WO2021141178A1 - Ensemble d'amorces pour analyse simultanée d'une séquence génomique entière de quatre sérotypes du virus de la dengue et procédé de synthèse d'adnc l'utilisant - Google Patents

Ensemble d'amorces pour analyse simultanée d'une séquence génomique entière de quatre sérotypes du virus de la dengue et procédé de synthèse d'adnc l'utilisant Download PDF

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WO2021141178A1
WO2021141178A1 PCT/KR2020/002061 KR2020002061W WO2021141178A1 WO 2021141178 A1 WO2021141178 A1 WO 2021141178A1 KR 2020002061 W KR2020002061 W KR 2020002061W WO 2021141178 A1 WO2021141178 A1 WO 2021141178A1
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cdna
dengue virus
dengue
primer set
whole genome
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PCT/KR2020/002061
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Korean (ko)
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황응수
김정헌
김지연
이상목
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서울대학교산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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

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  • the present invention provides a primer set capable of simultaneously producing cDNA (complementary DNA) for whole genome sequence analysis of four serotypes (types 1, 2, 3, 4) of dengue virus, and
  • the present invention relates to a cDNA synthesis method using the same, and more particularly, a primer that specifically binds to the RNA of each dengue virus serotype and a plurality of serotypes even when a plurality of dengue virus serotypes are mixed in a sample using the same. It relates to a method capable of rapidly and accurately performing whole genome sequencing by producing cDNA for
  • 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 fever is widely distributed in tropical and subtropical regions, and 50 to 100 million people are infected every year in 100 countries around the world. In Korea, the number of patients returning to Korea after being infected while staying abroad, such as traveling abroad, living abroad, working or studying abroad, is increasing every year, and since 2000, it has been designated and managed as a legal infectious disease.
  • the rapid detection of dengue virus in samples and the analysis of the serotype and whole genome sequence of the detected virus can be used as data for treatment and vaccine development of dengue virus, as well as in establishing a surveillance system to prevent the spread of the virus. indispensable
  • the present inventors prepared a primer set capable of synthesizing cDNA (complementary DNA) by specifically binding to each of the four serotypes of the dengue virus, and using the primer set, the four sera of the dengue virus As a result of analyzing whole genome sequences by synthesizing cDNA of samples with mixed types, it was confirmed that the accuracy of regular common sequencing of the detected whole genome sequences was significantly superior.
  • an object of the present invention is to provide a primer set for dengue virus cDNA synthesis, comprising 20 primers consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 20.
  • Another object of the present invention is to provide a method for synthesizing cDNA of dengue virus using 20 primers consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 20.
  • Another object of the present invention is to provide a dengue virus whole genome sequence analysis method using 20 primers consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 20.
  • Another object of the present invention is to provide a composition for dengue virus cDNA synthesis.
  • the present invention provides a primer set capable of simultaneously producing cDNA (complementary DNA) for whole genome sequence analysis of four serotypes (types 1, 2, 3, 4) of dengue virus, and
  • the present invention relates to a cDNA synthesis method using the same, and the common nucleotide sequence of the whole genome sequence of the dengue virus serotype analyzed through the primer according to the present invention shows high accuracy.
  • the present inventors have found that the whole genome sequencing using cDNA synthesized through the primer set of SEQ ID NOs: 1 to 20 can be performed quickly and accurately even when a plurality of serotypes of dengue virus are mixed in the sample. Virus serotypes were confirmed, and it was confirmed that the effect of detecting the genetic information of each dengue virus serotype with high reliability was 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 is a primer set for dengue virus cDNA synthesis, comprising 20 primers consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 20.
  • the primer consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 5 may bind to dengue-1 type RNA.
  • the primer consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 6 to 10 may bind to dengue-2 RNA.
  • the primer consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 11 to 15 may bind to dengue-3 RNA.
  • the primer consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 16 to 20 may bind to dengue-4 RNA.
  • a primer consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 5 can bind to dengue-1 type RNA, and consisting of nucleotide sequences of SEQ ID NOs: 6 to 10
  • a primer consisting of a nucleotide sequence selected from the group can bind to dengue-2 RNA
  • a primer consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 11 to 15 binds to dengue-3 RNA
  • a primer consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 16 to 20 may bind to dengue-4 RNA.
  • 5 specific primers out of 20 primers consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 20 during cDNA synthesis are 4 types of dengue virus, respectively. It can bind specifically to RNA of a serotype at the same time, and accordingly, when each serotype is present in a sample using the primer set of the present invention, cDNA of each serotype can be simultaneously synthesized from the RNA of the sample. Therefore, even when a plurality of dengue virus serotypes exist in a sample, it is possible to accurately detect the genetic information of each serotype at the same time.
  • 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 is used to synthesize cDNA through reverse transcriptase by specifically binding to a target gene, and includes primers of each serotype to confirm the full-length genome sequence of dengue virus. means set.
  • primers that specifically bind to each serotype of dengue virus can each complementarily bind to a specific position of dengue virus RNA to synthesize cDNA.
  • the primer represented by SEQ ID NO: 1 may bind to the first segment position (DENV1 F1) of dengue virus serotype 1
  • the primer represented by SEQ ID NO: 7 is the second segment position of dengue virus serotype 2 (DENV2 F2) and can be used to synthesize cDNA (see FIG. 1 and Table 2).
  • IUB ambiguity codes are used to indicate the sequences of primers. Therefore, in the primer sequence, A is adenine, G is guanine, C is cytosine, T is thymine, R is adenine and guanine (A+G), and Y is Cytosine and thymine (C+T), W stands for adenine and thymine (A+T). This applies equally to presenting all sequences herein.
  • Another aspect of the present invention is a cDNA synthesis method for dengue virus whole genome sequencing comprising the following steps:
  • the cDNA synthesis method of the present invention can be applied to a sample expected to be infected with 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 separation step of isolating the RNA of the dengue virus from the sample may be further included.
  • RNA isolation and extraction As a method of isolating the RNA of dengue virus from a sample, any method known in the art such as RNA isolation and extraction may be used.
  • the AGPC method (Acid Guanidium-Phenol-Chloroform), RNA extraction kit, and cesium chloride density gradient centrifugation (Guanidium/Cesium Chloride (CsCl) extraction method) can be used as a method of isolating RNA from the sample, e.g.
  • a spin column type RNA extraction kit may be used, but is not limited thereto.
  • the spin column method uses a membrane to filter RNA.
  • Cells are lysed using an RNase-free buffer solution, 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.
  • a step of removing ribosomal RNA and remaining DNA after extracting dengue virus RNA from a sample may be additionally performed.
  • Ribosomal RNA and residual DNA may be removed by methods known in the art, for example, RNA may be removed using an RNA removal kit, and DNA may be removed by a DNA degrading enzyme, Dnase I.
  • the primer of the present invention is prepared as a mixed solution included with the isolated RNA, dNTP mixture, and reverse transcriptase, and then the secondary structure of RNA is denatured through an incubation process. Then, a complex of RNA and a primer is generated through a quenching process, and cDNA complementary to the isolated RNA is synthesized through incubation and cooling using the RNA strand as a template by reverse transcriptase. In addition, the complementary binding of RNA and cDNA is separated through heat denaturation to obtain single-stranded cDNA.
  • the above-described cDNA acquisition process is reverse transcription-polymerase chain reaction (RT-PCR), reverse transcription-quantitative PCR (RT-qPCR), real-time four Steed reverse transcription-polymerase chain reaction (realtime nested RT-PCR; rt-nRT-PCR), digital reverse transcription polymerase chain reaction (digital RT-PCR; dRT-PCR) may be performed by, but is not limited thereto.
  • RT-PCR reverse transcription-polymerase chain reaction
  • RT-qPCR reverse transcription-quantitative PCR
  • real-time four Steed reverse transcription-polymerase chain reaction realtime nested RT-PCR; rt-nRT-PCR
  • digital reverse transcription polymerase chain reaction digital reverse transcription polymerase chain reaction
  • dRT-PCR 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.
  • Another aspect of the present invention is a dengue virus whole genome sequencing method comprising the following steps:
  • the preparation and synthesis steps of the dengue virus whole genome sequencing method include the preparation and synthesis steps of the cDNA synthesis method for dengue virus whole genome sequencing. Each may be performed identically, and description is omitted to avoid the complexity of the present specification.
  • full genome refers to the whole of DNA bases storing genetic information of one species, and the whole genome to be analyzed in the present invention refers to the entire genome of dengue virus.
  • the analysis step is gel electrophoresis, next-generation sequencing (NGS), DNA sequencing, PCR-SSCP (single stranded conformation polymorphism) analysis, PCR-RFLP (restriction fragment length) polymorphism) analysis, allele-specific hybridization method, DNA chip method, SNaPShot analysis method, TDGS (Two-dimensional Gene Scanning), Taq-Man®, and TOGATM at least one selected from the group consisting of a variety of known in the art It may be performed by a method, for example, an NGS method may be used, but is not limited thereto.
  • the analysis step may be to prepare a cDNA library (cDNA Library) with the prepared cDNA and analyze the whole genome sequence.
  • cDNA Library cDNA Library
  • the cDNA library may be prepared through a cDNA library construction kit, but is not limited thereto, and various methods known in the art may be appropriately used.
  • the cDNA library may be prepared using 1D PCR barcoding cDNA Sequencing Protocol using SQK-LSK108 kit (Oxford Nanopore Technologies, UK) or other similar kits according to the manual, but is not limited thereto.
  • compositions for dengue virus cDNA synthesis comprising a primer set comprising 20 primers comprising a base sequence selected from the group consisting of base sequences of SEQ ID NOs: 1 to 20.
  • the composition for dengue virus cDNA synthesis includes a reverse transcriptase for synthesizing complementary cDNA from RNA of dengue virus, a DNA polymerase for amplifying complementary DNA, a tube or other suitable containers, reaction buffers, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC-water, sterile water, and the like.
  • dNTPs deoxynucleotides
  • the present invention provides a primer set capable of simultaneously producing cDNA for whole genome sequence analysis of four serotypes (types 1, 2, 3, and 4) of dengue virus and cDNA synthesis using the same
  • serotypes types 1, 2, 3, and 4
  • cDNA synthesis using the same
  • FIG. 1 is a diagram showing a position at which a primer according to an embodiment of the present invention specifically binds to each serotype of dengue virus.
  • FIG. 2 is a diagram comparing the common nucleotide sequence of the full-length genome nucleotide sequence of each serotype of dengue virus produced in Experimental Group 1 with the common nucleotide sequence of the full-length genome nucleotide sequence produced in Experimental Group 2 according to an embodiment of the present invention; to be.
  • FIG. 3 is a view showing the results of comparing the phylogenetic tree of the regular common nucleotide sequences obtained in Experimental Group 1 and Experimental Group 2 with five reference genomes of each dengue virus serotype according to an embodiment of the present invention.
  • a primer set for synthesizing dengue virus cDNA comprising 20 primers, consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 20.
  • 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). Vero E6 cells were infected with the dengue virus of Table 1 below, and the medium was collected after 7-10 days of culture. Dengue virus of Table 1 is a pathogen resource provided by the National Culture Collection for Pathogens (NCCP) (NCCP 43251, 43254, 43256, 43257), and the primer set efficiency of the present invention was evaluated using this.
  • NCCP National Culture Collection for Pathogens
  • Vero E6 cells in the harvested medium were centrifuged at 100,000 x g for 3 hours by ultra-centrifugation to purify each dengue virus serotype.
  • Purified dengue virus RNA was extracted using the Viral RNA Mini Kit (Qiagen, USA).
  • cDNA was synthesized from the extracted RNA by Reverse Transcription PCR (RT-PCR) using a SuperScript IV First-Strand Synthesis System (Life Technologies, USA) kit. And, as shown in FIG. 1, the primer set of the present invention synthesizes cDNA by specifically binding to a specific position of each serotype of dengue virus.
  • the primer consisting of SEQ ID NO: 1 may specifically bind to the first fragment of dengue-1 type (DENV F1) to synthesize cDNA.
  • DEV F1 dengue-1 type
  • human ribosomal RNA present in the sample was removed with NEBNext rRNA depletion kit (NEB), and DNA remaining in the sample was removed with Dnase I.
  • NEB NEBNext rRNA depletion kit
  • nucleotide sequences of the primer sets designed for cDNA synthesis in the present invention are shown in Table 2 below.
  • F1 to F5 indicate positions where the primers specifically bind to the dengue virus serotype RNA shown in FIG. 1 .
  • the cDNA library was prepared according to the manual of the 1D PCR barcoding cDNA Sequencing Protocol using SQK-LSK108 kit (Oxford Nanopore Technologies, UK).
  • nucleotide sequence information was compared with the reference genome, and Geneious Prime version 2019.0.4 (Genius, New Zealand) was used to calculate coverage and depth, and a regular consensus sequence was obtained.
  • experimental group 1 and experimental group 2 were constructed, and according to the above experimental method, RNA was extracted from dengue virus serotypes 1, 2, 3, and 4 in Experimental Group 1, respectively, and cDNA was synthesized with the primer of the present invention. Then, a regular common nucleotide sequence was obtained through whole genome sequencing.
  • Experimental group 2 extracted RNA from a sample in which four serotypes of dengue virus were mixed, synthesized cDNA of each serotype simultaneously with the primers of the present invention, and then obtained regular common nucleotide sequences through whole genome sequencing.
  • the identity was determined by comparing the regular common nucleotide sequence obtained from each dengue virus serotype of Experimental Group 1 with each regular common nucleotide sequence obtained from a sample in which the four serotypes of Experimental Group 2 were mixed.
  • the fact that the common nucleotide sequence of the two experimental groups is the same means that even if several serotypes are mixed in the sample, if cDNA is synthesized through the primer set of the present invention, the whole genome sequence of each dengue virus serotype can be accurately confirmed. means that
  • NCCP is a number assigned by the National Pathogen Resource Bank. '_S' at the end of the NCCP number is the common nucleotide sequence of the dengue virus obtained from Experimental Group 1, and '_M' is the common nucleotide sequence of the dengue virus obtained from Experimental Group 2.
  • NCCP 43256_S refers to the dengue-3 common nucleotide sequence obtained in Experimental Group 1.
  • the common nucleotide sequence of each serotype of dengue virus obtained in Experimental Group 1 and Experimental Group 2 is 99.9% identical. This is the result of obtaining the full genome sequence by isolating each serotype by synthesizing cDNA with the primers of the present invention to obtain the full genome sequence of the dengue virus even when multiple dengue virus serotypes are mixed in the sample. It means that sequencing is possible with the same accuracy as
  • Example 2 Comparison of the common nucleotide sequence of Experimental Group 1 and Experimental Group 2 with a reference genome
  • the regular common nucleotide sequences obtained in Experimental Group 1 and Experimental Group 2 were compared with 5 reference genomes of each serotype (see Tables 3 to 6).
  • Table 3 compares the regular common nucleotide sequence of dengue-1 obtained in experimental group 1 and experimental group 2 with 5 reference genomes
  • Table 4 shows the regular common nucleotide sequence of dengue-2 obtained in experimental group 1 and experimental group 2 is compared with 5 reference genomes
  • Table 5 compares the regular common nucleotide sequence of dengue-3 obtained in Experimental Group 1 and Experimental Group 2 with 5 reference genomes
  • Table 6 shows that obtained in Experimental Group 1 and Experimental Group 2
  • the regular common nucleotide sequence of dengue-4 was compared with five reference genomes.
  • the present invention provides a primer set capable of simultaneously producing cDNA (complementary DNA) for whole genome sequence analysis of four serotypes (types 1, 2, 3, 4) of dengue virus, and
  • the present invention relates to a cDNA synthesis method using the same, and more particularly, a primer that specifically binds to the RNA of each dengue virus serotype and a plurality of serotypes even when a plurality of dengue virus serotypes are mixed in a sample using the same. It relates to a method capable of rapidly and accurately performing whole genome sequencing by producing cDNA for

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Abstract

La présente invention concerne un ensemble d'amorces permettant de produire de l'ADNc pour l'analyse simultanée de la séquence du génome entier de quatre sérotypes (types 1, 2, 3 et 4) du virus de la dengue, ainsi qu'un procédé de synthèse d'ADNc l'utilisant, l'ensemble d'amorces pouvant détecter la séquence génomique entière de chaque sérotype avec une précision remarquable à partir d'un échantillon contenant les quatre sérotypes du virus de la dengue mélangés dans celui-ci et pouvant ainsi être utilisée en tant que données dans la recherche sur le traitement et sur un vaccin contre le virus de la dengue, ainsi que pour établir un système de surveillance pour empêcher la propagation du virus.
PCT/KR2020/002061 2020-01-09 2020-02-13 Ensemble d'amorces pour analyse simultanée d'une séquence génomique entière de quatre sérotypes du virus de la dengue et procédé de synthèse d'adnc l'utilisant WO2021141178A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113564281A (zh) * 2021-08-06 2021-10-29 广东省公共卫生研究院 一种基于pcr法的多种型别登革病毒引物组及应用该引物组的试剂盒

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

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113564281A (zh) * 2021-08-06 2021-10-29 广东省公共卫生研究院 一种基于pcr法的多种型别登革病毒引物组及应用该引物组的试剂盒

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