KR20230111359A - Chikungunya virus universal primer sets for whole genome amplification method and diagnosis kit - Google Patents
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Abstract
본 발명은 치쿤군야 바이러스(Chikungunya virus) 감염 여부를 판별하기 위한 전장 유전체 서열 획득 방법 및 이의 용도에 관한 것이다. 본 발명의 방법, 키트, 조성물은 치쿤군야 바이러스의 유전체 서열을 증폭할 수 있는 범용 프라이머 세트를 포함하고, 이에 따라 시료 내 목적 치쿤군야 바이러스의 존재 유무 및 관련 질환을 간편하고 빠르게 확인할 수 있다. 또한 전장 유전체 분석을 통해 치쿤군야 바이러스의 변이여부를 판별하여 치쿤군야 바이러스 동정에도 효과적으로 사용될 수 있다.The present invention relates to a method for obtaining a full-length genome sequence for determining whether or not infected with Chikungunya virus, and a use thereof. The method, kit, and composition of the present invention include a universal primer set capable of amplifying the genome sequence of Chikungunya virus, and accordingly, the presence or absence of the target Chikungunya virus in a sample and related diseases can be easily and quickly confirmed. In addition, it can be effectively used for identifying Chikungunya virus by determining whether the Chikungunya virus has been mutated through whole genome analysis.
Description
본 발명은 치쿤군야 바이러스(Chikungunya virus)의 전장유전체를 증폭할 수 있는 프라이머 세트를 활용하여 미량의 시료에서 바이러스 유전체를 증폭하고 염기서열을 분석하여 전장 유전체 정보를 얻어내는 기술에 관한 것이다.The present invention relates to a technology for amplifying the viral genome from a small amount of sample using a primer set capable of amplifying the entire genome of Chikungunya virus and analyzing the base sequence to obtain full-length genome information.
치쿤군야 바이러스(Chikungunya virus, CHIKV)는 Alphavirus 속과 Togaviridae family에 속하는 positive-sense single-stranded RNA 바이러스이다1,2. 이 바이러스는 3개의 비번역 영역(UTR), 4개의 비구조 단백질(NSP) 코딩 서열 및 5개의 구조 단백질 코딩 서열을 포함하는 약 12kb 길이의 게놈을 가지고 있다2,3. 1952-1953 탄자니아에서 대규모 열병으로 처음 인식되었다4,5. CHIKV는 주요 곤충 매개체 중 하나인 이집트숲모기(Aedes aegypti)에 의해 아프리카에 광범위하게 퍼졌다6,7. 동부 중앙 남부 아프리카(ECSA)와 일부 서아프리카(WA) 계통이 확인되었다. 동남아시아의 발병은 2004년 이전에 아시아 계통으로 자주 관찰되었다. 2004년 인도양 섬에서 ECSA 계통의 E1-A226V 변이체(E1 외피 당단백질의 위치 226에 있는 단일 아미노산 돌연변이)의 기초는 두 번째 주요 매개체인 Aedes albopictus의 높은 감수성을 유도했다9. 인도에서 동남아시아로 거대하고 반복적으로 발생했으며 2005-2007년 동안 아프리카와 일부 유럽에서도 발생했다9-11. 이 변이체는 ECSA의 하위 계통으로 첫 번째 ECSA 유전자형-인도양 계보(IOL)로 클러스터링되었다9,12,13. 2013년에 세인트 마틴의 카리브 섬은 아시아계 CHIKV 사례를 보고했으며 나중에 남미 국가와 미국으로 확대되었다14-16. 재출현은 오늘 현재까지 지속적으로 제기되었다17,18. 이전에 사례가 없었던 새로운 벡터 또는 새로운 국가를 암시할 가능성이 있다19-21.Chikungunya virus (CHIKV) is a positive-sense single-stranded RNA virus belonging to the genus Alphavirus and family Togaviridae 1,2 . This virus has a genome approximately 12 kb in length, containing 3 untranslated regions (UTRs), 4 non-structural protein (NSP) coding sequences and 5 structural protein coding sequences 2,3 . It was first recognized as a large-scale fever in Tanzania in 1952-1953 4,5 . CHIKV was widely spread in Africa by Aedes aegypti, one of the major insect vectors 6,7 . East Central South African (ECSA) and some West African (WA) lineages have been identified. Outbreaks in Southeast Asia were frequently observed with an Asian lineage prior to 2004. Basis of the E1-A226V variant (a single amino acid mutation at position 226 of the E1 envelope glycoprotein) of the ECSA line from an Indian Ocean island in 2004 induced high susceptibility of the second major vector, Aedes albopictus 9 . There were large and recurrent outbreaks from India to Southeast Asia, and also to Africa and parts of Europe during 2005-2007 9-11 . This variant is a sublineage of ECSA and clustered into the first ECSA genotype-Indian Ocean lineage (IOL) 9,12,13 . In 2013, the Caribbean island of Saint-Martin reported Asian CHIKV cases, which later expanded to South American countries and the United States 14-16 . Reappearance has been continuously raised to this day 17,18 . It is likely to imply a new vector or a new country where there has been no previous case 19-21 .
CHIKV 재출현의 위험에서 백신 및 치료 개발, 역학 및 진화 유전체학의 측면에서 전체 게놈 시퀀싱(WGS)의 중요성이 증가했다22. Whole Genome Sequencing은 완전한 염기서열 분석을 위한 염기서열분석이다. 그것은 샷건 접근 방식으로 전체 게놈 무작위 시퀀싱의 적용에서 발생한다23. 무작위 크기의 게놈 시퀀싱을 통해 각 게놈 단편('읽기'라고 함)은 시퀀스에 반복적인 영역을 가진다. 읽기(read)는 이러한 반복 영역을 식별하고 서로를 조합하여 하나의 연속 시퀀스를 만든다. 결과적으로 전체 게놈이 시퀀싱된다23,24. WGS는 이제 NGS 플랫폼에서 수행할 수 있다25.Whole genome sequencing (WGS) has grown in importance in terms of vaccine and treatment development, epidemiology and evolutionary genomics at risk of CHIKV reemergence 22 . Whole genome sequencing is sequencing for complete sequencing. It arises from the application of whole genome random sequencing as a shotgun approach 23 . With random-sized genome sequencing, each genome fragment (called a 'read') has a region that is repetitive in sequence. A read identifies these repeating regions and combines them into one continuous sequence. As a result, the entire genome is sequenced 23,24 . WGS can now be performed on the NGS platform 25 .
이전에 대규모 병렬 시퀀싱으로 알려진 NGS(next generation sequencing)는 Sanger 시퀀싱 이후에 등장한 대용량 시퀀싱 기술의 총칭이다. NGS는 실행 시간, 비용 및 출력 데이터를 개선하기 위해 Sanger 시퀀싱의 다양한 접근 방식을 기반으로 개발되었다. short-read 또는 long-read를 분석하기 위한 여러 플랫폼이 있다26-28. Illumina는 short-read NGS28을 나타내는 플랫폼 중 하나이다. Illumina 플랫폼은 2개의 가역 어댑터의 브리지 증폭으로 페어 엔드 시퀀싱을 수행한다. 서로 다른 어댑터 서열이 단일 가닥 DNA 단편의 각 끝과 연결되어 플로우 셀 표면에 부착된다. 다른 하나는 하나의 어댑터를 부착한 후 표면의 상보적인 올리고에 결합한다. 그런 다음 두 개의 어댑터에 의해 유도된 브리지 모양에 따라 새로운 가닥이 합성된다. 각 형광 연결 염기는 합성 주기 동안 다른 신호를 방출한다. 방출된 신호는 DNA 서열을 결정한다29. ONT(Oxford Nanopore Technology)는 PacBio 플랫폼을 탑재한 Long-Read NGS의 대표적인 플랫폼이다. ONT는 최대 수십만 kb의 읽기 길이를 처리할 수 있다28. 단일 단백질 나노포어를 이용하여 나노포어 시퀀싱을 수행한다. 나노포어는 플로우 셀의 지질 이중층에 위치하며 단일 가닥 DNA 단편이 통과할 때 염기의 전기 신호를 읽는다. 각 염기는 다른 잔류 이온 전류를 제공하며 그 순서는 시퀀스를 나타낸다30. 이러한 NGS 플랫폼의 등장은 WGS뿐만 아니라 RNA 시퀀싱, 메타게노믹스, 질병 전파 모니터링(추적) 및 생물 정보학에서도 우수한 연구를 가능하게 했다31-36.Next generation sequencing (NGS), previously known as massively parallel sequencing, is a collective term for large-scale sequencing technologies that emerged after Sanger sequencing. NGS has been developed based on the different approaches of Sanger sequencing to improve run time, cost and output data. There are several platforms for analyzing short-reads or long-reads 26-28 . Illumina is one of the platforms presenting the short-read NGS28. The Illumina platform performs pair-end sequencing with bridge amplification of two reversible adapters. A different adapter sequence connects to each end of the single-stranded DNA fragment and attaches it to the surface of the flow cell. The other binds to a complementary oligo on the surface after attaching one adapter. Then, a new strand is synthesized according to the bridge shape induced by the two adapters. Each fluorescently linked base emits a different signal during the synthesis cycle. The signal emitted determines the DNA sequence 29 . ONT (Oxford Nanopore Technology) is a representative platform for Long-Read NGS equipped with the PacBio platform. ONT can handle read lengths of up to hundreds of thousands of kilobytes 28 . Nanopore sequencing is performed using a single protein nanopore. Nanopores are located in the lipid bilayer of the flow cell and read the electrical signals of the bases as single-stranded DNA fragments pass through them. Each base gives a different residual ionic current, and the order represents the sequence 30 . The emergence of these NGS platforms has enabled excellent research not only in WGS but also in RNA sequencing, metagenomics, disease transmission monitoring (tracking), and bioinformatics 31-36 .
본 발명은 빠른 차세대 시퀀싱(NGS)을 위한 CHIKV 범용 프라이머 개발을 목표로 한다. 한국에서 분리되어 이전에 부분 또는 구조 CDS에 대해 연구된 균주 NCCP43132(Strain KNIH/2009/77)를 사용하였다. 첫째, 프라이머는 ECSA(IOL 포함) 및 아시아 계통의 전체 서열을 포함하도록 가능한 한 설계되었다. 다음으로, 우리는 전체 게놈 서열에 대해 이중 가닥 cDNA를 사용하여 illumine shotgun sequencing을 수행했다. 앰플리콘의 빠른 시퀀싱은 Oxford Nanopore Technique에 의해 수행되었으며 시퀀스는 CLC 워크벤치 소프트웨어를 사용하여 분석되었다. 프라이머의 효율성을 확인하기 위해 다른 국가의 여러 균주와 파키스탄의 임상 샘플을 동일한 실험을 수행했다.The present invention aims to develop CHIKV universal primers for rapid next-generation sequencing (NGS). Strain NCCP43132 (Strain KNIH/2009/77), isolated from Korea and previously studied for partial or structural CDS, was used. First, the primers were designed as far as possible to cover the entire sequence of ECSA (including IOL) and Asian lineages. Next, we performed illumine shotgun sequencing using double-stranded cDNA for the whole-genome sequence. Rapid sequencing of amplicons was performed by Oxford Nanopore Technique and sequences were analyzed using CLC Workbench software. To confirm the efficiency of the primers, the same experiment was performed with several strains from other countries and clinical samples from Pakistan.
한국등록특허 제2030245호는 치쿤군야 바이러스 검출용 올리고뉴클레오티드 세트 및 이의 용도에 관해 개시하고 있고, 한국공개특허 제2011-0118176호는 치쿤군야의 검출을 위한 프로브 및 프라이머에 관해 개시하고 있으며, 미국공개특허 제2019/0367998호는 치쿤군야 바이러스 검출 방법이 개시되어 있다.Korean Patent Registration No. 2030245 discloses an oligonucleotide set for detecting chikungunya virus and its use, Korean Patent Publication No. 2011-0118176 discloses a probe and primer for detecting chikungunya, and US Patent Publication No. 2019/0367998 discloses a chikungunya virus detection method.
하지만, 본 발명의 치쿤군야 바이러스의 전장 유전체 검출을 위한 범용 프라이머 세트에 대해서는 아직까지 개시된 바가 없다.However, the universal primer set for detecting the whole genome of Chikungunya virus of the present invention has not been disclosed yet.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 치쿤군야 바이러스에서 증폭이 가능한 범용 프라이머를 활용하여 미량의 유전체로부터 PCR 등을 통하여 증폭된 유전체를 염기서열분석법으로 빠르고 간편하게 바이러스의 전장 유전체 염기서열을 확보할 수 있음을 확인하여 본 발명을 완성하였다.The present invention was derived from the above request, and the present inventors utilized general-purpose primers capable of amplification in Chikungunya virus to quickly and easily perform genome sequencing on the genome amplified through PCR or the like from a small amount of genome. It was confirmed that it was possible to secure the genome sequence and completed the present invention.
상기 과제를 해결하기 위하여, 본 발명은 하기의 단계를 포함하는 분리된 시료 내 치쿤군야 바이러스의 전장 유전체 서열 확인 방법을 제공한다.In order to solve the above problems, the present invention provides a method for confirming the whole genome sequence of chikungunya virus in an isolated sample comprising the following steps.
상기 서열 확인 방법은 (a) 치쿤군야 바이러스 전장 유전체에 상보적인 하나 이상의 정방향 및 역방향 프라이머 세트를 제조하는 단계;The sequence confirmation method includes (a) preparing one or more sets of forward and reverse primers complementary to the full genome of Chikungunya virus;
(b) 시료에서 RNA를 분리하는 단계;(b) isolating RNA from the sample;
(c) 분리된 RNA를 사용하여 역전사 반응을 수행하는 단계;(c) performing a reverse transcription reaction using the isolated RNA;
(d) 역전사 반응이 수행된 시료에 프라이머 세트를 첨가하여 중합효소 연쇄반응을 실시하는 단계; 및(d) performing a polymerase chain reaction by adding a primer set to the sample in which the reverse transcription reaction was performed; and
(e) 증폭된 산물의 서열을 분석하는 단계를 포함한다.(e) sequencing the amplified product.
또한, 상기 프라이머 세트는 서열번호 1 및 서열번호 2; 서열번호 3 및 서열번호 4; 서열번호 5 및 서열번호 6; 및 서열번호 7 및 서열번호 8의 프라이머 세트를 포함하는 것이다.In addition, the primer set includes SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; and primer sets of SEQ ID NO: 7 and SEQ ID NO: 8.
본 발명은 치쿤군야 바이러스 전장 유전체에 상보적인 하나 이상의 정방향 및 역방향 프라이머 세트를 포함하는 치쿤군야 바이러스 진단 키트로서,The present invention is a chikungunya virus diagnostic kit comprising at least one forward and reverse primer set complementary to the full-length chikungunya virus genome,
상기 프라이머 세트는 서열번호 1 및 서열번호 2; 서열번호 3 및 서열번호 4; 서열번호 5 및 서열번호 6; 및 서열번호 7 및 서열번호 8의 프라이머 세트를 포함하는 치쿤군야 바이러스 진단 키트를 제공한다.The primer set is SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; and a chikungunya virus diagnostic kit comprising primer sets of SEQ ID NO: 7 and SEQ ID NO: 8.
본 발명은 치쿤군야 바이러스 전장 유전체에 상보적인 하나 이상의 정방향 및 역방향 프라이머 세트를 포함하는 알파 코로나 바이러스 유발 질환 진단용 조성물로서, The present invention is a composition for diagnosing an alpha corona virus-induced disease comprising at least one forward and reverse primer set complementary to the full-length chikungunya virus genome,
상기 프라이머 세트는 서열번호 1 및 서열번호 2; 서열번호 3 및 서열번호 4; 서열번호 5 및 서열번호 6; 및 서열번호 7 및 서열번호 8의 프라이머 세트를 포함하는 것인 치쿤군야 바이러스 유발 질환 진단용 조성물을 제공한다.The primer set is SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; And SEQ ID NO: 7 and SEQ ID NO: 8 provides a composition for diagnosing a disease caused by Chikungunya virus.
본 발명의 일 예에서, 상기 시료는 혈액, 혈청, 객담, 소변 또는 생체 조직으로부터 분리된 것이다. In one embodiment of the present invention, the sample is separated from blood, serum, sputum, urine or biological tissue.
본 발명의 다른 예에서, 상기 치쿤군야 바이러스는 KNIH/2009/77을 포함하는 것이다. In another example of the present invention, the chikungunya virus includes KNIH/2009/77.
본 발명의 또 다른 예에서, 상기 치쿤군야 바이러스 유발 질환은 돌발성 발열, 다발성 관절통 및 발진으로 이루어진 군에서 선택된 하나 이상인 것이다. In another example of the present invention, the chikungunya virus-induced disease is at least one selected from the group consisting of sudden fever, polyarthralgia, and rash.
본 발명의 특징 및 이점을 요약하면 다음과 같다.The features and advantages of the present invention are summarized as follows.
본 발명은 치쿤군야 바이러스 전장 유전체의 서열 획득 방법 및 이의 용도에 관한 것이다. The present invention relates to a method for obtaining the sequence of the whole genome of chikungunya virus and its use.
본 발명의 방법, 키트 및 조성물은 치쿤군야 바이러스의 전장유전체를 증폭시킬 수 있는 정방향 및 역방향 프라이머 세트를 이용하고, 유전체 증폭을 통하기 때문에 미량의 바이러스 시료 및 미배양 임상시료 등에서 치쿤군야 바이러스의 존재 유무 및 변종유무를 간편하고 빠르게 확인할 수 있다. The method, kit, and composition of the present invention use forward and reverse primer sets capable of amplifying the entire genome of Chikungunya virus, and because genome amplification is performed, the presence or absence of Chikungunya virus and the presence or absence of variants can be easily and quickly confirmed in small amounts of virus samples and uncultured clinical samples.
따라서, 본 발명의 방법, 키트 및 조성물은 치쿤군야 바이러스의 전장유전체 서열을 정확하고 신속하게 얻을 수 있게 해주며, 이를 통해 치쿤군야 바9..이러스로 유발된 질환의 진단에 효과적으로 적용될 수 있다.Therefore, the method, kit, and composition of the present invention enable accurate and rapid acquisition of the whole genome sequence of Chikungunya virus, and thus can be effectively applied to the diagnosis of diseases caused by Chikungunya virus 9..virus.
도 1은 본 발명의 일 구현 예에 따른 계통발생 분석결과를 나타낸 것이다. (1A) 치쿤군야 바이러스의 계통수는 3개의 계통으로 나뉜다; 서부 아프리카(WA), 동부 중남부 아프리카(ECSA), 아시아. (1B) 검은색 사각형은 KNIH/2009/77 균주와 다른 균주 사이의 계통발생적 거리를 보기 위해 (1A)의 빨간색 화살표를 기반으로 한 작은 나무 확대이다.1 shows the results of phylogenetic analysis according to an embodiment of the present invention. (1A) The phylogenetic tree of Chikungunya virus is divided into three families; West Africa (WA), East Central and South Africa (ECSA), and Asia. (1B) The black square is a small tree enlargement based on the red arrow in (1A) to see the phylogenetic distance between the KNIH/2009/77 strain and other strains.
이하, 본 발명의 바람직한 구현예에 대하여 상세히 설명한다. 또한, 하기의 설명에서는 구체적인 구성요소 등과 같은 많은 특정 사항들이 도시되어 있는데, 이는 본 발명의 보다 전반적인 이해를 돕기 위해서 제공된 것일 뿐 이러한 특정 사항들 없이도 본 발명이 실시될 수 있음은 이 기술분야에서 통상의 지식을 가진 자에게는 자명하다 할 것이다. 그리고, 본 발명을 설명함에 있어서, 관련된 공지 기능 혹은 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, in the following description, many specific details such as specific components are shown, which are provided to help a more general understanding of the present invention, and that the present invention can be practiced without these specific details. It will be apparent to those skilled in the art. And, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description will be omitted.
본 발명의 목적을 달성하기 위하여, 본 발명은 하기의 단계를 포함하는 분리된 시료 내 치쿤군야 바이러스의 전장 유전체 서열 확인 방법을 제공한다.In order to achieve the object of the present invention, the present invention provides a method for confirming the whole genome sequence of chikungunya virus in an isolated sample comprising the following steps.
상기 서열 확인 방법은 (a) 치쿤군야 바이러스 전장 유전체에 상보적인 하나 이상의 정방향 및 역방향 프라이머 세트를 제조하는 단계;The sequence confirmation method includes (a) preparing one or more sets of forward and reverse primers complementary to the full genome of Chikungunya virus;
(b) 시료에서 RNA를 분리하는 단계;(b) isolating RNA from the sample;
(c) 분리된 RNA를 사용하여 역전사 반응을 수행하는 단계;(c) performing a reverse transcription reaction using the isolated RNA;
(d) 역전사 반응이 수행된 시료에 프라이머 세트를 첨가하여 중합효소 연쇄반응을 실시하는 단계; 및(d) performing a polymerase chain reaction by adding a primer set to the sample in which the reverse transcription reaction was performed; and
(e) 증폭된 산물의 서열을 분석하는 단계를 포함한다.(e) sequencing the amplified product.
또한, 상기 프라이머 세트는 서열번호 1 및 서열번호 2; 서열번호 3 및 서열번호 4; 서열번호 5 및 서열번호 6; 및 서열번호 7 및 서열번호 8의 프라이머 세트를 포함하는 것이다.In addition, the primer set includes SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; and primer sets of SEQ ID NO: 7 and SEQ ID NO: 8.
상기 프라이머의 농도는 각각 0.1 내지 1.0 pM, 0.1 내지 0.8 pM, 0.1 내지 0.6 pM, 0.2 내지 1.0 pM, 0.2 내지 0.8 pM, 0.2 내지 0.6 pM, 0.3 내지 1.0 pM, 0.3 내지 0.8 pM, 0.3 내지 0.6 pM, 0.4 내지 1.0 pM 또는 0.4 내지 0.8 pM, 예를 들어, 0.4 내지 0.6 pM일 수 있으나, 이에 한정되는 것은 아니다.The concentration of the primer is 0.1 to 1.0 pM, 0.1 to 0.8 pM, 0.1 to 0.6 pM, 0.2 to 1.0 pM, 0.2 to 0.8 pM, 0.2 to 0.6 pM, 0.3 to 1.0 pM, 0.3 to 0.8 pM, 0.3 to 0.6 pM, 0.4 to 1. It may be 0 pM or 0.4 to 0.8 pM, for example, 0.4 to 0.6 pM, but is not limited thereto.
상기 중합효소 연쇄반응을 실시하는 단계는 30 내지 50 사이클, 30 내지 46 사이클, 30 내지 44 사이클, 33 내지 50 사이클, 33 내지 46 사이클, 33 내지 44 사이클, 36 내지 50 사이클, 36 내지 46 사이클, 36 내지 44 사이클, 38 내지 50 사이클 또는 38 내지 46 사이클, 예를 들어, 38 내지 44 사이클 조건으로 수행되는 것일 수 있으나, 이에 한정되는 것은 아니다.The step of carrying out the polymerase chain reaction is 30 to 50 cycles, 30 to 46 cycles, 30 to 44 cycles, 33 to 50 cycles, 33 to 46 cycles, 33 to 44 cycles, 36 to 50 cycles, 36 to 46 cycles, 36 to 44 cycles, 38 to 50 cycles or 38 to 46 cycles, for example, 38 to 44 cycles It may be performed under conditions, but is not limited thereto.
본 발명의 일 예에서, 상기 시료는 혈액, 혈청, 객담, 소변 또는 생체 조직으로부터 분리된 것이다. In one embodiment of the present invention, the sample is separated from blood, serum, sputum, urine or biological tissue.
본 발명의 다른 예에서, 상기 치쿤군야 바이러스는 KNIH/2009/77을 포함하는 것이다. In another example of the present invention, the chikungunya virus includes KNIH/2009/77.
상기 증폭된 산물의 서열을 분석하는 단계는 이 분야에 알려진 시퀀싱 방법이면 종류에 관계없이 사용할 수 있고, 바람직하게는 Sanger법 또는 차세대염기서열분석법(NGS; next generation sequencing)을 사용할 수 있으나, 이에 한정되는 것은 아니다. In the step of analyzing the sequence of the amplified product, any sequencing method known in the art may be used regardless of the type, preferably Sanger method or next generation sequencing (NGS), but is not limited thereto.
염색체의 일부가 결핍 또는 중복되어 나타나는 DNA복제수 변이(CNVs)를 포함한 염색체 이상을 확인하기 위해 핵형분석, 형광동소보합법, 염색체 마이크로어레이, NGS기반의 스크리닝 검사와 같이 다양한 검사가 이루어지고 있다 (Capalbo A, et al. 2017, Hum Reprod. Vol. 32(3), pp. 492-498). 핵형분석은 다른 검사들에 비해 5Mb 정도의 낮은 해상도를 보이며 그보다 작은 크기의 염색체 결실/중복은 검출이 불가능하다. 5Mb 미만의 작은 크기의 염색체 결실 및 중복을 미세결실/중복이라고 하며, 단일유전자에 의한 질환 중 미세결실/중복에 의한 비율이 전체 변이의 15%에 해당한다(Vissers LE, et al. 2005, Hum Mol Genet. Vol. 15;14 Spec No. 2:R215-23.).In order to identify chromosomal abnormalities, including DNA copy number mutations (CNVs) in which part of the chromosome is missing or duplicated, various tests such as karyotyping, fluorescence in situ hybridization, chromosome microarray, and NGS-based screening are being performed (Capalbo A, et al. 2017, Hum Reprod. Vol. 32(3), pp. 492-498). Compared to other tests, karyotyping has a lower resolution of about 5 Mb, and chromosomal deletions/duplications smaller than that cannot be detected. Small chromosomal deletions and duplications of less than 5 Mb are called microdeletion/duplication, and among diseases caused by a single gene, the ratio of microdeletion/duplication accounts for 15% of all mutations (Vissers LE, et al. 2005, Hum Mol Genet. Vol. 15;14 Spec No. 2:R215-23.).
이러한 미세결실/중복을 검출해내기 위해서 특정 염기서열에 상보적인 탐침자를 활용한 형광동소보합법(FISH)과 염색체 마이크로어레이 검사가 이루어지고 있다. 형광동소보합법은 확인하려는 염기서열에 상보적인 탐침자에 형광라벨을 붙여 염색체 내에 특정 염기서열의 여부를 확인하는 검사법이다. 100kb-1Mb의 해상도를 보이기 때문에 미세결실/중복의 검출이 가능하지만 탐침자 서열에 상보적인 부분만 확인이 가능하기 때문에 기존에 알려진 변이에 대해서만 검출이 가능하다는 단점이 있다.In order to detect these microdeletions/duplications, fluorescence in situ hybridization (FISH) using probes complementary to specific nucleotide sequences and chromosome microarray tests are being performed. Fluorescence in situ hybridization is a test method to confirm whether a specific nucleotide sequence is present in a chromosome by attaching a fluorescent label to a probe complementary to the nucleotide sequence to be identified. Since it shows a resolution of 100 kb-1 Mb, it is possible to detect microdeletion/duplication, but it has the disadvantage that it can detect only previously known mutations because only the part complementary to the probe sequence can be identified.
현재 염색체 미세결실/중복을 확인하는 가장 일반적인 검사법으로 마이크로어레이를 기반으로 하는 비교유전체 혼성화법(aCGH)이 활용되고 있다(Russo CD, et al. 2014, Cancer Discov. Vol. 4(1), pp. 19-21). 마이크로어레이를 통해 검출 가능한 CNV의 크기는 탐침자의 밀도에 의해 결정되며 대략 50kb 크기의 CNV까지 검출이 가능하다. (Watson CT, et al. 2014) 하지만 전좌 또는 역위와 같이 염색체 재배열에 의한 염색체 이상은 검출이 불가능하다.Currently, a microarray-based comparative genomic hybridization (aCGH) method is used as the most common test method for confirming chromosomal microdeletion/duplication (Russo CD, et al. 2014, Cancer Discov. Vol. 4(1), pp. 19-21). The size of CNV detectable by microarray is determined by the density of probes, and it is possible to detect CNVs up to approximately 50 kb in size. (Watson CT, et al. 2014) However, chromosomal abnormalities caused by chromosomal rearrangements such as translocations or inversions cannot be detected.
생명체를 구성하는 유전자의 염기서열 순서를 확정하는 기술은 1세대 생어(Sanger) 방식과 차세대 염기서열 분석법인 NGS(Next Generation Sequencing)로 나뉜다. 1977년경에 개발된 생어 서열분석(Sanger sequencing)이라 불리는 1세대 방법은 유전자 증폭 반응(Polymerase Chain Reaction; PCR) 과정 중 ddNTPs(di-deoxynucleotidetriphosphates)가 DNA 가닥(strand)의 합성을 중단시키는 연쇄-정지(chain-termination) 원리를 응용하여 증폭된 DNA 절편(fragment)을 모아 염기서열을 확인하는 방법이다. 이 방법은 정확도는 높지만, 염기서열 확정에 시간이 오래 걸리며, 높은 비용이 발생한다는 문제점을 가지고 있다.The technology for determining the nucleotide sequence of genes constituting living organisms is divided into the first-generation Sanger method and NGS (Next Generation Sequencing), a next-generation sequencing method. The first-generation method called Sanger sequencing, developed around 1977, is a method of verifying the base sequence by collecting amplified DNA fragments by applying the chain-termination principle in which di-deoxynucleotidetriphosphates (ddNTPs) stop the synthesis of DNA strands during the polymerase chain reaction (PCR) process. Although this method has high accuracy, it has a problem that it takes a long time to confirm the base sequence and high cost occurs.
이와 같은 문제점을 극복하기 위하여 차세대 염기 서열 분석법이 개발되었다. 수많은 유전체의 유전정보를 분절화한 후, 증폭하여 빅데이타를 얻은 다음 생물정보학을 이용하여 원하는 생물체의 전장 유전체 염기서열(Whole genome sequences; WGS)을 확보하는 것이다. 차세대염기서열분석법(NGS)은 염색체를 작은 조각으로 나누고 각 조각의 유전정보를 병렬적으로 분석하는 염기서열분석법이다. NGS는 유전자분석 기술이 발전하면서 상대적으로 검사의 소요시간과 비용이 적고 단일염기 다형성(SNP), 삽입-결실(INDELs)까지 검출 가능한 높은 해상도 때문에 신생아의 유전성 질환 선별검사로 활용되고 있다. 그러나 염색체를 작게 나누어 분석하는 NGS의 원리적 특성상 큰 규모의 염색체의 구조적 변이나 CNVs을 검출하는데 기술적 한계가 있다(Yohe S, Thyagarajan B. 2017, Arch Pathol Lab Med. Vol. 141(11), pp. 1544-1557).In order to overcome these problems, a next-generation sequencing method has been developed. After segmenting the genetic information of numerous genomes, amplifying them to obtain big data, and then using bioinformatics to secure the whole genome sequences (WGS) of the desired organism. Next-generation sequencing (NGS) is a sequencing method that divides chromosomes into small pieces and analyzes the genetic information of each piece in parallel. With the development of genetic analysis technology, NGS is used as a screening test for genetic diseases in newborns because of its relatively low test time and cost, and high resolution capable of detecting even single nucleotide polymorphisms (SNPs) and insertions and deletions (INDELs). However, due to the principle characteristics of NGS, which divides and analyzes chromosomes into small pieces, there are technical limitations in detecting structural variations or CNVs of large-scale chromosomes (Yohe S, Thyagarajan B. 2017, Arch Pathol Lab Med. Vol. 141(11), pp. 1544-1557).
하지만 NGS는 탐침자를 기반으로 하는 마이크로어레이에서 검출할 수 없는 염색체 재배열에 의한 염색체 이상과 기존에 알려지지 않은 새로운 CNV의 검출이 가능하다(Talkowski ME, et al. 2011, Am J Hum Genet. Vol. 88(4), pp. 469-81). 또한 염색체를 작게 조각 내어 염기서열을 분석하는 특성으로 마이크로어레이 보다 더 높은 coverage와 해상도를 보이고 염색체 이상이 시작되는 구획점(breakpoint) 검출이 가능하다는 장점이 있다(Zhao M, et al. 2013, BMC Bioinformatics. Vol. 14, Suppl 11:S1).However, NGS can detect chromosomal abnormalities caused by chromosomal rearrangements that cannot be detected by probe-based microarrays and new CNVs previously unknown (Talkowski ME, et al. 2011, Am J Hum Genet. Vol. 88(4), pp. 469-81). In addition, it has the advantage of showing higher coverage and resolution than microarrays due to the characteristics of sequencing by slicing chromosomes into small fragments, and detecting breakpoints where chromosomal abnormalities begin (Zhao M, et al. 2013, BMC Bioinformatics. Vol. 14, Suppl 11: S1).
본 발명의 목적을 달성하기 위하여, 본 발명은 치쿤군야 바이러스 전장 유전체에 상보적인 하나 이상의 정방향 및 역방향 프라이머 세트를 포함하는 치쿤군야 바이러스 진단 키트로서,In order to achieve the object of the present invention, the present invention is a chikungunya virus diagnostic kit comprising at least one forward and reverse primer set complementary to the chikungunya virus full-length genome,
상기 프라이머 세트는 서열번호 1 및 서열번호 2; 서열번호 3 및 서열번호 4; 서열번호 5 및 서열번호 6; 및 서열번호 7 및 서열번호 8의 프라이머 세트를 포함하는 치쿤군야 바이러스 진단 키트를 제공한다.The primer set is SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; and a chikungunya virus diagnostic kit comprising primer sets of SEQ ID NO: 7 and SEQ ID NO: 8.
또한, 본 발명은 치쿤군야 바이러스 전장 유전체에 상보적인 하나 이상의 정방향 및 역방향 프라이머 세트를 포함하는 알파 코로나 바이러스 유발 질환 진단용 조성물로서, In addition, the present invention is a composition for diagnosing an alpha corona virus-induced disease comprising at least one forward and reverse primer set complementary to the full-length chikungunya virus genome,
상기 프라이머 세트는 서열번호 1 및 서열번호 2; 서열번호 3 및 서열번호 4; 서열번호 5 및 서열번호 6; 및 서열번호 7 및 서열번호 8의 프라이머 세트를 포함하는 것인 치쿤군야 바이러스 유발 질환 진단용 조성물을 제공한다.The primer set is SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; And SEQ ID NO: 7 and SEQ ID NO: 8 provides a composition for diagnosing a disease caused by Chikungunya virus.
이때, 본 발명의 또 다른 예에서, 상기 치쿤군야 바이러스 유발 질환은 돌발성 발열, 다발성 관절통 및 발진으로 이루어진 군에서 선택된 하나 이상인 것일 수 있다. At this time, in another example of the present invention, the chikungunya virus-induced disease may be at least one selected from the group consisting of sudden fever, polyarthralgia, and rash.
본 발명의 이점 및 특징, 그리고 그것들을 달성하는 방법은 상세하게 후술되어있는 실시예들을 참조하면 명확해질 것이다. 그러나 본 발명은 이하에서 개시되는 실시예들에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 것이며, 단지 본 실시예들은 본 발명의 개시가 완전하도록 하고, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이며, 본 발명은 청구항의 범주에 의해 정의될 뿐이다The advantages and features of the present invention, and how to achieve them, will become clear with reference to the detailed description of the following embodiments. However, the present invention will not be limited to the embodiments disclosed below, but will be implemented in various different forms, and only these embodiments are provided to complete the disclosure of the present invention and to fully inform those skilled in the art of the scope of the invention to which the present invention belongs, and the present invention is only defined by the scope of the claims.
실시예 1. RNA standard and cDNA synthesisExample 1. RNA standard and cDNA synthesis
균주 NCCP 43132의 바이러스 RNA(KNIH/2009/77)는 대한민국 국립병원체문화원(NCCP)에서 제공했다. 표 1은 이 연구에 사용된 게놈 RNA 및 임상 샘플에 대한 세부 정보를 제공한다. 임상 샘플은 파키스탄에서 cDNA 상태로 전달되었다. 게놈 RNA의 초기 농도는 제조사 매뉴얼에 따라 QuantiFluor RNA System(Promega, USA)과 Quantus™ Fluorometer(Promega, USA)를 이용하여 정량하였다.Viral RNA (KNIH/2009/77) of strain NCCP 43132 was provided by the National Institute of Pathogens and Culture of Korea (NCCP). Table 1 provides details of the genomic RNA and clinical samples used in this study. Clinical samples were delivered in cDNA condition from Pakistan. The initial concentration of genomic RNA was quantified using the QuantiFluor RNA System (Promega, USA) and the Quantus™ Fluorometer (Promega, USA) according to the manufacturer's manual.
(서열번호 1)GTGAGACACACGTAGCCTAC
(SEQ ID NO: 1)
(서열번호 2)CACCTCCCACTCCTTAATAG
(SEQ ID NO: 2)
(서열번호 3)TCAGAGCACGTCAACGTACT
(SEQ ID NO: 3)
(서열번호 4)ACAGTTGGRTAGTTTCTAGC
(SEQ ID NO: 4)
(서열번호 5)AAGAAACTCCAGGAGARTGC
(SEQ ID NO: 5)
(서열번호 6)TGGGTGCGTACATGARTGAC
(SEQ ID NO: 6)
(서열번호 7)CAYGATTGGACCAAGCTGCG
(SEQ ID NO: 7)
(서열번호 8)TCCCTGTGGGTTCGGAGAAT
(SEQ ID NO: 8)
* Genomic position are based on the genomic sequence of strain S27-African prototype (NC_004162)* Genomic position are based on the genomic sequence of strain S27-African prototype (NC_004162)
단일 가닥 cDNA(ss cDNA)는 게놈 RNA에서 합성되었다. 20μl의 반응 혼합물은 4 μl의 5X LunaScript RT supermix(New England Biolabs, USA), 2 μl의 RNA 및 뉴클레아제가 없는 물로 구성된다. 다음 조건에서 반응되었다; 2분 동안 25℃(프라이머 어닐링); 55℃에서 10분 동안(cDNA 합성); 1분 동안 95℃(열 비활성화).Single-stranded cDNA (ss cDNA) was synthesized from genomic RNA. A 20 μl reaction mixture consisted of 4 μl of 5X LunaScript RT supermix (New England Biolabs, USA), 2 μl of RNA and nuclease-free water. Reacted under the following conditions; 25° C. for 2 min (primer annealing); 55° C. for 10 min (cDNA synthesis); 95° C. for 1 min (heat inactivated).
실시예 2. Primer design and ScreenExample 2. Primer design and Screen
CHIKV의 게놈 서열은 Virus Pathogen Resource(ViPR; www.viprbrc.org) 데이터베이스에서 검색되었다. ViPR을 사용하여 CHIKV의 총 767개 게놈 서열을 정렬하고 CHIKV의 보존 영역을 결정했다. 프라이머는 보존된 서열로 설계되었다. 프라이머는 이전 연구와 유사한 기준으로 설계되었다37. 모든 올리고머는 Macrogen(한국)에서 합성되었다.The genome sequence of CHIKV was retrieved from the Virus Pathogen Resource (ViPR; www.viprbrc.org) database. A total of 767 genome sequences of CHIKV were aligned using ViPR and conserved regions of CHIKV were determined. Primers were designed with conserved sequences. Primers were designed with criteria similar to previous studies 37 . All oligomers were synthesized by Macrogen (Korea).
PCR 반응은 TaKaRa Ex Taq® 키트(Takara, Japan)를 사용하여 96-Well Thermal Cycler(Veriti Instruments, INC., USA)로 수행되었다. 50μl의 Reaction은 1μl의 각 프라이머(10μM), 5μl의 Ex Taq Buffer(10X), 3μl의 dNTP(각 2.5mM), 2μl의 템플릿, 0.25μl의 TaKaRa Ex Taq Polymerase 및 멸균된 증류수를 포함했다. PCR 조건은 다음과 같다; 95℃에서 1분간(초기 변성); 95℃에서 15초(변성), 60℃에서 15초(어닐링), 72℃에서 3분(연장)의 30 사이클; 3분 동안 72℃(최종 연장). 생성된 앰플리콘은 100 전압에서 40분 동안 전기영동(ADVANCE CO., LTD., Japan)으로 확인하였다. 전체 게놈 시퀀싱을 위해 가장 개별적인 앰플리콘을 생성하는 프라이머 쌍을 선택했다. 선택한 프라이머 시퀀스는 표 1에 나와 있다. 선택한 쌍의 앰플리콘은 제조업체의 프로토콜에 따라 DNA Clean & Concetrator-25(ZYMOresearch, USA)를 사용하여 정제되었다.PCR reactions were performed in a 96-Well Thermal Cycler (Veriti Instruments, INC., USA) using the TaKaRa Ex Taq® kit (Takara, Japan). A 50 μl reaction contained 1 μl of each primer (10 μM), 5 μl of Ex Taq Buffer (10X), 3 μl of dNTP (2.5 mM each), 2 μl of template, 0.25 μl of TaKaRa Ex Taq Polymerase and sterile distilled water. PCR conditions were as follows; 95° C. for 1 minute (initial denaturation); 30 cycles of 95°C for 15 seconds (denaturation), 60°C for 15 seconds (annealing), and 72°C for 3 minutes (extension); 72° C. for 3 minutes (final extension). The resulting amplicons were identified by electrophoresis (ADVANCE CO., LTD., Japan) at 100 voltage for 40 minutes. Primer pairs that generated the most discrete amplicons were selected for whole-genome sequencing. Selected primer sequences are shown in Table 1. Amplicons of selected pairs were purified using DNA Clean & Concetrator-25 (ZYMOresearch, USA) according to the manufacturer's protocol.
실시예 3. Quantification of ampliconsExample 3. Quantification of amplicons
앰플리콘의 양은 실시간 정량적 PCR(qPCR) 및 디지털 액적 PCR(ddPCR) 분석으로 2단계로 측정되었다. 분석에 사용된 프라이머와 프로브는 표 238-40에 나열되어 있다. 분석 믹스는 사용 준비를 위해 하나의 튜브에서 각 프라이머와 프로브를 혼합하여 준비했다. 각 프라이머와 프로브는 qPCR의 경우 0.5μM, ddPCR의 경우 0.4μM로 혼합되었다. 각 분석 믹스의 다음 부피는 단일 반응에 사용되어 반응에서 0.2μM의 최종 농도를 만든다. 3μl의 nsp2-1, C, E1 분석 및 5μl의 nsp2-2 분석.The amount of amplicons was measured in two steps with real-time quantitative PCR (qPCR) and digital droplet PCR (ddPCR) assays. Primers and probes used in the assay are listed in Table 2 38-40 . Assay mixes were prepared by mixing each primer and probe in one tube ready for use. Each primer and probe was mixed at 0.5 μM for qPCR and 0.4 μM for ddPCR. The next volume of each assay mix is used in a single reaction to make a final concentration of 0.2 μM in the reaction. 3 μl of nsp2-1, C, E1 assay and 5 μl of nsp2-2 assay.
All Taq-man probes were labelled with 5' end FAM as reporter dye and 3' end BHQ1 as quencher dye. All Taq-man probes were labeled with 5' end FAM as reporter dye and 3' end BHQ1 as quencher dye.
* Genomic position are based on the genomic sequence of S27-African prototype (NC_004162)* Genomic position are based on the genomic sequence of S27-African prototype (NC_004162)
ª Oligonucleotides have one base change form original sequences for wide coverage spectrums against CHIKV. Changed base highlight by extrabold.ª Oligonucleotides have one base change form original sequences for wide coverage spectrums against CHIKV. Changed base highlight by extrabold.
+ Two Oligonucleotides locate in same site and can cover the different genome spectrum. + Two Oligonucleotides locate in same site and can cover the different genome spectrum.
qPCR의 반응 혼합물은 Maxima Probe/ROX qPCR Master Mix 2X(Thermo Scientific™, USA)를 사용하여 제조되었다. 각 반응 혼합물의 25μl는 12.5μl 2X Master Mix, 2μl 템플릿, 분석 믹스 및 뉴클레아제가 없는 물로 구성되었다. PCR 조건은 다음과 같다. 95℃에서 10분간 (초기 변성); 95℃ 15초(변성) 및 60℃ 60초(어닐링/신장)의 40 사이클러. 형광은 LightCycler® 96 기기(Roche, Switzerland)의 확장 단계에서 FAM의 동적 범위를 기록하고 LightCycler® 96 소프트웨어(버전 1.1)로 분석했다.The reaction mixture for qPCR was prepared using Maxima Probe/ROX qPCR Master Mix 2X (Thermo Scientific™, USA). 25 μl of each reaction mixture consisted of 12.5 μl 2X Master Mix, 2 μl template, assay mix and nuclease-free water. PCR conditions are as follows. 95° C. for 10 minutes (initial denaturation); 40 cyclers of 95°C 15 sec (denaturation) and 60°C 60 sec (anneal/extension). Fluorescence was recorded over the dynamic range of the FAM at the extension stage on a LightCycler® 96 instrument (Roche, Switzerland) and analyzed with LightCycler® 96 software (version 1.1).
ddPCR의 반응 혼합물은 부피가 20 μl이고 프로브용 ddPCR 슈퍼믹스(BioRad Laboratories, USA) 10 μl, 주형 2μl, assay mix 및 nuclease-free water로 구성되었다. 모든 유효한 사본 번호는 제조업체의 지침에 따라 동적 범위 FAM(470nM/514nM)에서 선택되었다. ddPCR 실험은 QX200 시스템(BioRad Laboratories, Hercules, CA, USA)을 사용하여 수행되었다. 원시 데이터는 처음에 QuantaSoft 소프트웨어(BioRad Laboratories, Hercules, CA, USA)를 사용하여 분석되었다.The reaction mixture of ddPCR had a volume of 20 μl and consisted of 10 μl of ddPCR supermix for probe (BioRad Laboratories, USA), 2 μl of template, assay mix and nuclease-free water. All valid copy numbers were selected from the dynamic range FAM (470 nM/514 nM) according to the manufacturer's instructions. ddPCR experiments were performed using the QX200 system (BioRad Laboratories, Hercules, CA, USA). Raw data were initially analyzed using QuantaSoft software (BioRad Laboratories, Hercules, CA, USA).
앰플리콘의 복제 수는 이전에 설명한 대로 ddPCR 결과로 선형 회귀 분석을 사용하여 qPCR 결과에서 계산되었다41.Copy numbers of amplicons were calculated from qPCR results using linear regression analysis with ddPCR results as previously described 41 .
실시예 4. Whole Genome Shotgun SequencingExample 4. Whole Genome Shotgun Sequencing
1) Illumina Library preparation and sequencing1) Illumina Library preparation and sequencing
RNA sequencing에는 2가지 방법이 있다. 하나는 이중 가닥 cDNA(ds cDNA)를 만드는 것이고 다른 하나는 RNA library prep kit를 사용하는 것이다. 본 발명에서는 단일 가닥 cDNA에서 두 번째 가닥 cDNA를 합성하여 ds cDNA를 만들었다. 단일 가닥 cDNA는 random hexamer와 oligo-dt primer를 모두 포함하는 LunaScript RT supermix kit(NEB, England)를 사용하여 합성되었다. 혼합물은 LunaScript RT SuperMix(5X) 4μl, 템플릿 4μl 및 최대 20μl의 뉴클레아제가 없는 물 부피로 구성되었다. 두 번째 가닥 cDNA 합성은 제조사의 프로토콜에 따라 Maxima™ H Minus Double-Stranded cDNA Synthesis kit(Thermo Fisher Scientific, USA)를 사용하여 수행되었다. 그런 다음 100U의 RNase I을 100㎕의 ds cDNA 반응물에 첨가하고 실온에서 5분 동안 인큐베이션하여 잔류 RNA를 제거하였다. DNA Clean & Concentrator Kits(Zymo Research, USA)로 정제된 ds cDNA는 QuantiFluor ds DNA System(Promega, USA) 및 Quantus™ Fluorometer를 사용하여 제조업체 설명서에 따라 정량화했다. There are two methods of RNA sequencing. One is to make double-stranded cDNA (ds cDNA) and the other is to use an RNA library prep kit. In the present invention, ds cDNA was prepared by synthesizing second-strand cDNA from single-strand cDNA. Single-stranded cDNA was synthesized using the LunaScript RT supermix kit (NEB, England) containing both random hexamer and oligo-dt primers. The mixture consisted of 4 μl LunaScript RT SuperMix (5X), 4 μl template and up to 20 μl nuclease-free water volume. Second-strand cDNA synthesis was performed using the Maxima™ H Minus Double-Stranded cDNA Synthesis kit (Thermo Fisher Scientific, USA) according to the manufacturer's protocol. Then, 100 U of RNase I was added to 100 μl of ds cDNA reaction and incubated at room temperature for 5 minutes to remove residual RNA. Purified ds cDNA with DNA Clean & Concentrator Kits (Zymo Research, USA) was quantified using the QuantiFluor ds DNA System (Promega, USA) and Quantus™ Fluorometer according to the manufacturer's instructions.
라이브러리 준비는 제조사 매뉴얼에 따라 300-350bp PCR 단편을 만드는 Nextera DNA Flex Library Prep Kit(Illumina, USA)를 사용하여 수행되었다. 실제 어댑터 연결 DNA의 정량화를 위해 프로토콜에 따라 LightCycler® 96 기기(Roche, Switzerland)에서 KAPA 라이브러리 정량화 키트(Roche, Switzerland)를 사용하여 라이브러리 정량화를 확인했다. 라이브러리 샘플 정량화를 위한 정량화 대조군으로 10nM PhiX Control v3(Illumina, USA)의 10배 희석 시리즈를 사용했다. 샘플 농도는 LightCycler® 96 Software(version 1.1)의 Absolute Quantitative Analysis 프로그램에 의해 Cq 값을 기준으로 분석되었다. 100pM의 최종 로딩 샘플은 시퀀싱 실행 중에 각 기본 신호를 고르게 돕는 5% PhiX 컨트롤을 추가하여 준비했다. 20μl의 최종 로딩 샘플을 로딩한 다음 기기의 지침에 따라 시퀀싱 프로그램을 실행하였다.Library preparation was performed using the Nextera DNA Flex Library Prep Kit (Illumina, USA) to create 300-350bp PCR fragments according to the manufacturer's manual. For quantification of real adapter-ligated DNA, library quantification was confirmed using the KAPA Library Quantification Kit (Roche, Switzerland) on a LightCycler® 96 instrument (Roche, Switzerland) according to the protocol. A 10-fold dilution series of 10 nM PhiX Control v3 (Illumina, USA) was used as a quantification control for library sample quantification. Sample concentrations were analyzed based on Cq values by the Absolute Quantitative Analysis program in LightCycler® 96 Software (version 1.1). A final loading sample of 100 pM was prepared by adding a 5% PhiX control to help even out each base signal during the sequencing run. A final loading sample of 20 μl was loaded and then the sequencing program was run according to the instructions of the instrument.
2) Sequence analysis2) Sequence analysis
CLC Genomic Workbench Software(20.0.4 버전, Qiagen, Denmark)를 판독의 트리밍 및 매핑에 사용했다. Illumina의 두 쌍 끝 판독(정방향-역방향)은 품질이 낮은 판독값을 제거하기 위해 다음 매개변수 조건으로 트리밍되었다. Phred 품질 점수 30점; 최대 모호성 수 중 1개; 호모폴리머(단말에서 20개 염기 중 9개 염기에 걸쳐 동일한 염기를 의미하는 호모폴리머); 5' 및 3' 터미널에서 각각 15 및 10개의 베이스를 제거했다. 길이가 60 미만인 읽기는 삭제한다. 트리밍된 읽기는 NCBI에서 다운로드한 FJ445484(11,790bp)에 매핑되었다. 매핑 조건은 다음과 같다. 경기 점수 1점; 불일치 점수 2점; 3 삽입 비용; 3 삭제 비용; 자동 감지 페어링 거리; 무작위로 매핑한다(정렬 점수 및 선형 간격 비용은 CLC 워크벤치 소프트웨어에서 제공하는 기본값임). CHIKV의 완전한 서열은 계통발생적 거리를 확인하기 위해 MEGA 소프트웨어(버전 7.0.26, MEGA, USA)에 의해 컨센서스 서열로 분석되었다(표 3). 균주 SG650(AF079456), Onyong-nyong 바이러스(ONNV)는 트리의 외부 그룹으로 사용되었다. Muscle 알고리즘을 이용하여 서열을 정렬하고, Neighbor-joining 알고리즘을 이용하여 계통수를 구성하였다. 합의 시퀀스는 NCBI(GenBank: - )에서 사용할 수 있다.CLC Genomic Workbench Software (version 20.0.4, Qiagen, Denmark) was used for trimming and mapping reads. Illumina's two-end reads (forward-reverse) were trimmed with the following parametric conditions to remove low-quality reads. Phred Quality Score of 30; 1 of the maximum number of ambiguities; homopolymers (homopolymers meaning identical bases over 9 out of 20 bases at the termini); 15 and 10 bases were removed from the 5' and 3' terminals, respectively. Delete reads with a length less than 60. Trimmed reads were mapped to FJ445484 (11,790 bp) downloaded from NCBI. The mapping condition is as follows. 1 match point; Discordance score of 2 points; 3 insertion cost; 3 deletion fees; auto-sensing pairing distance; Map randomly (alignment score and linear interval cost are defaults provided by CLC Workbench software). The complete sequence of CHIKV was analyzed as a consensus sequence by MEGA software (version 7.0.26, MEGA, USA) to confirm phylogenetic distance (Table 3). Strain SG650 (AF079456), Onyong-nyong virus (ONNV), was used as the outer group of the tree. The sequences were aligned using the muscle algorithm, and a phylogenetic tree was constructed using the neighbor-joining algorithm. A consensus sequence is available from NCBI (GenBank: - ).
* DRC ; Democratic Republic of the Congo, + CAR ; Central African Republic* DRC; Democratic Republic of the Congo, + CAR; Central African Republic
실시예 5. MinION nanopore SequencingExample 5. MinION nanopore sequencing
1) Library preparation and Sequencing Run1) Library preparation and sequencing run
라이브러리는 제조업체의 프로토콜에 따라 Native Barcoding Expansion 1-12(EXP-NBD104, Oxford Nanopore Technologies, England)와 함께 Ligation kit(SQK-LSK109, Oxford Nanopore Technologies, England)의 1D 앰플리콘/cDNA를 사용하여 수행되었다. 라이브러리 준비 후 1D 플로우 셀(FLO-MIN 106, Oxford Nanopore Technologies, England)을 MinION 기기(Oxford Nanopore Technologies, 영국)에 놓고 컴퓨터의 MinKNOW 소프트웨어에 연결했다. Flow cell Check 및 Loading Sample도 프로토콜에 따라 수행되었다. 시퀀싱은 base-calling으로 24시간 동안 실행되었고 결과적으로 FAST5 파일을 만들었다.Libraries were performed using 1D amplicons/cDNA from the Ligation kit (SQK-LSK109, Oxford Nanopore Technologies, England) with Native Barcoding Expansion 1-12 (EXP-NBD104, Oxford Nanopore Technologies, England) according to the manufacturer's protocol. After library preparation, a 1D flow cell (FLO-MIN 106, Oxford Nanopore Technologies, England) was placed in a MinION instrument (Oxford Nanopore Technologies, England) and connected to the MinKNOW software on a computer. Flow cell check and loading samples were also performed according to the protocol. Sequencing was run for 24 hours with base-calling and resulted in a FAST5 file.
2) Sequence Analysis 2) Sequence Analysis
분석을 위해 FAST5 파일을 FASTAQ 형식으로 전송하였다. 그런 다음 MinION GUI 소프트웨어에 의해 낮은 품질의 읽기가 트리밍되었다. 트리밍된 판독 데이터는 분석을 위해 CLC Genomic Workbench Software로 가져왔다. 두 번째 트리밍은 다음과 같은 조건으로 읽혀지도록 처리되었다. 최대 모호성 수 중 1개; 호모폴리머 제거; 5' 및 3' 터미널에서 각각 15 및 10개의 베이스를 제거했다. 길이가 60 미만인 판독은 폐기하였다. 트리밍된 판독은 샷건 시퀀싱에 의해 전체 게놈 서열에서 가져온 참조 서열에 매핑되었다. CHIKV의 완전한 서열은 MEGA 소프트웨어에 의해 컨센서스 서열로부터 분석되었다.FAST5 files were transferred in FASTAQ format for analysis. Low quality reads were then trimmed by the MinION GUI software. Trimmed read data were imported into CLC Genomic Workbench Software for analysis. The second trimming was processed to be read under the following conditions. 1 of the maximum number of ambiguities; homopolymer removal; 15 and 10 bases were removed from the 5' and 3' terminals, respectively. Reads less than 60 in length were discarded. Trimmed reads were mapped to a reference sequence taken from the whole genome sequence by shotgun sequencing. The complete sequence of CHIKV was analyzed from the consensus sequence by MEGA software.
실험예 1. Whole Genome Shotgun SequencingExperimental Example 1. Whole Genome Shotgun Sequencing
한국의 모든 치쿤군야 바이러스 사례는 여행 관련 환자를 확인한다. 균주 NCCP 43132(KNIH/2009/77)는 여행 관련 환자로부터 처음 분리되어 국립 병원균 수집(NCCP)에 기탁되었다42. 이 균주의 부분 코딩 서열(CDS) 또는 구조 단백질 CDS 영역만이 이전에 연구되었습니다42-44. 완전한 게놈 기반 추가 연구를 위한 기반을 제공하기 위해 먼저 이중 가닥 cDNA를 사용하여 이 균주의 전체 게놈 샷건 시퀀싱을 분석했다. pair-end 읽기 데이터는 Illumina 시퀀싱에 의해 만들어졌고 CLC 워크벤치에 입력되었다. 읽은 데이터는 품질 관리 보고서를 기반으로 트리밍되었다. 트리밍된 읽기는 SGEHICHD122508(FJ445502)에 매핑되어 이 균주를 분리한 첫번째 연구에서 가장 유사한 읽기를 보여주었다. 가장 상동성인 게놈을 찾기 위해 BLAST 프로그램(NCBI)을 사용하여 컨센서스 서열을 확인하였다. BLAST 결과는 서열이 ECSA 유전자형-인도양 계보(IOL)45의 균주 SGEHICHT077808(FJ445484)과 가장 높은 유사성(쿼리 커버의 100%, 동일성 백분율의 99.94%)을 갖는 것으로 나타났다. SGEHICHT077808(FJ445484)에 다시 매핑하여 11,790bp 길이 크기의 결과 컨센서스 시퀀스를 얻었다. 표 4 및 표 5는 분석 소프트웨어에서 제공하는 트리밍 및 매핑 보고서를 보여준다. 추가 검사를 통해 시퀀스의 끝인 11763-11790 사이트에서 발견된 100 임계값 미만의 범위를 읽는다. 합의 서열은 MEGA7의 근육 정렬에 의해 여러 계통의 완전한 서열과 정렬되었다. Consensus sequence에서 7개의 차이점이 확인되었고 Sanger sequencing을 통해 올바른 것으로 확인되었습니다. 계통 발생 분석은 IOL에 클러스터링된 균주 NCCP 43132의 계통 발생 거리를 확인했습니다(도 1). 균주 NCCP 43132는 IOL45,46에 클러스터링된 싱가포르, 말레이시아, 파푸아뉴기니의 균주와 유사하다.All Chikungunya virus cases in South Korea identify travel-related cases. Strain NCCP 43132 (KNIH/2009/77) was first isolated from a travel-related patient and deposited with the National Pathogen Collection (NCCP) 42 . Only the partial coding sequence (CDS) or structural protein CDS region of this strain has been previously studied 42–44 . To provide a basis for further full genome-based studies, we first analyzed whole-genome shotgun sequencing of this strain using double-stranded cDNA. Paired-end read data were generated by Illumina sequencing and entered into the CLC workbench. Read data were trimmed based on quality control reports. Trimmed reads mapped to SGEHICHD122508 (FJ445502), showing the most similar reads from the first study isolating this strain. Consensus sequences were identified using the BLAST program (NCBI) to find the most homologous genome. BLAST results showed that the sequence had the highest similarity (100% of query cover, 99.94% of percent identity) to strain SGEHICHT077808 (FJ445484) of the ECSA genotype-Indian Ocean lineage (IOL)45. Remapping to SGEHICHT077808 (FJ445484) resulted in a consensus sequence with a length of 11,790 bp. Tables 4 and 5 show the trimming and mapping reports provided by the analysis software. Further inspection reads ranges below the 100 threshold found at sites 11763-11790 at the end of the sequence. The consensus sequence was aligned with the complete sequence of several lineages by muscle alignment of MEGA7. Seven differences were identified in the Consensus sequence and confirmed correct by Sanger sequencing. Phylogenetic analysis confirmed the phylogenetic distance of strain NCCP 43132 clustered in the IOL (Figure 1). Strain NCCP 43132 is similar to strains from Singapore, Malaysia and Papua New Guinea clustered in IOLs 45,46 .
(3A) Chart indicate the result of trimming, mapping of shotgun sequencing based on the number of reads. (3A) Chart indicate the result of trimming, mapping of shotgun sequencing based on the number of reads.
(3B) Seven bases and its site presented difference between reference and consensus sequences. Genomic site are provided based on the reference sequence CHKV strain S27-African prototype (NC_004162). Note show the bases at same site in all whole genome database of CHIKV.(3B) Seven bases and its site presented difference between reference and consensus sequences. Genomic site are provided based on the reference sequence CHKV strain S27-African prototype (NC_004162). Note show the bases at same site in all whole genome database of CHIKV.
실험예 2. MinION nanopore SequencingExperimental Example 2. MinION nanopore sequencing
Chikungunya 바이러스에는 3개 또는 4개의 주요 혈통이 있다. 서아프리카(WA), 동부 중앙 남부 아프리카(ECSA), 아시아 및 인도양 계보(IOL). ECSA, Asian, IOL 혈통이 유행병에 크게 기여했지만 2004년 이전에는 WA 혈통이 Afica 대륙에 집중되었다. 다른 혈통과 확연히 다르다. 본 발명에서 프라이머는 WA 계통 이외의 다른 사람들을 대상으로 설계되었다. 디자인된 프라이머로 얻을 앰플리콘의 크기와 오버랩 크기는 각각 약 3Kb와 100bp를 기준으로 먼저 선정하였고, 최종적으로 젤 전기영동을 통해 비교적 깨끗한 단일 밴드를 나타내는 프라이머를 선정하였다. Amplicon 1의 경우 30여 개의 프라이머 조합에 걸쳐 강한 non-target 크기의 밴드가 연속적으로 형성되었다. 그래서 추가 스크리닝을 중단하고 프라이머 세트를 선택했다. There are three or four main lineages of Chikungunya virus. West African (WA), Eastern Central Southern African (ECSA), Asian and Indian Ocean lineages (IOL). ECSA, Asian, and IOL ancestry contributed significantly to the epidemic, but prior to 2004, WA ancestry was concentrated on the Afica continent. It is distinctly different from other lineages. In the present invention, primers were designed for people other than WA strains. The size of the amplicon to be obtained with the designed primer and the overlap size were first selected based on about 3Kb and 100bp, respectively, and finally, a primer showing a relatively clean single band was selected through gel electrophoresis. In the case of Amplicon 1, a strong non-target size band was continuously formed over 30 primer combinations. So further screening was discontinued and a primer set was selected.
실험예 2. MinION nanopore SequencingExperimental Example 2. MinION nanopore sequencing
Nanopore 시퀀싱 후 OxfordNamopore의 Guppy GPU basecalling 소프트웨어를 사용하여 15Gb 원시 데이터(fast5 형식)를 계산하고 fastq 파일로 변환했다. 변환하는 동안 base-calling, 어댑터 트리밍 및 Q-Score 7 이상으로 필터링하여 시퀀스 품질을 수정했다. CLC Workbench 소프트웨어를 이용하여 생성된 fastq 파일에 대해 추가적인 트리밍을 수행한 후 Illumina shot gun sequencing을 통해 얻은 전체 genome sequence(11,790bp)를 reference로 사용하였다. 획득한 읽기 커버리지 길이 그래프는 1Kb 간격으로 인위적으로 조정되었다. 그 결과 118,012개의 매핑된 읽기, 3,027.56bp의 평균 읽기 길이, 30,629.57bp 길이의 평균 커버리지 길이를 얻었다. Consensus sequence를 추출하여 11,784bp 길이의 전체 genome sequence를 얻었으며, MEGA7을 이용한 reference와 비교하여 돌연변이도 확인하였다. After Nanopore sequencing, 15 Gb raw data (fast5 format) was calculated using OxfordNamopore's Guppy GPU basecalling software and converted to fastq files. During conversion, sequence quality was corrected by base-calling, adapter trimming, and filtering with a Q-Score of 7 or higher. After additional trimming was performed on the fastq file generated using CLC Workbench software, the entire genome sequence (11,790 bp) obtained through Illumina shot gun sequencing was used as a reference. The obtained read coverage length graph was artificially adjusted at 1Kb intervals. As a result, 118,012 mapped reads, an average read length of 3,027.56 bp, and an average coverage length of 30,629.57 bp were obtained. The consensus sequence was extracted to obtain the entire genome sequence with a length of 11,784 bp, and mutations were also confirmed by comparing with the reference using MEGA7.
실험예 3. 고찰Experimental Example 3. Consideration
치쿤군야 바이러스는 2004년 이전에 3개의 큰 계보를 가지고 있다. 서아프리카(WA), 동부 중앙 남부 아프리카(ECSA) 및 아시아(아시아 도시라고도 함, AUL). 인도양 섬에서 발견된 바이러스는 ECSA47의 하위 그룹으로 인도양 섬(IOL) 하위 계통으로 분류되었다. 2013-2014년 카리브해와 남미의 치쿤군야 바이러스는 아시아 계통으로 분류되었으며 ECSA 계통의 새로운 미국 하위 그룹이 2016년에 제시되었다48. 오늘날 3개 또는 4개의 계통이 CHIKV의 주요 계통으로 사용되었다; WA, ECSA, 아시아 및 IOL46.Chikungunya virus had three major lineages prior to 2004. West Africa (WA), East Central South Africa (ECSA) and Asia (also known as Asian Cities, AUL). Viruses found in Indian Ocean Islands are a subgroup of ECSA47 and have been classified as Indian Ocean Islands (IOL) subfamily. In 2013–2014, Chikungunya viruses from the Caribbean and South America were classified as an Asian lineage, and a new American subgroup of the ECSA lineage was presented in 2016 48 . Today, three or four strains are used as the main strains of CHIKV; WA, ECSA, Asia and IOL 46 .
<참고문헌><References>
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<110> KRISS <120> Chikungunya virus universal primer sets for whole genome amplification method and diagnosis kit <130> M21-0000 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CHIK9F <400> 1 gtgagacaca cgtagcctac 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CHIK3018R <400> 2 cacctcccac tccttaatag 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CHIK2900F_1 <400> 3 tcagagcacg tcaacgtact 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CHIK6053F_1 <400> 4 acagttggrt agtttctagc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CHIK5813F_1 <400> 5 aagaaactcc aggagartgc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CHIK8906F <400> 6 tgggtgcgta catgartgac 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CHIK8725F <400> 7 caygattgga ccaagctgcg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CHIK11775F <400> 8 tccctgtggg ttcggagaat 20 <110> KRISS <120> Chikungunya virus universal primer sets for whole genome amplification method and diagnosis kit <130> M21-0000 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> artificial sequence <220> <223> CHIK9F <400> 1 gtgagacaca cgtagcctac 20 <210> 2 <211> 20 <212> DNA <213> artificial sequence <220> <223> CHIK3018R <400> 2 cacctcccac tccttaatag 20 <210> 3 <211> 20 <212> DNA <213> artificial sequence <220> <223> CHIK2900F_1 <400> 3 tcagagcacg tcaacgtact 20 <210> 4 <211> 20 <212> DNA <213> artificial sequence <220> <223> CHIK6053F_1 <400> 4 acagttggrt agtttctagc 20 <210> 5 <211> 20 <212> DNA <213> artificial sequence <220> <223> CHIK5813F_1 <400> 5 aagaaactcc aggagartgc 20 <210> 6 <211> 20 <212> DNA <213> artificial sequence <220> <223> CHIK8906F <400> 6 tgggtgcgta catgartgac 20 <210> 7 <211> 20 <212> DNA <213> artificial sequence <220> <223> CHIK8725F <400> 7 caygattgga ccaagctgcg 20 <210> 8 <211> 20 <212> DNA <213> artificial sequence <220> <223> CHIK11775F <400> 8 tccctgtggg ttcggagaat 20
Claims (10)
(a) 치쿤군야 바이러스 전장 유전체에 상보적인 하나 이상의 정방향 및 역방향 프라이머 세트를 제조하는 단계;
(b) 시료에서 RNA를 분리하는 단계;
(c) 분리된 RNA를 사용하여 역전사 반응을 수행하는 단계;
(d) 역전사 반응이 수행된 시료에 프라이머 세트를 첨가하여 중합효소 연쇄반응을 실시하는 단계; 및
(e) 증폭된 산물의 서열을 분석하는 단계
상기 프라이머 세트는 서열번호 1 및 서열번호 2; 서열번호 3 및 서열번호 4; 서열번호 5 및 서열번호 6; 및 서열번호 7 및 서열번호 8의 프라이머 세트를 포함하는 것임. Method for confirming the whole genome sequence of chikungunya virus in an isolated sample comprising the following steps
(a) preparing one or more sets of forward and reverse primers complementary to the full-length chikungunya virus genome;
(b) isolating RNA from the sample;
(c) performing a reverse transcription reaction using the isolated RNA;
(d) performing a polymerase chain reaction by adding a primer set to the sample in which the reverse transcription reaction was performed; and
(e) analyzing the sequence of the amplified product
The primer set is SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; and a primer set of SEQ ID NO: 7 and SEQ ID NO: 8.
상기 프라이머 세트는 서열번호 1 및 서열번호 2; 서열번호 3 및 서열번호 4; 서열번호 5 및 서열번호 6; 및 서열번호 7 및 서열번호 8의 프라이머 세트를 포함하는 치쿤군야 바이러스 진단 키트. A chikungunya virus diagnostic kit comprising one or more forward and reverse primer sets complementary to the chikungunya virus full-length genome,
The primer set is SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; and a chikungunya virus diagnostic kit comprising primer sets of SEQ ID NO: 7 and SEQ ID NO: 8.
상기 프라이머 세트는 서열번호 1 및 서열번호 2; 서열번호 3 및 서열번호 4; 서열번호 5 및 서열번호 6; 및 서열번호 7 및 서열번호 8의 프라이머 세트를 포함하는 것인 치쿤군야 바이러스 유발 질환 진단용 조성물 A composition for diagnosing an alpha corona virus-induced disease comprising at least one set of forward and reverse primers complementary to the full-length chikungunya virus genome,
The primer set is SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; And a composition for diagnosing diseases caused by Chikungunya virus comprising primer sets of SEQ ID NO: 7 and SEQ ID NO: 8
The composition of claim 7, wherein the chikungunya virus comprises KNIH/2009/77
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| PCT/KR2023/000830 WO2023140596A1 (en) | 2022-01-18 | 2023-01-18 | Chikungunya virus universal primer set for whole genome amplification and diagnostic kit using same |
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| KR20110118176A (en) | 2009-02-25 | 2011-10-28 | 빅텍 프라이빗 리미티드 | Probes and primers for detection of chikungunya |
| KR102030245B1 (en) | 2017-10-12 | 2019-10-08 | 고려대학교 산학협력단 | Oligonucleotide set for detection of chikungunya virus and uses thereof |
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| KR102201869B1 (en) * | 2020-01-09 | 2021-01-12 | 서울대학교 산학협력단 | Oligonucleotide and plasmid for simultaneous detection of four types of dengue virus, and the analysis method of dengue virus serotype using them |
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| KR20110118176A (en) | 2009-02-25 | 2011-10-28 | 빅텍 프라이빗 리미티드 | Probes and primers for detection of chikungunya |
| KR102030245B1 (en) | 2017-10-12 | 2019-10-08 | 고려대학교 산학협력단 | Oligonucleotide set for detection of chikungunya virus and uses thereof |
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