WO2021141179A1 - Oligonucléotide et plasmide pour la détection simultanée de quatre sérotypes du virus de la dengue, et procédé d'analyse du sérotype du virus de la dengue utilisant celui-ci - Google Patents

Oligonucléotide et plasmide pour la détection simultanée de quatre sérotypes du virus de la dengue, et procédé d'analyse du sérotype du virus de la dengue utilisant celui-ci Download PDF

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WO2021141179A1
WO2021141179A1 PCT/KR2020/002853 KR2020002853W WO2021141179A1 WO 2021141179 A1 WO2021141179 A1 WO 2021141179A1 KR 2020002853 W KR2020002853 W KR 2020002853W WO 2021141179 A1 WO2021141179 A1 WO 2021141179A1
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dengue virus
seq
nucleotide sequence
dengue
probe
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Korean (ko)
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황응수
김지연
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서울대학교산학협력단
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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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • 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
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/10—Detection mode being characterised by the assay principle
    • C12Q2565/101—Interaction between at least two labels

Definitions

  • a primer that binds to , a probe that specifically binds to each serotype of dengue virus, and a probe that specifically binds to each serotype of dengue virus can be used to quickly It relates to a method capable of specifically detecting each serotype of dengue virus and accurately measuring the amount of antigen present in a sample.
  • Dengue virus belonging to Flavivirus has four serotypes (types 1, 2, 3, and 4), and Aedes aegypti and Aedes albopictus are carriers of Dengue fever. fever) and dengue hemorrhagic fever (DHF).
  • Dengue virus one of the mosquito-borne infectious diseases, has increased in incidence by more than 30% over the past 50 years, and there were more than 1,000 cases of outbreaks reported in Southeast Asia in 2019.
  • ELISA and conventional PCR have been used as diagnostic methods, but these methods require several steps, take a long time, and have low accuracy such as false-positive results. For this reason, a diagnostic method using real-time amplification technology has recently been developed, and an attempt has been made to implement an accurate and sensitive detection method within a short time.
  • the present inventors prepared a primer that specifically binds to multiple serotypes of dengue virus and a probe that specifically binds to each serotype of dengue virus, and using the primer and probe, a plurality of dengue virus serotypes or dengue virus It was confirmed that the accuracy of specifically detecting each serotype of dengue virus in a sample in which other flaviviruses were mixed was very good.
  • a primer set comprising three primers consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 2 to 4; And to provide an oligonucleotide set for Dengue virus serotype analysis, comprising a probe set comprising four probes consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 5 to 8.
  • Another object of the present invention is a primer set comprising three kinds of primers consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 2 to 4; And to provide a composition for dengue virus serotype analysis, comprising a probe set comprising four probes consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 5 to 8.
  • Another object of the present invention is a primer set comprising three kinds of primers consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 2 to 4; And to provide a kit for dengue virus serotype analysis, comprising a probe set comprising four probes consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 5 to 8.
  • Another object of the present invention is a primer set comprising three kinds of primers consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 2 to 4; And to provide a dengue virus serotype analysis method using a probe set comprising four probes consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 5 to 8.
  • Dengue virus serotype analysis results using the primer and probe according to the present invention indicates high accuracy.
  • the present inventors thus provided a primer set comprising three kinds of primers consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 2 to 4; And dengue virus serotype analysis using a probe set comprising four types of probes consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 5 to 8 can rapidly and accurately analyze dengue virus serotypes present in a sample. possible effects were confirmed.
  • dengue virus refers to a virus belonging to the family Flaviviridae and the genus Flavivirus, and having a gene (+) single-stranded RNA having a length of about 11 Kb.
  • Dengue virus has four serotypes 1, 2, 3 and 4, and in the present specification, dengue-1, dengue-2, dengue-3 and dengue-4 are each dengue virus serotype 1 , type 2, type 3 and type 4.
  • One aspect of the present invention provides a primer set comprising three primers consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 2 to 4; And it is an oligonucleotide set for Dengue virus serotype analysis, including a probe set comprising four probes consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 5 to 8.
  • the term "primer” is a nucleic acid sequence having a short free 3' hydroxyl group, capable of forming a complementary template and a base pair, and serving as a starting point for template strand copying. single-stranded oligonucleotides that function.
  • the primer can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) in an appropriate buffer and temperature.
  • primer set refers to a set including a forward primer and a reverse primer, which is used to specifically bind to a target gene and amplify it through a polymerase chain reaction.
  • probe refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases as short as possible to achieve specific binding to a gene or mRNA, and oligonucleotides It may be manufactured in the form of a probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like, and may be labeled for easier detection.
  • the oligonucleotide set may further include a primer consisting of the nucleotide sequence of SEQ ID NO: 1.
  • the primer consisting of the nucleotide sequence of SEQ ID NO: 1 can synthesize complementary cDNA using RNA of four serotypes of dengue virus as a template, and the synthesis of RNA from cDNA is polymerase chain reaction (PCR), reverse transcription Reverse Transcription PCR (RT-PCR), quantitative PCR (qPCR), Reverse Transcription-quantitative PCR (RT-qPCR), Real-time nested reverse transcription- Polymerase chain reaction (realtime nested RT-PCR; rt-nRT-PCR), digital polymerase chain reaction (dPCR), digital reverse transcription polymerase chain reaction (digital RT-PCR; dRT-PCR) and may be performed, for example, by reverse transcription polymerase chain reaction, but is not limited thereto.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription Reverse Transcription PCR
  • qPCR quantitative PCR
  • RT-qPCR Reverse Transcription-quantitative PCR
  • each probe may be labeled with a fluorescence at one end and a quencher at the other end.
  • four types of probes consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 5 to 8 are labeled with a fluorescence at the 5' end and a quencher at the 3' end (quencher) may be labeled.
  • fluorophore refers to a fluorescent compound that absorbs light of a specific wavelength and emits light of another wavelength, and “quencher” acts on the excited state of the fluorophore to remove excitation energy and inhibit light emission. means a molecule that
  • the fluorophore is 2-chloro-7-phenyl-1,4-dichloro-6-carboxyfluorescein (2-chloro-7-phenyl-1,4-dichloro-6-carboxyfluorescein; VIC), 6-carboxyfluorescein (FAM), ABY, JUN, fluorescein, hexachloro-6-carboxyfluorescein (HEX), tetrachloro-6 -Carboxyfluorescein (tetrachloro-6-carboxyfluorescein; TET), 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein (2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein) ; JOE), 5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid (5-((2-aminoethyl)amino)naphthalene
  • the quencher is a minor groove binder (MGB), a QSY-based material, tetramethylrhodamine (TAMRA), 4-(4-dimethylaminophenylazo)benzoic acid (4 -(4-dimethylaminophenylazo)benzoic acid), 4-dimethylaminophenylazophenyl-4-maleimide, carboxytetramethylrhodamine and BHQ
  • the quencher of the probe consisting of the nucleotide sequence of SEQ ID NO: 5 may be a minor groove binder (MGB)
  • the quencher of the probe consisting of the nucleotide sequence of SEQ ID NO: 6 is QSY series material
  • the quencher of the probe consisting of the nucleotide sequence of SEQ ID NO: 7 may be a minor groove binder (MGB)
  • the quencher of the probe consisting of the nucleotide sequence of SEQ ID NO: 8 is
  • four probes consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 5 to 8 may specifically bind to cDNA of dengue virus serotype, respectively.
  • the probe consisting of the nucleotide sequence of SEQ ID NO: 5 may be one that specifically binds to cDNA of dengue-1 type.
  • the probe consisting of the nucleotide sequence of SEQ ID NO: 6 may specifically bind to cDNA of dengue-2 type.
  • the probe consisting of the nucleotide sequence of SEQ ID NO: 7 may specifically bind to cDNA of dengue-3 type.
  • the probe consisting of the nucleotide sequence of SEQ ID NO: 8 may specifically bind to dengue-4 cDNA.
  • the probe consisting of the nucleotide sequence of SEQ ID NO: 5 may specifically bind to cDNA of dengue-1 type
  • the probe consisting of the nucleotide sequence of SEQ ID NO: 6 is cDNA of dengue-2 type may specifically bind to
  • the probe consisting of the nucleotide sequence of SEQ ID NO: 7 may specifically bind to cDNA of dengue-3 type
  • the probe consisting of the nucleotide sequence of SEQ ID NO: 8 is dengue-4 type It may be that it specifically binds to cDNA of
  • the probe set according to an embodiment of the present invention can specifically bind to the cDNA of each dengue virus serotype, so that a plurality of dengue virus serotypes or Flaviviruses other than dengue virus can be added to the sample or specimen. ), each serotype of dengue virus can be specifically detected with high accuracy even when mixed (see FIGS. 1 to 5 ).
  • Another aspect of the present invention is a primer set comprising three primers consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 2 to 4; and a probe set comprising four probes consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 5 to 8. It is a composition for serotype analysis of dengue virus.
  • each probe may be labeled with a fluorescence at one end and a quencher at the other end.
  • the composition for dengue virus serotype analysis may further include a plasmid comprising the nucleotide sequence of SEQ ID NO: 9.
  • plasmid refers to DNA that exists independently of chromosomes in bacterial cells and can independently proliferate, has a ring shape, and is not an essential gene for the survival of bacteria.
  • the plasmid comprising the nucleotide sequence of SEQ ID NO: 9 may include all nucleotide sequences specific for each of the four dengue virus serotypes. Therefore, the plasmid according to one embodiment may be used to construct a standard curve for absolute quantification of four dengue virus serotypes together with a primer and a probe set, and accordingly, the amount of dengue virus antigen present in the sample or specimen It can be used for accurate measurement by absolute quantification (refer to FIGS. 6 to 9).
  • IUB ambiguity codes are used to indicate the sequences of primers. Therefore, in the primer sequence, A represents adenine, G represents guanine, C represents cytosine, and T represents thymine. This applies equally to presenting all sequences herein.
  • Another aspect of the present invention is a method for serotyping Dengue virus comprising the steps of:
  • a preparation step of preparing cDNA obtained through the sample A preparation step of preparing cDNA obtained through the sample.
  • a primer set comprising three primers consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 2 to 4 and a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 5 to 8
  • the dengue virus serotype analysis method of the present invention can be applied to a sample expected to be infected with a dengue virus.
  • the sample may include a sample obtained from a patient infected with or expected to be infected with dengue virus, and the sample is a biological sample obtained from a subject to be tested, for example, a biopsy, cultured cells, saliva, skin tissue, It may be any one or more selected from the group consisting of blood, feces and urine, but is not limited thereto.
  • the first nucleic acid amplification reaction is a polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (quantitative PCR); qPCR), Real-Time Polymerase Chain Reaction (RT-PCR), Reverse Transcription-quantitative PCR (RT-qPCR), Real-Time Nested Reverse Transcription-Polymerase Chain Reaction ( Realtime nested RT-PCR; rt-nRT-PCR), digital polymerase chain reaction (dPCR), digital reverse transcription polymerase chain reaction (digital RT-PCR; dRT-PCR), but limited thereto it's not going to be
  • the first nucleic acid amplification reaction may be a real-time polymerase chain reaction, and may be performed using a method or kit known in the art, for example, Taqman multiplex master mix 2X (Thermo Fisher Scientific, USA) kit. may be, but is not limited thereto.
  • RNA of dengue virus may be isolated from a sample for cDNA synthesis in the preparation step.
  • AGPC method Acid Guanidium-Phenol-Chloroform
  • RNA extraction kit cesium chloride density gradient centrifugation method (Guanidium/Cesium Chloride (CsCl) extraction method)
  • CsCl Guanidium/Cesium Chloride
  • the spin column method uses a membrane to filter RNA.
  • Cells are lysed using a buffer solution containing RNase, and then RNA is bound to a silica membrane. Thereafter, after washing so that only RNA binds to the silica membrane, RNA can be isolated by using an elution buffer to recover the RNA present in the filter membrane.
  • a Viral RNA mini kit (QIAGEN, USA) or other similar kit may be used to isolate a nucleic acid from a sample according to the corresponding manual.
  • cDNA can be synthesized using a primer consisting of the nucleotide sequence of SEQ ID NO: 1.
  • cDNA is polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), reverse transcription -Reverse Transcription-quantitative PCR (RT-qPCR), Real-Time Polymerase Chain Reaction (RT-PCR), realtime nested RT- PCR; rt-nRT-PCR), digital polymerase chain reaction (dPCR), digital reverse transcription polymerase chain reaction (digital RT-PCR; dRT-PCR), but is not limited thereto.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription polymerase chain reaction
  • qPCR quantitative polymerase chain reaction
  • RT-qPCR reverse transcription -Reverse Transcription-quantitative PCR
  • RT-PCR Real-Time Polymerase Chain Reaction
  • dPCR digital polymerase chain reaction
  • digital reverse transcription polymerase chain reaction digital reverse transcription polymerase chain reaction
  • the cDNA synthesis step may be performed by Reverse Transcription PCR (RT-PCR), and specifically, SuperScript IV First-Strand Synthesis System (Life Technologies, USA) or Other similar kits can be used to synthesize cDNA from isolated RNA according to the appropriate manual.
  • RT-PCR Reverse Transcription PCR
  • SuperScript IV First-Strand Synthesis System Life Technologies, USA
  • Other similar kits can be used to synthesize cDNA from isolated RNA according to the appropriate manual.
  • the primer consisting of the nucleotide sequence of SEQ ID NO: 1 may specifically bind to all four serotypes of the dengue virus, and thus each serotype RNA of the dengue virus isolated from the sample is a single Dengue virus cDNA can be quickly synthesized using only one primer.
  • the dengue virus serotyping method may further comprise the following steps:
  • preparing a standard curve by performing a second nucleic acid amplification reaction using a plasmid comprising the nucleotide sequence of SEQ ID NO: 9; and a detection step of detecting the amount of antigen contained in the sample by calculating the gene copy number by substituting the threshold cycle value (Ct) obtained in the amplification step into the prepared standard curve.
  • Ct threshold cycle value
  • standard curve refers to a linear relationship between the threshold cycle value and the gene copy number calculated by regression analysis of the threshold cycle value and gene copy number obtained through the nucleic acid amplification reaction using the plasmid, primer set, and probe set of the present invention. It means a function expression that indicates the relationship.
  • the plasmid may be a plasmid capable of containing all of the specific nucleotide sequences of each of the four serotypes of the dengue virus, and thus, using only one plasmid, the plasmid is used for the four serotypes of the dengue virus. It is possible to create a standard curve for the present invention, and the inventors of the present invention confirmed that the accuracy of the standard curve prepared through the plasmid of the present invention is very good (see FIGS. 6 to 6 ).
  • the amount of antigen present in the sample can be accurately detected by substituting the threshold cycle value of the result of nucleic acid amplification reaction of the cDNA of the sample using the primer set and probe set of the present invention.
  • the second nucleic acid amplification reaction is a polymerase chain reaction (PCR), a reverse transcription polymerase chain reaction (RT-PCR), a quantitative polymerase chain reaction (quantitative PCR).
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription polymerase chain reaction
  • quantitative PCR quantitative polymerase chain reaction
  • qPCR Reverse Transcription-quantitative PCR
  • RT-PCR Real-Time Polymerase Chain Reaction
  • Real-time Nested Reverse Transcription-Polymerase Chain Reaction realtime nested RT-PCR; rt-nRT-PCR
  • digital polymerase chain reaction digital PCR; dPCR
  • digital reverse transcription polymerase chain reaction digital RT-PCR; dRT-PCR
  • digital RT-PCR digital reverse transcription polymerase chain reaction
  • it may be a real-time polymerase chain reaction, but is not limited thereto.
  • Another aspect of the present invention is a primer set comprising three primers consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 2 to 4; And it is a kit for serotype analysis of Dengue virus, comprising a probe set comprising four types of probes consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 5 to 8.
  • the kit for dengue virus serotyping may include a DNA polymerase, a tube or other suitable container, a reaction buffer, deoxynucleotides (dNTPs), an enzyme, a dye, sterile water, etc. , but is not limited thereto.
  • dNTPs deoxynucleotides
  • each serotype of dengue virus can be quickly and specifically detected, and the amount of antigen present in a sample can be accurately measured using a plasmid, so dengue virus It can be used as data for treatment and research.
  • 1 is a graph showing the results of specifically detecting each dengue-1 type through a primer set and a probe set according to an embodiment of the present invention.
  • FIG. 2 is a graph showing a result of specifically detecting each dengue-2 type through a primer set and a probe set according to an embodiment of the present invention.
  • FIG. 3 is a graph showing the results of specifically detecting each dengue-3 type through a primer set and a probe set according to an embodiment of the present invention.
  • FIG. 4 is a graph showing the results of specifically detecting each dengue-4 type through a primer set and a probe set according to an embodiment of the present invention.
  • 5 is a graph showing the results of simultaneous detection of four dengue virus serotypes through a primer set and a probe set according to an embodiment of the present invention.
  • FIG. 6 is a graph showing a standard curve of dengue-1 type prepared through a plasmid according to an embodiment of the present invention.
  • FIG. 7 is a graph showing a standard curve of dengue-2 type prepared through a plasmid according to an embodiment of the present invention.
  • FIG. 8 is a graph showing a standard curve of dengue-3 prepared through a plasmid according to an embodiment of the present invention.
  • FIG. 9 is a graph showing a standard curve of dengue-4 prepared through a plasmid according to an embodiment of the present invention.
  • a primer set comprising three primers consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 2 to 4; and a probe set comprising 4 probes consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 5 to 8; Containing, Dengue virus (Dengue virus) oligonucleotide set for serotype analysis.
  • Dengue virus Dengue virus
  • Vero E6 ATCC number CRL-1586 cells were cultured with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in Dulbecco's minimal essential medium (DMEM). Thereafter, each of the viruses in Table 1 below was infected and used to evaluate the specificity of the primer set and probe of the present invention.
  • FBS fetal bovine serum
  • DMEM Dulbecco's minimal essential medium
  • Table 1 Dulbecco's minimal essential medium
  • Dengue virus Sales/purchase information remark Dengue virus 1 NCCP; 43251 Dengue type 1 Dengue virus 2 NCCP; 43253 Dengue type 2 Dengue virus 3 NCCP; 43256 Dengue type 3 Dengue virus 4 NCCP; 43257 Dengue-4 Chikungunya virus NCCP; 43132 Japanese encephalitis virus KBPV; VR27 yellow fever virus Bionner; plasmid synthesis Zika virus Kenya ATCC; 1838 Zika virus PuertoRico ATCC; 1843
  • NCCP National Culture Collection for Pathogens
  • KBPV Bank for Pathogenic Viruses
  • ATCC American Type Culture Collection
  • Virus RNA cultured in Vero E6 cells was extracted using the Viral RNA Mini Kit (Qiagen, USA). Using the extracted RNA as a template, using the SuperScript IV First-Strand Synthesis System (Life Technologies, USA) kit and the primers for cDNA synthesis in Table 2 below, each virus was subjected to reverse transcription PCR (RT-PCR). cDNA was synthesized.
  • Real-time polymerase chain reaction was performed to evaluate the specificity of primers and probes and the detection sensitivity of the plasmid.
  • the real-time polymerase chain reaction was performed with a target (Target) in Taqman multiplex master mix 2X (Thermo Fisher Scientific, USA) and a primer set of SEQ ID NOs: 2 to 4 in Table 3, SEQ ID NOs: 5 to 8 of dengue virus in Table 4 A probe that specifically binds to each serotype was introduced, and the target was used as a template.
  • the real-time polymerase chain reaction was initially performed for 2 minutes at 50°C and 10 minutes at 95°C, followed by a total of 40 times at 95°C for 15 seconds and at 55°C for 1 minute.
  • a plurality of target materials for quantification containing plasmids at different concentrations were prepared by diluting the plasmids in Table 5 below in stages, and the primer sets in Table 3 and Table 4 in each target material A probe was inserted and a real-time polymerase chain reaction was performed.
  • the degree of fluorescence according to the number of PCR cycles is measured, a site where the fluorescence signal is amplified exponentially is selected and set as a threshold line (Th), and the set threshold line and amplification curve are set.
  • a standard curve was calculated by regression analysis of the threshold cycle value (Ct) and the number of copies at this intersection.
  • RNA extraction method After extracting each RNA from dengue-1 type, dengue-2 type, dengue-3 type and dengue-4 type according to the above-described RNA extraction method, complementation using a primer for cDNA synthesis consisting of SEQ ID NO: 1 of Table 1 cDNA was synthesized.
  • a primer set consisting of nucleotide sequences of SEQ ID NOs: 2 to 4 of Table 2, and probes of SEQ ID NOs: 5 to 8 of Table 3 were added, and each dengue virus serotype was added.
  • the real-time polymerase chain reaction according to the above-described method was initially carried out at 50 °C for 2 minutes and 95 °C for 10 minutes, followed by a total of 40 cycles at 95 °C for 15 seconds and 55 °C for 1 minute.
  • the serotype of the dengue virus was detected by analyzing the fluorescence emitted by the probe in each real-time polymerase chain reaction.
  • RNA extraction method After extracting each RNA from dengue-1 type, dengue-2 type, dengue-3 type and dengue-4 type according to the above-described RNA extraction method, complementation using a primer for cDNA synthesis consisting of SEQ ID NO: 1 of Table 1 cDNA was synthesized.
  • the primer set consisting of the nucleotide sequences of SEQ ID NOs: 2 to 4 of Table 2 and the probes of SEQ ID NOs: 5 to 8 of Table 3 were all put into the target containing all of the dengue-1 to dengue-4 cDNAs, and the above-mentioned According to the method, the real-time polymerase chain reaction was initially performed at 50° C. for 2 minutes and 95° C. for 10 minutes, followed by a total of 40 cycles at 95° C. for 15 seconds and at 55° C. for 1 minute.
  • each serotype can be accurately detected without cross-reaction in a real-time polymerase chain reaction through the primer set and probe of the present invention, and a plurality of dengue virus serotypes are present in the target. It was confirmed that each serotype can be specifically and simultaneously detected without cross-reaction even when they are mixed. This suggests that even when a sample or specimen contains multiple dengue virus serotypes, each serotype can be accurately detected without cross-reaction.
  • the primer set and the probe of the present invention were all added, and a real-time polymerase chain reaction was performed to detect the amplification product.
  • the primer set and probe of the present invention did not cross-react with flaviviruses other than dengue virus even if flaviviruses other than dengue virus were mixed in the target of the polymerase chain reaction, which was a sample or specimen. It suggests that the serotype of dengue virus can be accurately detected even when a plurality of flaviviruses are mixed in .
  • Example 3 Evaluation of detection sensitivity of plasmids for quantitative analysis
  • the plasmid of Table 5 was diluted stepwise to prepare a plurality of target substances for quantification containing the plasmids at different concentrations, and the primer sets and Tables in Table 3 were added to the target substances. Insert the probe of No. 4 and perform a real-time polymerase chain reaction using the plasmid as a template according to the above-described method for an initial period of 50 °C for 2 minutes and 95 °C for 10 minutes, followed by a total of 40 cycles at 95 °C for 15 seconds and at 55 °C for 1 minute. was performed with
  • a threshold line (Th) for each dengue virus serotype is set through sequential real-time polymerase chain reaction, and the threshold cycle value (Threshold cycle; Ct) at the intersection of the set threshold line and the amplification curve is used.
  • a standard curve was calculated for each dengue virus serotype, and the results are shown in FIGS. 6 to 9 and Tables 7 to 10.
  • the plasmid of the present invention is used to detect dengue virus serotypes, it is possible to calculate a standard curve for each of the four serotypes of the dengue virus using one plasmid, and using the calculated standard curve for dengue It was confirmed that it can be used for absolute quantification of the serotypes of 4 viruses. That is, it is expected that the gene copy number of each serotype of dengue virus present in the sample can be accurately obtained by substituting the threshold cycle value obtained using the primer set and probe set of the present invention into the calculated standard curve during the nucleic acid amplification process. do.
  • a primer that binds to , a probe that specifically binds to each serotype of dengue virus, and a probe that specifically binds to each serotype of dengue virus can be used to quickly It relates to a method capable of specifically detecting each serotype of dengue virus and accurately measuring the amount of antigen present in a sample.

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Abstract

La présente invention se rapporte à un oligonucléotide et à un plasmide pour la détection simultanée de quatre sérotypes du virus de la dengue et à un procédé d'analyse de sérotypes du virus de la dengue l'utilisant. Même lorsqu'une pluralité de sérotypes du virus de la dengue ou de flavivirus autres que les virus de la dengue sont mélangés dans l'échantillon, chacun des sérotypes du virus de la dengue peut être détecté rapidement et spécifiquement, et la quantité d'antigènes présents dans l'échantillon peut être mesurée avec précision au moyen d'un plasmide, et ainsi la présente invention peut être utilisée en tant que données pour un procédé de traitement du virus de la dengue et la recherche.
PCT/KR2020/002853 2020-01-09 2020-02-28 Oligonucléotide et plasmide pour la détection simultanée de quatre sérotypes du virus de la dengue, et procédé d'analyse du sérotype du virus de la dengue utilisant celui-ci WO2021141179A1 (fr)

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KR10-2020-0002975 2020-01-09
KR20200002975 2020-01-09
KR10-2020-0020852 2020-02-20
KR1020200020852A KR102201869B1 (ko) 2020-01-09 2020-02-20 뎅기 바이러스의 4종류 혈청형 동시 검출용 올리고뉴클레오티드 및 플라스미드와 이를 이용한 뎅기 바이러스 혈청형 분석 방법

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KR20230111359A (ko) * 2022-01-18 2023-07-25 한국표준과학연구원 전장유전체 증폭을 위한 치쿤군야 바이러스 범용 프라이머 세트 및 이를 이용한 진단 키트

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130130235A1 (en) * 2010-07-29 2013-05-23 Bigtec Private Limited Probes and primers for detection of dengue
US20140315745A1 (en) * 2011-11-01 2014-10-23 The United States Of America, As Represented By The Secretary, Dept Of Health And Human Services Broad detection of dengue virus serotypes
KR20150000771A (ko) * 2013-06-25 2015-01-05 원광대학교산학협력단 ４가지 혈청형 뎅기 바이러스의 다중 동시 감별 진단용 올리고뉴클레오티드 및 이의 용도
KR20190041237A (ko) * 2017-10-12 2019-04-22 고려대학교 산학협력단 뎅기 바이러스 검출용 올리고뉴클레오티드 세트 및 이의 용도
KR20190121600A (ko) * 2018-04-18 2019-10-28 주식회사 코젠바이오텍 다중 실시간 중합효소연쇄반응을 이용한 뎅기 바이러스 혈청형 검출세트 및 검출방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130130235A1 (en) * 2010-07-29 2013-05-23 Bigtec Private Limited Probes and primers for detection of dengue
US20140315745A1 (en) * 2011-11-01 2014-10-23 The United States Of America, As Represented By The Secretary, Dept Of Health And Human Services Broad detection of dengue virus serotypes
KR20150000771A (ko) * 2013-06-25 2015-01-05 원광대학교산학협력단 ４가지 혈청형 뎅기 바이러스의 다중 동시 감별 진단용 올리고뉴클레오티드 및 이의 용도
KR20190041237A (ko) * 2017-10-12 2019-04-22 고려대학교 산학협력단 뎅기 바이러스 검출용 올리고뉴클레오티드 세트 및 이의 용도
KR20190121600A (ko) * 2018-04-18 2019-10-28 주식회사 코젠바이오텍 다중 실시간 중합효소연쇄반응을 이용한 뎅기 바이러스 혈청형 검출세트 및 검출방법

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