까지 검출되어, 매우 우수한 민감도를 확인하였다. 이의 결과를 하기 표 2에 나타내었다.The WSSV dual target gene diagnosis method of the present invention is sensitive to the conventional gene diagnosis method, the VP664 gene detection diagnosis method according to the International Bureau of Animal Services manual, the VP 28 gene detection diagnosis method designed through the present invention, and the mixed diagnosis method of VP664 and VP28 gene detection primers. As confirmed, detection of up to 10 times higher dilution in the detection diagnosis method in which VP28 and VP664 detection primers were mixed compared to the conventional genetic diagnosis method, the VP664 gene detection diagnosis method according to the International Animal Services Manual and the VP 28 gene detection diagnosis method designed through the present invention. , and very good sensitivity was confirmed. The results are shown in Table 2 below.
Figure PCTKR2022021054-appb-img-000002
Figure PCTKR2022021054-appb-img-000002
[실시예 3] 본 발명의 WSSV 유전자 진단법의 검출률과 종래의 유전자 진단법의 검출률 비교[Example 3] Comparison of the detection rate of the WSSV genetic diagnosis method of the present invention and the detection rate of the conventional genetic diagnosis method
본 발명의 WSSV 유전자 진단법의 검출률과 종래의 유전자 진단법의 검출률을 비교하기 위하여, 본 발명의 WSSV 유전자 진단법과 종래의 유전자 진단법의 검출률을 하와이(미국), 국내 양식업장에서 무작위로 샘플링하여 검출률 비교 평가를 수행하였다. In order to compare the detection rate of the WSSV gene diagnosis method of the present invention and the detection rate of the conventional genetic diagnosis method, the detection rate of the WSSV gene diagnosis method of the present invention and the conventional genetic diagnosis method were randomly sampled in Hawaii (USA) and a domestic aquaculture farm, and the detection rate was compared and evaluated. was performed.
구체적으로는, 무작위 샘플에 대해 바이러스성 DNA/RNA 추출 키트를 제조사의 매뉴얼에 따라 사용하여, 핵산을 추출하였다. 상기 실시예 2와 동일하게, 종래의 유전자 진단법(국제수역사무국 매뉴얼에 따른 VP664 유전자 검출)과 대만 GeneReach Biotechnology Corporation 사에서 개발한 IQ2000 WSSV detection kit(국제수역사무국 매뉴얼에 검사법으로 등재)를 사용하여 본 발명의 WSSV 듀얼타겟 유전자 진단법과 비교하였다.Specifically, nucleic acids were extracted from random samples using a viral DNA/RNA extraction kit according to the manufacturer's manual. In the same manner as in Example 2 above, using the conventional gene diagnosis method (detection of the VP664 gene according to the manual of the International Animal Health Service) and the IQ2000 WSSV detection kit developed by Taiwan's GeneReach Biotechnology Corporation (listed as a test method in the manual of the International Animal Health Service) It was compared with the WSSV dual target gene diagnosis method of the invention.
종래의 유전자 진단법과 본 발명의 듀얼타겟 유전자 진단법은 상기 실시예 2와 같이 실시하였으며, IQ2000 WSSV detection kit는 제품 매뉴얼에 따라 수행하였다. The conventional gene diagnosis method and the dual target gene diagnosis method of the present invention were performed as in Example 2, and the IQ2000 WSSV detection kit was performed according to the product manual.
각각의 유전자 진단법에 따라 측정된 WSSV 검출률을 비교하였으며, 이의 결과를 하기 표 3, 표 4와 도 3에 나타내었다.WSSV detection rates measured according to each genetic diagnosis method were compared, and the results are shown in Table 3, Table 4 and FIG. 3 below.
Figure PCTKR2022021054-appb-img-000003
Figure PCTKR2022021054-appb-img-000003
Figure PCTKR2022021054-appb-img-000004
Figure PCTKR2022021054-appb-img-000004
본 발명을 통해 흰반점 증후군 바이러스(white spot syndrom virus; WSSV)를 검출함에 있어 야기될 수 있는 위음성, 위양성에 대해 방지할 수 있는, 조성물, 키트 및 검출방법을 제공할 수 있다. 특히, WSSV 외피막 단백질을 발현하는 유전자 VP28과 VP664을 증폭하기 위한 프라이머 쌍을 이용하고, 단일 튜브에서 두 유전자를 같은 형광물질이 탑재된 각 프로브와 같이 사용하여 실시간중합효소연쇄반응을 수행할 경우, WSSV에 대한 민감도를 개선시켜주고 핵산 추출과정에서 하나의 유전자 파편이 발생이 되더라도 다른 유전자를 동시 검출하기에 위음성이 나타날 확률을 현저히 감소시킬 수 있다.Through the present invention, it is possible to provide a composition, kit, and detection method capable of preventing false negatives and false positives that may occur in detecting white spot syndrome virus (WSSV). In particular, when real-time polymerase chain reaction is performed using a pair of primers to amplify the genes VP28 and VP664 that express WSSV outer membrane proteins, and using both genes together with each probe loaded with the same fluorescent material in a single tube , it improves the sensitivity to WSSV, and even if one gene fragment is generated during the nucleic acid extraction process, it can significantly reduce the probability of false negatives by simultaneously detecting other genes.
이상으로 본 발명의 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As above, specific parts of the content of the present invention have been described in detail, and for those skilled in the art, these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. It will be clear. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
전자파일 첨부하였음.Electronic file attached.

Claims (12)

  1. 흰반점 증후군 바이러스 (white spot syndrome virus; WSSV)의 VP664 및/또는 VP28 유전자를 증폭하기 위한 프라이머 쌍을 포함하는, 흰반점 증후군 바이러스 검출용 조성물.A composition for detecting white spot syndrome virus, comprising a pair of primers for amplifying VP664 and/or VP28 genes of white spot syndrome virus (WSSV).
  2. 제1항에 있어서, 상기 프라이머 쌍은 서열번호 1 및 2의 서열 및/또는 서열번호 4 및 5의 서열인 것을 특징으로 하는 조성물.The composition according to claim 1, wherein the primer pairs are sequences of SEQ ID NOs: 1 and 2 and/or sequences of SEQ ID NOs: 4 and 5.
  3. 제1항에 있어서, 상기 프라이머 쌍에 의해 증폭된 산물에 결합하는 프로브를 추가로 포함하는 조성물.The composition according to claim 1, further comprising a probe that binds to a product amplified by the primer pair.
  4. 제3항에 있어서, 상기 프로브는 서열번호 3 및/또는 6의 서열인 것을 특징으로 하는 조성물.The composition according to claim 3, wherein the probe is a sequence of SEQ ID NO: 3 and/or 6.
  5. 흰반점 증후군 바이러스 (white spot syndrome virus; WSSV)의 VP664 및/또는 VP28 유전자를 증폭하기 위한 프라이머 쌍인 것을 특징으로 하는, 흰반점 증후군 바이러스 검출용 키트.A kit for detecting white spot syndrome virus, characterized in that it is a primer pair for amplifying VP664 and/or VP28 genes of white spot syndrome virus (WSSV).
  6. 제5항에 있어서, 상기 프라이머 쌍은 서열번호 1 및 2의 서열 및/또는 서열번호 4 및 5의 서열인 것을 특징으로 하는 키트.The kit according to claim 5, wherein the primer pairs are sequences of SEQ ID NOs: 1 and 2 and/or sequences of SEQ ID NOs: 4 and 5.
  7. 제5항에 있어서, 상기 프라이머 쌍에 의해 증폭된 산물에 결합하는 프로브를 추가로 포함하는 키트.The kit according to claim 5, further comprising a probe that binds to the product amplified by the primer pair.
  8. 제7항에 있어서, 상기 프로브는 서열번호 3 및/또는 6의 서열인 것을 특징으로 하는 키트.The kit according to claim 7, wherein the probe is a sequence of SEQ ID NO: 3 and/or 6.
  9. 검체에서 추출된 핵산을 흰반점 증후군 바이러스 (white spot syndrome virus; WSSV)의 VP664 및/또는 VP28 유전자를 증폭하기 위한 프라이머 쌍으로 증폭시키는 단계를 포함하는, 흰반점 증후군 바이러스 검출방법.A method for detecting white spot syndrome virus, comprising amplifying nucleic acids extracted from a sample with a primer pair to amplify VP664 and/or VP28 genes of white spot syndrome virus (WSSV).
  10. 제9항에 있어서, 상기 프라이머 쌍은 서열번호 1 및 2의 서열 및/또는 서열번호 4 및 5의 서열인 것을 특징으로 하는 검출방법.10. The detection method according to claim 9, wherein the primer pairs are sequences of SEQ ID NOs: 1 and 2 and/or sequences of SEQ ID NOs: 4 and 5.
  11. 제9항에 있어서, 상기 프라이머 쌍에 의해 증폭된 산물에 결합하는 프로브를 추가로 포함하는 검출방법.The detection method according to claim 9, further comprising a probe that binds to the product amplified by the primer pair.
  12. 제11항에 있어서, 상기 프로브는 서열번호 3 및/또는 6의 서열인 것을 특징으로 하는 검출방법.The detection method according to claim 11, wherein the probe is a sequence of SEQ ID NO: 3 and/or 6.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117512221A (en) * 2023-12-07 2024-02-06 拱北海关技术中心 Dual real-time fluorescent PCR detection primers, probes, kit and method for white spot syndrome virus and octopoda iridovirus 1

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20210119047A (en) * 2020-03-24 2021-10-05 이형우 A kit for diagnosing infection of white spot syndrome virus

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20210119047A (en) * 2020-03-24 2021-10-05 이형우 A kit for diagnosing infection of white spot syndrome virus

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FOUZI M, SHARIFF M, OMAR A R, MD YUSOFF F, TAN S W: "TaqMan real-time PCR assay for relative quantification of white spot syndrome virus infection in Penaeus monodon Fabricius exposed to ammonia", JOURNAL OF FISH DISEASES, OXFORD., GB, vol. 33, no. 12, 1 December 2010 (2010-12-01), GB , pages 931 - 938, XP093073678, ISSN: 0140-7775, DOI: 10.1111/j.1365-2761.2010.01200.x *
HAQUE MD. INJA-MAMUN, SIDDIQUE MOHAMMAD ANWAR, HOSSAIN ANWAR: "PCR Based Detection of White Spot Syndrome Virus (WSSV) in Shrimp Post Larvae (PL) of Bangladesh", INTERNATIONAL JOURNAL OF SCIENCES: BASIC AND APPLIED RESEARCH (IJSBAR), GLOBAL SOCIETY OF SCIENTIFIC RESEARCH AND RESEARCHERS, JO, vol. 51, no. 1, 1 January 2020 (2020-01-01), JO , pages 191 - 197, XP093073682, ISSN: 2307-4531 *
HOSSAIN A., NANDI S.P., SIDDIQUE M.A., SANYAL S.K., SULTANA M., HOSSAIN M.A.: "irus in cultured shrimp", LETTERS IN APPLIED MICROBIOLOGY, WILEY-BLACKWELL PUBLISHING LTD., GB, vol. 60, no. 2, 1 February 2015 (2015-02-01), GB , pages 128 - 134, XP093073676, ISSN: 0266-8254, DOI: 10.1111/lam.12353 *
LEU JIANN-HORNG, TSAI JYH-MING, WANG HAN-CHING, WANG ANDREW H.-J., WANG CHUNG-HSIUNG, KOU GUANG-HSIUNG, LO CHU-FANG: "The Unique Stacked Rings in the Nucleocapsid of the White Spot Syndrome Virus Virion Are Formed by the Major Structural Protein VP664, the Largest Viral Structural Protein Ever Found", JOURNAL OF VIROLOGY, THE AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 79, no. 1, 1 January 2005 (2005-01-01), US , pages 140 - 149, XP093073683, ISSN: 0022-538X, DOI: 10.1128/JVI.79.1.140-149.2005 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117512221A (en) * 2023-12-07 2024-02-06 拱北海关技术中心 Dual real-time fluorescent PCR detection primers, probes, kit and method for white spot syndrome virus and octopoda iridovirus 1

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