赵丽;古勤生;陈红运;史团省;田延平;胡京昂;

• 1:郑州大学生物工程系
• 2:中国农业科学院郑州果树研究所
• 3:厦门出入境检验检疫局

摘要(Abstract)：

小西葫芦黄花叶病毒(Zucchini yellowmosaic virus,ZYMV)、西瓜花叶病毒(Watermelon mosaic virus,WMV)和黄瓜花叶病毒(Cucumber mosaic virus,CMV)是危害葫芦科作物最严重、最广泛的3种病毒。研究建立了以18SrRNA为内标检测ZYMV、CMV和WMV侵染葫芦科作物3种主要病毒的多重RT-PCR方法,并对影响多重RT-PCR扩增的退火温度、Taq聚合酶浓度、dNTPs浓度和镁离子浓度4种主要因素进行优化;对多重RT-PCR的灵敏性进行了测定,并与单重RT-PCR灵敏性进行比较。结果表明:最佳的退火温度、Taq聚合酶量、dNTPs浓度和Mg2+浓度分别为60.7℃、1.0U、0.8mmol/L和2.5mmol/L。多重RT-PCR各病毒的灵敏度与单重RT-PCR相同。

关键词(KeyWords)： 西葫芦(Cucurbita moschata Duch);病毒病;多重RT-PCR

Abstract:

Keywords:

基金项目(Foundation): 河南省科技攻关项目(082102150012)

作者(Author): 赵丽;古勤生;陈红运;史团省;田延平;胡京昂;

Email:

参考文献(References)：

• [1]CHEN Jie-yun,CHEN Ji-shuang,CHAI Li-hong,WANG Huai-ji,YANG Chen-jie.Molecular detection and pathogenic testing of two viruses infecting cucurbitaceoud crops[J].Acta Phytopathologica Sinica,2003,33(5):449-455.陈洁云,陈集双,柴立红,王槐基,杨陈洁.两种葫芦科病毒的分子检测和致病性研究[J].植物病理学报,2003,33(5):449-455.
• [2]GU Qin-sheng,FAN Zai-feng,LI Huai-fang.List of cucurbits virus name[J].China Watermelon and Muskmelon,2002(1):45-47.古勤生,范在丰,李怀方.葫芦科作物病毒名录[J].中国西瓜甜瓜,2002(1):45-47.
• [3]XIAO Huo-gen,FAN Huai-zhong.Studies on the detection of pa-paya Ringspot Virus in infected tissue of plant byELISA[J].Virolog-ica Sinica,1994,9(3):249-255.肖火根,范怀忠.植物组织粗汁液中的番木瓜环斑病毒的ELISA检测技术[J].中国病毒学,1994,9(3):249-255.
• [4]GU Qin-sheng,ROGGERO P,LENZI R,FAN Zai-fang,LI Huai-fang,YU Zheng-wang.Detection of zucchini yellow mosaic virus in northern China and resistance test in some watermelon cultivars[J].Journal ofFruit Science,2002,19(3):184-187.古勤生,ROGGERO P,LENZI R,范在丰,李怀方,俞正旺.北方地区小西葫芦黄花叶病毒的酶联检测和西瓜品种抗病性鉴定[J].果树学报,2002,19(3):184-187.
• [5]PENG Jun,WANG Guo-fen,HUANG Jun-sheng,DAI Peng,XIE Yu-ping,LI Wei-dong.Development of a multiplex PCR protocol for the detection of two viruses of banana[J].Acta Horticuturae Sin-nica,2006,33(4):845-848.彭军,王国芬,黄俊生,代鹏,谢玉萍,李伟东.香蕉两种主要病毒多重PCR检测方法的建立[J].园艺学报,2006,33(4):845-848.
• [6]ROY A,FAYAD A,BARTHE G,BRLANSKY R H.A multiplex polymerase chain reaction method for reliable,sensitive and simul-taneous detection of multiple viruses in citrus trees[J].VirologicalMethods,2005(129):47-55.
• [7]ITOT,IEKI H,OZAKI K.Simultaneous detection of sixcitrus viroids and Apple stemgroovingvirus fromcitrus plants bymultiplexreverse transcription polymerase chain reaction[J].Journal of Virological Methods,2002,106:235-239.
• [8]BERTOLINI E,OLMOSA,CARMENMART1′NEZM,GORRISMT,CAMBRA M.Single-step multiplex RT-PCR for simultaneous and colourimetric detection ofsixRNAviruses in olive trees[J].Journal of Virological Methods,2001,96:33-41.
• [9]PERIASAMY M,NIAZI F R,MALATHI V G.Multiplex RT-PCR,a novel technique for the simultaneous detection of the DNA and RNA virusescausingrice tungrodisease[J].Journal ofVirological Methods,2006,134:230-236.
• [10]GAMBINOG,GNBAUDOI.Simultaneousdetection ofnine grapevine viruses bymultiplexreverse transcription-polymerase Chain reaction with coamplification of a plant RNA as internal control[J].Virology,2006,96(11):1223-1229.
• [11]WANG Chong,CHEN Ji-shuang,HONG Jian,DU Zhi-you,ZHANG Hua-rong,CHENShao-ning.Optimization and application of a mul-tiplex RT-PCR system for simultaneous detection of three lily virus using18SrRNAas internal control[J].Acta Phytopathologica Sinica,2006,36(3):204-211.王冲,陈集双,洪健,杜志游,张华荣,陈绍宁.以18S rRNA为内参照的多重RT-PCR检测3种百合病毒[J].植物病理学报,2006,36(3):204-211.
• [12]HASSAN M,MYRTA A,POLAK J.Simultaneous detection and i-dentification of four pome fruit viruses by one-tube pentaplex RT-PCR[J].Virological Methods,2006(133):124-129.
• [13]NIU Jian-xin,MA Bing-gang,HE Mei,LI Hai-sheng,ZHAO Ying,LI Si-ping,ZHOU Duo-jin.Multiple RT-PCR detection of viruses in pear[J].Acta Phytopathologica Sinica,2006,36(1):12-21.牛建新,马兵钢,何梅,李海生,赵英,李四平,周多进.库尔勒香梨主要病毒多重RT-PCR检测技术研究[J].植物病理学报,2006,36(1):12-21.
• [14]DING Fang,CAO Qing,WANG Guo-ping,YI Gan-jun,ZHONG Yun.Studies on the simultaneous detection of citrus Huanglongbing pathogen,Citrus exocortis viroid,Citrus tristeza virus by multiplex RT-PCR[J].ActaHorticuturae Sinnica,2006,33(5):947-952.丁芳,曹庆,王国平,易干军,钟云.柑橘黄龙病、裂皮病和衰退病病原的多重RT-PCR检测[J].园艺学报,2006,33(5):947-952.