KR20130049765A - Primer composition for loop-mediated isothermal amplification reaction for detecting tomato yellow leaf curl virus, and use thereof - Google Patents

Primer composition for loop-mediated isothermal amplification reaction for detecting tomato yellow leaf curl virus, and use thereof Download PDF

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KR20130049765A
KR20130049765A KR1020120124504A KR20120124504A KR20130049765A KR 20130049765 A KR20130049765 A KR 20130049765A KR 1020120124504 A KR1020120124504 A KR 1020120124504A KR 20120124504 A KR20120124504 A KR 20120124504A KR 20130049765 A KR20130049765 A KR 20130049765A
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이석찬
정유철
박정안
길의준
허노열
신용길
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Abstract

PURPOSE: A primer composition for loop-mediated isothermal amplification reaction for detecting tomato yellow leaf curl virus is provided to quickly diagnose the virus using high concentration SYBR Green I under natural light with naked eye. CONSTITUTION: A primer set for loop-mediated isothermal amplification reaction for detecting tomato yellow leaf curl virus has sequence numbers 1-4. The primer set has sequence numbers 11-14. The virus is selected from the group consisting of TYLCV Jordan1 mutant strains, Jordan3 mutant strains, Israel mutant strains, Spain mutant strains, USA mutant strains, and Korea mutant strains. A primer composition for loop-mediated isothermal amplification reaction for detecting the virus contains the primer set, DNA polymerase, dNTPs, and a reaction buffer.

Description

토마토황화잎말림바이러스를 검출하기 위한 등온증폭 반응용 프라이머 조성물, 및 이의 이용{Primer composition for loop-mediated isothermal amplification reaction for detecting Tomato yellow leaf curl virus, and use thereof}Primer composition for loop-mediated isothermal amplification reaction for detecting Tomato yellow leaf curl virus, and use according to the present invention.

본 발명은 토마토황화잎말림바이러스를 검출하기 위한 등온증폭반응용 프라이머 세트, 이를 포함하는 조성물, 및 상기 조성물을 이용한 토마토황화잎말림바이러스 검출방법에 관한 것으로, 보다 구체적으로는 토마토황화잎말림바이러스의 검출을 위한 등온증폭 반응용 4개의 프라이머 세트와 이를 포함하는 프라이머 조성물 및 검출방법에 관한 것이다.
The present invention relates to a primer set for isothermal amplification reaction for detecting tomato sulfate leaf virus, a composition comprising the same, and a method for detecting tomato sulfate leaf virus using the composition. It relates to a set of four primers for isothermal amplification reaction for detection, a primer composition comprising the same and a detection method.

우리나라에서 최초로 토마토 재배시설에서 발견된 제미니바이러스(Geminivirus)는, 2008년 통영지역에서 발견되어 본 발명자들에 의하여 보고된 베고모바이러스인 토마토황화잎말림바이러스(TYLCV; Tomato yellow leaf curl virus)로서 하나의 게놈을 갖는 것으로 확인되었다. 토마토황화잎말림바이러스는 베고모바이러스(Begomovirus) 중에서도 가장 많은 종류의 변이주(variant strain)들을 갖고 있는 것으로 보고되어 있으며, 담배가루이(Whitefly)에 의하여 매개 되는 것으로 알려져 있다. 담배가루이는 이미 국내에 토착화가 이루어진 상태로 방제가 매우 어려운 실정이기 때문에 토마토황화잎말림바이러스에 의한 감염의 근절은 매우 어려운 것으로 알려져 있다. 또한 담배가루이에 의해 매개되는 다양한 바이러스 변이주들이 지속적으로 보고되고 있기 때문에 새로운 바이러스 변이주들의 출현, 및 복합감염에 대한 가능성도 배제할 수 없다. 토마토황화잎말림바이러스는 원래 토마토가 기주식물인 것으로 알려져 있었으나, 최근 박과, 콩과, 고추과 등의 작물에도 감염이 가능한 것으로 보고된 바 있다. 토마토황화잎말림바이러스는 국내에서 매우 심각한 농작물 피해를 야기하고 있으며, 아직까지 국내에서 보고되지 않은 신규 토마토황화잎말림바이러스 변이주들 또한 수출입 과정에서 국내로 충분히 유입될 가능성이 있기 때문에 이에 대한 신속한 진단법의 개발이 반드시 필요하다.Geminivirus found in tomato cultivation facility for the first time in Korea is one of the tomato yellow leaf curl virus (TYLCV), a begomovirus found in the Tongyeong area in 2008 and reported by the present inventors. It was confirmed to have a genome of. Tomato sulfide leaf virus has been reported to have the largest variety of strains among the begomovirus (Begomovirus), and is known to be mediated by whitefly. Since the tobacco powder has already been indigenized in Korea and is very difficult to control, it is known that eradication of infection caused by tomato leaf blight virus is very difficult. In addition, since various viral variants mediated by tobacco powder have been reported continuously, the emergence of new viral variants and the possibility of multiple infection cannot be excluded. Tomato yellow leaf curl virus was originally known to be a host plant of tomato, but it has recently been reported that it can infect crops such as pak, soybean, and red pepper. The tomato sulphide leaf virus causes severe crop damage in Korea, and new tomato sulphide leaf virus strains, which have not been reported in Korea yet, are also likely to enter the country during the import and export process. Development is a must.

제미니바이러스는 게놈의 구조, 기주의 범위 및 매개충의 종류에 따라 네 개 속(Genus)인 마스트레바이러스(Mastrevirus), 컬토바이러스(Curtovirus), 토포쿠바이스(Topocuvirus), 및 베고모바이러스(Begomovirus)로 구분되며 세계적으로 주요 경제작물에 심각한 피해를 야기하고 있다. 제미니바이러스 속인 베고모바이러스는 제미니바이러스 중 가장 다양한 바이러스 변이주들을 포함하고 있으며 담배가루이 (Whitefly, Bemisia tabaci, Gennadius)에 의해 매개되는 특징을 갖고 있다. 따라서 대부분의 제미니바이러스에 관련된 연구는 담배가루이가 서식하기 알맞은 열대 또는 아열대 기후 지역에서 보고된 것이 많다. 아시아 지역에서는 1960년대에 중국의 토마토 재배 지역에서 제미니바이러스가 관찰되었다는 보고가 있었으나, 일부 지역에 국한되어 있었고 피해 정도가 경미해 연구가 거의 이루어지지 않았다. 그러나 최근 10년 사이 아시아 지역 주요 국가들의 토마토 재배 지역에서 담배가루이가 관찰되었으며, 이 후 제미니바이러스가 검출되었다는 보고가 급증하고 있다.Geminiviruses are four genus, Mastrevirus, Curtovirus, Topocuvirus, and Begomovirus, depending on the structure of the genome, the extent of the host, and the type of mediator. It is categorized as causing serious damage to major economic crops worldwide. Begomovirus , a genome of the geniviruses , contains the most diverse strains of virus among the Geminiviruses , Whitefly, Bemisia. tabaci , Gennadius). As a result, most studies on Geminivirus have been reported in tropical or subtropical climates where tobacco flour is suitable. In Asia, there was a report that Geminivirus was observed in tomato growing regions in China in the 1960s, but it was confined to some regions and the degree of damage was minimal. However, in recent decades, tobacco has been observed in tomato cultivation areas of major Asian countries, and there has been a rapid increase in the detection of gemini virus.

베고모바이러스는 게놈(genome)의 구조에 따라 크게 두 개의 게놈(DNA-A 및 DNA-B)을 가지는 바이러스와 하나의 게놈(DNA-A)을 가지는 바이러스로 분류할 수 있다. 두 개의 게놈(DNA-A 및 DNA-B)을 가지는 베고모바이러스에서 DNA-A는 복제 및 전사 기능을 제공하며, DNA-B는 세포 내에서 바이러스 유전체의 이동에 필요한 이동단백질을 암호화하는 기능을 제공한다. 반면에, 하나의 게놈(DNA-A)을 가지는 베고모바이러스 중 몇몇 바이러스들은 복제를 완성하기 위하여 하나의 게놈 이외에 단일가닥의 DNA 분자인 베타위성 DNA(Beta-satellite DNA: DNA β)을 필요로 한다. 현재까지의 문헌 및 보고에 따르면, 베고모바이러스는 아프리카, 아시아를 포함하는 구대륙(동반구, 유럽, 아프리카, 아시아 및 오스트레일리아)에서는 한 개의 게놈(DNA-A)을 갖는 바이러스가 주로 분포되어 있으며, 신대륙(아메리카)에는 두 개의 게놈(DNA-A 및 DNA-B)을 갖는 바이러스가 주로 분포하고 있는 것으로 알려져 있다. 이러한 현상이 나타나는 이유는 베고모바이러스는 한 개의 게놈을 갖는 바이러스에서 두 개의 게놈을 갖는 바이러스로 진화하고 있기 때문으로 예상되고 있다. 지금까지 국내에서 발견된 제미니바이러스는 대부분 하나의 게놈을 갖는 베고모바이러스일 가능성이 높은 것으로 확인되고 있지만, 두 개의 게놈 또는 베타위성 DNA를 갖는 바이러스일 가능성도 배제할 수 없다.Begomovirus can be classified into a virus having two genomes (DNA-A and DNA-B) and a virus having one genome (DNA-A) according to the structure of the genome. In begomoviruses with two genomes (DNA-A and DNA-B), DNA-A provides replication and transcriptional functions, and DNA-B provides the ability to encode the mobile proteins required for the migration of viral genomes within cells. to provide. On the other hand, some of the begomoviruses with one genome (DNA-A) require beta-satellite DNA (DNA β), a single-stranded DNA molecule, in addition to one genome to complete replication. do. According to the literature and reports to date, begomovirus mainly contains viruses with one genome (DNA-A) in the old continent (Eastern Europe, Europe, Africa, Asia and Australia) including Africa and Asia. (America) is known to mainly distribute viruses with two genomes (DNA-A and DNA-B). This phenomenon is expected because begomovirus is evolving from one genome to two genomes. Geminiviruses found in Korea so far have been confirmed to be most likely begomovirus having one genome, but the possibility of being a virus having two genomes or beta satellite DNA cannot be excluded.

한편, 종래의 바이러스 진단법으로는 전자현미경 또는 혈청학적 방법을 주로 사용하였다. 전자현미경을 이용한 방법은 바이러스의 존재를 확인할 수는 있지만 형태학적 특징으로 종을 진단하는 것은 거의 불가능하다. 혈청학적 방법 중 ELISA(enzyme-linked immunosorbent assay) 방법은 가장 일반적으로 사용되는 진단 방법이나 중합효소연쇄반응(polymerase chain reaction, 이하, PCR이라 함) 진단법보다 검출감도가 약 1,000배 정도 낮으며, 항체와 검사시료의 예상하지 못한 비특이적 반응으로 정확한 진단이 실패하는 경우가 자주 발생한다. 최근에는 바이러스를 진단하기 위하여 높은 검출감도와 편리성을 가지고 있는 PCR 방법을 일반적으로 많이 사용하고 있으나, PCR을 이용한 진단 방법은 특이적인 프라이머(primer)의 개발이 매우 중요하며, 증폭된 반응산물을 전기영동(electrophoresis)으로 확인하고, 최종적으로는 염기서열 분석(DNA sequencing)을 해야 하는 일련의 과정을 거쳐야한다. 더불어 이러한 방법은 중합효소연쇄반응기(Thermocycler)와 같은 전문적인 장비 및 이를 운용할 수 있는 전문 인력이 요구되며, 최종 확인을 위한 증폭산물의 염기서열 분석은 고비용 및 고기술력을 요구하는 과정이다. 또한 이러한 일련의 과정들은 수행하는데 있어서 많은 시간이 소요되며 육안으로 식별 가능한 검출법이 아니기 때문에 분석 장비가 갖춰지지 않은 현장에서의 활용력은 현저히 떨어진다. 이와 같이 바이러스를 단시간 내에 효과적으로 검출하기 위해서는, 전문장비 없이 현장에서 실시간으로 검출할 수 있는 방법의 개발이 요구되고 있는 실정이다.On the other hand, an electron microscope or a serological method was mainly used as a conventional virus diagnosis method. Electron microscopy can detect the presence of virus but it is almost impossible to diagnose the species as a morphological feature. Among the serological methods, the enzyme-linked immunosorbent assay (ELISA) is about 1,000 times lower in detection sensitivity than the most commonly used diagnostic methods or the polymerase chain reaction (PCR) And an unexpected nonspecific response of the test sample often leads to failure of accurate diagnosis. Recently, many PCR methods with high detection sensitivity and convenience have been commonly used to diagnose viruses. However, the development of specific primers is very important for the diagnostic method using PCR. It is confirmed by electrophoresis and finally undergoes a series of processes that require DNA sequencing. In addition, this method requires specialized equipment such as a polymerase chain reactor (Thermocycler) and a professional manpower to operate the same, and the sequencing of the amplification product for final confirmation is a process that requires high cost and high technology. In addition, this process is time-consuming to perform and is not visually detectable. Therefore, the ability to use analytical equipment in the field is significantly reduced. As described above, in order to effectively detect viruses in a short time, development of a method capable of real-time detection in the field without specialized equipment is required.

등온증폭법(loop-mediated isothermal amplification, LAMP)은 기존의 PCR 방법과 유사하나 기존 PCR 방법은 변성, 접합, 및 신장의 세 단계를 반복적으로 수행하면서 유전자의 증폭을 시행하기 때문에 반응과정 중 지속적으로 온도의 변화를 필요로 하는 반면, 등온증폭법은 고정된 일정 온도에서 접합 및 신장이 가능한 장점을 가지고 있다. 이는 기존 PCR 방법에 사용되는 DNA Taq 중합효소(DNA Taq polymerase)를 사용하는 대신, 핵산말단가수분해(exonuclease) 기능을 갖고 있는 Bst DNA 중합효소(Bst polymerase)를 사용함으로서 열에 의존하지 않고 DNA의 이중나선 구조의 변성을 유발할 수 있기 때문이다. 따라서 등온증폭법은 유전자를 증폭하는 동안 온도의 변화를 필요로 하지 않기 때문에 전문장비 없이 손쉽게 고정된 온도에서 유전자 증폭을 가능하게 한다.
Loop-mediated isothermal amplification (LAMP) is similar to the conventional PCR method, but the conventional PCR method continuously amplifies genes by repeatedly performing three steps of denaturation, conjugation, and kidney. While temperature change is required, isothermal amplification has the advantage of bonding and stretching at a fixed constant temperature. This is a DNA Taq polymerase (DNA Taq) used in conventional PCR methods. It is because the polymerase) may, instead, by using the Bst DNA polymerase (Bst polymerase) that has a nucleic acid-terminal hydrolysis (exonuclease) function does not depend column lead to denaturation of the double helix structure of DNA that use. Therefore, isothermal amplification does not require a change in temperature during gene amplification, thus enabling gene amplification at a fixed temperature easily without specialized equipment.

본 발명은 상기와 같은 종래 기술상의 문제점을 해결하기 위하여 안출된 것으로, 토마토황화잎말림바이러스를 검출하기 위한 등온증폭반응용 프라이머 조성물, 및 이를 이용한 토마토황화잎말림바이러스 검출방법을 제공하는 것을 그 목적으로 한다.
The present invention has been made to solve the above-mentioned problems in the prior art, to provide a primer composition for isothermal amplification reaction for detecting tomato sulfide leaf virus, and tomato sulfide leaf virus detection method using the same It is done.

그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.
However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

본 발명은 서열번호 1 내지 4로 구성되는, 토마토황화잎말림바이러스(Tomato yellow leaf curl virus)를 검출하기 위한 등온증폭 반응용 프라이머 세트를 제공한다. 또한 본 발명은 서열번호 11 내지 14로 구성되는, 토마토황화잎말림바이러스(Tomato yellow leaf curl virus)를 검출하기 위한 등온증폭 반응용 프라이머 세트를 제공한다.The present invention provides a primer set for isothermal amplification reaction for detecting tomato yellow leaf curl virus consisting of SEQ ID NOs: 1 to 4. In another aspect, the present invention provides a primer set for isothermal amplification reaction for detecting tomato yellow leaf curl virus consisting of SEQ ID NOs: 11 to 14.

본 발명의 일 구현예로, 상기 토마토황화잎말림바이러스는 TYLCV Jordan1 변이주(strain), Jordan3 변이주(strain), Israel 변이주(strain), Spain 변이주(strain), USA 변이주(strain), 및 Korea 변이주(strain)로 이루어진 군에서 선택되는 것을 특징으로 한다.In one embodiment, the tomato sulphide leaf virus is TYLCV Jordan1 strain (train), Jordan3 strain (strain), Israel strain (strain), Spain strain (strain), USA strain (strain), and Korea strain ( strain) is selected from the group consisting of.

또한 본 발명은 상기 프라이머 세트를 포함하는, 토마토황화잎말림바이러스(Tomato yellow leaf curl virus)를 검출하기 위한 등온증폭 반응용 프라이머 조성물을 제공한다.The present invention also provides a primer composition for isothermal amplification reaction for detecting tomato yellow leaf curl virus (Tomato yellow leaf curl virus) comprising the primer set.

본 발명의 일 구현예로, 상기 조성물은 등온증폭 반응용 DNA 중합효소, dNTPs, 및 반응버퍼를 더 포함하는 것을 특징으로 한다.In one embodiment of the invention, the composition is characterized in that it further comprises a DNA polymerase for isothermal amplification reaction, dNTPs, and the reaction buffer.

또한 본 발명은 토마토에서 게놈 DNA(genomic DNA)를 추출하는 단계; 상기 gDNA를 주형으로 상기 조성물을 이용하여 60℃ 내지 70℃에서 30분 내지 2시간 동안 등온증폭법을 수행하여 표적 서열을 증폭시키는 단계; 및 상기 증폭된 산물을 검출하는 단계를 포함하는 토마토황화잎말림바이러스(Tomato yellow leaf curl virus) 검출방법을 제공한다.The present invention also comprises the steps of extracting genomic DNA (genomic DNA) from tomatoes; Amplifying a target sequence by isothermal amplification at 60 ° C. to 70 ° C. for 30 minutes to 2 hours using the gDNA as a template; And it provides a tomato yellow leaf curl virus (Tomato yellow leaf curl virus) detection method comprising the step of detecting the amplified product.

본 발명의 일 구현예로, 상기 토마토황화잎말림바이러스는 TYLCV Jordan1 분리주(isolate), Jordan3 분리주(isolate), Israel 분리주(isolate), Spain 분리주(isolate), USA 분리주(isolate) 및 Korea 분리주(isolate)으로 이루어진 군에서 선택되는 것을 특징으로 한다.In one embodiment of the present invention, the tomato sulfide leaf virus is TYLCV Jordan1 isolate (isolate), Jordan3 isolate (isolate), Israel isolate (isolate), Spain isolate (isolate), USA isolate (isolate) and Korea isolate (isolate) It is characterized in that the selected from the group consisting of.

본 발명의 다른 구현예로, 상기 증폭 산물을 검출하는 단계는 전기영동(electrophoresis) 또는 SYBR Green I을 이용하여 증폭된 DNA를 확인하는 것을 특징으로 한다.In another embodiment, the step of detecting the amplification product is characterized by identifying the amplified DNA using electrophoresis or SYBR Green I.

본 발명의 또 다른 구현예로, 상기 SYBR Green I을 이용하여 증폭된 DNA를 확인하는 방법은 SYBR Green I을 1,000배 내지 10,000배 농도로 사용하여 자연광하에서 육안으로 관찰하는 것을 특징으로 한다.
In another embodiment of the present invention, the method for identifying amplified DNA using the SYBR Green I is characterized by observing the naked eye under natural light using SYBR Green I at a concentration of 1,000 to 10,000 times.

본 발명에 따른 토마토황화잎말림바이러스(Tomato yellow leaf curl virus)를 검출하기 위한 등온증폭 반응용 프라이머 세트, 상기 프라이머 세트를 포함하는 프라이머 조성물 및 이를 이용한 검출방법으로 인하여, 식물 검체로부터 단시간 내에 전문장비 없이 효과적으로 토마토황화잎말림바이러스를 검출할 수 있다. 또한 고농도의 SYBR Green I을 이용하여 자연광하에서 육안으로 신속하게 진단할 수 있다. 따라서 토마토재배농가에 막대한 피해를 줄 수 있는 토마토 감염성 바이러스를 조기에 검출 가능하게 하여 보다 신속하고 효율적인 토마토 바이러스 진단 시스템을 구축할 수 있을 뿐만 아니라, 이를 통해 바이러스 감염으로 인한 경제적 손실을 방지할 수 있을 것으로 기대된다.
Isothermal amplification reaction primer set for detecting tomato yellow leaf curl virus according to the present invention, a primer composition comprising the primer set and a detection method using the same, specialized equipment within a short time from plant specimens Can effectively detect tomato sulphide virus. In addition, high-density SYBR Green I can be used to quickly diagnose with the naked eye under natural light. Therefore, early detection of tomato infectious viruses, which can cause enormous damage to tomato cultivators, can lead to a faster and more efficient tomato virus diagnosis system, which will prevent economic losses from virus infection. It is expected.

도 1 은 토마토황화잎말림바이러스 Jordan1 분리주의 염기서열을 나타낸 도면이다.
도 2 는 토마토황화잎말림바이러스 Jordan3 분리주의 염기서열을 나타낸 도면이다.
도 3 은 토마토황화잎말림바이러스 Israel 분리주의 염기서열을 나타낸 도면이다.
도 4 는 토마토황화잎말림바이러스 Spain 분리주의 염기서열을 나타낸 도면이다.
도 5 는 토마토황화잎말림바이러스 USA 분리주의 염기서열을 나타낸 도면이다.
도 6 은 토마토황화잎말림바이러스 Korea 분리주의 염기서열을 나타낸 도면이다.
도 7 은 토마토황화잎말림바이러스 6분리주의 유전자 염기서열 유사 부위를 나타낸 도면이다.
도 8 은 토마토황화잎말림바이러스의 등온증폭을 위한 프라이머 세트의 염기서열을 나타낸 도면이다.
도 9 는 본 발명의 프라이머 세트를 이용하여 증폭된 유전자를 전기영동으로 확인한 결과를 보여주는 도면이다.
도 10 은 본 발명의 프라이머 세트를 이용하여 증폭된 유전자를 SYBR Green I을 이용하여 자연광원 하에서 확인한 결과를 보여주는 도면이다.
도 11 은 상기 도 10에서와 동일한 증폭산물을 UV 광원 하에서 확인한 결과를 보여주는 도면이다.1 is a diagram showing the nucleotide sequence of the tomato sulfide dried virus Jordan1 isolate.
2 is a diagram showing the nucleotide sequence of the tomato sulfide dried virus Jordan3 isolate.
Figure 3 is a view showing the nucleotide sequence of tomato sulphide leaf virus Israel isolate.
4 is a diagram showing the nucleotide sequence of the tomato sulfide leaf virus Spain isolate.
5 is a diagram showing the nucleotide sequence of tomato sulphide leaf virus USA isolate.
6 is a diagram showing the nucleotide sequence of tomato sulphide leaf virus Korea isolates.
7 is a diagram showing a gene sequence-like site of tomato sulfide leaf dried virus 6 isolates.
Figure 8 is a view showing the base sequence of the primer set for the isothermal amplification of tomato sulfide dried virus.
9 is a view showing the results of the electrophoresis of the amplified gene using the primer set of the present invention.
10 is a diagram showing the results of confirming the gene amplified using the primer set of the present invention under natural light using SYBR Green I.
FIG. 11 is a diagram illustrating a result of confirming the same amplification product as shown in FIG. 10 under a UV light source. FIG.

본 발명자들은 토마토황화잎말림바이러스를 단시간 내에 효과적으로 검출하기 위해 전문장비 없이 현장에서 실시간으로 검출할 수 있는 방법에 대하여 연구한 결과 본 발명을 완성하게 되었다.The present inventors have completed the present invention as a result of research on a method capable of detecting tomato sulphide leaf virus in real time in the field without specialized equipment in order to detect effectively in a short time.

본 발명은 서열번호 1 내지 4 또는 서열번호 11 내지 14로 구성되는, 토마토황화잎말림바이러스(Tomato yellow leaf curl virus)를 검출하기 위한 등온증폭 반응용 프라이머 세트를 제공한다.
The present invention provides a primer set for isothermal amplification reaction for detecting tomato yellow leaf curl virus consisting of SEQ ID NO: 1 to 4 or SEQ ID NO: 11 to 14.

본 발명자들은 단시간 내에 전문장비 없이 토마토황화잎말림바이러스를 검출하기 위하여 등온증폭법(loop-mediated isothermal amplification, LAMP)을 이용하였다. 등온증폭법은 기존의 PCR(polymerase chain reaction)과 달리 유전자를 증폭하기 위한 온도조절을 필요로 하지 않기 때문에 전문장비 없이 유전자를 증폭할 수 있으며 단시간 내에 고농도의 유전자 증폭이 가능하다. 등온증폭법(loop-mediated isothermal amplification, LAMP)을 이용하기 위해서는 4개의 프라이머(F3, B3, FIP, 및 BIP)가 하나의 세트로 작용하여야 하는데, 이중 F3와 FIP는 유전자의 5’ 방향에 결합하는 프라이머이며, B3와 BIP는 3’ 방향에서 역방향으로 결합하는 프라이머이다. 또한 FIP와 BIP는 F2(혹은 B2)와 F1c(혹은 B1c)의 염기서열을 포함하도록 하는 프라이머이다.The present inventors used loop-mediated isothermal amplification (LAMP) to detect tomato sulfide leaf virus without any specialized equipment in a short time. Unlike conventional PCR (polymerase chain reaction), isothermal amplification does not require temperature control to amplify genes, so it is possible to amplify genes without specialized equipment and to amplify high concentrations of genes in a short time. In order to utilize loop-mediated isothermal amplification (LAMP), four primers (F3, B3, FIP, and BIP) must function as one set. F3 and FIP , And B3 and BIP are primers that bind in the reverse direction in the 3 'direction. In addition, FIP and BIP are primers that contain the nucleotide sequence of F2 (or B2) and F1c (or B1c).

기존에 토마토황화잎말림바이러스의 등온증폭을 위한 방법에 관하여 국외에서 보고된 바가 있으나, 매우 한정적인 토마토황화잎말림바이러스의 유전자 정보만을 이용하였기 때문에 다양한 변이주를 검출하는데는 한계가 있다. 본 발명에서는 기존의 토마토황화잎말림바이러스의 기주식물인 토마토에서 감염성을 나타내는 변이주들 뿐만이 아니라 박과(EU143745, EF433426), 콩과(AF071228, X15656), 고추과(AY530931) 등에서도 감염성을 나타내는 것으로 보고된 토마토황화잎말림바이러스 유전자 정보들을 모두 이용하였다. 따라서, 본 발명의 서열번호 1 내지 4 또는 서열번호 11 내지 14로 구성되는 프라이머 세포를 포함하는 조성물은 기존에 국내에서 보고된 변이주들 뿐만이 아니라 새로운 변이주들의 국내 유입 또는 발생 시에도 진단이 가능하다. 상기 프라이머 세트는 서열번호 1 내지 4 또는 서열번호 11 내지 14로 이루어지는 것이 바람직하나, 토마토황화잎말림바이러스를 진단할 수 있는 등온증폭 반응용 프라이머 세트라면 이에 한정되지 않는다.Previously, there has been a report on the method for isothermal amplification of tomato sulphide leaf virus, but there are limitations in detecting various strains because only genetic information of tomato sulphide leaf virus is used. In the present invention, as well as mutant strains that show infectivity in tomato, which is a host plant of the tomato sulfide leaf virus, it is reported to show infectivity not only in the fruit family (EU143745, EF433426), legumes (AF071228, X15656), red pepper (AY530931), etc. The tomato sulphide leaf virus gene information was used. Therefore, the composition comprising the primer cells consisting of SEQ ID NO: 1 to 4 or SEQ ID NO: 11 to 14 of the present invention can be diagnosed at the time of domestic influx or development of new strains as well as mutants previously reported in Korea. Preferably, the primer set includes SEQ ID NOs: 1 to 4 or SEQ ID NOs: 11 to 14, but is not limited to any primer set for isothermal amplification reaction for diagnosing tomato sulfide leaf virus.

또한 본 발명은 토마토에서 게놈 DNA를 추출하는 단계; 상기 게놈 DNA를 주형으로 프라이머 세트, 등온증폭 반응용 DNA 중합효소, dNTPs, 및 반응버퍼를 포함하는 조성물을 이용하여 60℃ 내지 70℃에서 30분 내지 2시간 동안 등온증폭법을 수행하여 표적 서열을 증폭시키는 단계; 및 상기 증폭 산물을 검출하는 단계를 포함하는 토마토황화잎말림바이러스(Tomato yellow leaf curl virus) 검출방법을 제공한다. 토마토로부터 직접 게놈 DNA를 추출할 수도 있지만, 검출을 위한 임상 검체 및 배양 세포 등에도 적용가능하다.The present invention also comprises the steps of extracting genomic DNA from tomatoes; The target sequence was subjected to isothermal amplification at 60 ° C. to 70 ° C. for 30 minutes to 2 hours using a composition comprising a primer set, a DNA polymerase for isothermal amplification reaction, dNTPs, and a reaction buffer as a template. Amplifying; And it provides a tomato yellow leaf curl virus (Tomato yellow leaf curl virus) detection method comprising the step of detecting the amplification product. Genomic DNA can be extracted directly from tomatoes, but is also applicable to clinical specimens and cultured cells for detection.

본 발명의 일실시예에서는 universal 프라이머 세트를 이용하여 등온증폭법을 수행하여 6 종류의 토마토황화잎말림바이러스를 모두 검출 가능하다는 것을 확인하였으며, 또한 SYBR Green I을 1,000배 내지 10,000배 농도로 사용하면 자연광하에서 육안으로 검출 가능하다는 것을 확인하였다(실시예 4 참조).
In one embodiment of the present invention was confirmed that it is possible to detect all six kinds of tomato sulphide leaf virus by performing isothermal amplification using a universal primer set, and also using SYBR Green I at a concentration of 1,000 to 10,000 times It was confirmed that visual detection was possible under natural light (see Example 4).

이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.
Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[[ 실시예Example ]]

실시예Example 1.  One. 토마토황화잎말림바이러스Tomato Sulfate Leaf Virus 유전자 수집 Gene collection

토마토황화잎말림바이러스(Tomato yellow leaf curl virus)의 검출방법을 연구하기 위하여, 토마토황화잎말림바이러스를 수집하고 클로닝(cloning)하고자 하였다. 각각의 바이러스는 노지에서 이병된 토마토에서 PCR(polymerase chain reaction) 방법을 통하여 증폭한 후 pGEM-T easy 유전자 클로닝 벡터(Promega Corporation, USA)에 삽입하여 사용하였으며(HM856911), 국내 미 발병 분리주의 유전자 정보는 미국 National center for biotechnology information(NCBI)의 GenBank에 등록되어 있는 Jordan1의 정보 EU143745, Jordan2의 정보 EF433426, Israel의 정보 X15656, Spain의 정보 AF071228, 및 USA의 정보 AY530931을 이용하였다. 각각의 염기서열은 도 1 내지 도 6에 나타내었다.
In order to study the detection method of tomato yellow leaf curl virus, tomato yellow leaf curl virus was collected and cloned. Each virus was amplified by PCR (polymerase chain reaction) method in tomato diseased in open field and inserted into pGEM-T easy gene cloning vector (Promega Corporation, USA) and used (HM856911). The information used was Jordan1 information EU143745, Jordan2 information EF433426, Israel information X15656, Spain information AF071228, and USA information AY530931 registered in GenBank of the National Center for Biotechnology Information (NCBI). Each base sequence is shown in FIGS. 1 to 6.

실시예Example 2.  2. 프라이머primer 작성 write

토마토황화잎말림바이러스(Tomato yellow leaf curl virus)를 등온증폭법(loop-mediated isothermal amplification, LAMP)을 이용하여 검출하기 위해 PrimerExplorer V4 프로그램을 이용하여 프라이머(primer)를 작성하였다. 등온증폭법을 이용하기 위해서는 4개의 프라이머(F3, B3, FIP, 및 BIP)가 하나의 세트로 작용해야 한다. PrimerExplorer V4를 이용하여 작성된 프라이머 세트는 도 8에 나타내었다.
Primers were prepared using PrimerExplorer V4 program to detect tomato yellow leaf curl virus using loop-mediated isothermal amplification (LAMP). To use isothermal amplification, four primers (F3, B3, FIP, and BIP) must act as one set. Primer sets prepared using PrimerExplorer V4 are shown in FIG. 8.

실시예Example 3. 식물 검체의 채집 3. Collection of plant specimens

실시예 2에서 제작된 프라이머 세트를 토마토황화잎말림바이러스를 검출하는데 사용가능한지 확인하기 위하여, 토마토 재배 온실에서 식물 검체를 채집하였다. 식물 검체는 외형적으로 바이러스 증상이 나타나지 않은 건전주와 토마토황화잎말림바이러스의 증상을 보이는 이병주의 두 가지 집단으로 나누어 채집하였으며, 채집된 검체의 잎 조직 1g에서 전체 게놈 DNA(gDNA)를 추출하고 이후 등온증폭법의 주형으로 사용하였다.
In order to check whether the primer set prepared in Example 2 can be used to detect tomato sulphide leaf virus, plant samples were collected in a tomato growing greenhouse. Plant samples were divided into two groups: healthy strains with no visible viral symptoms and two diseased strains with symptoms of tomato sulphide leaf virus.The entire genome DNA (gDNA) was extracted from 1 g of the leaf tissue of the collected specimens. It was then used as a template for isothermal amplification.

실시예Example 4. 등온증폭법의 시행 및 유용성 확인 4. Isothermal amplification and validation

실시예 2에서 제작된 프라이머 세트를 토마토황화잎말림바이러스를 검출하는데 사용가능한지 확인하기 위하여, 증폭반응용 프라이머 조성물을 제조하였다. 상기 프라이머 조성물의 제조를 위하여 2ul의 10배(10X) Bst 중합효소(polymerase), 반응버퍼(20mM Tris-HCl, 10mM (NH4)2SO4, 10mM KCl, 2mM MgSO4, 0.1% Triton X-100), 1.6ul의 10mM dNTPs(dATP, dTTP, dGTP, dCTP 각각 10mM씩 섞인 혼합물), 0.4ul의 10uM F3와 B3 프라이머(각각 서열번호 1, 2), 1.6ul의 10uM FIP와 BIP 프라이머(각각 서열번호 3, 4), 1ul의 20mM MgSO4, 1ul(8 Unit) Bst 중합효소, 주형 gDNA 1ul, 및 증류수 11.5ul를 반응튜브에 첨가한 후 혼합하였다. 제조된 증폭 반응 조성물을 65℃ 반응 용기에서 1시간 30분동안 반응시켜 등온증폭하였다. 반응이 완료된 후에는 총 20ul의 반응물 중 8ul를 전기영동(electrophoresis)하여 유전자가 증폭되었는지 확인하였다. 그 결과는 도 9에 나타내었다. 이후 동일한 반응물로 재반응(65℃, 1시간 30분)시킨 후에 10,000배로 농축된 SYBR Green I을 반응물 20ul 당 1ul씩 첨가하여 발색 반응을 확인하였다. 그 결과는 도 10 및 도 11에 나타내었다. 상기 바이러스 유전자의 증폭된 양상을 관찰한 전기영동 결과와 SYBR Green I을 이용하여 확인한 결과를 비교하여 유의성을 검증하였다.In order to confirm whether the primer set prepared in Example 2 can be used to detect tomato sulfide leaf virus, a primer composition for amplification reaction was prepared. 2ul of 10 times (10X) Bst polymerase for the preparation of the primer composition, reaction buffer (20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 10mM KCl, 2mM MgSO 4 , 0.1% Triton X- 100), 1.6ul of 10mM dNTPs (mixture of 10mM each of dATP, dTTP, dGTP, dCTP), 0.4ul of 10uM F3 and B3 primers (SEQ ID NOs 1 and 2, respectively), 1.6ul of 10uM FIP and BIP primers (each SEQ ID NO: 3, 4), 1ul of 20mM MgSO 4 , 1ul (8 Unit) Bst polymerase, 1ul of template gDNA, and 11.5ul of distilled water were added to the reaction tube, followed by mixing. The prepared amplification reaction composition was subjected to isothermal amplification by reacting in a 65 ° C. reaction vessel for 1 hour 30 minutes. After the reaction was completed, 8ul out of a total of 20ul of reaction was electrophoresis (electrophoresis) to confirm that the gene was amplified. The results are shown in FIG. After re-reaction with the same reaction (65 ℃, 1 hour 30 minutes) was confirmed by the coloring reaction by adding 1ul per 20ul of SYBR Green I concentrated 10,000 times concentrated. The results are shown in FIGS. 10 and 11. The significance was verified by comparing the results of the electrophoresis of SYBR Green I with the results of amplification of the viral gene.

또한 본 발명자들은 0.4ul의 10uM F3와 B3 프라이머(각각 서열번호 11, 12), 1.6ul의 10uM FIP와 BIP 프라이머(각각 서열번호 13, 14)를 사용하고 증폭 반응 온도 및 재반응 온도를 63 ℃로 하고 나머지 조건은 상기 실험과 동일하게 하여 실험을 수행하였다.In addition, the inventors used 0.4ul of 10uM F3 and B3 primers (SEQ ID NOs: 11 and 12, respectively) and 1.6ul of 10uM FIP and BIP primers (SEQ ID NOs: 13 and 14, respectively), and the amplification reaction temperature and the reaction temperature were 63 ° C. The rest of the conditions were performed in the same manner as the above experiment.

도 9에 나타난 바와 같이, universal primer set를 사용한 경우에는 비감염균주(lane 2)에서는 유전자가 증폭되지 않은 반면, TYLCV Jordan1 변이주(lane 3), Jordan3 변이주(lane 4), Israel 변이주(lane 5), Spain 변이주(lane 6), USA 변이주(lane 7) 및 Korea 변이주(lane 8)을 주형으로 이용한 경우에는 모두 유전자가 증폭된 것을 확인할 수 있었다.As shown in FIG. 9, in the case of using the universal primer set, the gene was not amplified in the non-infected strain (lane 2), while the TYLCV Jordan1 strain (lane 3), Jordan3 strain (lane 4), Israel strain (lane 5), When the Spanish mutant strain (lane 6), USA mutant strain (lane 7) and Korea mutant strain (lane 8) were used as a template, all of the genes were confirmed to be amplified.

또한 도 10 및 도 11에 나타난 바와 같이, SYBR Green I을 고농도로 농축하여 증폭된 유전자(universal primer set sample)를 염색한 경우에도 변이주 모두 유전자가 증폭되어 자연광하에서 녹색을 띠는 것을 확인할 수 있었다.In addition, as shown in Figures 10 and 11, even in the case of staining the amplified gene (universal primer set sample) by concentrating the SYBR Green I to a high concentration it was confirmed that all the mutant strains are amplified green in natural light.

상기 결과들을 통하여 서열번호 1 내지 4의 프라이머 세트는 TYLCV Jordan1 변이주, Jordan3 변이주, Israel 변이주, Spain 변이주, USA 변이주, 및 Korea 변이주 모두 검출하는데 사용가능하다는 것을 확인할 수 있었으며, 또한 SYBR GreenI을 10,000배로 농축하여 사용할 경우 자연광하에서 육안으로 감염여부를 확인할 수 있다는 것을 확인할 수 있었다.
Through the above results, it was confirmed that the primer sets of SEQ ID NOS: 1 to 4 can be used to detect TYLCV Jordan1 strain, Jordan3 strain, Israel variant, Spain variant, USA variant, and Korea variant, and also concentrated SYBR GreenI by 10,000-fold. In case of using it, it was confirmed that the human eye could check the infection under natural light.

전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술 분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야 한다.The above description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration <120> Primer composition for loop-mediated isothermal amplification reaction for detecting Tomato yellow leaf curl virus, and use therof <130> PB12-11047 <150> KR 11/0114822 <151> 2011-11-04 <160> 14 <170> KopatentIn 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Universal TYLCV F3 primer <400> 1 cccatgtaaa gtccagtctt 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Universal TYLCV B3 primer <400> 2 cctgattagt gtgattctgc t 21 <210> 3 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Universal TYLCV FIP primer <400> 3 cagatccacg agtaacatca ctaacgcaac gggatgatat taagc 45 <210> 4 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Universal TYLCV BIP primer <400> 4 attactcaca gagtgggtaa gaggtcttga tattttcatc catccag 47 <210> 5 <211> 2791 <212> DNA <213> TYLCV Jordan 1 strain <400> 5 accggatggc cgcgcctttt tcttttatgt ggtccccacg agggttccac agacgtcact 60 gtcaaccaat caaattgcat cctcaaacgt tagataagtg tttatttgtc tttatatact 120 tggtccccaa gttttttgtc ttgcaatatg tgggacccac ttctaaatga atttcctgaa 180 tctgttcacg gatttcgttg tatgttagct attaaatatt tgcagtccgt tgaggaaact 240 tacgagccca atacattggg ccacgattta attagagatc ttatatctgt tgtaagggcc 300 cgtgactatg tcgaagcgac caggcgatat aatcatttcc acgcccgtct cgaaggttcg 360 ccgaaggctg aacttcgaca gcccatacag cagccgtgct gctgtcccca ttgtccaagg 420 cacaaacaag cgacgatcat ggacgtacag gcccatgtac cgaaagccca gaatatacag 480 aatgtatcga agccctgatg ttccccgtgg atgtgaaggc ccatgtaaag tccaatcttt 540 tgagcaacgg gatgatatta agcatactgg tattgttcgt tgtgttagtg atgttactcg 600 tggatctgga attactcaca gagtgggtaa gaggttctgt gttaaatcga tatatttttt 660 aggtaaagtc tggatggatg aaaatatcaa gaagcagaat cacactaatc aggtcatgtt 720 cttcttggtc cgtgatagaa ggccctatgg aaacagtcca atggattttg gacaggtttt 780 taatatgttc gataatgagc ccagtaccac aaccgtgaag aatgatttgc gggataggtt 840 tcaagtgatg aggaaatttc atgctacagt tattggtggg ccttctggaa tgaaggaaca 900 ggcattagtt aagagatttt ttaaaattaa cagtcatgta acttataatc atcaggaggc 960 agccaagtac gagaaccata ctgaaaacgc cttgttattg tatatggcat gtacgcatgc 1020 ctctaatcca gtgtatgcaa ctatgaaaat acgcatctat ttctatgatt caatatcaaa 1080 ttaataaaat ttatatttta tatcatgagt ttctgttaca tttattgtgt tttcaagtac 1140 atcatacaat acatgatcaa ctgctctgat tacattatta atagaaatta caccaagact 1200 atctaaatac ttaagaactt gatatctaaa tactcttaag aaacgaccag tctgaggctg 1260 taatgtcgtc caaattcgga agttgagaaa acatttgtga atccccatta ccttcctgat 1320 gttgtggttg aatcttatct gaatggaaat gatgtcgtgg ttcattagaa atggcctctg 1380 gctgtgttct gttatcttga aatagagggg attgtttatc tcccagataa aaacgccatt 1440 ctctgcctga ggagcagtga tgagttcccc tgtgcgtgaa tccatgatta ttgcagttga 1500 gatggaggta gtatgagcag ccacagtcta ggtctacacg cttacgcctt attggtttct 1560 tcttggctat cttgtgttgg accttgattg atacttgcga acagtggctc gtagagggtg 1620 acgaaggttg cattcttgag agcccaattt ttcaaggata tatttttttc ttcgtctaga 1680 taatccctat atgaggaggt aggtcctgga ttgcagagga agatagtggg aattccccct 1740 ttaatttgaa tgggcttccc gtactttgtg ttgctttgcc agtccctttg ggcccccatg 1800 aattccttga agtgctttaa gtaatgcggg tctacgtcat caatgacgtt gtaccacgca 1860 tcattattgt acacctttgg gcttaggtct agatgtccac ataaataatt atgtgggcct 1920 agagacctgg cccacattgt tttgcctgtt ctgctatcac cctcaatgac aatacttatg 1980 ggtctccatg gccgcgcagc ggaatacacg acgttctcgg acacccatac ttcaagctca 2040 tctggaactt gattaaaaga tgaagataaa aagggagaaa tataaggagc cggaggctcc 2100 tgaaaaattc tatctaaatt actatttaaa ttatgaaact gtaaaatata atctttcggt 2160 actaattctt taatgattct aagagcctct gacttactgc ctgagttaag agctgcggcg 2220 taagcgtcat tggctgactg ctgaccccca cgtgcagatc gtccgtcgat ctgaaactca 2280 ccccagtcga cggtgtctcc gtccttatcg acataagact tgacgtctga actggattta 2340 gctccctgaa tgtttggatg gaaatgtgct gccctgcttg gggataccag gtcgaagaat 2400 cgctgatttt ggcatttgaa tttcccttca aattggataa gcacatggag atgtggttcc 2460 ccattctcgt ggagttctct gcaaactttg atgtattttt tatttgttgg ggtttctagg 2520 ttttttaatt gggaaagtgc ttcttctttt gttaaggagc aatgaggata tgtgaggaaa 2580 taatttttgc aatttatttg gaagcgctta ggaggagcca tatggtcaat gagtaccgat 2640 tgaccaagat ttttacactt atccctggtg tatcggtact caacatatag tgagtaccaa 2700 atggcatttt ggtaataaca taaaagtaca ttgcaattca aaattcaaaa ttaaaaaatc 2760 aaatcattaa agcggccatc cgtataatat t 2791 <210> 6 <211> 2781 <212> DNA <213> TYLCV Jordan 3 strain <400> 6 accggatggc cgcgcctttt ccttttatgt ggtccccacg agggttacac agacgtcact 60 gtcaaccaat caaattgcat cctcaaacgt tagataagtg tgcatttgtc tttatatact 120 tggtccccaa gttttttgtc ttgcaatatg tgggacccac ttcttaatga atttcctgaa 180 tctgttcacg gatttcgttg tatgttagct attaaatatt tgcagtccgt tgaggaaact 240 tacgagccca atacattggg ccacgattta attagggatc ttatatctgt tgtaagggcc 300 cgtgactatg tcgaagcgac caggcgatat aatcatttcc acgcccgtct cgaaggttcg 360 ccgaaggctg aacttcgaca gcccatacag cagccgtgct gctgtcccca ttgtccaagg 420 cacaaacaag cgacgatcat ggacgtacag gcccatgtac cgaaagccca gaatatacag 480 aatgtatcga agccctgatg ttcctcgtgg atgtgaaggc ccatgtaaag tccagtctta 540 tgagcaacgg gatgatatta agcatactgg tattgttcgt tgtgttagtg atgttactcg 600 tggatctgga attactcaca gagtgggtaa gaggttctgt gttaaatcga tatatttttt 660 aggtaaagtc tggatggatg aaaatatcaa gaagcagaat cacactaatc aggtcatgtt 720 cttcttggtc cgtgatagaa ggccctatgg aagcagccca atggattttg gacaggtttt 780 taatatgttc gataatgagc ccagtaccgc aaccgtgaag aatgatttgc gggataggtt 840 tcaagtgatg aggaaatttc atgcttcagt tattggtggg ccctctggaa tgaaggaaca 900 ggcattagtt aagagatttt ttaaaattaa cagtcatgta acttataatc atcaggaggc 960 agccaagtac gagaaccata ctgaaaacgc cttgttattg tatatggcat gtacgcatgc 1020 ctctaatcca gtgtatgcaa ctatgaaaat acgcatctat ttctatgatt caatatcaaa 1080 ttaataaaat ttatatttta tatcatgagt ttctgttaca tttattgtgt tttcaagtac 1140 atcatacaat acatgatcca ctgctctgat tacattgtta atggaaatta caccaagact 1200 atctaaatac ttaagaactc catatctaaa tactcttaag aaacgaccag tctgaggctg 1260 taatgtcgtc caaattcgga agttgagaaa acatttgtga atccccatta ccttcctgat 1320 gttgtggttg aatcttatct gaatggaaat gatgtcgtgg ttcattagaa atggcctctg 1380 actgtgttct gttatcttga aatagagggg attgtttatc tcccagataa aaacgccatt 1440 ctctgcctga ggagcagtga tgagttcccc tgtgcgtgaa tccatgatta ttgcagttga 1500 ggtcgaggta gtatgagcat ccacagtcta ggtctacacg cttacgcctt attggtttct 1560 tcttggctat cttgtgttgg accttgattg atacttgcga acagtggctc gtagagggtg 1620 acgaaggttg cattcttgag agcccaattt ttcaaggata tgtttttttc ttcgtctaga 1680 tattccctat atgaggaggt aggtcctgga ttgcagagga agatagtggg aattccccct 1740 ttaatttgaa tgggcttccc gtactttgtg ttgctttgcc agtccctctg ggcccccatg 1800 aattccttta agtgttttaa attatgcggg tctacgtcat caatgacgtt ataccacgca 1860 tcattactgt acacctttgg gcttaggtct agatgtccac ataaataatt atgtgggcct 1920 agagacctgg cccacattgt tttgcctgtt ctgctatcac cctcaatgac aatacttatg 1980 ggtctccatg gccgcgcagc ggataacacg acgttctcgg cgacccactc ttcaagttca 2040 tctggaactt gattaaaaga agaagaaaga aatggagaaa cataaacttc taaaggagga 2100 ctaaaaatcc tatctaaatt tgaacttaaa ttatgaaatt gtaaaatata gtcctttggg 2160 gccttctctt ttaatatatt gagggcctcg gatatactgc ctgaattgag tgcttcggca 2220 tatgcgtcgt tggcagattg ctgacctcct ctagctgatc tgccatcgat ttggaaaact 2280 ccaaaatcaa tgaagtctcc gtctttctcc acgtaggtct tgacatctgt tgagctctta 2340 gctgcctgaa tgttcggatg gaaatgtgct gacctgttag gggataccag gtcgaagaac 2400 cgttggttct tacattggta ttttccttcg aattggataa gcacatggag atgtggttcc 2460 ccattctcgt ggagttctct gcaaactttg atgtattttt tatttgttgg ggtttctatg 2520 ttttttaatt gggaaagtgc ttcctcttta gagagagaac aattgggata tgttaggaaa 2580 taatttttgg catatatttt aaataaacga ggcatgttga tatgaatcgg tgtccctcaa 2640 agctctatgg caatcggtgt atcggtgtct tacttatacc tggacaccta atggctattt 2700 ggtaatttca taaatgttca ttgcaattca aaattcaaaa ttcaaaaatc aaatcattaa 2760 agcggccatc cgtataatat t 2781 <210> 7 <211> 2787 <212> DNA <213> TYLCV Israel strain <400> 7 gttgaaatga atcggtgtcc ctcaaagctc tatggcaatc ggtgtatcgg tgtcttactt 60 atacttggac acctaatggc tatttggtaa tttcataaat gttcatttca attcaaaatt 120 caaaattcaa aaatcaaatc attaaagcgg ccatccgtat aatattaccg gatggccgcg 180 ccttttcctt ttatgtggtc cccacgaggg ttacacagat gttattgtca accaatcaaa 240 ttgcattctc aaacgttaga taagtgttca tttgtcttta tatacttggt ccccaagttt 300 tttgtcttgc aatatgtggg acccacttct taatgaattt cctgaatctg ttcacggatt 360 tcgttgtatg ttagctatta aatatttgca gtccgttgag gaaacttacg agcccaatac 420 attgggccac gatttaatta gggatcttat atctgttgta agggcccgtg actatgtcga 480 agcgaccagg cgatataatc atttccacgc ccgtctcgaa ggttcgccga aggctgaact 540 tcgacagccc atacagcagc cgtgctgctg tccccattgt ccaaggcaca aacaagcgac 600 gatcatggac gtacaggccc atgtaccgaa agcccagaat atacagaatg tatcgaagcc 660 ctgatgttcc ccgtggatgt gaaggcccat gtaaagtcca gtcttatgag caacgggatg 720 atattaagca tactggtatt gttcgttgtg ttagtgatgt tactcgtgga tctggaatta 780 ctcacagagt gggtaagagg ttctgtgtta aatcgatata ttttttaggt aaagtctgga 840 tggatgaaaa tatcaagaag cagaatcaca ctaatcaggt catgttcttc ttggtccgtg 900 atagaaggcc ctatggaaac agcccaatgg attttggaca ggtttttaat atgttcgata 960 atgagcccag taccgcaacc gtgaagaatg atttgcgtga taggtttcaa gtgatgagga 1020 aatttcatgc tacagttatt ggtgggccct ctggaatgaa ggaacaggca ttagttaaga 1080 gattttttaa aattaacagt catgtaactt tatttatatt cattcaggag gcagcaaagt 1140 acgagaacca tactgaaaac gccttgttat tgtatatggc atgtacgcat gcctctaatc 1200 cagtgtatgc aactatgaaa atacgcatct atttctatga ttcaatatca aattaataaa 1260 atttatattt tatatcatga gtttctgtta catttattgt gttttcaagt acatcataca 1320 atacatgatc aactgctctg attacattgt taatggaaat tacaccaaga ctatctaaat 1380 acttaagaac ttcatatcta aatactctta agaaatgacc agtctgaggc tgtaatgtcg 1440 tccaaattcg gaagtcgaga aaacatttgt gaatccccat taccttcctg atgttgtggt 1500 tgaatcttat ctgaatggaa atgatgtcgt ggttcattag aaatggcctc tggctgtgtt 1560 ctgttatctt gaaatagagg ggattgttta tctcccagat aaaaacgcca ttctctgcct 1620 gaggagcagt gatgagttcc cctgtgcgtg aatccatgat tattgcagtt gaggtggagg 1680 tagtatgagc agccacagtc taggtctaca cgcttacgcc ttattggttt cttcttggct 1740 atcttgtgtt ggaccttgat tgatacttgc gaacagtggc tcgtagaggg tgacgaaggt 1800 tgcattcttg agagcccaat ttttcaagga tatgtttttt tcttcgtcta gatattccct 1860 atatgaggag gtaggtcctg gattgcagag gaagatagtg ggaattcccc ctttaatttg 1920 aatgggcttc ccgtactttg tgttgctttg ccagtccctc tgggccccca tgaattcctt 1980 gaagtgcttt aaataatgcg ggtctacgtc atcaatgacg ttgtaccacg catcattact 2040 gtacaccttt gggcttaggt ctagatgtcc acataaataa ttatgtgggc ctagagacct 2100 ggcccacatt gttttgcctg ttctgctatc accctcaatg acaatactta tgggtctcca 2160 tggccgcgca gcggaataca cgacgttctc ggcgacccac tcttcaagtt catctggaac 2220 ttgattaaaa gaagaagaaa gaaatggaga aacataaact tctaaaggag gactaaaaat 2280 cctatctaaa tttgaactta aattatgaaa ttgtaaaata tagtcctttg gggccttctc 2340 ttttaatata ttgagggcct cggatttact gcctgaattg agtgcttcgg catatgcgtc 2400 gttggcagat tgctgacctc ctctagctga tctgccatcg atttgggaaa ctccaaaatc 2460 aatgaagttt ccgtctttct ccacgtaggt cttgacatct gttgagctct tagctgcctg 2520 aatgttcgga tggaaatgtg ctgacctgtt tggggatacc aagtcgaaga accgttggtt 2580 cttacattgg tatttgcctt cgaattggat aagcacatgg agatgtggtt ccccattctc 2640 gtggagttct ttgcaaactt tgatgtattt tttatttgtt ggggtttcta gtttttttaa 2700 ttgggaaagt gcttcctctt tagagagaga acaattggga tatgttagga aataattttt 2760 ggcatatatt ttaaataaac gaggcat 2787 <210> 8 <211> 2791 <212> DNA <213> TYLCV Spain strain <400> 8 taatattacc ggatggccgc gccttttcct tttatgtggt ccccatgagg gttcaacaga 60 cgtcactgtc aaccaatcaa attgcatcct caaacgttag ataagtgttc atttgtcttt 120 atatacttgg tccccaagta gtttgtcttg caatatgtgg gatccacttc taaatgaatt 180 tcctgaatct gttcacggat ttcgttgtat gttagctatt aaatatttgc agtccgttga 240 ggaaacttac gagcccaata cattgggcca cgatttaatt agggatctta tatctgttgt 300 aagggcccgt gactatgtcg aagcgaccag gcgatataat catttccacg cccgcctcga 360 aggttcgccg aaggctgaac ttcgacagcc catacagcag ccgtgctgct gtccccattg 420 tccaaggcac aaacaagcga cgatcatgga cgtacaggcc catgtaccgg aagcccagaa 480 tatacagaat gtatcgaagc actgatgttc cccgtggatg tgaaggccca tgtaaagtac 540 agtcttatga gcaacgggat gatattaagc atactggtat tgttcgttgt gttagtgatg 600 ttactcgtgg atctggaatt actcacagag tgggtaagag gttctgtgtt aaatcgatat 660 attttttagg taaagtctgg atggatgaaa atatcaagaa gcagaaccac actaatcagg 720 tcatgttctt cttggtccgt gatagaaggc cctatggaaa cagcccaatg gattttggac 780 aggtttttaa tatgttcgat aatgagccca gtaccgcaac agtgaagaat gatttgcggg 840 ataggtttca agtgatgagg aaatttcatg ctacagttat tggtgggccc tctggaatga 900 aggaacaggc attagttaag aggtttttta gaattaacag tcatgtaact tataatcatc 960 aggaggcagc caagtacgag aaccatactg aaaacgcctt gttattgtat atggcatgta 1020 cgcatgcctc taatccagtg tatgctacta tgaaaatacg catctatttc tatgattcaa 1080 tatcaaatta ataaaattta tattttatat catgagtttc tgttacattt attgtgtttt 1140 caagtacatc atacaataca tgatcaactg ctctgattac attgttaatt gaaattacac 1200 caatactatc taaatactta agaacttgat gtctaaatac ttttaagaaa cgaccagtct 1260 gaggctgtaa tgtcgtccaa attcggaagt tgagaaaaca tttgtgaatc cccaatacct 1320 tcctgatgtt gtggttgaat cttatctgaa tggaaatgat gtcgtggttc attagaaatg 1380 gcctctggct gtgttctgtt atcttgaaat agaggggatt gtttatctcc cagataaaaa 1440 cgccattctc tgcctgagga gcagtgatga gttcccctgt gcgtgaatcc atgattgttg 1500 cagttgaggt gttggtagta tgagcagcca cagtctaggt ctatacgctt acgccttatt 1560 ggtttcttct tggctatctt gtgttggacc ttgattgata cttgcgaaca gtggctcgta 1620 gagggtgacg aaggttgcat tcttgagagc ccaatttttc aaggatatgt ttttttcttc 1680 gtctagatat tccctatatg aggaggtagg tcctggattg cagaggaaga tagtgggaat 1740 tcccccttta atttgaatgg gcttcccgta ctttgtgttg ctttgccagt ccctctgggc 1800 ccccatgaat tctttgaagt gctttaaata atgcgggtct acgtcatcaa tgacgttgta 1860 ccatgcatca ttactgtaca cctttgggct taggtctaga tgtccacata aataattatg 1920 tgggcctaga gacctggccc acattgtttt gcctgttctg ctatcaccct caatgacaat 1980 actattaggt ctccatggcc gcgcagcgga agacacgacg ttctcggaca cccatacttc 2040 aagttcatct ggaacttgat taaaagatga agataaaaag ggagaaatat aaggagccgg 2100 aggctcctga aaaattctat ctaaattact atttaaatta tgaaattgta aaatataatc 2160 tttcggtatt aattctttaa tgattctaag agcctctgac ttactgcctg agttaagagc 2220 tgcggcgtaa gcgtcattgg ctgactgctg acccccacgt gcagatcgtc cgtcgatctg 2280 aaactcaccc cagtcgacgg tgtctccgtc cttatcgaca taagacttga cgtctgaact 2340 ggatttagct ccctgaatgt ttggatggaa atgtgctgcc ctgcttgggg ataccaggtc 2400 gaagaatcgc tgattttggc acttgaattt cccttcaaat tggataagca catggagatg 2460 tggttcccca ttctcgtgga gttctctgca aactttgatg tattttttat ttgttggggt 2520 ttctaggttt tttaattggg aaagtgcttc ttctttcgtt aaggagcact taggatatgt 2580 gaggaaataa tttttgcaat ttatttggaa gcgcttaggg ggagccatat ggtcaatgag 2640 taccgattga ccaagatttt tacacttatc cctggtgtat cggtactcaa tatataggga 2700 gtaccaaatg gcatattggt aattttgtaa aagtacaatg caattcaaaa ttcaaaattc 2760 aaaaatcaaa tcattaaagc ggccatccgt a 2791 <210> 9 <211> 2781 <212> DNA <213> TYLCV USA strain <400> 9 accggatggc cgcgcctttt ccttttatgt ggtccccacg agggttacac agacgtcact 60 gtcaaccaat caaattgcat cctcaaacgt tagataagtg ttcatttgtc tttatatact 120 tggtccccaa gtattttgtc ttgcaatatg tgggacccac ttctaaatga atttcctgaa 180 tctgttcacg gatttcgttg tatgttagct attaaatatt tgcagtccgt tgaggaaact 240 tacgagccca atacattggg ccacgattta attagggatc ttatatctgt tgtaagggcc 300 cgtgactatg tcgaagcgac caggcgatat aatcatttcc acgcccgtct cgaaggttcg 360 ccgaaggctg aacttcgaca gcccatacag caaccgtgct gctgtcccca ttgtccaagg 420 cacaaacaag cgacgatcat ggacgtacag gcccatgtac cgaaagccca gaatatacag 480 aatgtatcga agccctgatg ttccccgtgg atgtgaaggc ccatgtaaag tccagtctta 540 tgagcagcgg gatgatatta agcacactgg tgttgttcgt tgtgttagtg atgttactcg 600 tggatctgga attactcaca gagtcggtaa gaggttctgt gttaaatcga tatatttttt 660 aggtaaagtc tggatggatg aaaatatcaa gaagcagaat cacactaatc aggtcatgtt 720 ctttttggtc cgtgatagaa ggccctatgg aagcagtcca atggattttg gacaggtttt 780 taatatgttc gataatgagc ccagtaccgc aactgtgaag aatgatttgc gtgataggtt 840 tcaagtgatg aggaaatttc atgctacagt tattggtggg ccctctggaa tgaaggaaca 900 ggcattagtt aagagatttt ttaaaattaa cagtcatgta acttataatc atcaggaggc 960 agccaagtac gagaaccata ctgaaaacgc cttgttattg tatatggcat gtacgcatgc 1020 ctctaatcca gtgtatgcaa ctatgaaaat acgcatctat ttctatgatt caatatcaaa 1080 ttaataaaat ttatatttta tatcatgagt ttctgttaca tttattgtgt tttcaagtac 1140 atcatacaat acatgatcca ctgctctgat tacattgtta atggaaatta caccaagact 1200 atctaaatac ttaagaactt catatctaaa tactcttaag aaacgaccag tctgaggctg 1260 taatgtcgtc caaattcgga agttgagaaa acatttgtga atccccatta ccttcctgat 1320 gttgtggttg aatcttatct gaatggaaat gatgtcgtgg ttcattagaa atggcctctg 1380 gctgtggtct gttatcttga aatagagggg attgtttatc tcccaaataa aaacgccatt 1440 ctctgcctga ggagcagtga tgagttcccc tgtgcgtgaa tccatgatta ttgcagttga 1500 ggtggaggta gtatgagcag ccacagtcta ggtctacacg cttacgcctt attggtttct 1560 tcttggctat cttgtgttgg accttgattg atacttgcga acagtggctc gtagagggtg 1620 acgaaggttg cattcttgag agcccaattt ttcaaggata tgtttttttc ttcgtctaga 1680 tattccctat atgaggaggt aggtcctgga ttgcagagga agatagtggg aattccccct 1740 ttaatttgaa tgggcttccc gtactttgtg ttgctttgcc agtccctctg ggcccccatg 1800 aattccttga agtgctttaa ataatgcggg tctacgtcat caatgacgtt gtaccacgca 1860 tcattactgt acacctttgg gcttaggtct agatgtccac ataaataatt atgtgggcct 1920 agagacctgg cccacattgt tttgcctgtt ctgctatcac cctcaatgac aatacttatg 1980 ggtctccatg gccgcgcagc ggaagacacg acgttctcgg cgacccactc ttcaagttca 2040 tctggaactt gattaaaaga agaagaaaga aatggagaaa cataaacttc taaaggagga 2100 ctaaaaatcc tatctaaatt tgaatttaaa ttatgaaatt gtaaaatata gtcctttggg 2160 gccttttctt ttaatatatt gagggcctcg gatttactgc ctgaattgag tgctccggcg 2220 tatgcgtcgt tggcagattg ctgacctcct ctagctgatc tgccatcgat ttggaaaact 2280 ccaaaatcaa tgaagtctcc gtctttctcc acgtaggtct tgacatctgt tgagctctta 2340 gctgcctgaa tgtttggatg gaaatgtgct gacctgtttg gggataccag gtcgaagaac 2400 cgttggttct tacattggta tttgccttcg aattggataa gcacatggag atgtggttcc 2460 ccattctcgt ggaattctct gcaaactttg atgtattttt tatttgttgg ggtttctatg 2520 ttttttaatt gggaaagtgc ttcctcttta gagagagaac aattgggata tgttaggaaa 2580 taattcttgg catatatttt aaataaacga ggcatgttga aatgaatcgg tgtccctcaa 2640 agctctatgg caatcggtgt atcggtgtct tactaatacc tcgacaccta atggcaattt 2700 ggtaatttca taaatgttca ttgcaattca aaattcaaaa ttcaaaaatc aaatcattaa 2760 agcggtcatc cgtataatat t 2781 <210> 10 <211> 2774 <212> DNA <213> TYLCV Korea strain <400> 10 accggatggc cgcgcctttt ccttttatgt ggtccccacg agggttacac agacgtcacc 60 gtcaaccaat caaattgcat cctcaaacgt tagataagtg ttcatttgtc tttatatact 120 tggtccccaa gttttttgtc ttgcaatatg tgggacccac ttcttaatga gtttcctgaa 180 tctgttcacg gatttcgttg tatgttagct attaaatatt tgcagtccgt tgaggaaact 240 tacgagccca atacattggg ccacgattta attagggatc ttatatctgt tgtaagggcc 300 cgtgactatg tcgaagcgac caggcgatat aatcatttcc acgcccgtct cgaaggttcg 360 ccgaaggctg aacttcgaca gcccatacag cagccgtgct gctgtcccca ttgtccaagg 420 cacaaacaag cgacgatcat ggacgtacag gcccatgtac cgaaagccca gaatatacag 480 aatgtatcga agccctgatg ttccccgtgg atgtgaaggc ccatgtaaag tccagtctta 540 tgagcaacgg gatgatatta agcatactgg tattgttcgt tgtgttagtg atgttactcg 600 tggatctgga attactcaca gagtgggtaa gaggttctgt gttaaatcga tatatttttt 660 agggaaagtc tggatggatg aaaatatcaa gaagcagaat cacactaatc aggtcatgtt 720 cttcttagtc cgtgatagaa ggccctatgg aagcagccca atggattttg gacaggtttt 780 taatatgttc gataatgagc ccagtaccgc aaccgtgaag aatgatttgc gggataggtt 840 tcaagtgatg aggaaatttc atgctacagt tattggtgga ccctctggaa tgaaggaaca 900 ggcattagtt aagagatttt ttaaaattaa cagtcatgta acttataatc atcaggaggc 960 agccaagtac gagaaccata ctgaaaacgc cttgttactg tatatggcat gtacgcatgc 1020 ctcgaatcca gtgtatgcaa ctatgaaaat acgcatctat ttctatgatt caatatcaaa 1080 ttaataaaat ttatatttta tatcatgagt ttctgttaca tttattgtgt tttcaagtac 1140 atcatacaat acatgatcaa ctgatctgat tacattgtta atggaaatta caccaagact 1200 atctaaatac ttaagaactt catatctaaa tactcttaag aaatgaccag tctgaggatg 1260 taatgtcgtc caaattcgga agttgagaaa acatttgtga atccccatga ccttcctgat 1320 gttgtggttg aatcttatct gaatggaaat gatgtcgtgg ttcattagaa atggcctctg 1380 gctgtgttct gttatcttga aatagagggg attgtttatc tcccagataa aaacgccatt 1440 ctctgcctga ggagcagtga tgagttcccc tgtgcgtgaa tccatgatta ttgcagttga 1500 ggtggaggta gtatgagcag ccacagtcta ggtctacacg cttacgcctt attggtttct 1560 tcttggctat cttgtgttgg accttgattg atacttgcga acagtggctc gtagagggtg 1620 acgaaggttg cattcttgag agtccaattt ttcaaggata tgtttttttc ttcgtctaga 1680 tattccttat atgaggaggt aggtccttga ttgcagagga agatagtggg aattccccct 1740 ttaatttgaa tgggcttccc gtactttgtg ttgctttgcc agtccctttg ggcccccata 1800 aattccttga agtgctttaa ataatgcggg tctacgtcat caatgacgtt gtaccacgca 1860 tcattgctgt acacctttgg gcttaggtct agatgtccac ataaataatt atgtgggcct 1920 agagacctgg cccacattgt tttccctgtt ctgctatcac cctcaatgac aatacttatg 1980 ggtctccatg gccgcgcagc ggaagacacg acgttctcgg cgacccactc ttcaagttca 2040 tctggaactt gattaaaaga agaagaaaga aatggagaaa cataaacttc taaaggagga 2100 ctaaaaatcc tatctaaatt tgaacttaaa ttatgaaatt gtaaaatata gtcctttggg 2160 gccttctctt ttaatatatt gagggcctcg gatttactgc ctgaattgag tgcttcggca 2220 tatgcgtcgt tggcagattg ctgacctcct ctagctgatc tgccatcgat ttggaaaact 2280 ccaaaatcaa tgaagtctcc gtctttctcc acgtaggtct tgacatctgt tgagctctta 2340 gctgcctgaa tgttcggatg gaaatgtgct gacctgtttg gggataccag gtcgaagaac 2400 cgttggttct tacattggta tttgccttcg aattggataa gcacatggag atgtggttcc 2460 ccattctcgt ggagttcttt gcaaactttg atgtattttt tatttgttgg ggtttctagg 2520 ttttttaatt gggaaagtgc ttcctcttta gagagagaac aattgggata tgttaggaaa 2580 taatttttgg catatatatt aaataaacga ggcatgttga aatgaatcgg tgtccctcaa 2640 agctctatgg caatcggtgt atcggtgtct tacttatacc tggacaccta atggctattt 2700 ggtaatttca tgaatgttca ttgtaattca aaattcaaaa atcaaatcat taaagcggcc 2760 atccgtataa tatt 2774 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Universal TYLCV F3 primer <400> 11 gtatcgaagc cctgatgtt 19 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Universal TYLCV B3 primer <400> 12 cctgattagt gtgattctgc t 21 <210> 13 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Universal TYLCV FIP primer <400> 13 cacaacgaac aataccagta tgcttgaagg cccatgtaaa gtcc 44 <210> 14 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Universal TYLCV BIP primer <400> 14 ctggaattac tcacagagtg ggtgatattt tcatccatcc agact 45 <110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration <120> Primer composition for loop-mediated isothermal amplification          reaction for detecting Tomato yellow leaf curl virus, and use          therof <130> PB12-11047 <150> KR 11/0114822 <151> 2011-11-04 <160> 14 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Universal TYLCV F3 primer <400> 1 cccatgtaaa gtccagtctt 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Universal TYLCV B3 primer <400> 2 cctgattagt gtgattctgc t 21 <210> 3 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Universal TYLCV FIP primer <400> 3 cagatccacg agtaacatca ctaacgcaac gggatgatat taagc 45 <210> 4 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Universal TYLCV BIP primer <400> 4 attactcaca gagtgggtaa gaggtcttga tattttcatc catccag 47 <210> 5 <211> 2791 <212> DNA <213> TYLCV Jordan 1 strain <400> 5 accggatggc cgcgcctttt tcttttatgt ggtccccacg agggttccac agacgtcact 60 gtcaaccaat caaattgcat cctcaaacgt tagataagtg tttatttgtc tttatatact 120 tggtccccaa gttttttgtc ttgcaatatg tgggacccac ttctaaatga atttcctgaa 180 tctgttcacg gatttcgttg tatgttagct attaaatatt tgcagtccgt tgaggaaact 240 tacgagccca atacattggg ccacgattta attagagatc ttatatctgt tgtaagggcc 300 cgtgactatg tcgaagcgac caggcgatat aatcatttcc acgcccgtct cgaaggttcg 360 ccgaaggctg aacttcgaca gcccatacag cagccgtgct gctgtcccca ttgtccaagg 420 cacaaacaag cgacgatcat ggacgtacag gcccatgtac cgaaagccca gaatatacag 480 aatgtatcga agccctgatg ttccccgtgg atgtgaaggc ccatgtaaag tccaatcttt 540 tgagcaacgg gatgatatta agcatactgg tattgttcgt tgtgttagtg atgttactcg 600 tggatctgga attactcaca gagtgggtaa gaggttctgt gttaaatcga tatatttttt 660 aggtaaagtc tggatggatg aaaatatcaa gaagcagaat cacactaatc aggtcatgtt 720 cttcttggtc cgtgatagaa ggccctatgg aaacagtcca atggattttg gacaggtttt 780 taatatgttc gataatgagc ccagtaccac aaccgtgaag aatgatttgc gggataggtt 840 tcaagtgatg aggaaatttc atgctacagt tattggtggg ccttctggaa tgaaggaaca 900 ggcattagtt aagagatttt ttaaaattaa cagtcatgta acttataatc atcaggaggc 960 agccaagtac gagaaccata ctgaaaacgc cttgttattg tatatggcat gtacgcatgc 1020 ctctaatcca gtgtatgcaa ctatgaaaat acgcatctat ttctatgatt caatatcaaa 1080 ttaataaaat ttatatttta tatcatgagt ttctgttaca tttattgtgt tttcaagtac 1140 atcatacaat acatgatcaa ctgctctgat tacattatta atagaaatta caccaagact 1200 atctaaatac ttaagaactt gatatctaaa tactcttaag aaacgaccag tctgaggctg 1260 taatgtcgtc caaattcgga agttgagaaa acatttgtga atccccatta ccttcctgat 1320 gttgtggttg aatcttatct gaatggaaat gatgtcgtgg ttcattagaa atggcctctg 1380 gctgtgttct gttatcttga aatagagggg attgtttatc tcccagataa aaacgccatt 1440 ctctgcctga ggagcagtga tgagttcccc tgtgcgtgaa tccatgatta ttgcagttga 1500 gatggaggta gtatgagcag ccacagtcta ggtctacacg cttacgcctt attggtttct 1560 tcttggctat cttgtgttgg accttgattg atacttgcga acagtggctc gtagagggtg 1620 acgaaggttg cattcttgag agcccaattt ttcaaggata tatttttttc ttcgtctaga 1680 taatccctat atgaggaggt aggtcctgga ttgcagagga agatagtggg aattccccct 1740 ttaatttgaa tgggcttccc gtactttgtg ttgctttgcc agtccctttg ggcccccatg 1800 aattccttga agtgctttaa gtaatgcggg tctacgtcat caatgacgtt gtaccacgca 1860 tcattattgt acacctttgg gcttaggtct agatgtccac ataaataatt atgtgggcct 1920 agagacctgg cccacattgt tttgcctgtt ctgctatcac cctcaatgac aatacttatg 1980 ggtctccatg gccgcgcagc ggaatacacg acgttctcgg acacccatac ttcaagctca 2040 tctggaactt gattaaaaga tgaagataaa aagggagaaa tataaggagc cggaggctcc 2100 tgaaaaattc tatctaaatt actatttaaa ttatgaaact gtaaaatata atctttcggt 2160 actaattctt taatgattct aagagcctct gacttactgc ctgagttaag agctgcggcg 2220 taagcgtcat tggctgactg ctgaccccca cgtgcagatc gtccgtcgat ctgaaactca 2280 ccccagtcga cggtgtctcc gtccttatcg acataagact tgacgtctga actggattta 2340 gctccctgaa tgtttggatg gaaatgtgct gccctgcttg gggataccag gtcgaagaat 2400 cgctgatttt ggcatttgaa tttcccttca aattggataa gcacatggag atgtggttcc 2460 ccattctcgt ggagttctct gcaaactttg atgtattttt tatttgttgg ggtttctagg 2520 ttttttaatt gggaaagtgc ttcttctttt gttaaggagc aatgaggata tgtgaggaaa 2580 taatttttgc aatttatttg gaagcgctta ggaggagcca tatggtcaat gagtaccgat 2640 tgaccaagat ttttacactt atccctggtg tatcggtact caacatatag tgagtaccaa 2700 atggcatttt ggtaataaca taaaagtaca ttgcaattca aaattcaaaa ttaaaaaatc 2760 aaatcattaa agcggccatc cgtataatat t 2791 <210> 6 <211> 2781 <212> DNA <213> TYLCV Jordan 3 strain <400> 6 accggatggc cgcgcctttt ccttttatgt ggtccccacg agggttacac agacgtcact 60 gtcaaccaat caaattgcat cctcaaacgt tagataagtg tgcatttgtc tttatatact 120 tggtccccaa gttttttgtc ttgcaatatg tgggacccac ttcttaatga atttcctgaa 180 tctgttcacg gatttcgttg tatgttagct attaaatatt tgcagtccgt tgaggaaact 240 tacgagccca atacattggg ccacgattta attagggatc ttatatctgt tgtaagggcc 300 cgtgactatg tcgaagcgac caggcgatat aatcatttcc acgcccgtct cgaaggttcg 360 ccgaaggctg aacttcgaca gcccatacag cagccgtgct gctgtcccca ttgtccaagg 420 cacaaacaag cgacgatcat ggacgtacag gcccatgtac cgaaagccca gaatatacag 480 aatgtatcga agccctgatg ttcctcgtgg atgtgaaggc ccatgtaaag tccagtctta 540 tgagcaacgg gatgatatta agcatactgg tattgttcgt tgtgttagtg atgttactcg 600 tggatctgga attactcaca gagtgggtaa gaggttctgt gttaaatcga tatatttttt 660 aggtaaagtc tggatggatg aaaatatcaa gaagcagaat cacactaatc aggtcatgtt 720 cttcttggtc cgtgatagaa ggccctatgg aagcagccca atggattttg gacaggtttt 780 taatatgttc gataatgagc ccagtaccgc aaccgtgaag aatgatttgc gggataggtt 840 tcaagtgatg aggaaatttc atgcttcagt tattggtggg ccctctggaa tgaaggaaca 900 ggcattagtt aagagatttt ttaaaattaa cagtcatgta acttataatc atcaggaggc 960 agccaagtac gagaaccata ctgaaaacgc cttgttattg tatatggcat gtacgcatgc 1020 ctctaatcca gtgtatgcaa ctatgaaaat acgcatctat ttctatgatt caatatcaaa 1080 ttaataaaat ttatatttta tatcatgagt ttctgttaca tttattgtgt tttcaagtac 1140 atcatacaat acatgatcca ctgctctgat tacattgtta atggaaatta caccaagact 1200 atctaaatac ttaagaactc catatctaaa tactcttaag aaacgaccag tctgaggctg 1260 taatgtcgtc caaattcgga agttgagaaa acatttgtga atccccatta ccttcctgat 1320 gttgtggttg aatcttatct gaatggaaat gatgtcgtgg ttcattagaa atggcctctg 1380 actgtgttct gttatcttga aatagagggg attgtttatc tcccagataa aaacgccatt 1440 ctctgcctga ggagcagtga tgagttcccc tgtgcgtgaa tccatgatta ttgcagttga 1500 ggtcgaggta gtatgagcat ccacagtcta ggtctacacg cttacgcctt attggtttct 1560 tcttggctat cttgtgttgg accttgattg atacttgcga acagtggctc gtagagggtg 1620 acgaaggttg cattcttgag agcccaattt ttcaaggata tgtttttttc ttcgtctaga 1680 tattccctat atgaggaggt aggtcctgga ttgcagagga agatagtggg aattccccct 1740 ttaatttgaa tgggcttccc gtactttgtg ttgctttgcc agtccctctg ggcccccatg 1800 aattccttta agtgttttaa attatgcggg tctacgtcat caatgacgtt ataccacgca 1860 tcattactgt acacctttgg gcttaggtct agatgtccac ataaataatt atgtgggcct 1920 agagacctgg cccacattgt tttgcctgtt ctgctatcac cctcaatgac aatacttatg 1980 ggtctccatg gccgcgcagc ggataacacg acgttctcgg cgacccactc ttcaagttca 2040 tctggaactt gattaaaaga agaagaaaga aatggagaaa cataaacttc taaaggagga 2100 ctaaaaatcc tatctaaatt tgaacttaaa ttatgaaatt gtaaaatata gtcctttggg 2160 gccttctctt ttaatatatt gagggcctcg gatatactgc ctgaattgag tgcttcggca 2220 tatgcgtcgt tggcagattg ctgacctcct ctagctgatc tgccatcgat ttggaaaact 2280 ccaaaatcaa tgaagtctcc gtctttctcc acgtaggtct tgacatctgt tgagctctta 2340 gctgcctgaa tgttcggatg gaaatgtgct gacctgttag gggataccag gtcgaagaac 2400 cgttggttct tacattggta ttttccttcg aattggataa gcacatggag atgtggttcc 2460 ccattctcgt ggagttctct gcaaactttg atgtattttt tatttgttgg ggtttctatg 2520 ttttttaatt gggaaagtgc ttcctcttta gagagagaac aattgggata tgttaggaaa 2580 taatttttgg catatatttt aaataaacga ggcatgttga tatgaatcgg tgtccctcaa 2640 agctctatgg caatcggtgt atcggtgtct tacttatacc tggacaccta atggctattt 2700 ggtaatttca taaatgttca ttgcaattca aaattcaaaa ttcaaaaatc aaatcattaa 2760 agcggccatc cgtataatat t 2781 <210> 7 <211> 2787 <212> DNA <213> TYLCV Israel strain <400> 7 gttgaaatga atcggtgtcc ctcaaagctc tatggcaatc ggtgtatcgg tgtcttactt 60 atacttggac acctaatggc tatttggtaa tttcataaat gttcatttca attcaaaatt 120 caaaattcaa aaatcaaatc attaaagcgg ccatccgtat aatattaccg gatggccgcg 180 ccttttcctt ttatgtggtc cccacgaggg ttacacagat gttattgtca accaatcaaa 240 ttgcattctc aaacgttaga taagtgttca tttgtcttta tatacttggt ccccaagttt 300 tttgtcttgc aatatgtggg acccacttct taatgaattt cctgaatctg ttcacggatt 360 tcgttgtatg ttagctatta aatatttgca gtccgttgag gaaacttacg agcccaatac 420 attgggccac gatttaatta gggatcttat atctgttgta agggcccgtg actatgtcga 480 agcgaccagg cgatataatc atttccacgc ccgtctcgaa ggttcgccga aggctgaact 540 tcgacagccc atacagcagc cgtgctgctg tccccattgt ccaaggcaca aacaagcgac 600 gatcatggac gtacaggccc atgtaccgaa agcccagaat atacagaatg tatcgaagcc 660 ctgatgttcc ccgtggatgt gaaggcccat gtaaagtcca gtcttatgag caacgggatg 720 atattaagca tactggtatt gttcgttgtg ttagtgatgt tactcgtgga tctggaatta 780 ctcacagagt gggtaagagg ttctgtgtta aatcgatata ttttttaggt aaagtctgga 840 tggatgaaaa tatcaagaag cagaatcaca ctaatcaggt catgttcttc ttggtccgtg 900 atagaaggcc ctatggaaac agcccaatgg attttggaca ggtttttaat atgttcgata 960 atgagcccag taccgcaacc gtgaagaatg atttgcgtga taggtttcaa gtgatgagga 1020 aatttcatgc tacagttatt ggtgggccct ctggaatgaa ggaacaggca ttagttaaga 1080 gattttttaa aattaacagt catgtaactt tatttatatt cattcaggag gcagcaaagt 1140 acgagaacca tactgaaaac gccttgttat tgtatatggc atgtacgcat gcctctaatc 1200 cagtgtatgc aactatgaaa atacgcatct atttctatga ttcaatatca aattaataaa 1260 atttatattt tatatcatga gtttctgtta catttattgt gttttcaagt acatcataca 1320 atacatgatc aactgctctg attacattgt taatggaaat tacaccaaga ctatctaaat 1380 acttaagaac ttcatatcta aatactctta agaaatgacc agtctgaggc tgtaatgtcg 1440 tccaaattcg gaagtcgaga aaacatttgt gaatccccat taccttcctg atgttgtggt 1500 tgaatcttat ctgaatggaa atgatgtcgt ggttcattag aaatggcctc tggctgtgtt 1560 ctgttatctt gaaatagagg ggattgttta tctcccagat aaaaacgcca ttctctgcct 1620 gaggagcagt gatgagttcc cctgtgcgtg aatccatgat tattgcagtt gaggtggagg 1680 tagtatgagc agccacagtc taggtctaca cgcttacgcc ttattggttt cttcttggct 1740 atcttgtgtt ggaccttgat tgatacttgc gaacagtggc tcgtagaggg tgacgaaggt 1800 tgcattcttg agagcccaat ttttcaagga tatgtttttt tcttcgtcta gatattccct 1860 atatgaggag gtaggtcctg gattgcagaga gaagatagtg ggaattcccc ctttaatttg 1920 aatgggcttc ccgtactttg tgttgctttg ccagtccctc tgggccccca tgaattcctt 1980 gaagtgcttt aaataatgcg ggtctacgtc atcaatgacg ttgtaccacg catcattact 2040 gtacaccttt gggcttaggt ctagatgtcc acataaataa ttatgtgggc ctagagacct 2100 ggcccacatt gttttgcctg ttctgctatc accctcaatg acaatactta tgggtctcca 2160 tggccgcgca gcggaataca cgacgttctc ggcgacccac tcttcaagtt catctggaac 2220 ttgattaaaa gaagaagaaa gaaatggaga aacataaact tctaaaggag gactaaaaat 2280 cctatctaaa tttgaactta aattatgaaa ttgtaaaata tagtcctttg gggccttctc 2340 ttttaatata ttgagggcct cggatttact gcctgaattg agtgcttcgg catatgcgtc 2400 gttggcagat tgctgacctc ctctagctga tctgccatcg atttgggaaa ctccaaaatc 2460 aatgaagttt ccgtctttct ccacgtaggt cttgacatct gttgagctct tagctgcctg 2520 aatgttcgga tggaaatgtg ctgacctgtt tggggatacc aagtcgaaga accgttggtt 2580 cttacattgg tatttgcctt cgaattggat aagcacatgg agatgtggtt ccccattctc 2640 gtggagttct ttgcaaactt tgatgtattt tttatttgtt ggggtttcta gtttttttaa 2700 ttgggaaagt gcttcctctt tagagagaga acaattggga tatgttagga aataattttt 2760 ggcatatatt ttaaataaac gaggcat 2787 <210> 8 <211> 2791 <212> DNA <213> TYLCV Spain strain <400> 8 taatattacc ggatggccgc gccttttcct tttatgtggt ccccatgagg gttcaacaga 60 cgtcactgtc aaccaatcaa attgcatcct caaacgttag ataagtgttc atttgtcttt 120 atatacttgg tccccaagta gtttgtcttg caatatgtgg gatccacttc taaatgaatt 180 tcctgaatct gttcacggat ttcgttgtat gttagctatt aaatatttgc agtccgttga 240 ggaaacttac gagcccaata cattgggcca cgatttaatt agggatctta tatctgttgt 300 aagggcccgt gactatgtcg aagcgaccag gcgatataat catttccacg cccgcctcga 360 aggttcgccg aaggctgaac ttcgacagcc catacagcag ccgtgctgct gtccccattg 420 tccaaggcac aaacaagcga cgatcatgga cgtacaggcc catgtaccgg aagcccagaa 480 tatacagaat gtatcgaagc actgatgttc cccgtggatg tgaaggccca tgtaaagtac 540 agtcttatga gcaacgggat gatattaagc atactggtat tgttcgttgt gttagtgatg 600 ttactcgtgg atctggaatt actcacagag tgggtaagag gttctgtgtt aaatcgatat 660 attttttagg taaagtctgg atggatgaaa atatcaagaa gcagaaccac actaatcagg 720 tcatgttctt cttggtccgt gatagaaggc cctatggaaa cagcccaatg gattttggac 780 aggtttttaa tatgttcgat aatgagccca gtaccgcaac agtgaagaat gatttgcggg 840 ataggtttca agtgatgagg aaatttcatg ctacagttat tggtgggccc tctggaatga 900 aggaacaggc attagttaag aggtttttta gaattaacag tcatgtaact tataatcatc 960 aggaggcagc caagtacgag aaccatactg aaaacgcctt gttattgtat atggcatgta 1020 cgcatgcctc taatccagtg tatgctacta tgaaaatacg catctatttc tatgattcaa 1080 tatcaaatta ataaaattta tattttatat catgagtttc tgttacattt attgtgtttt 1140 caagtacatc atacaataca tgatcaactg ctctgattac attgttaatt gaaattacac 1200 caatactatc taaatactta agaacttgat gtctaaatac ttttaagaaa cgaccagtct 1260 gaggctgtaa tgtcgtccaa attcggaagt tgagaaaaca tttgtgaatc cccaatacct 1320 tcctgatgtt gtggttgaat cttatctgaa tggaaatgat gtcgtggttc attagaaatg 1380 gcctctggct gtgttctgtt atcttgaaat agaggggatt gtttatctcc cagataaaaa 1440 cgccattctc tgcctgagga gcagtgatga gttcccctgt gcgtgaatcc atgattgttg 1500 cagttgaggt gttggtagta tgagcagcca cagtctaggt ctatacgctt acgccttatt 1560 ggtttcttct tggctatctt gtgttggacc ttgattgata cttgcgaaca gtggctcgta 1620 gagggtgacg aaggttgcat tcttgagagc ccaatttttc aaggatatgt ttttttcttc 1680 gtctagatat tccctatatg aggaggtagg tcctggattg cagaggaaga tagtgggaat 1740 tcccccttta atttgaatgg gcttcccgta ctttgtgttg ctttgccagt ccctctgggc 1800 ccccatgaat tctttgaagt gctttaaata atgcgggtct acgtcatcaa tgacgttgta 1860 ccatgcatca ttactgtaca cctttgggct taggtctaga tgtccacata aataattatg 1920 tgggcctaga gacctggccc acattgtttt gcctgttctg ctatcaccct caatgacaat 1980 actattaggt ctccatggcc gcgcagcgga agacacgacg ttctcggaca cccatacttc 2040 aagttcatct ggaacttgat taaaagatga agataaaaag ggagaaatat aaggagccgg 2100 aggctcctga aaaattctat ctaaattact atttaaatta tgaaattgta aaatataatc 2160 tttcggtatt aattctttaa tgattctaag agcctctgac ttactgcctg agttaagagc 2220 tgcggcgtaa gcgtcattgg ctgactgctg acccccacgt gcagatcgtc cgtcgatctg 2280 aaactcaccc cagtcgacgg tgtctccgtc cttatcgaca taagacttga cgtctgaact 2340 ggatttagct ccctgaatgt ttggatggaa atgtgctgcc ctgcttgggg ataccaggtc 2400 gaagaatcgc tgattttggc acttgaattt cccttcaaat tggataagca catggagatg 2460 tggttcccca ttctcgtgga gttctctgca aactttgatg tattttttat ttgttggggt 2520 ttctaggttt tttaattggg aaagtgcttc ttctttcgtt aaggagcact taggatatgt 2580 gaggaaataa tttttgcaat ttatttggaa gcgcttaggg ggagccatat ggtcaatgag 2640 taccgattga ccaagatttt tacacttatc cctggtgtat cggtactcaa tatataggga 2700 gtaccaaatg gcatattggt aattttgtaa aagtacaatg caattcaaaa ttcaaaattc 2760 Aaaaaatcaaa tcattaaagc ggccatccgt a 2791 <210> 9 <211> 2781 <212> DNA <213> TYLCV USA strain <400> 9 accggatggc cgcgcctttt ccttttatgt ggtccccacg agggttacac agacgtcact 60 gtcaaccaat caaattgcat cctcaaacgt tagataagtg ttcatttgtc tttatatact 120 tggtccccaa gtattttgtc ttgcaatatg tgggacccac ttctaaatga atttcctgaa 180 tctgttcacg gatttcgttg tatgttagct attaaatatt tgcagtccgt tgaggaaact 240 tacgagccca atacattggg ccacgattta attagggatc ttatatctgt tgtaagggcc 300 cgtgactatg tcgaagcgac caggcgatat aatcatttcc acgcccgtct cgaaggttcg 360 ccgaaggctg aacttcgaca gcccatacag caaccgtgct gctgtcccca ttgtccaagg 420 cacaaacaag cgacgatcat ggacgtacag gcccatgtac cgaaagccca gaatatacag 480 aatgtatcga agccctgatg ttccccgtgg atgtgaaggc ccatgtaaag tccagtctta 540 tgagcagcgg gatgatatta agcacactgg tgttgttcgt tgtgttagtg atgttactcg 600 tggatctgga attactcaca gagtcggtaa gaggttctgt gttaaatcga tatatttttt 660 aggtaaagtc tggatggatg aaaatatcaa gaagcagaat cacactaatc aggtcatgtt 720 ctttttggtc cgtgatagaa ggccctatgg aagcagtcca atggattttg gacaggtttt 780 taatatgttc gataatgagc ccagtaccgc aactgtgaag aatgatttgc gtgataggtt 840 tcaagtgatg aggaaatttc atgctacagt tattggtggg ccctctggaa tgaaggaaca 900 ggcattagtt aagagatttt ttaaaattaa cagtcatgta acttataatc atcaggaggc 960 agccaagtac gagaaccata ctgaaaacgc cttgttattg tatatggcat gtacgcatgc 1020 ctctaatcca gtgtatgcaa ctatgaaaat acgcatctat ttctatgatt caatatcaaa 1080 ttaataaaat ttatatttta tatcatgagt ttctgttaca tttattgtgt tttcaagtac 1140 atcatacaat acatgatcca ctgctctgat tacattgtta atggaaatta caccaagact 1200 atctaaatac ttaagaactt catatctaaa tactcttaag aaacgaccag tctgaggctg 1260 taatgtcgtc caaattcgga agttgagaaa acatttgtga atccccatta ccttcctgat 1320 gttgtggttg aatcttatct gaatggaaat gatgtcgtgg ttcattagaa atggcctctg 1380 gctgtggtct gttatcttga aatagagggg attgtttatc tcccaaataa aaacgccatt 1440 ctctgcctga ggagcagtga tgagttcccc tgtgcgtgaa tccatgatta ttgcagttga 1500 ggtggaggta gtatgagcag ccacagtcta ggtctacacg cttacgcctt attggtttct 1560 tcttggctat cttgtgttgg accttgattg atacttgcga acagtggctc gtagagggtg 1620 acgaaggttg cattcttgag agcccaattt ttcaaggata tgtttttttc ttcgtctaga 1680 tattccctat atgaggaggt aggtcctgga ttgcagagga agatagtggg aattccccct 1740 ttaatttgaa tgggcttccc gtactttgtg ttgctttgcc agtccctctg ggcccccatg 1800 aattccttga agtgctttaa ataatgcggg tctacgtcat caatgacgtt gtaccacgca 1860 tcattactgt acacctttgg gcttaggtct agatgtccac ataaataatt atgtgggcct 1920 agagacctgg cccacattgt tttgcctgtt ctgctatcac cctcaatgac aatacttatg 1980 ggtctccatg gccgcgcagc ggaagacacg acgttctcgg cgacccactc ttcaagttca 2040 tctggaactt gattaaaaga agaagaaaga aatggagaaa cataaacttc taaaggagga 2100 ctaaaaatcc tatctaaatt tgaatttaaa ttatgaaatt gtaaaatata gtcctttggg 2160 gccttttctt ttaatatatt gagggcctcg gatttactgc ctgaattgag tgctccggcg 2220 tatgcgtcgt tggcagattg ctgacctcct ctagctgatc tgccatcgat ttggaaaact 2280 ccaaaatcaa tgaagtctcc gtctttctcc acgtaggtct tgacatctgt tgagctctta 2340 gctgcctgaa tgtttggatg gaaatgtgct gacctgtttg gggataccag gtcgaagaac 2400 cgttggttct tacattggta tttgccttcg aattggataa gcacatggag atgtggttcc 2460 ccattctcgt ggaattctct gcaaactttg atgtattttt tatttgttgg ggtttctatg 2520 ttttttaatt gggaaagtgc ttcctcttta gagagagaac aattgggata tgttaggaaa 2580 taattcttgg catatatttt aaataaacga ggcatgttga aatgaatcgg tgtccctcaa 2640 agctctatgg caatcggtgt atcggtgtct tactaatacc tcgacaccta atggcaattt 2700 ggtaatttca taaatgttca ttgcaattca aaattcaaaa ttcaaaaatc aaatcattaa 2760 agcggtcatc cgtataatat t 2781 <210> 10 <211> 2774 <212> DNA <213> TYLCV Korea strain <400> 10 accggatggc cgcgcctttt ccttttatgt ggtccccacg agggttacac agacgtcacc 60 gtcaaccaat caaattgcat cctcaaacgt tagataagtg ttcatttgtc tttatatact 120 tggtccccaa gttttttgtc ttgcaatatg tgggacccac ttcttaatga gtttcctgaa 180 tctgttcacg gatttcgttg tatgttagct attaaatatt tgcagtccgt tgaggaaact 240 tacgagccca atacattggg ccacgattta attagggatc ttatatctgt tgtaagggcc 300 cgtgactatg tcgaagcgac caggcgatat aatcatttcc acgcccgtct cgaaggttcg 360 ccgaaggctg aacttcgaca gcccatacag cagccgtgct gctgtcccca ttgtccaagg 420 cacaaacaag cgacgatcat ggacgtacag gcccatgtac cgaaagccca gaatatacag 480 aatgtatcga agccctgatg ttccccgtgg atgtgaaggc ccatgtaaag tccagtctta 540 tgagcaacgg gatgatatta agcatactgg tattgttcgt tgtgttagtg atgttactcg 600 tggatctgga attactcaca gagtgggtaa gaggttctgt gttaaatcga tatatttttt 660 agggaaagtc tggatggatg aaaatatcaa gaagcagaat cacactaatc aggtcatgtt 720 cttcttagtc cgtgatagaa ggccctatgg aagcagccca atggattttg gacaggtttt 780 taatatgttc gataatgagc ccagtaccgc aaccgtgaag aatgatttgc gggataggtt 840 tcaagtgatg aggaaatttc atgctacagt tattggtgga ccctctggaa tgaaggaaca 900 ggcattagtt aagagatttt ttaaaattaa cagtcatgta acttataatc atcaggaggc 960 agccaagtac gagaaccata ctgaaaacgc cttgttactg tatatggcat gtacgcatgc 1020 ctcgaatcca gtgtatgcaa ctatgaaaat acgcatctat ttctatgatt caatatcaaa 1080 ttaataaaat ttatatttta tatcatgagt ttctgttaca tttattgtgt tttcaagtac 1140 atcatacaat acatgatcaa ctgatctgat tacattgtta atggaaatta caccaagact 1200 atctaaatac ttaagaactt catatctaaa tactcttaag aaatgaccag tctgaggatg 1260 taatgtcgtc caaattcgga agttgagaaa acatttgtga atccccatga ccttcctgat 1320 gttgtggttg aatcttatct gaatggaaat gatgtcgtgg ttcattagaa atggcctctg 1380 gctgtgttct gttatcttga aatagagggg attgtttatc tcccagataa aaacgccatt 1440 ctctgcctga ggagcagtga tgagttcccc tgtgcgtgaa tccatgatta ttgcagttga 1500 ggtggaggta gtatgagcag ccacagtcta ggtctacacg cttacgcctt attggtttct 1560 tcttggctat cttgtgttgg accttgattg atacttgcga acagtggctc gtagagggtg 1620 acgaaggttg cattcttgag agtccaattt ttcaaggata tgtttttttc ttcgtctaga 1680 tattccttat atgaggaggt aggtccttga ttgcagagga agatagtggg aattccccct 1740 ttaatttgaa tgggcttccc gtactttgtg ttgctttgcc agtccctttg ggcccccata 1800 aattccttga agtgctttaa ataatgcggg tctacgtcat caatgacgtt gtaccacgca 1860 tcattgctgt acacctttgg gcttaggtct agatgtccac ataaataatt atgtgggcct 1920 agagacctgg cccacattgt tttccctgtt ctgctatcac cctcaatgac aatacttatg 1980 ggtctccatg gccgcgcagc ggaagacacg acgttctcgg cgacccactc ttcaagttca 2040 tctggaactt gattaaaaga agaagaaaga aatggagaaa cataaacttc taaaggagga 2100 ctaaaaatcc tatctaaatt tgaacttaaa ttatgaaatt gtaaaatata gtcctttggg 2160 gccttctctt ttaatatatt gagggcctcg gatttactgc ctgaattgag tgcttcggca 2220 tatgcgtcgt tggcagattg ctgacctcct ctagctgatc tgccatcgat ttggaaaact 2280 ccaaaatcaa tgaagtctcc gtctttctcc acgtaggtct tgacatctgt tgagctctta 2340 gctgcctgaa tgttcggatg gaaatgtgct gacctgtttg gggataccag gtcgaagaac 2400 cgttggttct tacattggta tttgccttcg aattggataa gcacatggag atgtggttcc 2460 ccattctcgt ggagttcttt gcaaactttg atgtattttt tatttgttgg ggtttctagg 2520 ttttttaatt gggaaagtgc ttcctcttta gagagagaac aattgggata tgttaggaaa 2580 taatttttgg catatatatt aaataaacga ggcatgttga aatgaatcgg tgtccctcaa 2640 agctctatgg caatcggtgt atcggtgtct tacttatacc tggacaccta atggctattt 2700 ggtaatttca tgaatgttca ttgtaattca aaattcaaaa atcaaatcat taaagcggcc 2760 atccgtataa tatt 2774 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Universal TYLCV F3 primer <400> 11 gtatcgaagc cctgatgtt 19 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Universal TYLCV B3 primer <400> 12 cctgattagt gtgattctgc t 21 <210> 13 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Universal TYLCV FIP primer <400> 13 cacaacgaac aataccagta tgcttgaagg cccatgtaaa gtcc 44 <210> 14 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Universal TYLCV BIP primer <400> 14 ctggaattac tcacagagtg ggtgatattt tcatccatcc agact 45

Claims (9)

서열번호 1 내지 4로 구성되는, 토마토황화잎말림바이러스(Tomato yellow leaf curl virus)를 검출하기 위한 등온증폭 반응용 프라이머 세트.
A primer set for isothermal amplification reaction for detecting tomato yellow leaf curl virus consisting of SEQ ID NOs: 1 to 4.
서열번호 11 내지 14로 구성되는, 토마토황화잎말림바이러스(Tomato yellow leaf curl virus)를 검출하기 위한 등온증폭 반응용 프라이머 세트.
A primer set for isothermal amplification reaction for detecting tomato yellow leaf curl virus consisting of SEQ ID NOs: 11 to 14.
제 1 항 또는 제 2항에 있어서,
상기 토마토황화잎말림바이러스는 TYLCV Jordan1 변이주(strain), Jordan3 변이주(strain), Israel 변이주(strain), Spain 변이주(strain), USA 변이주(strain) 및 Korea 변이주(strain)로 이루어진 군에서 선택되는 것을 특징으로 하는, 프라이머 세트.
The method according to claim 1 or 2,
The tomato sulfide leaf virus is selected from the group consisting of TYLCV Jordan1 strain, Jordan3 strain, Israel strain, Spain strain, USA strain and Korea strain. Characterized by a primer set.
청구항 1 또는 청구항 2의 프라이머 세트를 포함하는, 토마토황화잎말림바이러스(Tomato yellow leaf curl virus)를 검출하기 위한 등온증폭 반응용 프라이머 조성물.
A primer composition for isothermal amplification reaction for detecting tomato yellow leaf curl virus, comprising the primer set of claim 1 or 2.
제 4 항에 있어서,
상기 조성물은 등온증폭 반응용 DNA 중합효소, dNTPs, 및 반응버퍼를 더 포함하는 것을 특징으로 하는, 조성물.
The method of claim 4, wherein
The composition is characterized in that it further comprises a DNA polymerase for isothermal amplification reaction, dNTPs, and the reaction buffer.
토마토에서 게놈 DNA(gDNA)를 추출하는 단계;
상기 gDNA를 주형으로 제4항에 따른 조성물을 이용하여 60℃ 내지 70℃에서 30분 내지 2시간 동안 등온증폭반응법을 수행하여 표적 서열을 증폭시키는 단계; 및
상기 증폭된 산물을 검출하는 단계를 포함하는 토마토황화잎말림바이러스(Tomato yellow leaf curl virus) 검출방법.
Extracting genomic DNA (gDNA) from tomato;
Amplifying a target sequence by isothermal amplification at 60 ° C. to 70 ° C. for 30 minutes to 2 hours using the composition according to claim 4 as a template of the gDNA; And
And detecting the amplified product. A method for detecting Tomato yellow leaf curl virus.
제 6 항에 있어서,
상기 토마토황화잎말림바이러스는 TYLCV Jordan1 변이주(strain), Jordan3 변이주(strain), Israel 변이주(strain), Spain 변이주(strain), USA 변이주(strain) 및 Korea 변이주(strain)로 이루어진 군에서 선택되는 것을 특징으로 하는, 검출방법.
The method according to claim 6,
The tomato sulfide leaf virus is selected from the group consisting of TYLCV Jordan1 strain, Jordan3 strain, Israel strain, Spain strain, USA strain and Korea strain. The detection method characterized by the above-mentioned.
제 6 항에 있어서,
상기 증폭 산물을 검출하는 단계는 전기영동(electrophoresis) 또는 SYBR Green I을 이용하여 증폭된 DNA를 확인하는 것을 특징으로 하는, 검출방법.
The method according to claim 6,
The detecting of the amplified product is characterized in that for identifying the amplified DNA using electrophoresis or SYBR Green I.
제 8 항에 있어서,
상기 SYBR Green I을 이용하여 증폭된 DNA를 확인하는 방법은 SYBR Green I을 1,000배 내지 10,000배 농도로 사용하여 자연광하에서 육안으로 관찰하는 것을 특징으로 하는, 검출방법.








The method of claim 8,
The method for identifying the amplified DNA using the SYBR Green I, characterized in that the observation using the SYBR Green I at a concentration of 1,000 to 10,000 times with the naked eye under natural light.








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KR101894308B1 (en) 2017-06-20 2018-09-04 충북대학교 산학협력단 Primer set for loop-mediated isothermal amplification reaction for detecting of Rosellinia necatrix, and uses thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101894308B1 (en) 2017-06-20 2018-09-04 충북대학교 산학협력단 Primer set for loop-mediated isothermal amplification reaction for detecting of Rosellinia necatrix, and uses thereof

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