식물병연구 Research Article Open Access Res. Plant Dis. 23(2): (2017) 국내의토마토주요바이러스진단을위한역전사중합반응법용프라이머세트 Sp

식물병연구 Research Article Open Access Res. Plant Dis. 23(2): 193-201 (2017) https://doi.org/10.5423/rpd.2017.23.2.193 국내의토마토주요바이러스진단을위한역전사중합반응법용프라이머세트 Specific Primer Sets for RT-PCR Detection of Major RNA Viruses of Tomato Plants in Korea 신준성 1 ㆍ한정헌 2 ㆍ신유주 1 ㆍ곽해련 3 ㆍ최홍수 3 * ㆍ김정수 4 * 1 바이오큐브시스템, 2 농촌진흥청국립농업과학원유전자공학과, 3 농촌진흥청국립농업과학원작물보호과, 4 안동대학교식물의학과 *Co-corresponding authors HS Choi Tel: +82-63-238-3300 Fax: +82-63-238-3838 E-mail: hschoi@korea.kr JS Kim Tel: +82-54-820-6224 Fax: +82-54-820-6320 E-mail: plvirus@anu.ac.kr These authors contributed equally to this work as first authors. Jun-Sung Shin 1, Jung-Heon Han 2, Yu-Ju Shin 1, Hae-Ryun Kwak 3, Hong-Soo Choi 3 *, and Jeong-Soo Kim 4 * 1 BioCube System, Inc., Gwacheon 13820, Korea 2 Gene Engineering Division, National Institute of Agricultural Sciences, Rural Development Administration, Jeonju 54874, Korea 3 Crop Protection Division, National Institute of Agricultural Sciences, Rural Development Administration, Wanju 55365, Korea 4 Department of Plant Medicals, Andong National University, Andong 36729, Korea Received September 12, 2016 Revised April 16, 2017 Accepted April 17, 2017 Major tomato viruses in Korea are Tomato chlorosis virus (ToCV), Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Pepper mottle virus (PepMoV), and Tomato mosaic virus (ToMV). RT-PCR conditions for the viruses were examined, especially in primer set and RT-PCR mixture. Total 46 primer sets from the unique sequence of the viruses were tested for nonspecific background products in a RT-PCR mixture without template. Among them 16 primer sets were applied to healthy tomato RNA, resulting the compatibility between RT-PCR mixture and primer set influenced RT-PCR to reduce nonspecific background products. Based on the combinations among cdna synthesis parameters and RT-PCR mixtures, two reaction mixtures were finally selected for ToCV detection. The condition allowed to determine more specific primer sets; C029 (ToCV), C072 (TSWV), C070 (CMV), C048 (PepMoV), and C065 (ToMV). These primer sets are expected to be of use to specific detection of the major viruses in tomato plants. Keywords: Detection, Primer, RT-PCR, Tomato, Virus 서론 국내의토마토에서발생하는주요바이러스는 Tomato Research in Plant Disease pissn 1598-2262, eissn 2233-9191 www.online-rpd.org chlorosis virus (ToCV), Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Pepper mottle virus (PepMoV), Tomato mosaic virus (ToMV), Tomato yellow leaf curl virus (TYLCV) 이며, TYLCV를제외한나머지는모두 RNA 바이러스이다 (Ji 등, 2008; Kataya 등, 2008; Kim 등, 2011). ToCV 는 2013년에논산, 익산, 함평, 화순, 제주에서처음발생하였고, 감염된식물체는잎갈변과엽맥퇴록병징을보이며, 7종이상의잡초에서감염이확인되었다 (Kil 등, 2015). TSWV는 2004년안양 The Korean Society of Plant Pathology cc This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 193

194 Research in Plant Disease Vol. 23 No. 2 관양동지역에서대발생하였고, 감염식물에괴사및다중원형반점을일으킨다 (Cho 등, 2005). CMV는국내채소류에서가장많이발생하는바이러스로토마토에서는잎에모자이크와고사리잎 (fern leaf) 모양병징을일으킨다 (Kim 등, 2011). 이밖에 PepMoV와 ToMV는토마토품종에따라황화모자이크, 엽맥괴사, 퇴록반점등을일으키는것으로알려져있다 (Kim 등, 2008). 식물바이러스진단은바이러스피해를최소화하기위한초기대응책이며 (Aboul-Ata 등, 2011), 효소면역항체법 (enzyme-linked immunosorbent assay, ELISA) 에서부터차세대염기서열분석 (next generation sequencing, NGS) 법까지다양한방법이이용된다 (Boonham 등, 2014; Jeong 등, 2014). 특히, 역전사중합반응 (RT-PCR) 법은혈청학적진단법에비해검출감도와특이도가높다 (Hu 등, 1995; Mumford 등, 1994; Park과 Kim, 2004; Webster 등, 2004). 또한실험절차가간편하고진단에필요한프라이머설계및제작이용이하여식물바이러스진단에널리이용된다 (Webster 등, 2004). 일반적으로식물바이러스진단용프라이머는대상바이러스자체또는그와유사한계통의유전체서열을참고하여설계한다. RT-PCR 진단에서기주의유전체서열을고려하지않고설계된프라이머를사용할경우, 프라이머염기서열과상동성이있는기주유래비특이산물의생성을완전히회피하는것은매우어려운일이다. 따라서 RT-PCR의진단효율을높이기위해서는목적바이러스에특이적인프라이머선발, 주형준비법과사용량, cdna 합성온도와시간, RT-PCR 반응액의종류, 프라이머및 PCR 증폭조건등을고려해야한다 (Chen 등, 2011; Hassani-Mehraban 등, 2016; Kumar 등, 2011; Lee 등, 2011; Okuda와 Hanada, 2001; Webster 등, 2004). 이들가운데프라이머는진단의특이성에가장많은영향을주는요인이다 (Hassani-Mehraban 등, 2016; Kumar 등, 2011; Mumford 등, 1994; Webster 등, 2004). 오늘날에는 NGS 기술발달로과거 10여년전에비해식물바이러스뿐만아니라다양한작물의유전체정보가풍성해졌다 (Thottathil 등, 2016). 2012년에는국내과학자가포함된국제협력연구를통해토마토의전장유전체서열이완전히분석되었다 (The Tomato Genome Consortium, 2012). 이는기주의전장유전체정보를고려해토마토에발생하는바이러스에특이적인프라이머설계가가능함을의미한다. 따라서본연구에서는토마토전장유전체서열, expressed sequence tag (EST), 식물바이러스염기서열간상동성분석을통해바이러스특이적인프라이머후보군을탐색하고, one step RT- PCR mix와 cdna 합성조건을달리하여토마토주요바이러스 진단에필요한특이프라이머세트를선발하고자하였다. 재료및방법 식물및바이러스. 바이러스증식및건전 RNA 채취를위해국내에시판되는토마토두품종 ( 서광과주옥 ) 을사용하였다. 바이러스는농촌진흥청작물보호과식물바이러스실에서유지하고있는 ToCV-tom, PepMoV-tom, ToMV-n1을사용하였고, TSWV-old와 CMV-P1은농우바이오 (Nongwoo Bio, Suwon, Korea) 이장하박사로부터분양받았다. 바이러스증식및주형준비. 감염 RNA 채취를위해각각의바이러스가감염된토마토또는담배잎을접종원으로사용하여 TSWV는주옥토마토에, 나머지바이러스는서광토마토에증식하였다. 접종액은멸균한막자사발에감염잎 (1 g) 과접종액 (0.1 M Tris-HCl, ph 8.0, 1% sodium sulfite) 3 ml를널고마쇄한다음, 일반두루마리화장지 4겹으로여과하여준비하였다. 접종개체는적정량의 carborundum (600 mesh) 을혼합한여과액을묻힌붓으로떡잎또는본엽 1 2매전개된잎을수회문질러준비하였고, 흐르는물로곧바로세척한다음과천소재실험농장에서재배하였다. 접종후 25일경접종엽을제외한나머지잎을일괄채취하고, 필요한양만큼알루미늄호일로포장한다음 20 C에보관하면서필요시꺼내어사용하였다. 식물체전체 RNA 는제품사용설명서에따라 BCS Plant RNA Prep Kit (BioCube System, Gwacheon, Korea) 로정제하였고, 정제한 RNA는 20 C에보관하면서분리후 7일이내에사용하였다. RNA 농도는 Nanodrop ND2000 (Thermo Scientific, Waltham, MA, USA) 를이용하여 100 600 ng/µl 농도가되도록필요한농도로조절하여사용하였다. 검정용프라이머분석및제작. National Center for Biotechnology Information (NCBI) 에등록된바이러스염기서열을분석한다음전장염기서열이보고된대표바이러스를선발하고 (Table 1), 생물정보분석회사인씨더스 (Seeders, Daejeon, Korea) 에분석의뢰하였다. 분석방법을간단히요약하면, 바이러스유전체서열과토마토전장유전체서열, 토마토 EST, 2014년 11월까지 NCBI에등록된모든식물바이러스유전체염기서열 (plant virus genome sequence data, PVS data) 간의상동성분석을통해 5가지바이러스특이서열을 1차로선발하였다. 프라이머는선발서열과 Primer3 프로그램 (Koressaar와 Remm, 2007) 으로예상증폭산물의크

Research in Plant Disease Vol. 23 No. 2 195 Table 1. Description of reference viruses for primer design Virus NCBI accession no. Genome Genome size (bp) Isolate Country ToCV NC_007340.1 RNA 1 8,595 Florida USA NC_007341.1 RNA 2 8,247 Florida USA TSWV NC_002052.1 RNA L 8,897 CNPH1 USA NC_002050.1 RNA M 4,821 Almeria Spain NC_002051.1 RNA S 2,916 CPNH9 USA PepMoV NC_001517.1 RNA 9,640 California USA CMV NC_002034.1 RNA 1 3,357 Fny USA NC_002035.1 RNA 2 3,050 Fny USA NC_001440.1 RNA 3 2,216 Fny USA ToMV NC_002692.1 RNA 6,383 Queensland Australia ToCV, Tomato chlorosis virus; TSWV, Tomato spotted wilt virus; PepMoV, Pepper mottle virus; CMV, Cucumber mosaic virus; ToMV, Tomato mosaic virus. 기가 500 1,500 bp, Tm값은 60 C, 나머지조건은프로그램의기본값에적합하도록설계한다음, 마크로젠사 (Macrogen, Seoul, Korea) 에의뢰하여합성하였다. RT-PCR 반응액과증폭산물확인. RT-PCR 반응액은 Taq DNA polymerase의농도 (1 U/10 µl 또는 2 U/10 µl) 와활성억제방식을달리하여 cdna 합성과 DNA 증폭이동일반응액에서일어나도록 one-step 방식으로제조하였다. Taq DNA polymerase 활성억제방식은 RT-PCR mix1과 2는 chemical inhibition 방식, RT-PCR mix3과 4는 antibody inhibition 방식이며, 반응액에포함된 Taq DNA polymerase의농도는 RT- PCR mix1과 3은 1 U/10 µl, RT-PCR mix2와 4는 2 U/10 µl가되도록제작하였다. cdna 합성및 DNA 증폭은 PCR기 (Life Eco; Bioer Technology, Hangzhou, China) 에서수행하였다. 증폭산물가운데 10 µl를 EtBr 혹은 GelGreen (BioCube System) 이첨가된 1% agarose gel에서 100 V 5 A 조건으로 30 60분간전기영동하고, 반응결과는 gel-documentation system GDS200D (intron, Seongnam, Korea) 로확인하였다. RT-PCR mix와프라이머조합별특이도비교. 첫번째실험에서는식물 RNA 연구용으로시판되고있는 RT- PCR mix3과공시프라이머조합간반응양상을조사하였다. RT-PCR 반응액은주형을첨가하지않고, 2 RT-PCR mix 10 µl, 멸균수 8 µl, 10 pmol/µl 농도의정방향과역방향프라이머를각각 1 µl씩첨가하여제조하였다. 반응은 42 C/20분, 95 C/10분, 35회 (95 C/1분, 56.0 C, 59.6 C, 63.7 C 처리구에서 1분, 72 C/1분 ) 로수행하였으며, 반응양상은비특이증폭산물유무와정도 ( 약함과강함 ) 에따라판별하였다. 두번째실험에서는첫번째실험에서선발한프라이머조합을대상으로특이성을심층조사하였다. 건전한서광토마토의 RNA (100 ng/µl) 1 µl를주형으로사용하였고, RT-PCR mix 종류와 Tm값구배조건 (51.0 C, 52.2 C, 53.9 C, 56.0 C, 58.5 C, 60.7 C, 62.4 C), 멸균수 7 µl 첨가를제외한나머지실험조건은첫번째실험과동일하게하였다. 비특이정도는비특이증폭산물의총개수, 밴드진하기, dimer의진하기와넓이등을고려하여육안으로판별하였다. 선발한프라이머개수가적었던 ToCV와 TSWV 경우는조합별결과를젤그림상에서비교하였고, PepMoV, ToMV, CMV 프라이머조합은다음과같은조건으로비교하였다. 프라이어별로세가지 Tm값 (56.0 C, 59.6 C, 63.7 C) 에서발생한비특이증폭산물의총개수 (A), 1 kb DNA ladder의 500 bp 밴드의진하기를 1로했을때증폭산물의상대적인진하기 (B), 1 kb DNA ladder의 100 200 bp 사이의구간넓이를 3으로했을때 dimer의상대적인진하기와넓이 (0: 없음, 1: 얇거나흐림, 5: 넓거나아주진함 ) (C) 를 A B+C 형식으로수치화하였다. 그리고이들값을바이러스별로묶어평균값으로환산하고오차값을계산한다음그값이작을수록특이도가높은것으로판독하였다. RT-PCR mix 종류와주형농도, cdna 합성온도및시간별반응양상비교. 본실험에서는 4가지 RT-PCR mix 와 ToCV 진단용프라이머조합가운데주형을넣지않은상태에서가장특이도가높았던 C029 프라이머조합을사용

196 Research in Plant Disease Vol. 23 No. 2 하였다. 특이성은이전처럼비특이증폭산물을 A B+C 형식으로수치화한다음판별하였다. 주형농도, cdna 합성온도와시간변화에따른특이도비교는다음의조건 1, 2, 3에따라각각수행하였다. 조건 1: RT-PCR 반응액은 2 RT-PCR mix 10 µl, 멸균수 7 µl, 10 pmol/µl 농도의정방향과역방향프라이머를각각 1 µl, 농도를달리한 RNA (100, 200, 400, 800 ng/µl) 1 µl를첨가하여제조하였다. cdna 합성은 42 C/20분, PCR 반응은 95 C/10분, 35회 (95 C/1분, 58 C/1분, 72 C/1분 ) 로수행하였다. 조건 2: RT-PCR 반응액은조건 1의주형농도를 100 ng/ µl로고정하여제조하였고, cdna는 42 C, 45 C, 48 C, 51 C, 56 C 온도처리구에서 20분간합성하였으며, PCR 반응은조건 1과동일하게수행하였다. 조건 3: cdna는 48 C에서각각 5, 10, 15, 20분간합성하였고, 나머지는조건 2와동일하게실험하였다. 선발프라이머검출감도및특이도비교. 건전 RNA 와감염 RNA는토마토와주옥 (TSWV의경우 ) 토마토잎에서분리하였는데, 원액과이를 diethylpyrocarbonate (DEPC)- water로 10배씩연속희석한용액을주형으로사용하였다. RT-PCR 반응액은 2 RT-PCR mix3 10 µl, 멸균수 7 µl, 10 pmol/µl 농도의정방향과역방향프라이머를각각 1 µl, RNA ( 약 200 ng/µl) 1 µl를첨가하여제조하였으며, cdna 합성은 48 C/5분, PCR 반응은 95 C/10분, 35회 (95 C/1분, 58 C/1분, 72 C/1분 ), 72 C/1분 1회로수행하였다. 결과 프라이머조합별특이도비교. 시판중인 RT-PCR mix3 에주형을넣지않고, 세가지 Tm값 (56.0 C, 59.6 C, 63.7 C) 에서 46개프라이머조합 (Table 2) 의증폭양상을조사하였다. C041 (ToCV), C044 (ToCV), C073 (TSWV) 프라이머조합에서는 Tm값변화에상관없이비특이산물이증폭되지않았다. 반면에 ToCV 의 C029, C032, C040, PepMoV 의 C048, C054, C055, ToMV의 C065, C066, CMV의 C068, C069, C070, C071, TSWV의 C072 조합에서는비특이산물이약하게증폭되었고, 나머지프라이머조합에서는결과판독에영향을줄정도로비특이산물이많이증폭되었다 (Table 2). 한편, 프라이머이외에다른반응조건을최적화한다면비특이산물이조금생성된다할지라도목적바이러스진단에사용할수있을것으로판단되었다. 따라서비특이산물이증폭되지않거나약하게증폭된프라이머조합을이 후실험재료로선발하였다. 진단용 RT-PCR mix 선발. 이전실험에서선발한프라이머조합과건전한서광토마토 RNA를이용하여 RT- PCR mix별비특이산물생성정도를조사하였다. ToCV 진단용프라이머의경우 RT-PCR mix별증폭양상은비슷하였다. C041의경우주형을넣지않은상태에서는비특이산물이증폭되지않았으나 (Table 2), 건전한 RNA 주형이존재할때는특이도가다소떨어졌다 (Fig. 1). TSWV 진단용프라이머조합은 RT-PCR mix 간에비특이산물생성정도가다르게나타났는데, C072는 RT-PCR mix1과 4에서목적산물범위에비특이산물이많이생성되었고, C073은 RT-PCR mix4에서비특이정도가가장심하였다 (Fig. 1). 대체적으로 ToCV와 TSWV 프라이머조합에서는 RT-PCR mix2와 3이 RT-PCR mix1 과 4에비해다소양호한결과를보였으나, C072의경우 RT- PCR mix2와 3 간의특이도정도를 gel 그림으로세부비교하는것에는다소어려움이있었다 (Fig. 1). 따라서 PepMoV, ToMV, CMV 프라이머조합에서는재료및방법에서술한것처럼프라이머조합의비특이정도를바이러스별평균값과오차값의크기로비교하였다. PepMoV 와 CMV 프라이머조합의경우 ToCV 및 TSWV용프라이머조합에서처럼대체적으로 RT-PCR mix3에서양호한결과를보였으며, ToMV용프라이머조합에서는 RT-PCR mix1이가장특이도가높은것으로나타났다 (Fig. 2). 한편, Taq DNA polymerase 활성억제방식에따른비특이정도를비교했을때 chemical inhibition 방식의반응액 (RT-PCR mix1과 2) 이 antibody inhibition 방식 (RT-PCR mix3과 4) 보다다소높게나타났으며, 동일방식의반응액간에는 Taq DNA polymerase 의농도가 1 U/10 µl일때 2 U/10 µl보다비특이정도가낮게나타났다 (Fig. 2). cdna 합성조건최적화및선발프라이머적용. RT- PCR을이용한진단에서 cdna 합성은진단의특이성을결정짓는중요한단계이다. cdna 합성에적합한조건을찾고자공시한 4종의 RT-PCR mix에서주형의농도, cdna 합성온도및반응시간변화에따른비특이정도를비교하였다. 이를위해주형을넣지않은 RT-PCR mix3에서비특이정도가낮고, 건전한 RNA 주형첨가시비특이산물이약하게생성된 ToCV용 C041이적합하다고판단되어본실험에사용하였다. 각각의 RT-PCR 반응액에서주형 ( 서광토마토건전주의 RNA) 을농도별로첨가하고비특이정도를조사한결과, 비특이정도는 RT-PCR mix1, 3, 2, 4 순으로낮게나타났으며,

Research in Plant Disease Vol. 23 No. 2 197 Table 2. Parameters and suitability of primers used in this study Virus Code Forward primer Reverse primer Amplicon size (bp) Nonspecific background product* ToCV C029 ATCGGACATTATGTTCAAGG AAATCCTCATTACCACATCG 281 Weak C030 CCTGATTGGTTCTAAACTGC CCACACCTACCACGATACTT 198 Strong C031 ATTGCTTAGACCGATTCAGA TCACCCATTTTAGCGTACTT 372 Strong C032 TATGTGTCAGGCCATTGTAA TTCATAAGCAGGTTCGAGAT 348 Weak C033 TGATGTTTCCAGTGTTTTCA TGGTAAATGAAATTCCCAAC 418 Strong C034 GTGTTACGATGGGTTTGTTT CTTGAGTTTGTCCCAAAGAG 559 Strong C035 TTCCAAGTTCGATAGGAAGA CGAAACATATAACGACGAATC 565 Strong C036 TCGACTTTTCGAGGTTGTAT CCACACCTACCACGATACTT 556 Strong C037 CATGTTTTCCAACAGGAGAT GGGATTATCATATTGCCTCA 832 Strong C038 CTCTTTGGGACAAACTCAAG ATCGAACTTGGAAACGAGTA 980 Strong C039 GATGAAACCGAAGATGGATA AACAACAGTCAAATCGCTCT 943 Strong C040 TCTGATGCCTTGGTTACTTT AAGCAACAAACCTACCTTGA 564 Weak C041 TGATGTTTCCAGTGTTTTCA TAGTTTGCACCAAAGGAACT 527 No C042 ATCTCGAACCTGCTTATGAA TCAGACTCGGGTTAGGTAAA 657 Strong C043 TTTACCTAACCCGAGTCTGA TAGTTTGCACCAAAGGAACT 713 Strong C044 TACAGCTTAACCCGTTGAAT CATTGTCAATACAGTGGCAG 518 No C045 TAGACACATGGCAATGGTAA AAAACGCACTGTCTGTACCT 1,055 Strong PepMoV C046 GATGGATTTGGCTACAACAT CAGCTAATTCAAAAGCCCTA 443 Strong C047 AATAGTGTGTTTGGGTCGTC CTACCTGTGCCTTGACTCTC 308 Strong C048 TGTTCACTAGGCTCAGGAGT GACGACCCAAACACACTATT 461 Weak C049 GTGATGAATGGCTTAATGGT CCAAATAACCTTGTTTGAGC 371 Strong C050 ACACTGGCAATAATGCTTTT TCCAGTGGATTTACCAGAAC 390 Strong C051 TATCTGGTGGTCCATTTTTC CTACCTGTGCCTTGACTCTC 355 Strong C052 TGTTCACTAGGCTCAGGAGT GAAAAATGGACCACCAGATA 414 Strong C053 GTGGTCTCTGGGATGAAATA CCAACTACGATCACCTCCT 249 Strong C054 GAATGCTAGTTTTCTGTGGG ATGTTGTAGCCAAATCCATC 557 Weak C055 CACCATTCCAAGAATCAAAT CCAAATAACCTTGTTTGAGC 576 Weak C056 ATGATGAAAAACAGGTCGTC GACGACCCAAACACACTATT 613 Strong C057 AAGAGGAGCGAATTGTATCA ACCATTAAGCCATTCATCAC 694 Strong C058 GTTTTGCTGATAGAACCGAC TGAATTCGTTCTCCGTAACT 740 Strong C059 GAAGCTGCTCTGTATTGCTT CAAGAGAAGACTTGGACTGG 769 Strong C060 GACGCTCTAAGACGAAAAGA ATCAGGTTTGGCACTCTAAA 772 Strong C061 AAAGAATTCAGGCATTGAAA ATCAGGTTTGGCACTCTAAA 757 Strong C062 GGCTCTAAAAGCTGAACTGA TGATACAATTCGCTCCTCTT 745 Strong C063 AGTGGTTGGAATCTCAAATG CAGGGAATTTCATTATAGCG 815 Strong ToMV C064 TAGTTCAGCAAAGGTGGTTT ACGTCTGCATAAGTCTCACC 681 Strong C065 TTCACACAGTCTGACAAGGA GGAAGATCCACAAAATCAAA 824 Weak C066 AGATTGCTCAAAGATGGAGA AATTTCATGGTAACAGCGTC 1,064 Weak

198 Research in Plant Disease Vol. 23 No. 2 Table 2. Continued Virus Code Forward primer Reverse primer Amplicon size (bp) Nonspecific background product* CMV C067 CAGACTTATTCGTACCGAGG GTAGATGATTTCGCGGATAG 323 Strong C068 ACTCTCTGACGAGTTCGGTA CAATTCCATAATTCTTTCGC 392 Strong C069 GTCGAGTCATGGACAAATCT ACTGACCATTTTAGCCGTAA 803 Weak C070 ACATCAATGAATTGGTAGCC CTCGGTACGAATAAGTCTGG 876 Weak C071 CTATCCGCGAAATCATCTAC TCAACTTGACTAATGGTCCC 959 Weak TSWV C072 GCAATTCTGGTTCTCTATGC CATTCTTCAGAGTGTCCCAT 375 Weak C073 CTAATGAAATCGGAAAATCG AGAATAATCGGATAGTCGCA 197 No C074 GACAGCCTGGAACATAAAAG GAAAATGTGGAACACAAGGT 673 Strong ToCV, Tomato chlorosis virus; PepMoV, Pepper mottle virus; ToMV, Tomato mosaic virus; CMV, Cucumber mosaic virus; TSWV, Tomato spotted wilt virus. *RT-PCR was performed with each primer combination in the RT-PCR mix3 without template RNA. The condition was as follows cdna synthesis (42 C/20 min), denaturation (95 C/10 min), amplification (35 cycles of 95 C/1 min, 58.5 C/1 min, 60.7 C/1 min, 62.4 C/1 min, and 72 C/1 min), and final extension (72 C/10 min). The result was determined by visual observation for the average amount or intensity of nonspecific background products of each gel. C029 /281 RT-PCR mix1 RT-PCR mix2 RT-PCR mix3 RT-PCR mix4 M 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7 TSWV ToCV C032 /348 C041 /527 C072 /375 C073 /197 Fig. 1. Gel images of nonspecific background products on four different RT-PCR mixtures. RT-PCR was performed using 1 μl of RNA (100 ng/μl) from healthy tomato of Seogwang as template. The condition was as follows; cdna synthesis (42 C/20 min), denaturation (95 C/10 min), amplification (35 cycles of 95 C/1 min, 51.0 C 62.4 C/1 min, and 72 C/1 min), and final extension (72 C/10 min). Each gel is divided into eight lanes; lane M for 100 bp DNA ladder, lane 1 7 for 51.0 C, 52.2 C, 53.9 C, 56.0 C, 58.5 C, 60.7 C, and 62.4 C in order. The label at left side of each gel indicates target virus name and primer combination/expected size of amplicon. ToCV, Tomato chlorosis virus; TSWV, Tomato spotted wilt virus. RT-PCR mix4를제외한나머지반응액에서는주형의농도변화에관계없이일정하였다. 특이한것은 RT-PCR mix4의경우주형의농도가 200 ng/µl 이하일때보다 400 ng/µl 이상처리구에서비특이정도가더높게나타났다 (Fig. 3A). 주형의농도와 cdna 합성시간을 100 ng/µl와 20분으로고정하고온도변화에따른비특이정도를조사했을때, RT- PCR mix4를제외한나머지반응액에서는권장온도인 48 C 이하에서비슷한경향을보였으나, 역전사효소의활성이떨어지는 51 C 이상에서는 RT-PCR mix3이다른반응액에비 해영향을많이받았다 (Fig. 3B). 한편, cdna 합성시간에따른비특이정도는 RT-PCR mix1 을제외한나머지반응액에서합성시간이길어질수록증가하는경향을보였다. 비특이정도는 RT-PCR mix1이전시간대에서가장낮았고, 합성시간 10분에서 RT-PCR mix3은 RT-PCR mix1과비슷한결과를보였다 (Fig. 3C). RT-PCR mix1의경우 RT-PCR mix3에비해기능이다소우수하였으나, 최적화된 RT-PCR 조건에서는큰차이가없을것으로판단되어 RT-PCR mix3을이용해다음실험을진행

Research in Plant Disease Vol. 23 No. 2 199 하였다. RT-PCR mix3 에적합한조건을이전실험에서비특 이산물이생성되지않거나 (C041, C044, C073) 그정도가약 했던 (C029, C032, C040, C048, C054, C055, C065, C066, C069, C070, C071, C072) 프라이머조합에적용했을때, 바이러스 별로 ToCV 는 C029, TSWV 는 C072, CMV 는 C070, PepMoV 는 C048, ToMV C065 의특이도와민감도가가장좋았다 (data not shown). 이를기준으로바이러스진단을위해선발한 프라이머조합의전체적인결과를보면 C029 (ToCV), C048 Average value for non-specificity 8 7 6 5 4 3 2 1 0 A B C RT-PCR mix1 RT-PCR mix2 RT-PCR mix3 RT-PCR mix4 PepMoV ToMV RT-PCR mixtures CMV Fig. 2. Bar graph for average value of non-specificity in different RT-PCR mixtures. The average value was calculated from amount and intensity of nonspecific background products in each primer combinations for target virus. RT-PCR was performed at the condition described in Fig. 1 except for Tm value (56.0 C, 59.6 C, and 63.7 C). (A) PepMoV, Pepper mottle virus. (B) ToMV, Tomato mosaic virus. (C) CMV, Cucumber mosaic virus. (PepMoV), C070 (CMV) 은건전과감염 RNA 원액과희석액모두에서비특이산물이전혀생성되지않았고, 결과에영향을줄정도는아니었으나 C072 (TSWV) 와 C065 (ToMV) 는고농도시료에서비특이산물이증폭되었다 (Fig. 4). 고찰 본연구에서는바이러스특이프라이머조합을선발하고자토마토와대상바이러스의염기서열상동성을기초로바이러스특이염기서열을선발하고프라이머를제작하였다. 그럼에도불구하고주형을넣지않은 RT-PCR 반응에서프라이머조합에따라비특이산물이생성됨을확인하였다 (Table 2). 일반적으로 PCR mix에는 Taq DNA polymerase 정제방법에따라발현용벡터 DNA 잔존물이남게되는데 (Chen 등, 2015), 반응액내에존재하는벡터 DNA 잔존물과건전 RNA 간의간섭현상등이비특이산물생성의주요원인가운데하나일것으로판단되었다. 다만본실험결과는비특이산물이많이생성될수있도록 RT-PCR 검정조건이최적화되지않은상태에서수행하였다. 그러므로 C072처럼주형이없거나건전한 RNA 첨가조건에서일부비특이산물이생성된다고해서진단에활용할수없는것은아니다 (Fig. 3). 진단대상이되는바이러스에보다특이적인프라이머를선발하기위해서는기주식물과바이러스뿐만아니라 Taq DNA polymerase 단백질발현시스템인대장균유전체도함께분석할필요가있다 Average value for non-specificity 6 5 4 3 2 1 0 RT-PCR mix1 RT-PCR mix2 RT-PCR mix3 RT-PCR mix4 A B C 100 200 400 800 42 45 48 51 56 5 10 15 20 RNA concentration (100 ng/ l) Temperature(C) Incubation time (min) Parameters for cdna synthesis Fig. 3. Bar graph for average value of non-specificity according to the combinations between cdna synthesis parameter and reaction mixture in RT-PCR. The average value was calculated from amount and intensity of nonspecific background products in each primer combination. RT-PCR was performed with C029 primer combination of Tomato chlorosis virus at the condition described in Fig. 1 with exceptions; cdna synthesis (48 C/20 min) (A), template RNA (100 ng/μl) from healthy tomato of Seogwang (B), and cdna synthesis (20 min) (C). Tm value for PCR is 58 C over all test.

200 Research in Plant Disease Vol. 23 No. 2 ToCV C029/281 TSWV C072/375 CMV C070/876 PepMoV C048/461 ToMV C065/824 고판단된다. 한편, 프라이머종류와함께 RT-PCR mix 의조성과종류에 따라진단의특이성에차이를보였는데, TSWV 진단용 C072 는 RT-PCR mix1 과이와조성이다른 RT-PCR mix4 에서비특 이정도가심하고 (Fig. 1), 다른바이러스프라이머조합에 서는 RT-PCR mix2 와 4 에서그정도가심하였다 (Fig. 2). 이는 Taq DNA polymerase 농도가높을때비특이산물이많이생 성된다는이전의보고와관련이있을것으로판단되었다 (Usta 등, 2005). 반면에 ToCV 진단용프라이머는주형의농 도, cdna 합성온도와시간을달리했을때 RT-PCR mix 1 의 특이도가가장높은것으로나타났다 (Fig. 3). 이는 C072 와 C029 의결과가상충되는것으로 RT-PCR 반응액과선택한 프라이머간의친화성이진단의특이성에중요한요인임 을암시한다. RNA dilution Healthy Diseases M 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7 Fig. 4. RT-PCR detection of five tomato viruses using finally selected primer combinations for each virus. Each gel is divided into eight lanes; lane M for 100 bp DNA ladder, lane 1 for initial sap of purified total RNA, and lanes of 2 7 for 10-fold serial dilutions. The label at left side of each gel indicates target virus name and primer combination/expected size of amplicon. Template RNA was prepared from Seogwang tomato plant except for TSWV ( Juok tomato). RT-PCR was performed as follows: cdna synthesis (47 C/5 min), denaturation (95 C/10 min), amplification (35 cycles of 95 C/1 min, 65 C/1 min, and 72 C/1 min), and final extension (72 C/10 min). ToCV, Tomato chlorosis virus; TSWV, Tomato spotted wilt virus; CMV, Cucumber mosaic virus; PepMoV, Pepper mottle virus; ToMV, Tomato mosaic virus. 그러나 RT-PCR mix3 은 cdna 합성온도를역전사효소의 내성온도인 50 C 이하와합성시간 5 분이내에서는 RT-PCR mix1 과큰차이를보이지않았다 (Fig. 3). 결과적으로 RT-PCR mix3 은 RT-PCR mix1 에비해적용성이다소높은것으로보 인다. 끝으로 RT-PCR mix3 을이용해선발한프라이머의진 단특이성을보면, ToMV 용 C065 의경우주형이고농도일때 비특이산물이강하게증폭되었다 (Fig. 4). 이는경우에따라프라이머자체만으로비특이산물생성을억제하는것에는한계가있으며, 기주식물의전장유전체서열정보나분석방법이아직완전하지않음을의미한다고도할수있다. 예로써전장유전체조립시생물정보연구자들은대장균을포함한미생물염기서열을모두제거한다고하는데 (personal communication), 이것이본실험에어떤영향을주었는지는아직논의되어야할소지가많다고하겠다. 이런상황에서비특이산물생성을피하기위해서는 Taq DNA polymerase 정제시 DNA 오염이되지않은반응액을사용하던지 (Lawyer 등, 1993) 또는단백질프라이머종류를바꾸는것도한방법이지만, 당장은 RNA 주형준비시 DNase 처리를통한기주 DNA 제거가많은도움이될것으로판단된다. 요약 국내의토마토에서발생하는주요바이러스는 Tomato chlorosis virus (ToCV), Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Pepper mottle virus (PepMoV), Tomato mosaic virus (ToMV) 이다. 이들바이러스를진단하기위해프라이머세트와반응액을포함하는역전사중합반응 (RT- PCR) 법의조건을조사하였다. 공시한바이러스에특이적인염기서열로부터모두 46개프라이머세트를설계하고, 이를이용해주형을넣지않은 RT-PCR에서비특이반응을조사하였다. 이들가운데 16개조합을건전한토마토 RNA에적용한결과프라이머세트와 RT-PCR 반응액간의친화성이비특이반응감소에영향을주었다. cdna 합성과관련된인자와 RT-PCR 반응액사이의조합을근거로 ToCV 진단을위한두종류의반응액을선발하였다. ToCV 진단시수립된조건을나머지바이러스진단에적용했을때, 특이성이높은프라이머세트 C029 (ToCV), C072 (TSWV), C070 (CMV), C048 (PepMoV), C065 (ToMV) 를선발할수있었다. 이들프라이머세트는공시한바이러스를특이적으로진단하는데유용할것으로판단된다. Conflicts of Interest No potential conflict of interest relevant to this article was reported.

Research in Plant Disease Vol. 23 No. 2 201 Acknowledgement This research was supported by Agri-Bio industry Technology Development Program, Ministry of Agricµlture, Food and Rural Affairs (114069-02-2-WT011). References Aboul-Ata, A. E., Mazyad, H., El-Attar, A. K., Soliman, A. M., Anfoka, G., Zeidan, M., Gorovits, R., Sobol, I. and Czosnek, H. 2011. Diagnosis and control of cereal viruses in the Middle East. Adv. Virus Res. 81: 33-61. Boonham, N., Kreuze, J., Winter, S., van der Vlugt, R., Bergervoet, J., Tomlinson, J. and Mumford, R. 2014. Methods in virus diagnostics: from ELISA to next generation sequencing. Virus Res. 186: 20-31. Chen, S., Gu, H., Wang, X., Chen, J. and Zhu, W. 2011. Multiplex RT-PCR detection of Cucumber mosaic virus subgroups and Tobamoviruses infecting tomato using 18S rrna as an internal control. Acta Biochim. Biophys. Sin. (Shanghai) 43: 465-471. Chen, S., Zheng, X., Cao, H., Jiang, L., Liu, F. and Sun, X. 2015. A simple and efficient method for extraction of Taq DNA polymerase. Electron. J. Biotechnol. 18: 355-358. Cho, J. D., Kim, J. S., Kim, J. Y., Kim, J. H., Lee, S. H., Choi, G. S., Kim, H. R. and Chung, B. N. 2005. Occurrence and symptoms of Tomato spotted wilt virus on vegetables in Korea. Res. Plant Dis. 11: 213-216. (In Korean) Hassani-Mehraban, A. Westenberg, M., Verhoeven, J. T., van de Vossenberg, B. T., Kormelink, R. and Roenhorst, J. W. 2016. Generic RT-PCR tests for detection and identification of tospoviruses. J. Virol. Methods 233: 89-96. Hu, J. S., Li, H. P., Barry, K., Wang, M. and Jordan, R. 1995. Comparison of dot blot, ELISA, and RT-PCR assays for detection of two cucumber mosaic virus isolates infecting banana in Hawaii. Plant Dis. 79: 902-906. Ji, J., Oh, T. K., Lee, H. J., Kim, S. H., Rajangam, U., Kim, S. C., Kim, Y. S. and Choi, C. W. 2008. Molecular characterization of tomato infecting Tobacco leaf curl geminivirus isolated from Jeju island. Res. Plant Dis. 24: 238. (Abstract) Jeong, J. J., Ju, H. J. and Noh, J. 2014. A review of detection methods for the plant viruses. Res. Plant Dis. 20: 173-181. (In Korean) Kataya, A. R. A., Stavridou, E., Farhan, K. and Livieratos, I. 2008. Nucleotide sequence analysis and detection of a Greek isolate of Tomato chlorosis virus. Plant Pathol. 57: 819-824. Kil, E. J., Lee, Y. J., Cho, S., Auh, C. K., Kim, D., Lee, K. Y., Kim, M. K., Choi, H. S., Kim, C. S. and Lee, S. 2015. Identification of natural weed hosts of Tomato chlorosis virus in Korea by RT-PCR with root tissues. Eur. J. Plant Pathol. 142: 419-426. Kim, J. S., Lee, S. H., Choi, H. S., Kim, M. K., Kwak, H. R., Nam, M., Kim, J. S., Choi, G. S., Cho, J. D., Cho, I. S. and Chung, B. N. 2011. Occurrence of virus diseases on major crops in 2010. Res. Plant Dis. 17: 334-341. (In Korean) Kim, M. K., Kwak, H. R., Han, J. H., Ko, S. J., Lee, S. H., Park, J. W., Jonson, M. G., Kim, K. H., Kim, J. S., Choi, H. S. and Cha, B. J. 2008. Isolation and characterization of Pepper mottle virus infecting tomato in Korea. Plant Pathol. J. 24: 152-158. Kumar, S., Udaya, S. A. C., Nayaka, S. C., Lund, O. S. and Prakash, H. S. 2011. Detection of Tobacco mosaic virus and Tomato mosaic virus in pepper and tomato by multiplex RT-PCR. Lett. Appl. Microbiol. 53: 359-363. Koressaar, T. and Remm, M. 2007. Enhancements and modifications of primer design program Primer3. Bioinformatics 23: 1289-1291. Lawyer, F. C., Stoffel, S., Saiki, R. K., Chang, S. Y., Landre, P. A., Abramson, R. D. and Gelfand, D. H. 1993. High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5 to 3 exonuclease activity. PCR Methods Appl. 2: 275-287. Lee, J. S., Cho, W. K., Lee, S. H., Choi, H. S. and Kim, K. H. 2011. Development of RT-PCR based method for detecting five nonreported quarantine plant viruses infecting the family Cucurbitaceae or Solanaceae. Plant Pathol. J. 27: 93-97. Mumford, R. A., Barker, I. and Wood, K. R. 1994. The detection of tomato spotted wilt virus using the polymerase chain reaction. J. Virol. Methods 46: 303-311. Okuda, M. and Hanada, K. 2001. RT-PCR for detecting five distinct Tospovirus species using degenerate primers and dsrna template. J. Virol. Methods 96: 149-156. Park, M. R. and Kim, K. H. 2004. RT-PCR detection of three nonreported fruit tree viruses useful for quarantine purpose in Korea. Plant Pathol. J. 20: 147-154. Tomato Genome Consortium. 2012. The tomato genome sequence provides insights into fleshy fruit evolution. Nature 485: 635-641. Thottathil, G. P., Jayasekaran, K. and Othman, A. S. 2016. Sequencing crop genomes: a gateway to improve tropical agriculture. Trop. Life Sci. Res. 27: 93-114. Usta, M., Sipahioglu, H. M. and Polat, B. 2005. Studies on parameters influencing the performance of reverse transcriptase polymerase chain reaction (RT-PCR) in detecting Prunus necrotic ringspot virus (PNRSV). Phytopathol. Mediterr. 44: 189-194. Webster, C. G., Wylie, S. J. and Jones, M. G. K. 2004. Diagnosis of plant viral pathogens. Curr. Sci. 86: 1604-1607.