KR20050015063A - Screening system of reassortant influenza viruses using primer dependent multiplex RT-PCR - Google Patents
Screening system of reassortant influenza viruses using primer dependent multiplex RT-PCRInfo
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Abstract
본 발명은 재조합 독감 바이러스(influenza virus)를 제조하는 과정 중 프라이머(primer)와 주형간의 결합특이성에 따른 멀티플렉스 역전사 중합효소 연쇄반응(multiplex RT-PCR)의 차이를 이용하여 재조합 독감 바이러스(influenza virus)의 유전자를 스크리닝하는 방법에 관한 발명이다. 보다 상세하게는, 약독화 독감 바이러스주의 유전자와 독성 바이러스주의 대응하는 유전자의 염기서열 중 상호간의 변이가 많은 부분에 해당하는 프라이머를 제작하고 하나의 반응에서 2 내지 3개의 중합효소 연쇄반응이 동시에 진행되도록 함으로써 약독화 독감 바이러스주의 6개의 유전자와 독성바이러스의 2개의 유전자를 갖는 6:2 재조합 바이러스를 효과적으로 스크리닝하는 방법에 관한 발명이다. The present invention utilizes the difference of multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) according to the binding specificity between the primer and the template during the production of influenza virus (influenza virus). The invention relates to a method of screening for genes. More specifically, a primer corresponding to a large portion of the mutated sequence among the gene sequences of the attenuated flu virus strain and the corresponding gene of the toxic virus strain is prepared, and two to three polymerase chain reactions are simultaneously performed in one reaction. The invention relates to a method for effectively screening a 6: 2 recombinant virus having six genes of attenuated flu virus strain and two genes of toxic virus.
Description
독감 바이러스의 감염에 의해 유발되는 독감은 상,하부 호흡기 증상과 함께 발열, 두통, 근육통과 무력감 등이 동반되는 급성호흡기 질환이다. 독감은 보통 겨울철에 발병하는데 유행되는 아형(subtype)에 따라 다양한 증상과 중증도를 나타내며, 전염력이 강하고 폐렴 및 심폐질환의 합병증을 일으켜 높은 치사율을 보일 수 있으므로, 노인이나 소아 뿐만 아니라 성인의 경우에도 독감백신의 접종이 적극 권장되고 있다.Influenza caused by the influenza virus is an acute respiratory disease accompanied by fever, headache, muscle pain and weakness along with upper and lower respiratory symptoms. Influenza has a variety of symptoms and severity depending on the subtypes that are commonly prevalent in winter, and can be highly contagious and cause high mortality due to complications of pneumonia and cardiopulmonary disease. Vaccination is highly recommended.
독감 바이러스(influenza virus)는 올소믹소비리데 (Orthomyxoviridae)에 속하며 PB2, PB1, PA, HA, NP, NA, M 및 NS의 여덟 개의 네가티브 센스 RNA 단편을 갖고 있는 바이러스이다. 이 중 외피 단백질을 이루고 있는 두개의 단백질 헤마글루티닌 (hemagglutinin: 이하 "HA"라 약칭함)과 뉴라미니다아제 (neuraminidase: 이하 "NA"라 약칭함)는 면역항체를 유도하는 중요한 면역원이며, 이들은 항원의 대/소변이(antigenic shift and drift) 과정을 통해 변형되는 특징을 갖고 있다. 이런 독감 바이러스의 변화는 동일 아종내의 다른 독감 바이러스에 대하여 형성된 면역도 회피할 수 있게 할 수 있으며, 일반적으로 독감 바이러스에 의해 유도된 면역은 단기간에 소실되기 때문에 매 시즌에 유행이 예측되는 바이러스에 대하여 새로이 면역을 유도해야 한다.Influenza virus belongs to Orthomyxoviridae and is a virus with eight negative sense RNA fragments of PB2, PB1, PA, HA, NP, NA, M and NS. The two proteins hemagglutinin (abbreviated as "HA") and neuraminidase (referred to as "NA") which make up the envelope protein are important immunogens that induce immune antibodies. They are characterized by modification through antigenic / antigenic shift and drift. Such flu virus changes can also avoid immunity against other influenza viruses in the same subspecies. In general, immunity induced by influenza virus is lost in a short period of time, and for viruses that are expected to be epidemic every season. Induce new immunity
독감백신의 경우, 세계보건기구(WHO)에서 유행을 예측하는 독감 바이러스주(A 형 두 종류와 B 형 한 종류)에 대해 제조된 불활화 백신이 일반적으로 사용되고 있다. 불활화 백신으로는 불활화한 독감 바이러스 전체를 사용하는 인플루엔자 백신과 독감 바이러스 입자를 항원성이 유지되도록 분쇄 및 불활화하여 제조한 인플루엔자 분할백신, 그리고 독감 바이러스의 헤마글루티닌만을 분리하여 제조한 인플루엔자 에이취 에이 백신의 세 종류가 많이 사용되고 있다. 이들 불활화 백신에 소요되는 바이러스는 수정란에서 배양되는 것이 일반적이며 최근에는 세포배양을 통해서 생산하는 연구도 활발히 진행되고 있다.Influenza vaccines are commonly used inactivated vaccines prepared by the World Health Organization (WHO) for influenza virus strains (two types A and one type B). Inactivated vaccines were prepared by separating only influenza vaccines using the entire inactivated influenza virus, influenza split vaccine prepared by crushing and inactivating influenza virus particles to maintain antigenicity, and hemagglutinin of the flu virus. Three types of influenza HIV vaccine are commonly used. Viruses required for these inactivating vaccines are generally cultured in fertilized eggs, and recently, studies are being actively conducted to produce them through cell culture.
그러나, 세계보건기구(WHO)에서 유행을 예측하는 독감 바이러스주의 경우 수정란에서의 증식력이 낮아 생산성이 높지 않은 경우가 있으므로, 생산성 향상을 위해 수정란에서 고생산성을 보이는 바이러스주와 재조합하여 바이러스의 표면 항원인 HA와 NA의 유전자 단편은 예측된 바이러스의 것을 포함하며 나머지 여섯 개의 내부 유전자 단편은 고생산성 바이러스주의 것을 포함하는 6:2 재조합 바이러스를 제조하여 수정란에서의 생산성을 높이는 방법이 오래 전부터 사용되어 왔다 (Influenza, Plenum Medical Book Company, 291, 1987). However, influenza virus strains predicting epidemics by the World Health Organization (WHO) may not have high productivity due to low proliferative capacity in fertilized eggs. Therefore, the surface antigen of the virus may be recombined with high yielding virus strains in fertilized eggs to improve productivity. Gene fragments of phosphorus HA and NA contain those of the predicted virus, and the remaining six internal gene fragments have long been used to increase productivity in fertilized eggs by producing a 6: 2 recombinant virus containing a highly productive viral strain. (Influenza, Plenum Medical Book Company, 291, 1987).
불활화 백신은 젊은 성인의 경우 70 % 이상의 예방효과를 보이며, 호흡기질환에 의한 입원, 2차 감염인 폐렴 등에 의한 입원율을 감소시키는 효과가 있지만, 면역 지속기간이 짧으며 근육주사로 접종하고 바이러스의 초기 감염을 방어할 수 있는 점막분비항체(sIgA)를 유도할 수 없는 단점을 가지고 있다. 또한 감염된 세포를 사멸시킬 수 있는 CTL 면역이 잘 유도되지 못하는 한계를 가지고 있다. Inactivated vaccines have a protective effect of more than 70% in young adults and reduce the rate of hospitalization due to respiratory disease and secondary infections such as pneumonia.However, the duration of immunization is short and the vaccine is given by intramuscular injection. Mucosal secreted antibodies (sIgA) that can protect against early infection has the disadvantage that can not be induced. In addition, CTL immunity, which can kill infected cells, is limited.
불활화 백신이 가지는 이런 단점들을 보완하면서 면역력을 높이며 코점막을 통한 투여를 가능케하는 독감 생백신 개발이 여러 연구진에 의해서 시도되어 왔으며, 1965년 구소련에서의 저온적응된 약독화 바이러스주를 사용한 생백신 바이러스주의 개발을 시작으로 현재까지 많은 연구가 진행되고 있다 (Rev Roum Inframicrobiol 2, 179-89, 1965). 구소련의 경우, A/Leningrad/134/47/57(H2N2) 바이러스를 생백신의 공여 바이러스주로 하여 재조합 바이러스를 제조하여 개발하였으며, 미국에서는 A/Ann Arbor/6/60 (H2N2)를 공여바이러스주로 하여 제조한 재조합 바이러스주를 사용한 생백신이 임상실험을 끝내고 미국 FDA에서 49세 이하와 5세 이상에서 예방접종이 허가된 상태이다. To overcome these shortcomings of inactivated vaccines, several researchers have attempted to develop live vaccines that boost immunity and allow administration through the nasal mucosa, and in 1965, live vaccines using cold-adapted attenuated virus strains from the former Soviet Union. Since the beginning of development, much research has been carried out (Rev Roum Inframicrobiol 2, 179-89, 1965). In the former Soviet Union, A / Leningrad / 134/47/57 (H2N2) virus was used as a live vaccine donor virus strain and a recombinant virus was developed. In the United States, A / Ann Arbor / 6/60 (H2N2) as donor virus strain was developed. Live vaccines using recombinant viral lines prepared have been approved by the US FDA for immunizations under 49 and 5 years of age.
불활화 백신과 생백신의 경우, 백신의 생산성 또는 안정성의 확보를 위해 수정란에서 생산성이 높은 균주 또는 약독화된 바이러스주와의 재조합 바이러스 제조 과정이 필요하다. 재조합 독감 바이러스의 제조과정 중 독감 바이러스의 8개의 분절된 RNA 단편들의 조합은 무작위적으로 일어나기 때문에 생성될 수 있는 재조합 바이러스의 종류는 최대 28가지, 즉 256가지이다. 이들 바이러스 중 공여 바이러스의 약독화 특성을 갖고 있으면서 독성 바이러스의 외피 단백질인 HA와 NA를 가진 6:2 재조합 바이러스를 신속하게 효율적으로 선별하는 방법의 확립이 매우 중요하다.In the case of inactivated vaccines and live vaccines, it is necessary to prepare recombinant viruses with highly productive strains or attenuated virus lines in fertilized eggs to ensure the productivity or stability of the vaccine. Since the combination of eight fragmented RNA fragments of the flu virus occurs randomly during the production of the recombinant flu virus, there are a maximum of 2 8 or 256 kinds of recombinant viruses that can be generated. It is very important to establish a method for rapidly and efficiently selecting a 6: 2 recombinant virus having HA and NA, which are envelope proteins of a toxic virus, having attenuated characteristics of a donor virus among these viruses.
6:2 재조합 바이러스를 제조하는 방법은 우선 독성 바이러스와 공여 바이러스를 일정 비율로 혼합한 후 11일간 발육한 수정란에 접종하고 저온에서 배양하여 무작위적인 조합으로 이루어진 재조합 바이러스를 얻는다. 이들 바이러스들 중 공여바이러스의 특성인 저온 적응성을 가진 바이러스와 독성 바이러스의 외피단백질을 갖는 바이러스만을 선별하기 위해서 저온 배양과 공여바이러스 특이 항체 존재하에 수정란에서의 계대배양을 수행한다. 공여바이러스 특이 항체 존재 하에서의 플라크 분리 (plaque isolation)을 통해 단일 바이러스 클론을 얻은 후 다시 수정란에서의 배양으로 바이러스를 증폭시킨다. 증폭된 바이러스의 HA와 NA는 항체를 이용한 헤마그루티닌 억제 분석법(hemagglutinin inhibition assay; 이하 "HI"라 약칭함)와 뉴라미니다제(neuraminidase inhibition assay; 이하 "NI"라 약칭함)를 통하여 빠른 분석이 가능하지만, 나머지 내부 유전자 6개의 경우에는 유전자 차원에서의 분석이 수행되어져야만 한다. In the method of producing a 6: 2 recombinant virus, first, a toxic virus and a donor virus are mixed at a predetermined ratio, and then inoculated into fertilized eggs developed for 11 days and cultured at low temperature to obtain a recombinant virus consisting of random combinations. Among these viruses, in order to select only viruses having low temperature adaptability, which are characteristic of donor viruses and envelope proteins of toxic viruses, passaging in fertilized eggs is carried out in the presence of cold culture and donor virus specific antibodies. Single virus clones are obtained through plaque isolation in the presence of donor virus specific antibodies and then amplified by culture in fertilized eggs. HA and NA of the amplified virus were obtained through hemagglutinin inhibition assay (abbreviated as "HI") and neuraminidase inhibition assay (abbreviated as "NI") using antibodies. Rapid analysis is possible, but for the remaining six internal genes, gene-level analysis must be performed.
재조합 독감 바이러스의 유전자를 RNA상에서 분석하는 방법으로는 PAGE (Polyacrylamide gel electrophoreses) 젤 상에서 RNA 서열의 차이에 의한 단일 가닥 RNA의 이동성의 차이를 비교하는 방법 (J. Virol. 29,1142-1148,1979)과 노던 블랏팅 (Northern bloting)과 크롯 분석 (Crot analysis: RNA-DNA hybridization) 방법이 사용되어져 왔다 (J. Gen. Virol. 64, 2611-2620, 1983: Vaccine 3, 267-273, 1985). 그러나, 이들 방법들은 분해되기 쉬운 RNA를 분석에 직접 사용하여야 하며 PAGE 젤을 사용하여 장시간 전개(running)해야 하는 번거로움이 존재하며, 젤 상에서의 유전자 이동의 차이로 유전자 염기 서열의 차이를 분석하기 때문에 불확실한 결과가 나오는 경우도 흔히 발견되었다.Recombinant flu virus genes can be analyzed on RNA using PAGE (Polyacrylamide gel electrophoreses) to compare the differences in mobility of single-stranded RNA due to differences in RNA sequence (J. Virol. 29,1142-1148,1979). ) And Northern bloting and Crot analysis: RNA-DNA hybridization methods have been used (J. Gen. Virol. 64, 2611-2620, 1983: Vaccine 3, 267-273, 1985) . However, these methods require the use of vulnerable RNA directly for analysis and the hassle of running for a long time using a PAGE gel, and the differences in gene sequence on the gel can be used to analyze differences in gene sequences. As a result, uncertain results are often found.
이런 단점들을 보완하기 위해 역전사 중합효소 연쇄반응 (Reverse Transcriptation-Polymerase Chain Reaction; 이하 RT-PCR이라 약칭함)과 제한효소의 특이성을 조합하여 유전자를 분석하는 RFLP (Restriction Fragment Length Polymorphism) 등의 방법이 도입되기 시작하였다 (J. Virol. Methods 52, 41-49, 1995). 이들 RFLP 방법은 공여바이러스와 독성 바이러스의 게놈이 제한효소에 의해 절단되어 생기는 양상을 비교하는 방법으로 현재 가장 많이 쓰이고 있으나, 독감 바이러스의 여덟 개 RNA 게놈을 각각 분리하여 RT-PCR로 증폭한 후 절단효소로 반응을 수행해야 하며, 분석에 적당한 절단효소를 찾기 위해서 독성 바이러스의 염기서열을 먼저 분석해야 하고, 공여 바이러스와 독성 바이러스를 구분할 수 있는 적당한 절단효소를 찾는데 많은 시간이 소요되며, 절단효소가 인식하는 염기서열 부분에 변이가 생길 경우 결과가 불확실해지는 등의 단점이 있다. To overcome these shortcomings, methods such as Reverse Transcriptation-Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR) and Restriction Fragment Length Polymorphism (RFLP), which analyze genes by combining the specificity of restriction enzymes, It began to be introduced (J. Virol. Methods 52, 41-49, 1995). These RFLP methods are the most widely used method to compare the genome of donor virus and virulence virus by restriction enzymes. However, eight RNA genomes of influenza virus are isolated and amplified by RT-PCR. The reaction must be carried out with an enzyme, and in order to find a suitable cleavage enzyme for analysis, the nucleotide sequence of the toxic virus must be analyzed first, and it takes a long time to find a suitable cleavage enzyme that can distinguish a donor virus from a toxic virus. If a mutation occurs in the part of the nucleotide sequence to be recognized there is a disadvantage that the result is uncertain.
또한, RT-PCR 방법은 상기 목적 외에 독감 바이러스의 검출, 서브유형(subtype)의 구별 등에 사용되어 왔으나, 재조합 바이러스의 스크리닝에 이를 도입하는 것은 공여 바이러스와 독성 바이러스의 효율적인 구별과 신속한 반응에 한계를 보여 왔다. In addition, the RT-PCR method has been used for the detection of influenza virus, subtype distinction, etc. in addition to the above purpose, the introduction of it in the screening of recombinant virus has a limitation in the efficient identification and rapid response of donor virus and toxic virus Has been shown.
이에, 본 발명자들은 다수의 공여바이러스 특이 프라이머의 설계와 PCR 방법의 개선을 통해 다양한 조합으로 구성된 바이러스 중 생백신 바이러스로 사용될 수 있는 재조합 바이러스만을 빠른 시간에 분석하는 방법을 개발하게 되었으며, 2개 내지 4개의 유전자를 동일 반응에서 증폭시켜 실제 재조합 바이러스의 스크리닝에 응용하여 확인함으로써 본 발명을 완성하였다. Accordingly, the present inventors have developed a method for analyzing only a recombinant virus that can be used as a live vaccine virus among a variety of combinations by designing a plurality of donor virus specific primers and improving PCR methods in a short time. The present invention was completed by amplifying dog genes in the same reaction and applying them to screening of actual recombinant viruses.
첫째, 본 발명은 저온적응 독감 바이러스 HTCA-A101 바이러스(수탁번호 : KCTC 0400 BP)와 A형 독성 바이러스와의 재조합 바이러스의 신속한 스크리닝에 필요한 올리고뉴클레오타이드 프라이머를 제공한다.First, the present invention provides oligonucleotide primers necessary for the rapid screening of recombinant viruses of a cold-acting influenza virus HTCA-A101 virus (Accession Number: KCTC 0400 BP) and a type A toxic virus.
둘째, 본 발명은 상기 올리고뉴클레오타이드 프라이머 세트를 이용한 멀티플렉스 역전사 PCR 방법을 사용하여 재조합 바이러스를 스크리닝하는 방법을 제공한다.Second, the present invention provides a method for screening recombinant viruses using a multiplex reverse transcription PCR method using the oligonucleotide primer set.
공여 바이러스의 약독화 특성과 독성 바이러스의 외피 단백질을 갖는 재조합 바이러스를 제조하기 위해서는, 얻어진 재조합 바이러스들의 유전자를 분석하여 그 유래를 밝혀 공여 바이러스의 내부 유전자들과 독성 바이러스의 외피 단백질 유전자들을 포함하는 바이러스주를 선별하는 것이 가장 중요하다.In order to prepare a recombinant virus having the attenuated characteristics of the donor virus and the envelope protein of the toxic virus, the genes of the obtained recombinant viruses are analyzed and found to be derived, and the virus includes the internal genes of the donor virus and the envelope protein genes of the toxic virus. The most important thing is to select states.
그러나, 공여 바이러스와 독성 바이러스를 구별하는 프라이머의 설계에 많은 시간이 소요되며, 다양한 조합으로 만들어진 다수의 재조합 바이러스에서 RNA들을 분리하여 분석하는 작업은 많은 노력과 시간을 요구한다. 이런 작업은 북반구의 경우 매년 2월에 WHO에서 권장되는 바이러스를 입수하여 단기간(약 1~2 개월)에 생백신 바이러스주를 제조하여 생산에 도입해야 하는 과정을 고려할 경우 가장 집중적으로 연구되어져야 하며 단기간에 수행될 수 있는 방법이 개발되어져야 하는 시점이다.However, designing a primer that distinguishes a donor virus from a toxic virus takes a lot of time, and the task of separating and analyzing RNAs from a plurality of recombinant viruses made of various combinations requires a lot of effort and time. This work should be studied most intensively, considering that the Northern Hemisphere receives the virus recommended by the WHO every February and manufactures live virus vaccines in the short term (about 1 to 2 months). It is time to develop a method that can be performed.
이에, 본 발명자들은 현재까지 축적된 독감 바이러스 데이터 베이스를 분석하여 약독화된 공여 바이러스에 특이적인 프라이머를 다수 제작하였고, 매트릭스 형태로 공여 바이러스와 독성 바이러스에 대한 RT-PCR을 수행하여 두 바이러스를 구별할 수 있는 프라이머들을 선별할 수 있었으며, 이들 프라이머를 사용한 멀티플렉스 PCR (multiplex PCR) 방법을 도입하여 3 또는 4개 반응만으로 하나의 바이러스를 신속하게 판별하는 방법을 고안할 수 있었다.Accordingly, the present inventors analyzed a flu virus database accumulated to date to produce a plurality of primers specific to the attenuated donor virus, and distinguishes the two viruses by performing RT-PCR on the donor virus and the toxic virus in a matrix form. Primers could be selected, and multiplex PCR (multiplex PCR) methods using these primers could be introduced to devise a method for rapidly identifying a virus with only three or four reactions.
따라서, 한 가지 양태에서, 본 발명의 목적은 공여 바이러스와 독감 바이러스 A형의 6:2 재조합 바이러스를 찾는 스크리닝 과정 중, 공여 바이러스인 HTCA-A101에 특이적으로 반응하는 프라이머 세트 및 그들을 사용한 PCR 방법을 제공한다.Accordingly, in one embodiment, an object of the present invention is to screen a primer set that specifically reacts with the donor virus HTCA-A101 during the screening process for a 6: 2 recombinant virus of donor virus and influenza virus type A and a PCR method using them. To provide.
추가의 양태에서, 본 발명의 추가의 목적은 상기 PCR 방법을 동시에 다중 방식으로 수행하는 보다 간단하고 경제적인 멀티플렉스 PCR 방법을 제공하는 것이다.In a further aspect, a further object of the present invention is to provide a simpler and more economical multiplex PCR method which simultaneously performs the above PCR method in multiple manner.
또한, 본 발명에 따른 멀티플렉스 PCR 방법을 사용함으로써 6:2 재조합 바이러스를 신속하게 스크리닝할 수 있다.In addition, by using the multiplex PCR method according to the present invention, it is possible to quickly screen for 6: 2 recombinant virus.
보다 구체적으로, 본 발명은 독감 바이러스의 8개 유전자 RNA 게놈 절편으로부터 약독화 공여 바이러스주 게놈에 대해서는 PCR 산물을 생성하지만 독성 바이러스 게놈에 대해서는 PCR 산물을 생성하지 않는 PCR 프라이머 세트를 디자인하는 단계; 독성 바이러스와 약독화 공여 바이러스를 수정란에 감염시켜 두 종의 바이러스의 무작위 재조합을 통한 재조합 바이러스를 생산하는 단계; 재조합 바이러스의 RNA 게놈의 역전사를 통해 cDNA를 수득하는 단계; 상기 재조합 바이러스의 cDNA와 함께, 각각의 PCR 산물들이 구별될 수 있는 크기를 고려하여 선택된 8개 유전자 RNA 절편별로 한 가지 프라이머 세트를 사용하여 8개 유전자에 대한 PCR 반응을 수행하는 단계; 약독화 공여 바이러스에 특이적인 프라이머 세트에 의해 6개 내부 유전자(internal genes, PB2, PB1, PA, NP, M, NS) RNA 절편은 증폭되지만 외피 단백질(HA 및 NA)을 암호화하는 2개 RNA 절편은 증폭되지 않는 재조합 바이러스를 선택하는 단계를 포함함을 특징으로 하여, 독감 바이러스 생백신 생산을 위한 공여 바이러스와 독성 바이러스간의 6:2 재조합 바이러스를 스크리닝하기 위한 PCR 방법을 제공한다. More specifically, the present invention comprises the steps of designing a set of PCR primers that generate a PCR product for an attenuated donor virus line genome from an 8 gene RNA genome segment of influenza virus but no PCR product for a toxic viral genome; Infecting toxic and attenuated donor viruses with fertilized eggs to produce recombinant virus through random recombination of two viruses; Obtaining cDNA through reverse transcription of the RNA genome of the recombinant virus; Performing a PCR reaction on 8 genes using one primer set for each of 8 gene RNA fragments selected in consideration of the size of each PCR product, together with the cDNA of the recombinant virus; Six internal genes (PB2, PB1, PA, NP, M, NS) RNA fragments are amplified by primer sets specific for the attenuated donor virus, but two RNA fragments encoding envelope proteins (HA and NA). The method includes selecting a recombinant virus that is not amplified, thereby providing a PCR method for screening a 6: 2 recombinant virus between a donor virus and a toxic virus for production of live influenza virus.
보다 바람직하게는, 본 발명은 약독화 공여 바이러스로서 HTCA-A101 균주 (KCTC 0400 BP)를 사용하는 PCR 방법을 제공한다.More preferably, the present invention provides a PCR method using HTCA-A101 strain (KCTC 0400 BP) as an attenuated donor virus.
또한, 본 발명에서 PCR을 사용하여 스크리닝하고자 하는 독성 바이러스로서 A형 독감 바이러스인 A/Moscow/10/99, A/New/Calenonia/20/99, A/Shangdong/9/93 및 A/Singapore/6/86 을 예시적으로 사용하지만, 이들 바이러스에 국한되지는 않는다. In addition, in the present invention, as a virulent virus to be screened using PCR, type A influenza viruses A / Moscow / 10/99, A / New / Calenonia / 20/99, A / Shangdong / 9/93 and A / Singapore / 6/86 is used as an example, but is not limited to these viruses.
본 발명은 또한, 상기한 PCR 방법에서 8개 유전자에 대한 PCR 반응이 3 내지 4개의 튜브에서 2 내지 3개의 반응이 동시에 수행됨을 특징으로 하는 멀티플렉스 PCR 방법을 제공한다. 이러한 다중 PCR 방법은 보다 빠르고 보다 저렴하게 원하는 재조합 바이러스에 대한 스크리닝을 가능하게 할 것이다.The present invention also provides a multiplex PCR method, characterized in that the PCR reaction for 8 genes in the above-described PCR method is performed at the same time 2 to 3 reactions in 3 to 4 tubes. This multiplex PCR method will allow for faster and cheaper screening for the desired recombinant virus.
보다 구체적으로, 본 발명은 제1 튜브에는 PB2, PB1 및 NP에 대한 프라이머 세트, 제2 튜브에는 M, NS 및 PA에 대한 프라이머 세트 및 제3 튜브에는 HA 및 NA에 대한 프라이머 세트를 넣어 재조합 바이러스에 대해서 제1 및 제2 튜브에서만 PCR 생성물이 증폭됨을 특징으로 하는 멀티플렉스 PCR 방법을 제공한다. 이때, HA 및 NA는 주된 면역원성 단백질이기 때문에 불활화 백신 또는 생백신으로서 재조합 바이러스를 사용하는 경우 이들은 독성 바이러스 유래의 것이어야 한다.More specifically, the present invention is a recombinant virus by putting a primer set for PB2, PB1 and NP in the first tube, a primer set for M, NS and PA in the second tube and a primer set for HA and NA in the third tube. For a multiplex PCR method, characterized in that the PCR product is amplified only in the first and second tubes. At this time, since HA and NA are the main immunogenic proteins, when using recombinant viruses as inactivating vaccines or live vaccines, they should be from toxic viruses.
따라서, 바람직하게는, 본 발명은 약독화 공여 바이러스와 독성 바이러스 간의 6:2 재조합 바이러스를 스크리닝하는 멀티플렉스 PCR 방법을 제공한다.Thus, preferably, the present invention provides a multiplex PCR method for screening a 6: 2 recombinant virus between an attenuated donor virus and a toxic virus.
본 발명에 따른 PCR 및 RT-PCR 반응은 통상적인 공지된 PCR 방법에 의해 수행될 수 있으며, 바람직하게는 94℃에서 5분간 1회 변성시킨 후, 94℃에서 30초간 변성, 55℃에서 1분간 어닐링 및 72℃에서 1분 20초간 연장시키는 반응을 30회 수행함으로써 수행될 수 있으나, 이러한 PCR 조건으로 제한하고자 하는 것은 아니다.PCR and RT-PCR reaction according to the present invention can be carried out by a conventional known PCR method, preferably denatured once at 94 ℃ for 5 minutes, modified at 94 ℃ for 30 seconds, 1 minute at 55 ℃ It can be carried out by performing annealing and the reaction 30 times extending for 1 minute and 20 seconds at 72 ° C., but are not intended to be limited to these PCR conditions.
본 발명은 또한, 표 1에서 제시된 바와 같은 상기 PCR 방법에서 사용하기 위한 약독화 공여 바이러스 특이적인 내부 유전자 6개 및 외피 단백질 유전자 2개를 포함하는 8개 유전자 RNA에 대한 프라이머 세트를 제공한다.The invention also provides primer sets for eight gene RNAs comprising six attenuated donor virus specific internal genes and two envelope protein genes for use in the PCR method as set forth in Table 1.
본 발명에서 언급되는 바와 같은 독감 바이러스의 비면역원성 6개 유전자는 PB2, PB1, PA, NP, M 및 NS이고 면역원성 2개 유전자는 HA 및 NA이며, 본 발명에 따른 PCR 방법에서는 약독화 공여 바이러스 HTCA-A101에 특이적으로 PCR 산물을 생성하지만 맹독성 바이러스에 대해서는 PCR 산물을 생성되지 않는 특이적인 프라이머 세트를 사용함으로써, 불활화 백신 또는 생백신으로 사용하기 위한 6:2 재조합 바이러스를 PCR 방법으로 신속하게 스크리닝하는 방법을 제공한다.The six non-immunogenic genes of the flu virus as mentioned in the present invention are PB2, PB1, PA, NP, M and NS and the two immunogenic genes are HA and NA, and in the PCR method according to the present invention attenuated donation By using a specific primer set that generates a PCR product specifically for the virus HTCA-A101 but no PCR product for a virulent virus, the 6: 2 recombinant virus for use as an inactivated vaccine or live vaccine can be rapidly It provides a method for screening.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
1. 올리고뉴클레오타이드 프라이머의 제작1. Preparation of Oligonucleotide Primers
독감 바이러스 염기서열의 데이터 베이스를 이용하여 공여 바이러스와 독성 바이러스와의 상동성 비교를 수행한 후, 상동성이 낮은 부분에 대한 프라이머를 제작하였다. 프라이머 제작 시 프라이머의 길이는 19-21 mer 정도로 하였으며, 그 중 최소한 2-3 mer는 공여 바이러스와 독성 바이러스에서 연속된 차이를 보이는 부분을 선택하여 그 부분을 프라이머의 3' 말단으로 하였으며, 어닐링 온도가 58-60℃ 정도가 되도록 하여 멀티플렉스 PCR 수행시 유리하도록 설계하였다. 또한, 중간중간에 유전자 변이가 있는 것을 우선적으로 선택하였으며 한가지 염기의 서열이 연속되는 부분이 있는 곳은 선택에서 제외하여 공여 바이러스에 특이적인 프라이머를 제작하였다. 이렇게 제작된 상동성이 낮은 부분은 스크리닝을 위한 프라이머로 사용되었다. 반대로 상동성이 높은 부분은(데이터 베이스 에 기초하였을 때 100% 상동성) 포지티브 표준 프라이머(positive control)로서 RT-PCR에 사용하였다.구체적으로, 본 발명에서는 A/X-31 바이러스를 저온적응시킨 HTCA-A101 (이하 A101이라 약칭함)를 공여 바이러스로 사용하였다. 공여바이러스와의 상동성 비교를 위한 A형 바이러스들의 염기서열은 독감 바이러스 전문 데이터 베이스 사이트인 ()에서 얻었으며, 그 중 사람을 숙주로 하는 H3N2와 H1N1 서브타입의 바이러스들의 염기서열만을 사용하여 상동성을 비교하였다. 상동성 비교는 A101과 독성 바이러스간의 1:1 비교가 아니라 데이터베이스에 있는 바이러스들과 A101간의 비교이며, 데이터베이스에 있는 염기서열 중에서 전체 염기서열이 분석되어져 있으며 사람에게 감염되는 바이러스의 서열을 전부 추출하여 비교하였다. 프라이머의 선정에 있어서 각 절편(segment)별로 상동성에 차이가 있으므로 일률적인 기준은 적용하기가 어려웠으며, PCR 수행시의 산물의 크기도 고려해서 가장 큰 차이를 보이는 부분들을 선정해서 프라이머를 제작하였다.After comparing the homology between the donor virus and the toxic virus using a database of the influenza virus sequence, primers were prepared for the low homology. When constructing the primer, the primer length was about 19-21 mer, and at least 2-3 mer were selected to show the continuous difference between the donor virus and the toxic virus, and the portion was the 3 'end of the primer. It was designed to be advantageous when performing multiplex PCR by making it be about 58-60 ℃. In addition, the gene mutations were selected first, and the primers specific to the donor virus were prepared by excluding the regions where the sequence of one base sequence was selected. The low homology portion thus prepared was used as a primer for screening. Conversely, high homology moieties (100% homology based on database) were used for RT-PCR as positive standard primers. Specifically, in the present invention, the A / X-31 virus was cold-adapted. HTCA-A101 (hereinafter abbreviated as A101) was used as the donor virus. The base sequence of type A viruses for homology comparison with donor virus was obtained from the influenza virus database site (), using only the sequences of H3N2 and H1N1 subtype viruses that host humans. The same sex was compared. The homology comparison is not a 1: 1 comparison between A101 and toxic viruses, but a comparison between viruses in the database and A101. The entire nucleotide sequence is analyzed from the nucleotide sequences in the database. Compared. Since the homology of each segment differs in the selection of the primer, it is difficult to apply a uniform standard. The primers were prepared by selecting the parts having the greatest difference in consideration of the size of the product when PCR was performed.
2. A101과 독성 바이러스의 주형에 따른 RT-PCR 경향성 확인 2. Identification of RT-PCR tendency according to template of A101 and virulent virus
본 발명에서는 우선 A101의 여덟 개의 RNA 게놈에 대하여 상기에서 제작한 프라이머 세트를 이용하여 모든 PCR 산물이 나오는 조건을 확립하였으며, 같은 조건 하에서 독성 바이러스의 PCR 산물을 비교 분석하였다.In the present invention, first, the conditions for all PCR products were established using the primer sets prepared above for eight RNA genomes of A101, and the PCR products of toxic viruses were compared and analyzed under the same conditions.
3. 멀티플렉스 RT-PCR 조건 확립3. Establish Multiplex RT-PCR Conditions
공여 바이러스주의 게놈에 대해서는 PCR 산물을 생성하지만 독성 바이러스에 대해서는 생성하지 않는 프라이머를 검색하여 찾은 다음, 그 중 유전자별로 한 세트씩을 선택하여 8개의 PCR 반응이 3개의 튜브에서 이루어지도록 고안하였다. We searched for primers that generate PCR products for the genome of donor virus lines but not for toxic viruses, and then select one set for each gene to design eight PCR reactions in three tubes.
멀티플렉스 RT-PCR 조건에 관한 선행기술들은 하나의 주형에서 여러 개의 PCR 산물을 얻기 위하여 산물과 프라이머 간의 상호작용을 줄이기 위한 조건들에 대한 것들이 대부분인데, 본 발명에서는 독감 바이러스의 8개의 주형에서 일어나는 각각의 PCR 반응이 동시에 일어나는 것을 특징으로 한다. 또한, 아가로오스 젤 상에서 PCR 산물들을 구별하기 위해서는 PCR 산물들의 크기를 고려한 조합을 설정해야만 했다. 멀티플렉스 RT-PCR을 통해 재조합된 바이러스의 유전자 유래를 파악할 수 있었으며 염기서열의 부분 분석에 의한 결과와 얻은 결과가 일치함을 확인할 수 있었다.Prior arts relating to multiplex RT-PCR conditions are mostly for conditions for reducing the interaction between the product and the primer to obtain multiple PCR products in one template. Each PCR reaction is characterized in that it occurs at the same time. In addition, to distinguish PCR products on agarose gels, one had to set a combination that considered the size of PCR products. Multiplex RT-PCR was able to determine the gene origin of the recombinant virus, and the results obtained by partial analysis of the sequencing sequences were confirmed to be in agreement.
이하, 실시예에 의하여 본 발명을 상세히 설명한다. 하기 실시예는 본 발명을 구체적으로 예시하는 것이며 본 발명의 내용이 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of examples. The following examples illustrate the invention in detail and are not intended to limit the scope of the invention.
<실시예 1> 올리고 뉴클레오타이드 프라이머의 설계 및 합성Example 1 Design and Synthesis of Oligonucleotide Primer
A형 독감 바이러스는 8개의 네가티브 센스 RNA 가닥으로 이루어져 있으며, 각각은 PB2, PB1, PA, HA, NP, NA, M, NS 단백질을 암호화하고 있다. 이들 8개의 유전자에 대해서 독감 바이러스 염기 서열 전문 사이트(http://www.flu.lanl.gov)에서 사람을 숙주로 하는 H3N2와 H1N1 서브유형 바이러스에 해당하는 염기서열과 약독화된 바이러스 HTCA-A101의 상동성 비교를 수행하였다. 다중 상동성 비교의 경우 VECTOR NTI의 얼라이먼트 프로그램(Aligment program)을 이용하였으며, 상동성이 낮은 부분을 선택하여 5' 프라이머 3개와 3' 프라이머 3개씩 각 유전자별 여섯 부분을 선택하였다. 이러한 유전자별 프라이머 조합으로 PB2, PB1, PA, NP, M, NS 각각에 대해서 9개 프라이머 세트를 디자인하였다. HA 및 NA에 대해서는 5 프라이머 2개와 3 프라이머 2개씩 각 유전자별로 네 부분이 선택되었으며, 각각 4개의 프라이머 세트를 디자인하였다. 이 프라이머 세트들을 사용하여 HTCA-A101 바이러스의 주형에 특이적으로 증폭되는 PCR 산물을 얻었다. 반면 포지티브 표준 (positive control)으로서 사용하기 위해 상동성이 높은 부분의 프라이머를 제작하였으며, PB2, PB1, PA, NP, M, NS에 대해서는 각각 1개의 포지티브 표준 프라이머를 디자인하고, HA 및 NA에 대해서는 2개의 포지티브 표준 프라이머를 디자인하였다. 각 프라이머의 염기서열 및 증폭산물의 크기는 표 1에 나타내었다.Influenza A virus consists of eight negative sense RNA strands, each encoding PB2, PB1, PA, HA, NP, NA, M, NS proteins. For these 8 genes, the nucleotide sequence corresponding to the human-host H3N2 and H1N1 subtype viruses and the attenuated virus HTCA-A101 were obtained from the flu virus sequencing site ( http://www.flu.lanl.gov ). A homology comparison of was performed. In the case of multiple homology comparisons, VECTOR NTI's alignment program was used, and six parts of each gene were selected by three 5 'primers and three 3' primers by selecting parts with low homology. Nine primer sets were designed for each of PB2, PB1, PA, NP, M, and NS using these gene-specific primer combinations. For HA and NA, four parts were selected for each gene, two 5 primers and two 3 primers, and four primer sets were designed. These primer sets were used to obtain PCR products that were specifically amplified for the template of HTCA-A101 virus. On the other hand, primers with high homology were prepared for use as positive control, and one positive standard primer was designed for PB2, PB1, PA, NP, M, and NS, and for HA and NA. Two positive standard primers were designed. The base sequence of each primer and the size of the amplification product are shown in Table 1.
<실시예 2> 독감 바이러스에서 RNA의 분리Example 2 Isolation of RNA from Flu Virus
RNA의 분리는 제조원(GibcoBRL사)의 TRIZOL LS 시약을 이용하였으며, 바이러스 배양액에서 바로 RNA를 분리하였다. 바이러스 배양액 300 ul에 TRIZOL LS 시약 900 ul를 가한 후, 200 ul의 클로로포름을 가하고 10초간 강하게 진탕한 다음 12000 rpm에서 10분간 원심분리하였다. 상등액을 취하여 새 튜브에 옮긴 다음 동일 부피의 이소프로필 알코올(isopropanol)을 가하고 상온에서 10분간 정치시키고 12000 rpm에서 5분간 원심분리하여 RNA를 침전시켰다. 상등액을 제거하고 75% 에탄올 1ml을 가하여 진탕한 후 다시 12000 rpm에서 10분간 원심분리하여 RNA 침전을 얻었다. RNase를 함유하지 않은 물로 RNA 침전물을 용해시키고 소량씩 분주하여 70℃에 보관하여 사용하였다.RNA was isolated using TRIZOL LS reagent of the manufacturer (GibcoBRL Co., Ltd.), and RNA was immediately isolated from the virus culture. After adding 900 ul of TRIZOL LS reagent to 300 ul of the virus culture, 200 ul of chloroform was added thereto, followed by vigorous shaking for 10 seconds, followed by centrifugation at 12000 rpm for 10 minutes. The supernatant was taken, transferred to a new tube, and then the same volume of isopropyl alcohol (isopropanol) was added and allowed to stand for 10 minutes at room temperature and centrifuged at 12000 rpm for 5 minutes to precipitate RNA. The supernatant was removed, shaken with 1 ml of 75% ethanol, and then centrifuged at 12000 rpm for 10 minutes to obtain RNA precipitation. The RNA precipitate was dissolved in water containing no RNase, aliquoted in small portions, and stored at 70 ° C. for use.
독감 바이러스주로는 공여 바이러스주 외에 최근 유행한 독성 바이러스주 4종 A/New Caledonia/20/99 (H1N1), A/Moscow/10/99 (H3N2), A/Singapore/6/86 (H1N1), A/Shangdong/9/93 (H3N2)을 선택하였다. Influenza virus strains include four donor virus strains, A / New Caledonia / 20/99 (H1N1), A / Moscow / 10/99 (H3N2), A / Singapore / 6/86 (H1N1), A / Shangdong / 9/93 (H3N2) was selected.
<실시예 3> 독감 바이러스 RNA에서 cDNA 제조Example 3 cDNA Preparation from Flu Virus RNA
생성된 RNA의 농도를 260nm에서의 흡광도에 근거하여 결정한 다음, 전체 2ug의 RNA를 주형으로 하여 퀴아젠(QIAGEN)사의 옴니스크립트(Omniscript) RT 키트를 사용하여 RT을 수행하였다. 반응 혼합액의 조성은 RNA 용액 12ul, 5mM dNTP 2ul, 10배 RT 완충액 2ul, 10pmol/ul 프라이머 1ul(독감 바이러스 8개 유전자의 네가티브 센스 RNA 3'에 공통적으로 보존되어 있는 염기서열 5'-AGCGAAAGCAGG-3'를 프라이머로 이용) 및 역전사 효소(Reverse transcriptase) 1ul를 혼합하였다. 혼합액을 37℃에서 60분간 반응시켜 cDNA를 합성한 후 94℃에서 5분간 반응시켜 역전사 효소를 불활화하였다. 합성된 cDNA는 8개 유전자의 모든 PCR 반응에 사용되었다. The concentration of the generated RNA was determined based on the absorbance at 260 nm, and then RT was performed using QIAGEN's Omniscript RT kit using a total of 2 ug of RNA as a template. The composition of the reaction mixture was 12ul of RNA solution, 2ul of 5mM dNTP, 2ul of 10-fold RT buffer, 1ul of 10 pmol/ul primer (nucleotide sequence 5'-AGCGAAAGCAGG-3 which is commonly conserved in negative sense RNA 3 'of 8 flu virus genes). 'As a primer) and 1 ul of reverse transcriptase were mixed. The mixed solution was reacted at 37 ° C. for 60 minutes to synthesize cDNA, and then reacted at 94 ° C. for 5 minutes to inactivate reverse transcriptase. The synthesized cDNA was used for all PCR reactions of eight genes.
<실시예 4> 공여 바이러스와 독성 바이러스의 PCR 산물 생성 확인Example 4 Confirmation of PCR Product Generation of Donor Viruses and Toxic Viruses
프라이머 특이성에 의한 독성 바이러스와 공여 바이러스의 PCR 산물 생성 여부를 알아보기 위하여, 실시예 3에서 제조한 cDNA 용액 1ul와 실시예 1에서 합성한 각 유전자별 프라이머 세트를 각각 혼합하고 94℃에서 5분간 변성시킨 다음, 변성 (94℃에서 30초), 결합 (55℃에서 1분) 및 연장 (72℃에서 1분20초)을 총 30회 반복하는 PCR을 수행하였다.In order to determine whether the PCR product of the toxic virus and the donor virus was generated by primer specificity, 1 ul of the cDNA solution prepared in Example 3 and primer sets for each gene synthesized in Example 1 were mixed and denatured at 94 ° C. for 5 minutes. PCR was then performed for a total of 30 repetitions of denaturation (30 seconds at 94 ° C.), binding (1 minute at 55 ° C.), and extension (1 minute 20 seconds at 72 ° C.).
제조된 PCR 산물을 아가로스 젤 상에서 분리한 결과, 공여 바이러스의 경우 각 유전자별 상동성이 낮은 프라이머 조합 즉 내부 유전자 6개에 대한 9가지 프라이머 세트와 외피 단백질 HA와 NA의 경우 4가지 프라이머 세트에 대한 PCR 산물이 모두 수득되었다. 이에 반해 독성 바이러스의 경우 양성 대조반응의 확인으로 cDNA가 존재함에도 불구하고 PCR 산물 검출빈도가 낮았으며, 아래 표 2와 같은 결과를 얻었다. 도 2는 이중에 PB2를 실례로 보여주고 있다. 이로써 같은 조건 하에서의 RT-PCR 수행시 프라이머의 공여 바이러스에 대한 특이적인 결합에 의해 공여 바이러스와 독성 바이러스의 PCR 결과가 현저한 차이가 있음을 확인할 수 있었다.As a result of separating the prepared PCR product on agarose gel, a primer combination with low homology for each gene, i.e., 9 primer sets for 6 internal genes and 4 primer sets for coat proteins HA and NA, were used for the donor virus. All PCR products were obtained . In contrast, in the case of toxic viruses, despite the presence of cDNA as a positive control reaction, the frequency of PCR product detection was low, and the results are shown in Table 2 below. 2 illustrates PB2 as an example. As a result, it was confirmed that the PCR results of the donor virus and the toxic virus were significantly different due to the specific binding of the primer to the donor virus when performing RT-PCR under the same conditions.
<실시예 5> 멀티플렉스 RT-PCR의 수행Example 5 Performing Multiplex RT-PCR
실시예 4를 통하여 공여 바이러스에서는 PCR 산물을 생성하고 독성 바이러스에서는 PCR 산물을 생성하지 않았던 프라이머 세트를 이용하여 멀티플렉스 RT-PCR을 수행하였다. 프라이머 세트는 PB2-2 (1002bp), NP-4 (853bp), PB1-6 (559bp)를 한 튜브에 넣었으며, PA-5 (730bp), NS-5 (521bp), M-6 (326bp)를 한 튜브에 넣었고, HA-4 (1086bp), NA-4 (504 bp)를 한 튜브에 넣고 반응을 수행하였다. 독감 바이러스의 경우 유전자가 결절되어 있음에 착안하여 프라이머의 양이 3배 내지 2배로 첨가되는 것 외에는 모노플렉스(Monoplex) PCR 조건과 동일하게 하여 멀티플렉스 RT-PCR을 수행한 결과 공여 바이러스의 경우, 8개 유전자를 세 튜브의 반응에서 재현성 있게 모두 얻을 수 있었으며, 독성 바이러스 A/New Caledonia/20/99 (H1N1)와 A/Moscow/10/99 (H3N2)의 경우에는 8개 유전자에 대한 양성 대조군의 PCR 산물은 생성되나 멀티플렉스 PCR 튜브에서 PCR이 이루어지지 않았다(도 3 참조). 이에 반해, 공여 바이러스주의 주형에서는 8개 유전자 모두에 해당하는 멀티플렉스 PCR 산물을 확인 할 수 있다(도 3).In Example 4, multiplex RT-PCR was performed using a primer set that produced a PCR product in a donor virus and a PCR product in a toxic virus. The primer set contained PB2-2 (1002bp), NP-4 (853bp), PB1-6 (559bp) in one tube, PA-5 (730bp), NS-5 (521bp), M-6 (326bp) Was put into one tube, HA-4 (1086bp), NA-4 (504 bp) was put in one tube and the reaction was carried out. In the case of the influenza virus, except for the fact that the gene is missing, the amount of the primers is added 3 to 2 times, except that the multiplex RT-PCR is performed in the same manner as the monoplex PCR conditions. All eight genes were reproducibly obtained in three tube reactions, and positive controls for eight genes for the toxic viruses A / New Caledonia / 20/99 (H1N1) and A / Moscow / 10/99 (H3N2). PCR products were produced but no PCR was done in the multiplex PCR tubes (see Figure 3). On the contrary, in the template of the donor virus strain, multiplex PCR products corresponding to all eight genes can be identified (FIG. 3).
<실시예 6> 재조합 바이러스의 유전자 분석Example 6 Genetic Analysis of Recombinant Virus
공여 바이러스와 독성 바이러스간의 재현성 있는 멀티플렉스 PCR 결과를 얻은 후, 실제 재조합 바이러스 분석을 수행하였다.After reproducible multiplex PCR results between the donor virus and the toxic virus were obtained, actual recombinant virus analysis was performed.
독성 바이러스 A/New Caledonia/20/99(H1N1)와 공여 바이러스를 동시에 수정란에 감염시켜 생장시킨 결과, 두 종의 바이러스의 RNA 단편들의 무작위적인 조합을 통해 다양한 재조합 바이러스가 만들어졌으며, 재조합 바이러스들 중 저온에서의 배양과 공여 바이러스에 대한 항체를 이용하는 선별과정을 거쳐서 독성 바이러스의 HA와 NA를 갖으며 저온적응 특성을 갖는 바이러스를 선별하였다. 독성 바이러스도 저온에서 일부 생장하는 특성을 보이므로 결국 유전자의 분석과정을 거쳐야 공여 바이러스의 내부 유전자들을 포함하는 목적하는 재조합 바이러스를 얻을 수 있는 것이다.Toxic virus A / New Caledonia / 20/99 (H1N1) and donor virus were simultaneously infected with fertilized eggs and grown to produce a variety of recombinant viruses through random combinations of RNA fragments of two viruses. After culturing at low temperature and screening using antibody against donor virus, viruses with HA and NA of toxic virus and low temperature adaptation characteristics were selected. Toxic viruses also exhibit some characteristics of growth at low temperatures, and thus must be analyzed to obtain the desired recombinant virus that includes the internal genes of the donor virus.
재조합 바이러스들의 유전자 분석은 상기 실시예 5의 방법에 따라 수행하였으며, 그 결과는 표 3 및 도 4에 나타내었다. 분석 결과, 재조합 바이러스 7번은 내부유전자 6개에 대해서는 PCR 산물이 생성되고 외피 단백질을 암호화하는 2개 유전자에서는 PCR 산물이 생성되지 않는 6:2(PB2, PB1, PA, NP, M, NS는 공여 바이러스 유래, HA, NA는 독성 바이러스 유래)의 재조합 바이러스로 예상되었으며, 재조합 바이러스 2번은 5:3(PB2, PB1, NP, M, NS는 공여 바이러스주 유래, PA, HA, NA는 독성 바이러스 유래)의 재조합 바이러스로 예상되었고, 그 외 4:4((PA, PB1, NP, M는 공여 바이러스주 유래, PB2, NS, HA, NA는 독성 바이러스 유래)와 7:1((PB2, PB1, PA, NP, M, NS, NA는 공여 바이러스주 유래, HA는 독성 바이러스 유래) 등의 여러 종류의 재조합 바이러스를 얻을 수 있었다.Genetic analysis of recombinant viruses was performed according to the method of Example 5, the results are shown in Table 3 and FIG. As a result, the recombinant virus No. 7 generated a PCR product for six internal genes and a gene of 6: 2 (PB2, PB1, PA, NP, M, NS, which does not generate a PCR product in two genes encoding the envelope protein). Virus-derived, HA, NA from toxic viruses) Recombinant virus No. 2 is 5: 3 (PB2, PB1, NP, M, NS from donor virus lines, PA, HA, NA from toxic viruses) ) Were expected to be recombinant viruses, and other 4: 4 ((PA, PB1, NP, M from donor virus lines, PB2, NS, HA, NA from toxic viruses) and 7: 1 ((PB2, PB1, PA, NP, M, NS, and NA were derived from donor virus lines, and HA was derived from toxic viruses.
또한 6:2의 조합인 재조합 바이러스 7번의 유전자를 부분 염기서열을 분석해 보거나 전체 유전자에 대해 9개의 프라이머 세트를 이용하여 PCR하여 그 결과를 독성 바이러스 및 공여 바이러스와 비교한 결과 (표 4 참조), 4 개의 튜브에서 반응하여 얻은 결과 (도 4를 참조)와 일치하는 결과를 얻었으며, 이로써 하나의 재조합 바이러스의 8개 유전자 유래 분석을 3 또는 4 튜브의 PCR을 통해 수행하는 것이 가능하였으며 빠른 시간에 많은 수의 재조합 바이러스 분석이 용이함을 알 수 있었다.In addition, the genes of recombinant virus No. 7, which is a combination of 6: 2, were analyzed by partial sequencing or PCR using 9 primer sets for all genes, and the results were compared with toxic and donor viruses (see Table 4). Results consistent with the results obtained from reactions in four tubes (see FIG. 4) resulted in eight gene-derived analyzes of one recombinant virus via PCR of three or four tubes, and in a short time. It can be seen that a large number of recombinant virus analysis is easy.
본 발명에 따라 디자인된 공여 바이러스주에 특이적인 프라이머 세트를 이용하는 멀티플렉스 역전사 중합효소 반응을 통한 바이러스 스크리닝 방법은 재조합 바이러스의 제조과정 중 다양한 조합으로 형성된 중간 단계의 바이러스들로부터 적은 노동력과 짧은 시간 동안에 정확한 조합을 가진 바이러스를 선별 가능하게 하여, 독성 바이러스를 입수하여 단기간에 재조합 바이러스를 제조함으로써 생백신 바이러스의 원활한 생산 및 공급을 가능하게 하는 효과가 있다. The virus screening method using a multiplex reverse transcriptase reaction using a primer set specific to a donor virus line designed according to the present invention can be carried out in a short time and with less labor from intermediate viruses formed by various combinations during the production of recombinant viruses. By selecting a virus having the correct combination, by obtaining a toxic virus and producing a recombinant virus in a short time there is an effect that enables the smooth production and supply of live vaccine virus.
도 1는 약독화 바이러스주 HTCA-A101의 8개 RNA 단편 중 PB2에 대하여 수행한 표 1에 기재된 PB2 프라이머 세트를 사용한 RT-PCR 결과를 나타낸다. 이때, 상단에 표시된 M은 분자량 마커를 나타내며, 레인 1번 내지 9번은 본 발명에 따른 PB2의 프라이머 세트 1 내지 9번을 의미하며, C는 포지티브 PB2 프라이머 세트를 나타낸다.1 shows the results of RT-PCR using the PB2 primer set described in Table 1 performed on PB2 among the eight RNA fragments of the attenuated virus line HTCA-A101. At this time, M indicated at the top represents a molecular weight marker, lanes 1 to 9 means primer sets 1 to 9 of PB2 according to the present invention, and C represents a positive PB2 primer set.
도 2는 네 종류의 독성 바이러스 [A/New Caledonia/20/99 (H1N1), A/Moscow/10/99 (H3N2), A/Beijing/262/95 (H1N1), A/Shangdong/9/93 (H3N2)]의 8개 RNA 단편 중 PB2에 대하여 표 1에 기재된 PB2 프라이머 세트를 사용한 수행한 RT-PCR 결과를 나타낸다. 이때, 상단의 숫자 및 문자는 도 1의 설명에서와 동일하다.Figure 2 shows four types of toxic viruses [A / New Caledonia / 20/99 (H1N1), A / Moscow / 10/99 (H3N2), A / Beijing / 262/95 (H1N1), A / Shangdong / 9/93 (H3N2)] shows the RT-PCR results performed using the PB2 primer set described in Table 1 for PB2 among the 8 RNA fragments. At this time, the numbers and letters at the top are the same as in the description of FIG.
도 3은 두 종류의 독성 바이러스 [A/New Caledonia/20/99 (H1N1), A/Moscow/10/99 (H3N2)]와 약독화 바이러스주의 cDNA를 주형으로 하여 동일 조건에서 동시에 멀티플렉스 PCR을 수행한 결과를 나타낸다. 도 3의 A는 독성 바이러스 A/New Caledonia/20/99 (H1N1)에 대한 결과를 나타내며 이때, 상단에 표시된 M은 분자량 마커를 나타내며, 약독화 바이러스주의 8개 RNA 에 대한 결과는 레인 1은 PB2, PB1, NP, 레인 2 는 M, NS, PA, 레인 3 은 HA, NA 에 대한 프라이머 세트, 보다 구체적으로는 레인 1 은 PB2-2 (1002bp), NP-4 (853bp), PB1-6 (559bp), 레인 2 는 PA-5 (730bp), NS-5 (521bp), M-6 (326bp), 레인 3 은 HA-4 (1086bp), NA-4 (504 bp)를 사용한 결과이고, 레인 4 내지 6은 레인 1 내지 3에서 사용한 프라이머와 동일한 프라이머 세트를 사용한 결과를 나타내며, 레인 7 내지 14는 8개 RNA 각각에 대한 포지티브 대조군의 결과이며 이중 레인 13 및 14는 각각 A/New Caledonia/20/99 (H1N1)의 H1 아형 및 N1 아형에 특이적인 프라이머 세트를 사용한 결과이다. 도 3의 B는 독성 바이러스 A/Moscow/10/99 (H3N2)에 대한 결과를 나타내며 이때, 상단에 표시된 M은 분자량 마커를 나타내며, 약독화 바이러스주의 8개 RNA 에 대한 결과는 레인 1은 PB2, PB1, NP, 레인 2 는 M, NS, PA, 레인 3 은 HA, NA 에 대한 프라이머 세트, 보다 구체적으로는 레인 1 은 PB2-2 (1002bp), NP-4 (853bp), PB1-6 (559bp), 레인 2 는 PA-5 (730bp), NS-5 (521bp), M-6 (326bp), 레인 3 은 HA-4 (1086bp), NA-4 (504 bp)를 사용한 결과이고, 레인 4 내지 6은 레인 1 내지 3에서 사용한 프라이머와 동일한 프라이머 세트를 사용한 결과를 나타내며, 레인 7 내지 14는 8개 RNA 각각에 대한 포지티브 대조군의 결과이며 이중 레인 13 및 14는 각각 A/Moscow/10/99 (H3N2)의 H3 아형 및 N2 아형에 특이적인 프라이머 세트를 사용한 결과이다.Figure 3 shows the multiplex PCR under the same conditions using two types of toxic viruses [A / New Caledonia / 20/99 (H1N1), A / Moscow / 10/99 (H3N2)] and attenuated virus strains as a template. The results obtained are shown. 3A shows the results for the toxic virus A / New Caledonia / 20/99 (H1N1), where M at the top represents the molecular weight marker and the results for 8 RNAs of the attenuated virus strain are lanes 1 to PB2. , PB1, NP, lane 2 is M, NS, PA, lane 3 is a primer set for HA, NA, more specifically lane 1 is PB2-2 (1002bp), NP-4 (853bp), PB1-6 ( 559 bp), lane 2 is PA-5 (730 bp), NS-5 (521 bp), M-6 (326 bp), lane 3 is the result using HA-4 (1086 bp), NA-4 (504 bp) 4 to 6 show results using the same primer set as the primers used in lanes 1 to 3, lanes 7 to 14 are the results of the positive control for each of the 8 RNAs, of which lanes 13 and 14 are A / New Caledonia / 20, respectively. The result is the use of primer sets specific for H1 subtype and N1 subtype of / 99 (H1N1). 3B shows the results for the toxic virus A / Moscow / 10/99 (H3N2), where M at the top represents the molecular weight marker, and the results for the eight RNAs of the attenuated virus strain are lanes 1 to PB2, PB1, NP, lane 2 is M, NS, PA, lane 3 is a primer set for HA, NA, more specifically lane 1 is PB2-2 (1002bp), NP-4 (853bp), PB1-6 (559bp ), Lane 2 is PA-5 (730bp), NS-5 (521bp), M-6 (326bp), lane 3 is the result using HA-4 (1086bp), NA-4 (504 bp), lane 4 6 to 6 show results using the same primer set as the primers used in lanes 1 to 3, lanes 7 to 14 are the results of the positive control for each of the 8 RNAs, of which lanes 13 and 14 are A / Moscow / 10/99, respectively. The result is the use of primer sets specific for the H3 and N2 subtypes of (H3N2).
도 4는 독성 바이러스 A/New Caledonia/20/99 (H1N1)와 약독화 바이러스주를 하나의 수정란에 동시에 감염시켜 생장시킨 뒤 얻은 바이러스를 약독화 바이러스주에 대한 항체와 반응시킨 후 플라크 분리(plaque isolation)하여 얻은 바이러스를 본 발명의 방법으로 분석한 결과로, 4개의 레인이 하나의 바이러스를 분석한 것으로 레인 1 내지 3 은 8개 유전자 유래를 알기 위한 멀티플렉스 PCR이며 레인 4는 RNA 준비 과정과 RT-PCR 과정에서의 유전자의 존재유무를 보여주기 위한 포지티브 대조군(positive control)으로서 여덟 개 유전자 중에서 M(649bp)에 대하여 PCR을 수행한 결과이다. 레인 1 은 PB2, PB1, NP, 레인 2 는 M, NS, PA, 레인 3 HA, NA, 레인 4는 M 에 대한 프라이머 세트, 보다 구체적으로는 레인 1 은 PB2-2 (1002bp), NP-4 (853bp), PB1-6 (559bp), 레인 2는 PA-5 (730bp), NS-5 (521bp), M-6 (326bp), 레인 3는 HA-4 (1086bp), NA-4 (504bp), 레인 4는 Positive M (649bp)를 사용한 결과이다.FIG. 4 shows that plaque is isolated after reacting the virus obtained with the attenuated virus A / New Caledonia / 20/99 (H1N1) and the attenuated virus line at the same time with one fertilized egg and reacting the virus with the antibody against the attenuated virus line As a result of analyzing the virus obtained by the isolation method according to the method of the present invention, four lanes were analyzed for one virus, and lanes 1 to 3 were multiplex PCR to know the origin of eight genes. As a positive control to show the presence or absence of genes in the RT-PCR process, PCR was performed on M (649 bp) among eight genes. Lane 1 is PB2, PB1, NP, lane 2 is M, NS, PA, lane 3 HA, NA, lane 4 is primer set for M, more specifically lane 1 is PB2-2 (1002bp), NP-4 (853bp), PB1-6 (559bp), lane 2 is PA-5 (730bp), NS-5 (521bp), M-6 (326bp), lane 3 is HA-4 (1086bp), NA-4 (504bp ), Lane 4 is the result of using Positive M (649bp).
<110> CJ Corporation Protheon <120> Screening system of reassortant influenza viruses using primer dependent multiplex RT-PCR <160> 145 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB2-1 sense primer designed from HTCA-A101 <400> 1 aatgacaaat acagttcag 19 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB2-1 antisense primer designed from HTCA-A101 <400> 2 gaagaagttt tattatctgt gc 22 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB2-2 sense primer designed from HTCA-A101 <400> 3 cacacagatt ggtggaatt 19 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB2-2 antisense primer designed from HTCA-A101 <400> 4 gaagaagttt tattatctgt gc 22 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB2-3 sense primer designed from HTCA-A101 <400> 5 cgacatgact ccaagcatc 19 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB2-3 antisense primer designed from HTCA-A101 <400> 6 gaagaagttt tattatctgt gc 22 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB2-4 sense primer designed from HTCA-A101 <400> 7 aatgacaaat acagttcag 19 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PB2-4 antisense primer designed from HTCA-A101 <400> 8 gaatgaggaa tcccctcaga 20 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB2-5 sense primer designed from HTCA-A101 <400> 9 cacacagatt ggtggaatt 19 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PB2-5 antisense primer designed from HTCA-A101 <400> 10 gaatgaggaa tcccctcaga 20 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB2-6 sense primer designed from HTCA-A101 <400> 11 cgacatgact ccaagcatc 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PB2-6 antisense primer designed from HTCA-A101 <400> 12 gaatgaggaa tcccctcaga 20 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB2-7 sense primer designed from HTCA-A101 <400> 13 aatgacaaat acagttcag 19 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PB2-7 antisense primer designed from HTCA-A101 <400> 14 ctcttgtctt ctttgcccag 20 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB2-8 sense primer designed from HTCA-A101 <400> 15 cacacagatt ggtggaatt 19 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PB2-8 antisense primer designed from HTCA-A101 <400> 16 ctcttgtctt ctttgcccag 20 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB2-9 sense primer designed from HTCA-A101 <400> 17 cgacatgact ccaagcatc 19 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PB2-9 antisense primer designed from HTCA-A101 <400> 18 ctcttgtctt ctttgcccag 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Positive PB2 sense primer designed from HTCA-A101 <400> 19 catcgtcaat gatgtgggag 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Positive PB2 antisense primer designed from HTCA-A101 <400> 20 tggctgtcag taagtatgct 20 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB1-1 sense primer designed from HTCA-A101 <400> 21 aaatgttcta agtattgctc ca 22 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PB1-1 antisense primer designed from HTCA-A101 <400> 22 aggttcgata aaacctgtcg 20 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB1-2 sense primer designed from HTCA-A101 <400> 23 gagagcaaga gtatgaaact ta 22 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PB1-2 antisense primer designed from HTCA-A101 <400> 24 aggttcgata aaacctgtcg 20 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PB1-3 sense primer designed from HTCA-A101 <400> 25 ctagcaagca tcgatttgaa a 21 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PB1-3 antisense primer designed from HTCA-A101 <400> 26 aggttcgata aaacctgtcg 20 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB1-4 sense primer designed from HTCA-A101 <400> 27 aaatgttcta agtattgctc ca 22 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB1-4 antisense primer designed from HTCA-A101 <400> 28 gtcacctcta tggcatcgg 19 <210> 29 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB1-5 sense primer designed from HTCA-A101 <400> 29 gagagcaaga gtatgaaact ta 22 <210> 30 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB1-5 antisense primer designed from HTCA-A101 <400> 30 gtcacctcta tggcatcgg 19 <210> 31 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PB1-6 sense primer designed from HTCA-A101 <400> 31 ctagcaagca tcgatttgaa a 21 <210> 32 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB1-6 antisense primer designed from HTCA-A101 <400> 32 gtcacctcta tggcatcgg 19 <210> 33 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB1-7 sense primer designed from HTCA-A101 <400> 33 aaatgttcta agtattgctc ca 22 <210> 34 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB1-7 antisense primer designed from HTCA-A101 <400> 34 catcatcact gcattgttca tt 22 <210> 35 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB1-8 sense primer designed from HTCA-A101 <400> 35 gagagcaaga gtatgaaact ta 22 <210> 36 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB1-8 antisense primer designed from HTCA-A101 <400> 36 catcatcact gcattgttca tt 22 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PB1-9 sense primer designed from HTCA-A101 <400> 37 ctagcaagca tcgatttgaa a 21 <210> 38 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB1-9 antisense primer designed from HTCA-A101 <400> 38 catcatcact gcattgttca tt 22 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Positive PB1 sense primer designed from HTCA-A101 <400> 39 atgatgatgg gcatgttcaa 20 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Positive PB1 antisense primer designed from HTCA-A101 <400> 40 ggaacagatc ttcatgatct 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-1 sense primer designed from HTCA-A101 <400> 41 taatcgaggg aagagatcgc 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-1 antisense primer designed from HTCA-A101 <400> 42 tgtgttcaat tggagccaca 20 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-2 sense primer designed from HTCA-A101 <400> 43 acaacaccac gaccacttag 20 <210> 44 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-2 antisense primer designed from HTCA-A101 <400> 44 tgtgttcaat tggagccaca 20 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-3 sense primer designed from HTCA-A101 <400> 45 gatggaagga acccaatgtt 20 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-3 antisense primer designed from HTCA-A101 <400> 46 tgtgttcaat tggagccaca 20 <210> 47 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-4 sense primer designed from HTCA-A101 <400> 47 taatcgaggg aagagatcgc 20 <210> 48 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PA-4 antisense primer designed from HTCA-A101 <400> 48 tttatgatga aaccaacaag c 21 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-5 sense primer designed from HTCA-A101 <400> 49 acaacaccac gaccacttag 20 <210> 50 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PA-5 antisense primer designed from HTCA-A101 <400> 50 tttatgatga aaccaacaag c 21 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-6 sense primer designed from HTCA-A101 <400> 51 gatggaagga acccaatgtt 20 <210> 52 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PA-6 antisense primer designed from HTCA-A101 <400> 52 tttatgatga aaccaacaag c 21 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-7 sense primer designed from HTCA-A101 <400> 53 taatcgaggg aagagatcgc 20 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-7 antisense primer designed from HTCA-A101 <400> 54 gaacatgggc cttgaaacct 20 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-8 sense primer designed from HTCA-A101 <400> 55 acaacaccac gaccacttag 20 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-8 antisense primer designed from HTCA-A101 <400> 56 gaacatgggc cttgaaacct 20 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-9 sense primer designed from HTCA-A101 <400> 57 gatggaagga acccaatgtt 20 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-9 antisense primer designed from HTCA-A101 <400> 58 gaacatgggc cttgaaacct 20 <210> 59 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Positive PA sense primer designed from HTCA-A101 <400> 59 gtagtctgcc tttgtggcc 19 <210> 60 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Positive PA antisense primer designed from HTCA-A101 <400> 60 tcttgtctta tggtgaatag 20 <210> 61 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NP-1 sense primer designed from HTCA-A101 <400> 61 acctatatac aggagagtaa ac 22 <210> 62 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NP-1 antisense primer designed from HTCA-A101 <400> 62 aaagcttccc tcttgggagc 20 <210> 63 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NP-2 sense primer designed from HTCA-A101 <400> 63 gaacaatggt gatggaattg g 21 <210> 64 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NP-2 antisense primer designed from HTCA-A101 <400> 64 aaagcttccc tcttgggagc 20 <210> 65 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NP-3 sense primer designed from HTCA-A101 <400> 65 gtgggtacga ctttgaaagg 20 <210> 66 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NP-3 antisense primer designed from HTCA-A101 <400> 66 aaagcttccc tcttgggagc 20 <210> 67 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NP-4 sense primer designed from HTCA-A101 <400> 67 acctatatac aggagagtaa ac 22 <210> 68 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NP-4 antisense primer designed from HTCA-A101 <400> 68 agtgtacttg attccatagt c 21 <210> 69 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NP-5 sense primer designed from HTCA-A101 <400> 69 gaacaatggt gatggaattg g 21 <210> 70 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NP-5 antisense primer designed from HTCA-A101 <400> 70 agtgtacttg attccatagt c 21 <210> 71 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NP-6 sense primer designed from HTCA-A101 <400> 71 gtgggtacga ctttgaaagg 20 <210> 72 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NP-6 antisense primer designed from HTCA-A101 <400> 72 agtgtacttg attccatagt c 21 <210> 73 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NP-7 sense primer designed from HTCA-A101 <400> 73 acctatatac aggagagtaa ac 22 <210> 74 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NP-7 antisense primer designed from HTCA-A101 <400> 74 tgctgccata atggtggttc 20 <210> 75 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NP-8 sense primer designed from HTCA-A101 <400> 75 gaacaatggt gatggaattg g 21 <210> 76 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NP-8 antisense primer designed from HTCA-A101 <400> 76 tgctgccata atggtggttc 20 <210> 77 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NP-9 sense primer designed from HTCA-A101 <400> 77 gtgggtacga ctttgaaagg 20 <210> 78 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NP-9 antisense primer designed from HTCA-A101 <400> 78 tgctgccata atggtggttc 20 <210> 79 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Positive NP sense primer designed from HTCA-A101 <400> 79 taaggcgaat ctggcgccaa 20 <210> 80 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Positive NP antisense primer designed from HTCA-A101 <400> 80 taagatcctt cattactcat 20 <210> 81 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-1 sense primer designed from HTCA-A101 <400> 81 acgtactctc tatcatcccg 20 <210> 82 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> M-1 antisense primer designed from HTCA-A101 <400> 82 tctagcctga ctagcaacc 19 <210> 83 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-2 sense primer designed from HTCA-A101 <400> 83 ccatggggcc aaagaaatct 20 <210> 84 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> M-2 antisense primer designed from HTCA-A101 <400> 84 tctagcctga ctagcaacc 19 <210> 85 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> M-3 sense primer designed from HTCA-A101 <400> 85 tctagcctga ctagcaacc 19 <210> 86 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-3 antisense primer designed from HTCA-A101 <400> 86 acgtactctc tatcatcccg 20 <210> 87 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-4 sense primer designed from HTCA-A101 <400> 87 tgatatttgc ggcaatagtg 20 <210> 88 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-4 antisense primer designed from HTCA-A101 <400> 88 ccatggggcc aaagaaatct 20 <210> 89 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-5 sense primer designed from HTCA-A101 <400> 89 tgatatttgc ggcaatagtg 20 <210> 90 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-5 antisense primer designed from HTCA-A101 <400> 90 tgatatttgc ggcaatagtg 20 <210> 91 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> M-6 sense primer designed from HTCA-A101 <400> 91 attgctgact cccagcatc 19 <210> 92 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-6 antisense primer designed from HTCA-A101 <400> 92 tgatatttgc ggcaatagtg 20 <210> 93 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-7 sense primer designed from HTCA-A101 <400> 93 acgtactctc tatcatcccg 20 <210> 94 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-7 antisense primer designed from HTCA-A101 <400> 94 cttccctcat agactttggc 20 <210> 95 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-8 sense primer designed from HTCA-A101 <400> 95 ccatggggcc aaagaaatct 20 <210> 96 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-8 antisense primer designed from HTCA-A101 <400> 96 cttccctcat agactttggc 20 <210> 97 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> M-9 sense primer designed from HTCA-A101 <400> 97 attgctgact cccagcatc 19 <210> 98 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-9 antisense primer designed from HTCA-A101 <400> 98 cttccctcat agactttggc 20 <210> 99 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Positive M sense primer designed from HTCA-A101 <400> 99 gcagcgtaga cgctttgtc 19 <210> 100 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Positive M antisense primer designed from HTCA-A101 <400> 100 ccttccgtag aaggccctc 19 <210> 101 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-1 sense primer designed from HTCA-A101 <400> 101 agttgcagac caagaactag 20 <210> 102 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-1 antisense primer designed from HTCA-A101 <400> 102 gcaatattag agtctccagc 20 <210> 103 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-2 sense primer designed from HTCA-A101 <400> 103 atcaagacag ccacacgtgc 20 <210> 104 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-2 antisense primer designed from HTCA-A101 <400> 104 gcaatattag agtctccagc 20 <210> 105 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-3 sense primer designed from HTCA-A101 <400> 105 ggaaagcaga tagtggagcg 20 <210> 106 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-3 antisense primer designed from HTCA-A101 <400> 106 gcaatattag agtctccagc 20 <210> 107 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-4 sense primer designed from HTCA-A101 <400> 107 agttgcagac caagaactag 20 <210> 108 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NS-4 antisense primer designed from HTCA-A101 <400> 108 ctaattgttc ccgccatttc t 21 <210> 109 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-5 sense primer designed from HTCA-A101 <400> 109 atcaagacag ccacacgtgc 20 <210> 110 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NS-5 antisense primer designed from HTCA-A101 <400> 110 ctaattgttc ccgccatttc t 21 <210> 111 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-6 sense primer designed from HTCA-A101 <400> 111 ggaaagcaga tagtggagcg 20 <210> 112 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NS-6 antisense primer designed from HTCA-A101 <400> 112 ctaattgttc ccgccatttc t 21 <210> 113 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-7 sense primer designed from HTCA-A101 <400> 113 agttgcagac caagaactag 20 <210> 114 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-7 antisense primer designed from HTCA-A101 <400> 114 attctctgtt atcttcagtt 20 <210> 115 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-8 sense primer designed from HTCA-A101 <400> 115 atcaagacag ccacacgtgc 20 <210> 116 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-8 antisense primer designed from HTCA-A101 <400> 116 attctctgtt atcttcagtt 20 <210> 117 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-9 sense primer designed from HTCA-A101 <400> 117 ggaaagcaga tagtggagcg 20 <210> 118 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-9 antisense primer designed from HTCA-A101 <400> 118 attctctgtt atcttcagtt 20 <210> 119 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Positive NS sense primer designed from HTCA-A101 <400> 119 agcaaaagca gggtgacaaa 20 <210> 120 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Positive NS antisense primer designed from HTCA-A101 <400> 120 cagagactcg aactgtgtta 20 <210> 121 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HA-1 sense primer designed from HTCA-A101 <400> 121 ggtttcactt ggactggggt 20 <210> 122 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HA-1 antisense primer designed from HTCA-A101 <400> 122 tgagaattcc ttttcgattt ga 22 <210> 123 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HA-2 sense primer designed from HTCA-A101 <400> 123 tgaccaaatc aggaagca 18 <210> 124 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HA-2 antisense primer designed from HTCA-A101 <400> 124 tgagaattcc ttttcgattt ga 22 <210> 125 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HA-3 sense primer designed from HTCA-A101 <400> 125 ggtttcactt ggactggggt 20 <210> 126 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HA-3 antisense primer designed from HTCA-A101 <400> 126 gcatccagtc tttgtatcca 20 <210> 127 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HA-4 sense primer designed from HTCA-A101 <400> 127 tgaccaaatc aggaagca 18 <210> 128 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HA-4 antisense primer designed from HTCA-A101 <400> 128 gcatccagtc tttgtatcca 20 <210> 129 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Positive H1 control sense primer designed from HTCA-A101 <400> 129 atgaaagcaa aactactagt tca 23 <210> 130 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Positive H1 control antisense primer designed from HTCA-A101 <400> 130 agacgggtga tgaacacccc at 22 <210> 131 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Positive H2 control sense primer designed from HTCA-A101 <400> 131 cctcaacagg tagaatatgc gg 22 <210> 132 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Positive H2 control antisense primer designed from HTCA-A101 <400> 132 aggagcaatt agattccctg tg 22 <210> 133 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NA-1 sense primer designed from HTCA-A101 <400> 133 agatatgccc caaattagtg 20 <210> 134 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> NA-1 antisense primer designed from HTCA-A101 <400> 134 cgattgttag ccagcccatg cca 23 <210> 135 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NA-2 sense primer designed from HTCA-A101 <400> 135 tgcgtttgta tcaatgggac 20 <210> 136 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> NA-2 antisense primer designed from HTCA-A101 <400> 136 cgattgttag ccagcccatg cca 23 <210> 137 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NA-3 sense primer designed from HTCA-A101 <400> 137 agatatgccc caaattagtg 20 <210> 138 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NA-3 antisense primer designed from HTCA-A101 <400> 138 ctgcgatttg gaattaggtg 20 <210> 139 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NA-4 sense primer designed from HTCA-A101 <400> 139 tgcgtttgta tcaatgggac 20 <210> 140 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NA-4 antisense primer designed from HTCA-A101 <400> 140 ctgcgatttg gaattaggtg 20 <210> 141 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Positive N1 control sense primer designed from HTCA-A101 <400> 141 agcaaaagca ggagtttaaa a 21 <210> 142 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Positive N1 control antisense primer designed from HTCA-A101 <400> 142 cgattgttag ccagcccatg cca 23 <210> 143 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Positive N2 control sense primer designed from HTCA-A101 <400> 143 atgaatccaa atcaaaagat aa 22 <210> 144 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Positive N2 control antisense primer designed from HTCA-A101 <400> 144 ttgccaggat cgcatgacac at 22 <210> 145 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> Uni12 primer designed from HTCA-A101 <400> 145 agcaaagcag g 11<110> CJ Corporation Protheon <120> Screening system of reassortant influenza viruses using primer dependent multiplex RT-PCR <160> 145 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB2-1 sense primer designed from HTCA-A101 <400> 1 aatgacaaat acagttcag 19 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB2-1 antisense primer designed from HTCA-A101 <400> 2 gaagaagttt tattatctgt gc 22 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB2-2 sense primer designed from HTCA-A101 <400> 3 cacacagatt ggtggaatt 19 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB2-2 antisense primer designed from HTCA-A101 <400> 4 gaagaagttt tattatctgt gc 22 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB2-3 sense primer designed from HTCA-A101 <400> 5 cgacatgact ccaagcatc 19 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB2-3 antisense primer designed from HTCA-A101 <400> 6 gaagaagttt tattatctgt gc 22 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB2-4 sense primer designed from HTCA-A101 <400> 7 aatgacaaat acagttcag 19 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PB2-4 antisense primer designed from HTCA-A101 <400> 8 gaatgaggaa tcccctcaga 20 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB2-5 sense primer designed from HTCA-A101 <400> 9 cacacagatt ggtggaatt 19 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PB2-5 antisense primer designed from HTCA-A101 <400> 10 gaatgaggaa tcccctcaga 20 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB2-6 sense primer designed from HTCA-A101 <400> 11 cgacatgact ccaagcatc 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PB2-6 antisense primer designed from HTCA-A101 <400> 12 gaatgaggaa tcccctcaga 20 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB2-7 sense primer designed from HTCA-A101 <400> 13 aatgacaaat acagttcag 19 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PB2-7 antisense primer designed from HTCA-A101 <400> 14 ctcttgtctt ctttgcccag 20 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB2-8 sense primer designed from HTCA-A101 <400> 15 cacacagatt ggtggaatt 19 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PB2-8 antisense primer designed from HTCA-A101 <400> 16 ctcttgtctt ctttgcccag 20 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB2-9 sense primer designed from HTCA-A101 <400> 17 cgacatgact ccaagcatc 19 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PB2-9 antisense primer designed from HTCA-A101 <400> 18 ctcttgtctt ctttgcccag 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Positive PB2 sense primer designed from HTCA-A101 <400> 19 catcgtcaat gatgtgggag 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Positive PB2 antisense primer designed from HTCA-A101 <400> 20 tggctgtcag taagtatgct 20 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB1-1 sense primer designed from HTCA-A101 <400> 21 aaatgttcta agtattgctc ca 22 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PB1-1 antisense primer designed from HTCA-A101 <400> 22 aggttcgata aaacctgtcg 20 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB1-2 sense primer designed from HTCA-A101 <400> 23 gagagcaaga gtatgaaact ta 22 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PB1-2 antisense primer designed from HTCA-A101 <400> 24 aggttcgata aaacctgtcg 20 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PB1-3 sense primer designed from HTCA-A101 <400> 25 ctagcaagca tcgatttgaa a 21 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PB1-3 antisense primer designed from HTCA-A101 <400> 26 aggttcgata aaacctgtcg 20 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB1-4 sense primer designed from HTCA-A101 <400> 27 aaatgttcta agtattgctc ca 22 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB1-4 antisense primer designed from HTCA-A101 <400> 28 gtcacctcta tggcatcgg 19 <210> 29 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB1-5 sense primer designed from HTCA-A101 <400> 29 gagagcaaga gtatgaaact ta 22 <210> 30 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB1-5 antisense primer designed from HTCA-A101 <400> 30 gtcacctcta tggcatcgg 19 <210> 31 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PB1-6 sense primer designed from HTCA-A101 <400> 31 ctagcaagca tcgatttgaa a 21 <210> 32 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PB1-6 antisense primer designed from HTCA-A101 <400> 32 gtcacctcta tggcatcgg 19 <210> 33 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB1-7 sense primer designed from HTCA-A101 <400> 33 aaatgttcta agtattgctc ca 22 <210> 34 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB1-7 antisense primer designed from HTCA-A101 <400> 34 catcatcact gcattgttca tt 22 <210> 35 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB1-8 sense primer designed from HTCA-A101 <400> 35 gagagcaaga gtatgaaact ta 22 <210> 36 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB1-8 antisense primer designed from HTCA-A101 <400> 36 catcatcact gcattgttca tt 22 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PB1-9 sense primer designed from HTCA-A101 <400> 37 ctagcaagca tcgatttgaa a 21 <210> 38 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PB1-9 antisense primer designed from HTCA-A101 <400> 38 catcatcact gcattgttca tt 22 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Positive PB1 sense primer designed from HTCA-A101 <400> 39 atgatgatgg gcatgttcaa 20 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Positive PB1 antisense primer designed from HTCA-A101 <400> 40 ggaacagatc ttcatgatct 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-1 sense primer designed from HTCA-A101 <400> 41 taatcgaggg aagagatcgc 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-1 antisense primer designed from HTCA-A101 <400> 42 tgtgttcaat tggagccaca 20 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-2 sense primer designed from HTCA-A101 <400> 43 acaacaccac gaccacttag 20 <210> 44 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-2 antisense primer designed from HTCA-A101 <400> 44 tgtgttcaat tggagccaca 20 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-3 sense primer designed from HTCA-A101 <400> 45 gatggaagga acccaatgtt 20 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-3 antisense primer designed from HTCA-A101 <400> 46 tgtgttcaat tggagccaca 20 <210> 47 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-4 sense primer designed from HTCA-A101 <400> 47 taatcgaggg aagagatcgc 20 <210> 48 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PA-4 antisense primer designed from HTCA-A101 <400> 48 tttatgatga aaccaacaag c 21 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-5 sense primer designed from HTCA-A101 <400> 49 acaacaccac gaccacttag 20 <210> 50 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PA-5 antisense primer designed from HTCA-A101 <400> 50 tttatgatga aaccaacaag c 21 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-6 sense primer designed from HTCA-A101 <400> 51 gatggaagga acccaatgtt 20 <210> 52 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PA-6 antisense primer designed from HTCA-A101 <400> 52 tttatgatga aaccaacaag c 21 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-7 sense primer designed from HTCA-A101 <400> 53 taatcgaggg aagagatcgc 20 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-7 antisense primer designed from HTCA-A101 <400> 54 gaacatgggc cttgaaacct 20 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-8 sense primer designed from HTCA-A101 <400> 55 acaacaccac gaccacttag 20 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-8 antisense primer designed from HTCA-A101 <400> 56 gaacatgggc cttgaaacct 20 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-9 sense primer designed from HTCA-A101 <400> 57 gatggaagga acccaatgtt 20 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PA-9 antisense primer designed from HTCA-A101 <400> 58 gaacatgggc cttgaaacct 20 <210> 59 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Positive PA sense primer designed from HTCA-A101 <400> 59 gtagtctgcc tttgtggcc 19 <210> 60 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Positive PA antisense primer designed from HTCA-A101 <400> 60 tcttgtctta tggtgaatag 20 <210> 61 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NP-1 sense primer designed from HTCA-A101 <400> 61 acctatatac aggagagtaa ac 22 <210> 62 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NP-1 antisense primer designed from HTCA-A101 <400> 62 aaagcttccc tcttgggagc 20 <210> 63 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NP-2 sense primer designed from HTCA-A101 <400> 63 gaacaatggt gatggaattg g 21 <210> 64 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NP-2 antisense primer designed from HTCA-A101 <400> 64 aaagcttccc tcttgggagc 20 <210> 65 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NP-3 sense primer designed from HTCA-A101 <400> 65 gtgggtacga ctttgaaagg 20 <210> 66 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NP-3 antisense primer designed from HTCA-A101 <400> 66 aaagcttccc tcttgggagc 20 <210> 67 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NP-4 sense primer designed from HTCA-A101 <400> 67 acctatatac aggagagtaa ac 22 <210> 68 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NP-4 antisense primer designed from HTCA-A101 <400> 68 agtgtacttg attccatagt c 21 <210> 69 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NP-5 sense primer designed from HTCA-A101 <400> 69 gaacaatggt gatggaattg g 21 <210> 70 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NP-5 antisense primer designed from HTCA-A101 <400> 70 agtgtacttg attccatagt c 21 <210> 71 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NP-6 sense primer designed from HTCA-A101 <400> 71 gtgggtacga ctttgaaagg 20 <210> 72 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NP-6 antisense primer designed from HTCA-A101 <400> 72 agtgtacttg attccatagt c 21 <210> 73 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NP-7 sense primer designed from HTCA-A101 <400> 73 acctatatac aggagagtaa ac 22 <210> 74 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NP-7 antisense primer designed from HTCA-A101 <400> 74 tgctgccata atggtggttc 20 <210> 75 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NP-8 sense primer designed from HTCA-A101 <400> 75 gaacaatggt gatggaattg g 21 <210> 76 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NP-8 antisense primer designed from HTCA-A101 <400> 76 tgctgccata atggtggttc 20 <210> 77 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NP-9 sense primer designed from HTCA-A101 <400> 77 gtgggtacga ctttgaaagg 20 <210> 78 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NP-9 antisense primer designed from HTCA-A101 <400> 78 tgctgccata atggtggttc 20 <210> 79 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Positive NP sense primer designed from HTCA-A101 <400> 79 taaggcgaat ctggcgccaa 20 <210> 80 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Positive NP antisense primer designed from HTCA-A101 <400> 80 taagatcctt cattactcat 20 <210> 81 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-1 sense primer designed from HTCA-A101 <400> 81 acgtactctc tatcatcccg 20 <210> 82 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> M-1 antisense primer designed from HTCA-A101 <400> 82 tctagcctga ctagcaacc 19 <210> 83 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-2 sense primer designed from HTCA-A101 <400> 83 ccatggggcc aaagaaatct 20 <210> 84 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> M-2 antisense primer designed from HTCA-A101 <400> 84 tctagcctga ctagcaacc 19 <210> 85 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> M-3 sense primer designed from HTCA-A101 <400> 85 tctagcctga ctagcaacc 19 <210> 86 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-3 antisense primer designed from HTCA-A101 <400> 86 acgtactctc tatcatcccg 20 <210> 87 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-4 sense primer designed from HTCA-A101 <400> 87 tgatatttgc ggcaatagtg 20 <210> 88 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-4 antisense primer designed from HTCA-A101 <400> 88 ccatggggcc aaagaaatct 20 <210> 89 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-5 sense primer designed from HTCA-A101 <400> 89 tgatatttgc ggcaatagtg 20 <210> 90 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-5 antisense primer designed from HTCA-A101 <400> 90 tgatatttgc ggcaatagtg 20 <210> 91 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> M-6 sense primer designed from HTCA-A101 <400> 91 attgctgact cccagcatc 19 <210> 92 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-6 antisense primer designed from HTCA-A101 <400> 92 tgatatttgc ggcaatagtg 20 <210> 93 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-7 sense primer designed from HTCA-A101 <400> 93 acgtactctc tatcatcccg 20 <210> 94 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-7 antisense primer designed from HTCA-A101 <400> 94 cttccctcat agactttggc 20 <210> 95 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-8 sense primer designed from HTCA-A101 <400> 95 ccatggggcc aaagaaatct 20 <210> 96 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-8 antisense primer designed from HTCA-A101 <400> 96 cttccctcat agactttggc 20 <210> 97 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> M-9 sense primer designed from HTCA-A101 <400> 97 attgctgact cccagcatc 19 <210> 98 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-9 antisense primer designed from HTCA-A101 <400> 98 cttccctcat agactttggc 20 <210> 99 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Positive M sense primer designed from HTCA-A101 <400> 99 gcagcgtaga cgctttgtc 19 <210> 100 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Positive M antisense primer designed from HTCA-A101 <400> 100 ccttccgtag aaggccctc 19 <210> 101 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-1 sense primer designed from HTCA-A101 <400> 101 agttgcagac caagaactag 20 <210> 102 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-1 antisense primer designed from HTCA-A101 <400> 102 gcaatattag agtctccagc 20 <210> 103 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-2 sense primer designed from HTCA-A101 <400> 103 atcaagacag ccacacgtgc 20 <210> 104 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-2 antisense primer designed from HTCA-A101 <400> 104 gcaatattag agtctccagc 20 <210> 105 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-3 sense primer designed from HTCA-A101 <400> 105 ggaaagcaga tagtggagcg 20 <210> 106 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-3 antisense primer designed from HTCA-A101 <400> 106 gcaatattag agtctccagc 20 <210> 107 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-4 sense primer designed from HTCA-A101 <400> 107 agttgcagac caagaactag 20 <210> 108 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NS-4 antisense primer designed from HTCA-A101 <400> 108 ctaattgttc ccgccatttc t 21 <210> 109 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-5 sense primer designed from HTCA-A101 <400> 109 atcaagacag ccacacgtgc 20 <210> 110 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NS-5 antisense primer designed from HTCA-A101 <400> 110 ctaattgttc ccgccatttc t 21 <210> 111 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-6 sense primer designed from HTCA-A101 <400> 111 ggaaagcaga tagtggagcg 20 <210> 112 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NS-6 antisense primer designed from HTCA-A101 <400> 112 ctaattgttc ccgccatttc t 21 <210> 113 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-7 sense primer designed from HTCA-A101 <400> 113 agttgcagac caagaactag 20 <210> 114 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-7 antisense primer designed from HTCA-A101 <400> 114 attctctgtt atcttcagtt 20 <210> 115 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-8 sense primer designed from HTCA-A101 <400> 115 atcaagacag ccacacgtgc 20 <210> 116 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-8 antisense primer designed from HTCA-A101 <400> 116 attctctgtt atcttcagtt 20 <210> 117 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-9 sense primer designed from HTCA-A101 <400> 117 ggaaagcaga tagtggagcg 20 <210> 118 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS-9 antisense primer designed from HTCA-A101 <400> 118 attctctgtt atcttcagtt 20 <210> 119 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Positive NS sense primer designed from HTCA-A101 <400> 119 agcaaaagca gggtgacaaa 20 <210> 120 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Positive NS antisense primer designed from HTCA-A101 <400> 120 cagagactcg aactgtgtta 20 <210> 121 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HA-1 sense primer designed from HTCA-A101 <400> 121 ggtttcactt ggactggggt 20 <210> 122 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HA-1 antisense primer designed from HTCA-A101 <400> 122 tgagaattcc ttttcgattt ga 22 <210> 123 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HA-2 sense primer designed from HTCA-A101 <400> 123 tgaccaaatc aggaagca 18 <210> 124 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HA-2 antisense primer designed from HTCA-A101 <400> 124 tgagaattcc ttttcgattt ga 22 <210> 125 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HA-3 sense primer designed from HTCA-A101 <400> 125 ggtttcactt ggactggggt 20 <210> 126 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HA-3 antisense primer designed from HTCA-A101 <400> 126 gcatccagtc tttgtatcca 20 <210> 127 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HA-4 sense primer designed from HTCA-A101 <400> 127 tgaccaaatc aggaagca 18 <210> 128 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HA-4 antisense primer designed from HTCA-A101 <400> 128 gcatccagtc tttgtatcca 20 <210> 129 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Positive H1 control sense primer designed from HTCA-A101 <400> 129 atgaaagcaa aactactagt tca 23 <210> 130 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Positive H1 control antisense primer designed from HTCA-A101 <400> 130 agacgggtga tgaacacccc at 22 <210> 131 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Positive H2 control sense primer designed from HTCA-A101 <400> 131 cctcaacagg tagaatatgc gg 22 <210> 132 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Positive H2 control antisense primer designed from HTCA-A101 <400> 132 aggagcaatt agattccctg tg 22 <210> 133 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NA-1 sense primer designed from HTCA-A101 <133> 133 agatatgccc caaattagtg 20 <210> 134 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> NA-1 antisense primer designed from HTCA-A101 <400> 134 cgattgttag ccagcccatg cca 23 <210> 135 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NA-2 sense primer designed from HTCA-A101 <400> 135 tgcgtttgta tcaatgggac 20 <210> 136 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> NA-2 antisense primer designed from HTCA-A101 <400> 136 cgattgttag ccagcccatg cca 23 <210> 137 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NA-3 sense primer designed from HTCA-A101 <400> 137 agatatgccc caaattagtg 20 <210> 138 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NA-3 antisense primer designed from HTCA-A101 <400> 138 ctgcgatttg gaattaggtg 20 <139> <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NA-4 sense primer designed from HTCA-A101 <400> 139 tgcgtttgta tcaatgggac 20 <210> 140 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NA-4 antisense primer designed from HTCA-A101 <400> 140 ctgcgatttg gaattaggtg 20 <210> 141 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Positive N1 control sense primer designed from HTCA-A101 <400> 141 agcaaaagca ggagtttaaa a 21 <210> 142 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Positive N1 control antisense primer designed from HTCA-A101 <400> 142 cgattgttag ccagcccatg cca 23 <210> 143 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Positive N2 control sense primer designed from HTCA-A101 <400> 143 atgaatccaa atcaaaagat aa 22 <210> 144 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Positive N2 control antisense primer designed from HTCA-A101 <400> 144 ttgccaggat cgcatgacac at 22 <210> 145 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> Uni12 primer designed from HTCA-A101 <400> 145 agcaaagcag g 11
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| KR1020030053534A KR100632429B1 (en) | 2003-08-01 | 2003-08-01 | Screening system of reassortant influenza viruses using primer dependent multiplex RT-PCR |
| PCT/KR2004/001903 WO2005012572A1 (en) | 2003-08-01 | 2004-07-28 | Screening method of reassortant influenza viruses using primer dependent multiplex rt-pcr |
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| US20040121335A1 (en) | 2002-12-06 | 2004-06-24 | Ecker David J. | Methods for rapid detection and identification of bioagents associated with host versus graft and graft versus host rejections |
| US7226739B2 (en) | 2001-03-02 | 2007-06-05 | Isis Pharmaceuticals, Inc | Methods for rapid detection and identification of bioagents in epidemiological and forensic investigations |
| US20030027135A1 (en) | 2001-03-02 | 2003-02-06 | Ecker David J. | Method for rapid detection and identification of bioagents |
| US7666588B2 (en) | 2001-03-02 | 2010-02-23 | Ibis Biosciences, Inc. | Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy |
| US7217510B2 (en) | 2001-06-26 | 2007-05-15 | Isis Pharmaceuticals, Inc. | Methods for providing bacterial bioagent characterizing information |
| US8073627B2 (en) | 2001-06-26 | 2011-12-06 | Ibis Biosciences, Inc. | System for indentification of pathogens |
| JP2006516193A (en) | 2002-12-06 | 2006-06-29 | アイシス・ファーマシューティカルス・インコーポレーテッド | Rapid identification of pathogens in humans and animals |
| US8057993B2 (en) | 2003-04-26 | 2011-11-15 | Ibis Biosciences, Inc. | Methods for identification of coronaviruses |
| US8158354B2 (en) | 2003-05-13 | 2012-04-17 | Ibis Biosciences, Inc. | Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture |
| US7964343B2 (en) | 2003-05-13 | 2011-06-21 | Ibis Biosciences, Inc. | Method for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture |
| US8097416B2 (en) | 2003-09-11 | 2012-01-17 | Ibis Biosciences, Inc. | Methods for identification of sepsis-causing bacteria |
| US8546082B2 (en) | 2003-09-11 | 2013-10-01 | Ibis Biosciences, Inc. | Methods for identification of sepsis-causing bacteria |
| US20120122096A1 (en) | 2003-09-11 | 2012-05-17 | Rangarajan Sampath | Compositions for use in identification of bacteria |
| US8163895B2 (en) | 2003-12-05 | 2012-04-24 | Ibis Biosciences, Inc. | Compositions for use in identification of orthopoxviruses |
| US7666592B2 (en) | 2004-02-18 | 2010-02-23 | Ibis Biosciences, Inc. | Methods for concurrent identification and quantification of an unknown bioagent |
| ES2641832T3 (en) | 2004-05-24 | 2017-11-14 | Ibis Biosciences, Inc. | Mass spectrometry with selective ion filtration by setting digital thresholds |
| US20050266411A1 (en) | 2004-05-25 | 2005-12-01 | Hofstadler Steven A | Methods for rapid forensic analysis of mitochondrial DNA |
| US7811753B2 (en) | 2004-07-14 | 2010-10-12 | Ibis Biosciences, Inc. | Methods for repairing degraded DNA |
| US8084207B2 (en) | 2005-03-03 | 2011-12-27 | Ibis Bioscience, Inc. | Compositions for use in identification of papillomavirus |
| EP1869180B1 (en) | 2005-03-03 | 2013-02-20 | Ibis Biosciences, Inc. | Compositions for use in identification of polyoma viruses |
| CA2616281C (en) | 2005-07-21 | 2014-04-22 | Isis Pharmaceuticals, Inc. | Methods for rapid identification and quantitation of mitochondrial dna variants |
| US20070087338A1 (en) * | 2005-10-17 | 2007-04-19 | Rangarajan Sampath | Compositions for use in identification of influenza viruses |
| EP2064332B1 (en) | 2006-09-14 | 2012-07-18 | Ibis Biosciences, Inc. | Targeted whole genome amplification method for identification of pathogens |
| US8871471B2 (en) | 2007-02-23 | 2014-10-28 | Ibis Biosciences, Inc. | Methods for rapid forensic DNA analysis |
| WO2008151023A2 (en) | 2007-06-01 | 2008-12-11 | Ibis Biosciences, Inc. | Methods and compositions for multiple displacement amplification of nucleic acids |
| US8354230B2 (en) | 2007-12-21 | 2013-01-15 | Quest Diagnostics Investments Inc. | Multiplex detection assay for influenza and RSV viruses |
| US8550694B2 (en) | 2008-09-16 | 2013-10-08 | Ibis Biosciences, Inc. | Mixing cartridges, mixing stations, and related kits, systems, and methods |
| EP2344893B1 (en) | 2008-09-16 | 2014-10-15 | Ibis Biosciences, Inc. | Microplate handling systems and methods |
| EP2347254A2 (en) | 2008-09-16 | 2011-07-27 | Ibis Biosciences, Inc. | Sample processing units, systems, and related methods |
| EP2396803A4 (en) | 2009-02-12 | 2016-10-26 | Ibis Biosciences Inc | Ionization probe assemblies |
| WO2010128396A2 (en) * | 2009-05-08 | 2010-11-11 | Novartis Ag | Generic assays for detection of influenza viruses |
| WO2011008971A1 (en) | 2009-07-17 | 2011-01-20 | Ibis Biosciences, Inc. | Lift and mount apparatus |
| WO2011008972A1 (en) | 2009-07-17 | 2011-01-20 | Ibis Biosciences, Inc. | Systems for bioagent identification |
| EP2957641B1 (en) | 2009-10-15 | 2017-05-17 | Ibis Biosciences, Inc. | Multiple displacement amplification |
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